2011 (SBTE) 25th Annual Meeting Proceedings - International ...
2011 (SBTE) 25th Annual Meeting Proceedings - International ...
2011 (SBTE) 25th Annual Meeting Proceedings - International ...
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<strong>Proceedings</strong> of the 25 th <strong>Annual</strong> <strong>Meeting</strong> of the<br />
Brazilian Embryo Technolo<br />
echnology Societ<br />
ciety (<strong>SBTE</strong>)<br />
The impact of reproductive biotechnologies on<br />
animal health and production<br />
Cumbuc<br />
umbuco, CE, Brazil<br />
August 18 to 20, <strong>2011</strong>.
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
From the President<br />
Dear colleagues,<br />
Welcome to Cumbuco! It is with a great pleasure that we bring, for the second time in the <strong>SBTE</strong>’s<br />
history, our <strong>Annual</strong> <strong>Meeting</strong> to Ceará State. The called “Land of Light” is one of the most beautiful areas in<br />
the Northeast of Brazil and has magnificent beaches like this in Cumbuco. There could not be a more appropriate<br />
stage for a scientific meeting and a very special moment for the professionals in the embryo technology.<br />
This year, we decided to bring to our scientific program, in addition to technologies already in use,<br />
emerging issues in reproductive technology. Also, with great satisfaction, <strong>SBTE</strong> brings the first meeting of the<br />
Innovation Network on Animal Reproduction (EMBRAPA), which presents and discusses important issues to<br />
the development of our activity. The <strong>Annual</strong> <strong>Meeting</strong>, as usual, has a high level scientific program. We hope<br />
you all enjoy.<br />
Trusting in the relevance of our meeting to the scientific progress and professional qualification, we<br />
received the institutional support from CNPq and CAPES, traditional <strong>SBTE</strong>’s partners, and now from CRMV-<br />
CE, additionally. I also thank the support of condominium of companies, which demonstrate the importance<br />
of the interaction between science and commercial activity.<br />
At the end of our administration (2010-<strong>2011</strong>), I would like to thank all members of the current board:<br />
Pietro Baruselli, Alexsandra Pereira, Rodrigo Alonso, Dárcio Teixeira, Guilherme Nogueira, Marcelo Bertolini<br />
e Luciana Melo. These people faced the great challenge that was to continue the excellent job made by the<br />
previous administrations, seeking to contribute to the advancement of our society. In the spirit of renewal, we<br />
wish success to the new board, which can count on our full support.<br />
Vicente José de Figueirêdo Freitas<br />
<strong>SBTE</strong> President - 2010-<strong>2011</strong><br />
II
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
From the Scientific Committe<br />
Greetings!<br />
It has been a great pleasure to serve you and our society at the scientific committee for the past two<br />
years.With the combined efforts of intensive and productive collaboration among ourselves, along with the<br />
tireless support from our associates, post-docs and graduate and undergraduate students, we have strived to<br />
meet our commitment to you, and to bring you the most excellent scientific events and best opportunities for<br />
technical advancement as possible during our annual meetings.<br />
Our Brazilian society has grown and matured into a well respected organization worldwide. Our<br />
annual meeting has attained international proportions, and we are pleased to have many Latin-American<br />
colleagues as faithful <strong>SBTE</strong> members and regular attendees in our audience, as well as the presence of<br />
participants from many countries throughout North America and Europe. Carrying out annual meetings at<br />
an international scale has been a tradition for <strong>SBTE</strong>, with the continuous presence of distinguished invited<br />
speakers from Brazil and abroad, who come to us to address issues that are of broad relevance to our country<br />
and economy. The scientific contents of our annual meetings have been directed towards promoting progress<br />
and development to every corner where we may exert influence as professionals and/or members of academia<br />
and research institutes. To keep up with the tradition, we have carefully analyzed the topics and themes<br />
discussed at our past events over the years, with the goal of maintaining those ideas that have been greatly<br />
accepted and anticipated by the public. In addition, by envisioning the present and future of our society, we<br />
attempted to uncover fields and areas of interest that have been less frequently addressed at the meetings. In<br />
doing so, we hope to maintain aspects of the program that have been successful in our scientific discussions<br />
over the years, as well as to expand the scope of the scientific program by including advanced topics that<br />
inexorably have relevance in what we all do, either directly or indirectly, currently or in the near future to us<br />
all.<br />
We must all be aware that the scientific contents of our meetings cannot possibly cover all aspects in<br />
animal reproduction at once. However, as <strong>SBTE</strong> looks forward into long-term planning, all areas and themes N<br />
are envisioned to be covered over time. That is the reason we try to have a healthy, productive and transparent<br />
transition between administrations. Also, as <strong>SBTE</strong> is characterized by such wide diversity in terms of<br />
backgrounds, interests, and activities, the programs in the past two annual meetings (24 th and 25 th ) have been<br />
organized in sections blocked as common subjects so that you could focus your attention on specific sections.<br />
This design also allows you to use the time during sections of lower personal interest to carry out other<br />
activities related or unrelated to our event, such as for leisure and entertainment. The simultaneous preconference<br />
symposia offer you a choice of six themes that range from in-depth scientifically oriented topics to<br />
very practical and applied subjects. Following a similar pattern, the main conferences are organized in<br />
sessions covering either basic scientific topics or practical aspects, centered on the application of current<br />
biotechnology of reproduction in an array of animal species, with the bovine species as the main axis.<br />
On behalf of the scientific committee, we would like to take this opportunity to thank: (a) the members<br />
of the Executive Board, for their trust in our ability to carry out our tasks at the Scientific Committee, and for<br />
the organization of this terrific event as a whole; (b) the invited speakers, for their great insights, outstanding<br />
knowledge, and high quality of the scientific material shared with us, along with their kind patience and<br />
willingness to work hard to meet our short deadlines; (c) the Chairs of the pre-conference symposia, for<br />
organizing such exceptional sessions; (d) each member of the Scientific Committee, Section Editors, and<br />
abstract reviewers, for their tremendous efforts to help us in preparing the proceedings in time; (e) the individuals<br />
who worked relentlessly on the never-ending task of formatting and editing the proceedings, along with the<br />
support from the Editor and staff at the Journal Acta Scientiae Veterinariae; (f) the Section Chairs at the main<br />
event, for volunteering to host our invited speakers; and (g) the <strong>SBTE</strong> staff, composed of the <strong>SBTE</strong> secretary<br />
and our own students and scholars, who tirelessly helped us all during the events.<br />
Finally, your participation in the scientific program has been the very soul of our conferences. In both<br />
annual meetings (24 th and 25 th ), close to 500 abstracts have been accepted and presented at the meeting as<br />
III
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
posters. Also, we had more than 60 initial abstract entries for the <strong>SBTE</strong> Student Competition Award, and<br />
numerous entries for the other three important categories of awards given by <strong>SBTE</strong>. This has been a true<br />
demonstration of your engagement to our society and trust in our efforts. For those important reasons, we<br />
thank YOU, for your faithful support to our society and for coming to our most important venue seeking novel<br />
knowledge, social and scientific interactions, open-minded discussions, and fruitful collaborations during<br />
our meeting. Again, it was truly a privilege to serve you and our society at both the 25 th anniversary (2010)<br />
and the 25 th annual meeting (<strong>2011</strong>) of the Brazilian Embryo Technology Society.<br />
I hope you enjoy this meeting. We look forward to an exciting experience shared by all!<br />
Marcelo Bertolini<br />
Chair of the Scientific Committee - 2010-<strong>2011</strong><br />
www.ufrgs.br/actavet<br />
Acta Scientiae Veterinariae. 39(Suppl 1)<br />
<strong>2011</strong><br />
IV
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
25 th ANNUAL AL MEETING OF THE BRAZILIAN EMBRYO TECHNOLOGY OGY SOCIETY (<strong>SBTE</strong>)<br />
August 18 to 20, <strong>2011</strong> - Cumbuco, CE, Brazil<br />
Executive Board<br />
Vicente José de Figueirêdo Freitas,<br />
President<br />
Pietro Sampaio Baruselli, Vice-<br />
President<br />
Dárcio Ítalo Alves Teixeira, 1 st Treasurer<br />
Guilherme de Paula Nogueira, 2 nd<br />
Treasurer<br />
Alexsandra Fernandes Pereira, 1 st Secretary<br />
Rodrigo Vitório Alonso, 2 nd Secretary<br />
Luciana Magalhães Melo, Director of<br />
Communications<br />
Marcelo Bertolini, Scientific Committee<br />
Scientific Committee<br />
Marcelo Bertolini, Chair<br />
Carlos Eduardo Ambrósio<br />
Dárcio Ítalo Alves Teixeira<br />
Fabiana Forell<br />
Fernanda da Cruz Landin-Alvarenga<br />
Fidel Ovídio Castro<br />
Flávio Vieira Meirelles<br />
João Henrique Moreira Viana<br />
Jorge Luis Zambrano Varón<br />
José Luiz Rodrigues<br />
José Buratini Júnior<br />
Mário Binelli<br />
Matthew B. Wheeler<br />
Ricardo C. Chebel<br />
Section Editors<br />
Marcelo Bertolini, Editor-in-Chief<br />
Carlos Eduardo Ambrósio<br />
Fernanda da Cruz Landin-Alvarenga<br />
Flávio Vieira Meirelles<br />
João Henrique Moreira Viana<br />
José Buratini Jr.<br />
Mário Binelli<br />
Ricardo C. Chebel<br />
Manuscripts and abstract<br />
reviewers<br />
Alejandro Gibbons<br />
Alexandre Henryli de Souza<br />
Andre Penido de Oliveira<br />
Anibal Ballarotti do Nascimento<br />
Anna Carolina Denicol<br />
Anthony Cesar Souza Castilho<br />
Antônio Chaves de Assis Neto<br />
Barbara Loureiro<br />
Bruno Campos Carvalho<br />
Carlos Antônio Carvalho Fernandes<br />
Carlos Eduardo Ambrósio<br />
Carlos Jose Hoff de Souza<br />
Celina Furlanetto Mançanares<br />
Christina Ramires Ferreira<br />
Ciro Moraes Barros<br />
Claudia Lima Verde Leal<br />
Claudia Maria Bertan Membrive<br />
Daniele dos Santos Martins<br />
Dárcio Ítalo Alves Teixeira<br />
Denise Lopes<br />
Eduardo Kenji N. Arashiro<br />
Eduardo Ramos de Oliveira<br />
Erica Zimberknopf<br />
Fabiana Bressan<br />
Fabiana Forell<br />
Fabiola Freitas Paula Lopes<br />
Felipe Perecin<br />
Felipe Zandonadi<br />
Fernanda da Cruz Landin-Alvarenga<br />
Fernando Silveira Mesquita<br />
Flavia Lombardi Lopes<br />
Flávio Vieira Meirelles<br />
Frederico Ozanam Papa<br />
Gisele Zoccal Mingoti<br />
Guilherme de Paula Nogueira<br />
Hymerson Costa Azevedo<br />
Ian Martin<br />
Ines Cristina Giometti<br />
Jeferson Ferreira da Fonseca<br />
João Henrique Moreira Viana<br />
Jorge Luis Zambrano Varón<br />
José Antonio Dell’Aqua Jr.<br />
José Buratini Junior<br />
José Carlos Ferrugem Moraes<br />
Jose Luiz Moraes Vasconcelos<br />
José Luiz Rodrigues<br />
José Nélio S. Sales<br />
José Roberto Viana Silva<br />
Juliana Lopes Almeida<br />
Klibs N. Galvao<br />
Ligia Garcia Mesquita<br />
Ligia Pegoraro<br />
Lilian de Jesus Oliveira<br />
Lilian Tamy Iguma<br />
Lúcia Daniel Machado da Silva<br />
Luciana Relly Bertolini<br />
Luciano Andrade Silva<br />
Luiz Gustavo Bruno Siqueira<br />
Luiz Sergio Almeida Camargo<br />
Manoel Francisco de Sá Filho<br />
Mara Iolanda Batistella Rubin<br />
Marcelo Bertolini<br />
Marcelo Fabio Gouveia Nogueira<br />
Marcelo Marcondes Seneda<br />
Marcelo Rezende Luz<br />
Marco Aurélio Carneiro Meira Bergamaschi<br />
Marcos R Chiaratti<br />
Margot Alves Nunes Dode<br />
Maria Angelica Miglino<br />
Mário Binelli<br />
Maurício Machaim Franco<br />
Mayra Elena Ortiz D‘Avila Assumpção<br />
Michele Munk Pereira<br />
Miller Pereira Palhão<br />
Milton Passipieri<br />
Paula de Carvalho Papa<br />
Paulo Bayard Dias Gonçalves<br />
Rafael Augusto Satrapa<br />
Raquel Varella Serapião<br />
Reno Roldi de Araujo<br />
V<br />
Ricardo C. Chebel<br />
Roberto Sartori Filho<br />
Robson Fortes Giglio<br />
Rogério Ferreira<br />
Ronaldo Luis Aoki Cerri<br />
Rui Machado<br />
Sabine Wohlres Viana<br />
Sony Dimas Bicudo<br />
Vilceu Bordignon<br />
<strong>Proceedings</strong> formatting and<br />
editing<br />
N<br />
Molecular and Developmental Biology Lab<br />
University of Fortaleza (UNIFOR)<br />
Marcelo Bertolini, Editor-in-Chief<br />
Juliana Lopes Almeida, Coordinator<br />
Luciana Relly Bertolini<br />
Ana Karoline Freire da Rocha<br />
Cristiano Feltrin<br />
Felipe de Jesus Moraes Júnior<br />
Igor de Sá Carneiro<br />
Kaio César Simiano Tavares<br />
Leonardo Tondello Martins<br />
Luís Fernando Schütz<br />
Maurício Barbosa Salviano<br />
Saul Gaudêncio Neto<br />
Victor Hugo Vieira Rodrigues<br />
Chairs of the Pre-Conference<br />
Symposia<br />
Carlos Eduardo Ambrósio<br />
Cezinande de Meira<br />
Jéferson Fonseca<br />
José Buratini Jr.<br />
Maurício Machain Franco<br />
Pietro Sampaio Baruselli<br />
Acta Scien<br />
enti<br />
tiae Veterin<br />
eterinari<br />
ariae<br />
ae<br />
Laerte Ferreiro, Editor<br />
Itabajara da Silva Vaz Jr<br />
Bruna Crespo Barbosa
CONTENTS<br />
ISSN 1679-9216 (Online)<br />
Acta Scientiae Veterinariae. 39 (Suppl 1): s1-s467<br />
<strong>2011</strong><br />
PRE-CONFERENCE SYMPOSIA<br />
<strong>SBTE</strong> <strong>2011</strong><br />
SYMPOSIUM 1: STRATEGIES TO OPTIMIZE FERTILITY AND GENETIC IMPROVEMENT IN CATTLE<br />
What is producing the dramatic improvement in reproductive efficiency in U.S. dairy herds from 2000 until now? ....... s1<br />
Milo Wiltbank, Alexandre H. Souza, Jerry N. Guenther, Mary M. Herlihy & Roberto Sartori .<br />
How FTAI and FTET impact reproductive efficiency of Brazilian dairy herds ................................................................... s3<br />
Carlos Alberto Rodrigues, Roberta Machado Ferreira, Lais Mendes Vieira, Andressa L. Ranieri, Péricles R.L. Silva & Pietro<br />
Sampaio Baruselli<br />
Fixed time artificial insemination and embryo transfer programs in Brazil ..................................................................... s15<br />
Luis Fernando Nasser, Luciano Penteado, Carlos R. Rezende, Manoel F. Sá Filho & Pietro Sampaio Baruselli<br />
Implementation of DNA markers to produce genomically-enhanced EPDs in Nellore cattle ........................................ s23<br />
Raysildo Barbosa Lôbo, Donald Nkrumah, Daniela do Amaral Grossi, Priscila Sales de Barros, Pablo Paiva, Luiz Antônio<br />
Framatino Bezerra, Henrique Nunes de Oliveira & Marcos Vinícius Barbosa da Silva<br />
SYMPOSIUM 2: ADVANCES IN EMBRYO PRODUCTION AND EMBRYO TRANSFER TECHNOLOGY IN SMALL RUMINANTS<br />
State-of-the-art in the superovulation of ewes ................................................................................................................ s29<br />
Maria Emilia Franco Oliveira<br />
State-of-the-art in the transcervical embryo collection in goats and sheep ................................................................... s37<br />
Alberto Lopes Gusmão<br />
In vitro culture of goat preantral follicles ......................................................................................................................... s43<br />
José Ricardo de Figueiredo, Ana Paula Ribeiro Rodrigues & Valdevane Rocha Araújo<br />
Use of embryo transfer for the preservation of small ruminants in risk of extinction ...................................................... s45<br />
Edilson Soares Lopes Júnior & Vicente José de Figueirêdo Freitas<br />
SYMPOSIUM 3: THE INTERFACE BETWEEN HUMAN AND ANIMAL REPRODUCTION<br />
State-of-the-art in human assisted reproduction ............................................................................................................. s47<br />
Eduardo Leme Alves da Motta, Marcilio Nichi & Paulo Cesar Serafini<br />
The equine model to study the influence of obesity and insulin resistance in human ovarian function ........................ s57<br />
Eduardo Leite Gastal, Melba de Oliveira Gastal, Áurea Wischral & Jeremy Davis Schimidt<br />
Using animals to develop assisted human reproduction ............................................................................................... s71<br />
Carlos Gilberto Almodin<br />
The role of veterinarians in human in vitro embryo production ...................................................................................... s73<br />
Daniela Paes de Almeida Ferreira Braga & Edson Borges Jr.<br />
SYMPOSIUM 4: STEM CELLS AND GAMETE AND EMBRYO EPIGENETICS<br />
Veterinary applications in regenerative medicine; development of induced pluripotential stem cells (iPSC) in dogs .. s81<br />
Sehwon Koh, Steve Bischoff, Shengdar Tsai & Jorge A. Piedrahita<br />
Reprogramming somatic cells: pluripotency through gene induction and nuclear transfer ........................................... s83<br />
Fabiana Fernandes Bressan, Felipe Perecin, Juliano Rodrigues Sangalli & Flávio Vieira Meirelles<br />
Fetal membranes stem cells aplication in small animals ............................................................................................... s97<br />
Carlos Eduardo Ambrósio, Cristiane Valverde Wenceslau, José Luiz Nogueira, Dilayla Kelly de Abreu, Elaine Aparecida<br />
Fernandes Rodrigues, Thais Borges Lessa, Daniele dos Santos Martins, Luciana Relly Bertolini & Maria Angelica Miglino<br />
Reprogramming of genomic imprints by in vitro culture and cloning procedures in cattle .......................................... s103<br />
Lawrence Charles Smith, João Suzuki Jr., Felipe Perecin & Flávio Vieira Meirelles<br />
WORKSHOP 5: THE USE OF DOPPLER ULTRASONOGRAPHY ON EQUINE REPRODUCTION<br />
Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares ........................... s105<br />
Jair Camargo Ferreira, Fernanda Saules Ignácio & Cezinande de Meira<br />
VII
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
Mare’s folliculogenesis: assessment of ovarian and perifollicular vascular perfusion by Doppler ultrasound ........... s113<br />
Renata Cristina Uliani, Luciano Andrade Silva & Marco Antonio Alvarenga<br />
Uterine and luteal hemodynamic evaluation of the non pregnant mare ...................................................................... s117<br />
Fernanda Saules Ignácio, Jair Camargo Ferreira & Cezinande de Meira<br />
Local effect of the conceptus on uterine vascular perfusion and remodeling during early pregnancy in mares - new<br />
findings by Doppler ultrasonography .................................................................................................................... s123<br />
Luciano Andrade Silva<br />
WORKSHOP 6: RESEARCH NETWORK IN ANIMAL REPRODUCTION<br />
The innovation network in animal reproduction: EMBRAPA’s experience in organizing a research project on<br />
reproductive biotechnology ................................................................................................................................... s135<br />
Maurício Machain Franco, Aiesca Pellegrin, Ricardo Alamino Figueiredo, Alexandre Rossetto Garcia, Luiz Sergio Almeida<br />
Camargo, Hymerson Costa Azevedo & João Henrique Moreira Viana<br />
MAIN CONFERENCE<br />
<strong>SBTE</strong> <strong>2011</strong><br />
The year 2009 worldwide statistics of embryo transfer in domestic farm animals summary of the <strong>International</strong> Embryo<br />
Transfer (IETS) Data Retrieval Committee Report ................................................................................................ s139<br />
Brad Stroud & Gabriel A. Bó<br />
Temporal historical observations, rapidly expanding technological tools, and integration of scientific disciplines to<br />
enhance reproductive performance of lactating dairy cows: the foster mothers of the human race .................... s147<br />
William W. Thatcher<br />
SESSION 1: THE FEMALE EFFECT ON ANIMAL REPRODUCTION<br />
Mechanisms involved in selection of a single dominant follicle and regression of the corpus luteum: Does a common<br />
differentiating cell type, the granulosa/large luteal cell, underlie these two disparate physiological events? .... s171<br />
Milo Wiltbank, Brian W. Kirkpatrick, Michele R. Bastos, Paulo D. Carvalho, Gulnaz Yilmazbas-Mecitaglu, Chris A. Burke,<br />
Alexandre H. Souza & Roberto Sartori<br />
Deciphering early sensor and driver properties of the endometrium: contribution of the uterus to pregnancy outcome . s173<br />
Olivier Sandra<br />
Use of applied reproductive technologies (FTAI, FTET) to improve the reproductive efficiency in dairy cattle ........... s183<br />
Ricardo C. Chebel<br />
SESSION 2: DEVELOPMENTAL BIOLOGY: FROM NORMAL TO ABNORMAL<br />
From hatching into fetal life in the pig ............................................................................................................................ s203<br />
Poul Hyttel, Kristian M. Kamstrup & Sara Hyldig<br />
Abnormalities in bovine conceptus development during the embryonic phase after in vitro fertilization (IVF) and cloning<br />
by nuclear transfer (NT) ......................................................................................................................................... s223<br />
Antônio Chaves de Assis Neto, Álvaro Riveiros Galdos, Ana Carolina Furlanetto Mançanares, Míryan Lança Vilia Alberto,<br />
Carlos Eduardo Ambrósio, Phelipe Oliveira Favaron, Flavio Vieira Meirelles & Maria Angelica Miglino<br />
The placenta of bovine clones ...................................................................................................................................... s227<br />
Pascale Chavatte-Palmer, Rita Sau Fong Lee, Sylvaine Camous, Nicolas Le Cleac’h, Hélène Jammes & Michel Guillomot<br />
Clinical disorders observed during the first 30 days of life of cloned Zebu calf ........................................................... s243<br />
Eduardo Harry Birgel Junior, Flávio Vieira Meirelles, Eliza Rossi Komninou, Mariana Tikuma Nunes, Fábio Celidonio<br />
Pogliani, Paulo Fantinato Neto, Melina Marie Yasuoka, José Rodrigo V. Pimentel, Flávia Saldanha Kubrusly & Maria Angélica<br />
Miglino<br />
SESSION 3: ADVANCED REPRODUCTIVE AND SUPPORTIVE TECHNOLOGIES<br />
Reproductive technologies and epigenetics their implications for genomic selection in cattle ................................... s253<br />
Patrice Humblot<br />
Application of functional genomic approaches to the study of placental and fetal function ......................................... s263<br />
Jorge A. Piedrahita<br />
VIII
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
Derivation and potential applications of pluripotent stem cells for regenerative medicine in horses .......................... s273<br />
Olivia Eilers Smith, Bruce Douglas Murphy & Lawrence Charles Smith<br />
Recent advances in micromanipulation and transgenesis in domestic mammals ...................................................... s285<br />
Daniel Salamone, Romina Bevacqua, Federico Pereyra Bonnet, Andrés Gambini, Natalia Canel, M. Inés Hiriart, Gabriel<br />
Vichera, Lucia Moro & Javier Jarazo<br />
Genetic engineering of livestock to improve human health: The human lysozyme transgenic goat model ................ s295<br />
Elizabeth Ann Maga & James Donald Murray<br />
SESSION 4: MULTI-SPECIES<br />
Use of Color-Doppler Ultrasonography to Monitor Follicle Dynamics in Horses ......................................................... s301<br />
Eduardo Leite Gastal<br />
Biotechnology of reproduction in the canine species: where do we go? ..................................................................... s303<br />
John Verstegen, Karine Verstegen-Onclin & Karine Reynaud<br />
Increasing ovulation quota: more than a matter of energy ............................................................................................ s305<br />
Carlos G. Gutierrez, Silene Ferraro, Victor Martinez, Adriana Saharrea, Clarisa Cortez, Arantzatzu Lassala, Héctor Basurto &<br />
Joel Hernandez<br />
Ovum pick-up and in vitro embryo production in buffalo species: an update ............................................................... s317<br />
Bianca Gasparrini<br />
ABSTRACTS<br />
<strong>SBTE</strong> <strong>2011</strong><br />
STUDENT COMPETITION<br />
A001 Changes on Oocyte Quality of Repeat Breeder Holstein Cows May Explain Their Reduced Fertility ............... s337<br />
R.M. Ferreira, M.R. Chiaratti, H. Ayres, M.L. Ferraz, C.A. Rodrigues, et al.<br />
A002 Effect of Zona Pellucida Deficiency on Early Development of Bovine Embryos ................................................. s337<br />
A.E. Velasquez, J.R. Manriquez, F.O. Castro, L. Rodriguez<br />
A003 Effect of Ethanol Extract of Azadirachta indica in the Synchronization of the Cell Cycle of Bovine Fibroblasts s338<br />
N.C. Rabelo, C. Capobiango, R. Quintão, A.P. Moreira, S.G. Brito, et al.<br />
A004 Influence of High or Low Intake of Dry Matter/Energy on In Vitro Production of Bovine Embryos ...................... s338<br />
A.B. Prata, R.S. Surjus, M. Borsato, M.C.M. Silveira, M.C.C. Mattos, et al.<br />
A005 Oocyte Recovery in Queens after Contraceptive Treatment with Deslorelin Acetate (Suprelorin) .................... s339<br />
C.L. Ackermann, E. Trevisol, R. Volpato, A.A.P. Derussi, C.R.F. Guatolini, et al.<br />
A006 The Methylation Patterns of the IGF2 And IGF2r Genes in Bovine Spermatozoa are not Affected by Flow<br />
Cytometry Sex Sortin ............................................................................................................................................. s339<br />
J.O. Carvalho Neto, V.A. Michalczechen-Lacerda, R. Sartori, F.C. Rodrigues4; O. Bravim, et al.<br />
MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
A007 High Fat Diet and Genetic Dyslipidemia Impaired Testicle Morphometry in Nice: Preliminary Results ............. s340<br />
P.A.B.Cordeiro, A.C.S. Figueiredo, M.P. Palhão, I.C.S. Loyola, M.M.Gioso, et al.<br />
A008 Recommended Antibiotic for Ram Semen Cryopreservation Extender ............................................................. s340<br />
E.M. Madeida, K.L. Goularte, J. Pradieé, I. Bianchi, F.P.L. Leite, et al.<br />
A009 Qualitative Analysis of the Established Spermatogenesis in Cavia Porcellus (Linnaeus 1758) ....................... s341<br />
A. Gradela, A.K.R. Nunes, L.F.T. Martins, J.M. Santos, B.B. Gouveia, et al.<br />
A010 Glycerol and Dimethylformamide Cryopretectans Association for the Ovine Semen Cryopreservation ........... s341<br />
R.F. Bittencourt, A.L. Ribeiro Filho, G.F.O. Menezes, R.C. Andrade, L.O. Mascarenhas, et al.<br />
A011 Echogenicity Evaluation of the Testicular Parenchyma in Prepubertal Ovines .................................................. s342<br />
P.P.M. Teixeira, D.J. Cardilli, L.C. Padilha, C.C. D´Amato, M.E.F. Oliveira, et al.<br />
A012 Evaluation of Protamine and Transition Protein Gene Expression in Bovine Sperm Cells and Testicles .......... s342<br />
R. Simões, M.P. Milazzotto, F.R.O. Barros, M.A.M.M. Ferraz, M. Nichi, et al.<br />
A013 Age at Puberty in Boar Submitted to Porcine Somatotropin (PST) Treatment ..................................................... s343<br />
C. Pizoni, C.F. Demarco, I.M. Cavazini, V.R. Rabassa, A. Schneider, et al.<br />
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Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A015 Evaluation of Different Extenders in the Cryopreservation of Bovine Spermatozoa From Cauda Epididymides<br />
Stored for 24 hours ................................................................................................................................................ s344<br />
I.C. Giometti, P.M. Papa, F.O. Papa, L.A. Oliveira<br />
A016 Evaluation of Two Different Percoll Gradients for Bovine Sperm Selection ........................................................ s344<br />
R.C. Nishimura, J.O. Carvalho Neto, M.A.N. Dode<br />
A017 Action of Nitric Oxide Routes CGMP in In Vitro Capacitation of Cryopreserved Bovine Spermatozoa .............. s345<br />
A.C.M.S. Leal, M.C.Caldas-Bussiere, C.S.P. Carvalho, C.R. Quirino, P.A.P.M. Silva<br />
A018 Testis Size, Semen Criteria and Seminal Plasma Proteins of Rams Subjected to Scrotal Insulation ................ s345<br />
A.A. Moura, D.R. Rocha, C.E.A. Souza, A.A. Araújo, M.F.Van Tilburg, et al.<br />
A019 Glass Wool Colunm vs Mini-Percoll® Gradient in Bovine Sperm Separation – Preliminary Results ................ s346<br />
S.M.P. Souza, G.M. Mican, J.A. Narváez, R.S. Fontes, C.R. Quirino<br />
A020 Comparison of Bi and Tridimensional Culture System of Equine Testicle Samples ........................................... s346<br />
I. Martin, L. Maia, C.C. Macedo, B.Vita, G.A. Monteiro, et al.<br />
A021 Efficiency Comparison of Media for the Equine Sperm Capacitation through Flow Cytometry Analysis ........... s347<br />
T.P. Gardes, R.P. Arruda, D.F. Silva, R.N.R. Cardoso, J. Nascimento, et al.<br />
A022 Comparison of Different Methods for Evaluation of Morphology, Plasm Membrane and Acrosome Integrity of<br />
Cryopreserved Bovine Semen Imported ............................................................................................................... s347<br />
E.R. Cunha, C.G. Silva, A.M. Cunha, L.O.F. Rezende, G.H.L. Martins, et al.<br />
A023 Correlation Among Osmotic Test and Different Procedures of Post-Thaw Sperm Viability Evaluation .............. s348<br />
G.F.O. Menezes, R.F. Bittencourt, L.R.A. Santos, R.C. Andrade, L.O. Mascarenhas, et al.<br />
A024 Canine Semen Cryopreservation: Identification of Critical Points ....................................................................... s348<br />
C.F. Lucio, F.M. Regazzi, L.G. Silva, D.S.R. Angrimani, M. Nichi, et al.<br />
A025 Detection of Pathogens in Bovine Semen Using Fluorescent Multiplex PCR .................................................... s349<br />
F.E.F. Dias, T.V. Cavalcante, A.K.F. Lima, C.M. Nunes, J.F. Sousa, et al.<br />
A026 Different Protocols of Thawing Ram Semen Cryopreserved in Extendes Containing Low-DensityLipoprotein s349<br />
L.C.O. Moura, M.C. Silva, A.C.D. Oliveira, M.M.Neves, P.P.N. Snoeck, et al.<br />
A027 Effect of Porcine Somatotropin (PST) on Testicular Morphological and Functional Characteristics from<br />
Prepubertal Boars ................................................................................................................................................. s350<br />
D. Perazzoli, D.F. Frigotto, C.M. Pereira, V.R. Rabassa, I. Bianchi, et al.<br />
A028 Effect of Different Concentrations of Reduced Glutathione (GSH) for Cryopreservation of Canine Sperm ....... s350<br />
C.I. Vannuchi, C.F. Lucio, L.G. Silva, F.M. Regazzi, D.S.R. Agrimani, et al.<br />
A029 Effects of Selenium and Chromium on Seminal Quality of Buffaloes Supplemented with Byproducts of<br />
Amazonian Agroindustry ....................................................................................................................................... s351<br />
A.X. Santos, A.R. Garcia, C. Faturi, B.S. Nanhúm, J.B. Lourenço Junior, et al.<br />
A030 Efficiency of the Capacitation Media on Motility Maintenance and Induction of Equine Spermatozoa<br />
Hyperactivation ...................................................................................................................................................... s351<br />
D.F. Silva, R.P.Arruda, T.P. Gardes, R.N.R. Cardoso, J. Nascimento, et al.<br />
A031 Improvement of Sperm and Seminal Quality in Nelore vs Canchim Bulls after Inclusion in a Feedlot System . s352<br />
M.M. Guardieiro, J.O. Carvalho Neto, F.L.M. Silva, M.C.M. Silveira, T.N. Rodrigues, et al.<br />
A032 Influence of Sexing on Bovine Sperm Nanoroughness Measured by Atomic Force Microscopy ...................... s352<br />
S.R. Moita, J.O. Carvalho Neto, M.A.N. Dode, L.P.Silva<br />
A033 The Influence of Culture Medium on Oviduct Cells Sperm Viability in Cattle ..................................................... s353<br />
P.S. Valleriote, H.F. Gomes, C.S.P. Carvalho, F.P. Carvalho, A.J.B. Dias<br />
A034 Influence of Period of the Year in Seminal Quality of Captive Collared Pecaries (Pecari tajacu) Raised in the State<br />
of Para ................................................................................................................................................................... s353<br />
P.R. Kahwage, A.R. Garcia, D.A.A. Guimarães, O.M. Ohashi, N.I. Albuquerque, et al.<br />
A035 Influence of Storage Container (Cylindrical and Flats) on Semen Equine Cryopreserved ................................ s354<br />
B. Fagundes, J.A. Narváez, M.F. Van Tilburg, M.A. Barreto, W. Martins, et al.<br />
A036 Improving the Quality of RNA from Bovine Semen for Use in PCR Q-Real Time ............................................... s354<br />
V.T.F. Cipriano, A. Mioranza, A. Renzi, E.S. Ramos, R.B. Lôbo<br />
A037 Experimental Model to Preserve Wild Canine Sperm Through the Freeze-Drying Process .............................. s355<br />
L.C.O. Magalhães, C.M.M. Oña, M.J. Sudano, L.F. Crocomo, D.M. Paschoal, et al.<br />
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Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A038 Spermatic Profile of Ejaculated and Epididymal Semen from Dogs Epididymides after Cooling ...................... s355<br />
D.S.R. Angrimani, C.F. Lucio, G.A.L. Veiga, A. Dalmazzo, M. Nichi, et al.<br />
A039 Lyophilized Seminal Plasma Impoves the Motility of Frozen Ram Semen and Increases the Cleavage in<br />
Heterologous IVF ................................................................................................................................................... s356<br />
R. Casali, F.C. Zago, M. Federle, J.N. Drum, M. Sponchiado, et al.<br />
A040 Use of the Powdered Coconut Water as an Alternative Extender for the Cryopreservation of Collared Peccaries<br />
(Tayassu tajacu) Semen ........................................................................................................................................ s356<br />
M.A. Silva, L.B. Campos, J.A.B. Bezerra, G.L. Lima, G.C.X. Peixoto, et al.<br />
FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
A041 Addition of Insulin to the In Vitro Culture Medium Promotes Survival and Development of Follicles Preantral<br />
Goats...................................................................................................................................................................... s357<br />
R.N Chaves, A.M.C.V. Alves, L.R. Faustino, C.A.P. Lopes, K.P.L. Oliveira, et al.<br />
A042 Analysis of Reference Genes and Expression of mRNAs for the Bmp System in Goat Ovarian Follicles Grown In<br />
Vitro and In Vivo .................................................................................................................................................... s357<br />
J.J.N.Costa, M.J. Passos, G.L. Vasconcelos, A.M.L.R. Portela, A.W.B. Silva, et al.<br />
A043 Apoptosis in Fetal Bovine and Buffaloes Ovaries by Tunel Method .................................................................... s358<br />
T.V.G. Silva, S.S.D. Santos, B.B. Silva, B.L.G. Bittencourt, R.V. Sampaio, et al.<br />
A044 Evaluation of a FSH Protocol for Transvaginal Follicular Aspiration ................................................................... s358<br />
T.C.C. Sovernigo, É.S. Santos, R. Zanin, P.R. Adona, S. Guemera, et al.<br />
A045 Comparison Between Luteal Phase Period and the Number of Follicular Waves in Bos Taurus, Bos Indicus and<br />
Bubalus Bubalis Heifers Maintained on the Same Nutritional and Environmental Status .................................. s359<br />
J.M. Baldrighi, M.F.S. Filho, J.A. Visintin, P.S. Baruselli & M.E. Assumpção<br />
A046 Behavior of a New Polymorphism on Gdf-9 Gene (Fecgsi) in Ewes of Santa Inês Breed in Front of Superovulation<br />
Protocols ................................................................................................................................................................ s359<br />
B.D.M. Silva, T.A.S.N. Silva & J.P. Neves<br />
A047 Canine Preantral Follicles Culture at Different Concentrations of Insulin in Presence of Follicle-Stimulating<br />
Hormone (FSH) ..................................................................................................................................................... s360<br />
M.K. Brasil, G.M. Silva, A.B.G. Duarte, V.R. Araújo, A.K.F. Lima, et al.<br />
A048 Follicular Dynamics in Nelore Donors Superstimulated with eCG Administered in a Single Dose or Fractioned<br />
s360<br />
L.U. Gimenes, G.F.C. Grabert, M.G.Lourenço, J. Tojal, A.M.S. Chanquetti, et al.<br />
A049 The Effect of Ecg in Multiple Ovulation Protocols for High Production Holstein Cows Embryo Donors ............. s361<br />
C.AL. Rodrigues, L.M. Vieira, A.L. Ranieri, P.R.L. Silva, H. Ayres, et al.<br />
A050 Effect of Activin-a on The Development of Bovine Secondary Follicles In Vitro Cultured .................................. s361<br />
J.R.S. Passos, A.W.B. Silva, G.L. Vasconcelos, M.J. Passos, J.J.N. Costa, et al.<br />
A051 Effect of Low Level Laser Irradiation on Mitochondrial Membrane Potential and Cycle of Bovine Cumullus Cells s362<br />
C.A. Soares, K. Annes, T.R. Dreyer, H.S. Martinho, S.S. Nicolau, et al.<br />
A052 Effects of Ovarian Ortotopic Autotransplantation in Reproductive Behavior in Goats (Capra hircus) ................ s362<br />
N.A. Carmo, S.A. Marcondes, F.A. Santos, G.H.F. Moura, T.L. Nunes, et al.<br />
A053 Effect of Ecg on Ovulation and Conception Rates in Red Sindhi Cows (Bos taurus indicus) Treated with Two<br />
Synchronization of Ovulation Protocols ................................................................................................................ s363<br />
R.R.C. Mello, J.E. Ferreira, A.P.T.B. Silva, L.M. Mascarenhas, B.J.F. Silva, et al.<br />
A054 Timing Effect of Insemination Using Sex-Sorted Sperm in Embryo Production with Nelore (Bos indicus)<br />
Superovulated Donors .......................................................................................................................................... s363<br />
J.G. Soares, N.A.T. Carvalho, P.S. Baruselli, C.M. Martins, M. Nichi, et al.<br />
A055 Efficiency of Different Treatments of Ovarian Cysts in Dairy Cows ..................................................................... s364<br />
T.A. Miyauchi, C.A.C. Fernandes, M.R.P. Portinari, M.M. Gioso, B.F.L. Alves, et al.<br />
A056 Stability of Reference Genes and Levels of mRNA for The IGF System in Bovine Ovarian Follicles Grown In Vitro<br />
and In Vivo ............................................................................................................................................................. s364<br />
G.L. Vasconcelos, A.W.B. Silva, J.J.N. Costa, M.J. Passos, R.O.D.S. Rossi, et al.<br />
A057 Expression of mRNA Encoding Fgfr1b and Fgfr2b in Bovine Fetal Ovaries during Gestation .......................... s365<br />
R.B.Silva, E.S. Caixeta, C. Price & J.B. Junior<br />
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Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A058 Leukemia Inhibitory Factor Stimulates the In Vitro Development of Sheep Preantral Follicles and the Production<br />
Of Embryos ............................................................................................................................................................ s365<br />
V.B. Luz, T.F.P. Silva, V.R. Araújo, A.B.G. Duarte, J.J.H. Celestino, et al.<br />
A059 Effects of FSH and Growth Differentiation Factor -9 (GDF-9) on In Vitro Growth of Bovine Secondary Follicless366<br />
R.P. Ribeiro, G.L. Vasconcelos, A.W.B. Silva, M.J. Passos, F.T.G. Bezerra, et al.<br />
A060 Influence of Protocols for Superovulation on Induced Stress of Brown Brocket Deer (Mazama Gouazoubira) s366<br />
E.S. Zanetti & J.M.B. Duarte<br />
A061 Steady-State Levels of Vasoactive Intestinal Peptide mRNA in Goat Ovaries and Its Effect on the In Vitro<br />
Development of Isolated Preantral Follicles ......................................................................................................... s367<br />
J.B. Bruno, V.R. Araújo, J.J.H. Celestino, M.V.A. Saraiva, R.M.P. Rocha, et al.<br />
A062 Standard of Emergency Follicular Waves in Long Protocols (With or Without CIDR Replacement) and<br />
Seasonality Effect in Santa Ines Ewes .................................................................................................................. s367<br />
M.E.F. Oliveira, C.C. D’amato, H. Ayres, L.G. Oliveira, P.P.M. Teixeira, et al.<br />
A063 Behavioral Parameters of Dairy Goats Subjected to Synchronization with PGF2a ............................................ s368<br />
A.F. Silva, L.V. Esteves, F. Zandonadi, R.C. Cruz & J.F. Fonseca<br />
A064 Expression Profile of Candidate Genes for the Acquisition of Competence during Oocyte Growth in Bovine .. s368<br />
I.R. Bessa, R.C. Nishimura, F. Paulini, M.M. Franco & M.A.N. Dode<br />
A065 Antral and Preantral Follicular Population in Nelore Ovaries with High and Low Antral Follicular Counting:<br />
Preliminary Results ............................................................................................................................................... s369<br />
G.M.G. Santos, K.C. Silva-Santos, C. L. Schneider, L.S. Siloto, F. Morotti, et al.<br />
A066 Follicular Proportion and Population of Rats Submitted to Different Procedures of Ovarian Autologous<br />
Transplantation ...................................................................................................................................................... s369<br />
M.F. Macedo, M.B. Bezerra, N.A. Carmo, F.F.P.C. Barros & W.R.R. Vicente<br />
A067 First Successful Estrous Synchronization Using a Prostaglandin Analogue in Collared Peccaries (Tayassu<br />
Tajacu) ................................................................................................................................................................... s370<br />
A.R. Silva, K.M. Maia, M.A. Silva, G.C.X. Peixoto, J.A.B. Bezerra, et al.<br />
A068 Cellular Proliferation of Poliovular Follicles in Adult Nelore Ovaries .................................................................. s370<br />
K.C. Silva-Santos, G.M.G. Santos, R.L. Oliveira, L.S. Siloto, A.P.F.R.L. Bracarense, et al.<br />
A069 Superovulatory Response and Pregancy Rates of Holstein Cows In Vivo Embryo Production Within Different<br />
Catagories During Summer and Winter ................................................................................................................ s371<br />
L.M. Vieira, C.A. Rodrigues, P.R.L. Silva, J.N.S. Sales, M.F.S. Filho, et al.<br />
A070 Superovulation of Nelore Heifers Using Single Injection of Folicle Stimulant Hormone Carried Into Polimeric<br />
Matrix ..................................................................................................................................................................... s371<br />
E.M. Mogollon-Waltero, H.J. Narvaez, R. Costa, C.R. Quirino & R.S. Fontes<br />
A071 Energy Supplementation in Santa Inês Sheep ................................................................................................... s372<br />
S.S. Venturi, I.R. Soares, A.F. Silva, E.C. Cardoso, A.R. Rodrigues, et al.<br />
A072 Variation on Vascularization of Follicles Induced to Ovulate with HCG and GnRH Evaluated by Doppler<br />
Ultrasonography - Preliminary Results ................................................................................................................. s372<br />
R.C. Uliani, L A Silva & M.A. Alvarenga.<br />
A073 Viability of Canine Preantral Follicles Cultured In Vitro after Conservation in an Oocyte Transporter ............... s373<br />
R.J.S. Gonçalves, V.R.P. Barros, A.P. Almeida, A.B.G.Duarte, V.R. Araújo, et al.<br />
A074 Ascorbic Acid Improves the Survival and In Vitro Growth of Isolated Caprine Preantral Follicles ..................... s373<br />
G.M. Silva, C.H. Lobo, V.R. Araújo, A.B.G. Duarte, A.A. Moura, et al.<br />
FTAI, FTET AND AI<br />
A075 Follicular and Reproductive Traits in Short-Term Progesterone and PGF2alpha Based Estrous- Synchronization<br />
Protocol in Sheep .................................................................................................................................................. s374<br />
R.V. Allende, F.R. Saravia, E.N. Lara, A.F. Leiva; T. Díaz, et al.<br />
A076 Effect of CIDR® Reutilization in a Progesterone-PGF2alpha Based Estrous-Synchronization Protocol on Estrous<br />
Presentation and Ovulatory Performance in Sheep .............................................................................................. s374<br />
F.R. Saravia, R.V. Allende, E.N. Lara, A.F. Leiva, T. Diaz, et al.<br />
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Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A077 Effect of Estradiol Benzoate at CIDR Insertion on Ovarian Traits after Short-Term Progesterone and PGF2alpha<br />
Based Estrous-Synchronization Protocol in Sheep .............................................................................................. s375<br />
J.F. Cox, E.N. Lara, A.F. Leiva, H. Muñoz, R.V. Allende, et al.<br />
A078 TAI Increases the Reproductive Performance of Lactating Primiparous Acyclic Nelore Cows .......................... s375<br />
R.R. Cunha, M.M. Gioso, R.J. Carvalho, C.A.C. Fernandes, M.P. Palhão, et al.<br />
A079 Occurrence of Estrus after Synchronization Improve the Use of Sex Semen in Timed Artificial Insemiantion<br />
Protocol .................................................................................................................................................................. s376<br />
P.S. Baruselli, E.K. Abe, R.V. Sala, M. Nichi, E.P. Campos Filho, et al.<br />
A080 The Use of Bioabortogen® and Bioleptogen® Vaccines may Improve Reproductive Efficiency of Beef Cows . s376<br />
M. Sivieri, R.M. Ferreira, D.P. Borges, T.S. Antonio, R.L. Gonçalves, et al.<br />
A081 Evaluation of Artificial Insemination in Girolando Heifers with Sexed Semen ................................................... s377<br />
M.F. Mendanha, S. Ferrari & D.S. Silva<br />
A082 Evaluation of Different Doses of eCG in the TAI Conception Rate in Cycling Nelore Heifers Synchronized with<br />
Intravaginal Progesterone Device Previously Used For 9 Days .......................................................................... s377<br />
E.R. Carvalho, A.D.P. Rodrigues, T. Martins, H.B. Graff, R.F.G. Peres, et al.<br />
A083 Evaluation of the Effects of Heat Stress on Reproductive Performance of Nelore Heifers during the Breeding<br />
Season................................................................................................................................................................... s378<br />
M. Maturana Filho, N.A. Teixeira, S.C. Scolari, A. Kehrle, P.H.P. Miguez, et al.<br />
A084 The Characteristics of the Folicular Dynamics Over the Diameter of the Corpus Luteum in Fixed Time Embryo<br />
Transfer Recipients ................................................................................................................................................ s378<br />
A.L. Ribeiro Filho, B.H.A. AndradeT.P. Coutinho, L.M.C. Feitosa, M.V.G. Loiola, et al.<br />
A085 Follicular Features in Murrah, Mediterranean and Crossbred Buffalo Heifers Submitted to Exogenous Control of<br />
Ovulation................................................................................................................................................................ s379<br />
A.R. Garcia, G.R. Silva, B.S. Nahúm, J.S. Pessoa, A.A. Gonçalves, et al.<br />
A086 Comparative Pregnancy Rates of High-Producing Holstein Cows Synchronized for FTAI with Progesterone<br />
Estradiol-Based Protocol or Double...................................................................................................................... s379<br />
A.L. Ranieri, C.A. Rodrigues, L.M. Vieira, P.R.L. Silva, G.B. Gouvea, et al.<br />
A087 Comparison of the Conception Rates of Nelore Lactating Cows Submitted to FTAI and Treated with Porcine<br />
Pituitary Extract and Equine Chorionic Gonadotropin .......................................................................................... s380<br />
S.M. González, J.T. Campos, W. Blaschi, T.R.R. Barreiros & M.M. Seneda<br />
A088 Comparison of Rates of Conception and Pregnancy Losses in Different Years in Nelore Cows under FTAI<br />
Vaccinated or not Against BVDV and BoHV-1....................................................................................................... s380<br />
T.R.R. Barreiros, A.A.O. Friedel, R.A.F. Stellato, S.M. González, W. Blaschi, et al.<br />
A089 Conception Rates in Postpartum Nelore Cows Treated with eCG Before and/or after Fixed-Time Artificial<br />
Insemination .......................................................................................................................................................... s381<br />
A.P. Lemes, A.D.P. Rodrigues, R.F.G. Peres, H.B. Graff, E.R. Carvalho, et al.<br />
A090 Intracornual Insemination with Sex Sorted Sperm does not affect the Pregnancy per AI after Timed Artificial<br />
Insemination .......................................................................................................................................................... s381<br />
R.V. Sala, M.F. Sá Filho, R.W. Girotto, M. Nichi, E.P. Campos Filho, et al.<br />
A091 Effect of New and Reused Vaginal Inserts on Plasma Progesterone Profile and Follicular Size at the End of<br />
Progesterone and Prostaglandin F2alpha Estrous Synchronization Treatment in Beef Cows ............................ s382<br />
E.N. Lara, A.F. Leiva, C. Baeza, A. Rodríguez, P. Rojas, et al.<br />
A092 Dose Effect of Estradiol Benzoate Associated with Progesterone on the Synchronization of Follicular Wave<br />
Emergence in Bos Indicus and Bos Taurus Cows ................................................................................................ s382<br />
M.R. Bastos, R.S. Surjus, A.B. Prata, M.A.P. Meschiatti, M. Borsato, et al.<br />
A093 Effect of Estrous Detection on Pregnancy Rate in Multiparous Nelore (Bos Indicus) Cows Submitted to a Timed AI<br />
in the Brazilian South Pantanal ............................................................................................................................. s383<br />
J.C. Borges, M.R. Silva, I.C. Canavari, R.R. Raineri, J.F. Massoneto, et al.<br />
A094 Effect of hCG and/or CIDR in Lactating Dairy Cattle on Circulating Progesterone and Conception Rates ....... s383<br />
A.B. Nascimento, A.H. Souza, M. Herlihy, A. Keskin, F.P. Dalla Costa, et al.<br />
A095 Effect of the Ovulatory Follicle Diameter on the Pregnancy Rate of Lactating Cows Fixed Time Inseminated with<br />
Low Progesterone Protocols ................................................................................................................................. s384<br />
L.F.M. Pfeifer, S.C.B.S. Leal, A. Schneider, E. Schmitt & M.N. Corrêa<br />
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Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A096 Effect of Synchronization Follicular Wave Method on Bovine Superovulatory Response ................................. s384<br />
J.E. Ferreira, R.R.C. Mello, A.P.T.B. Silva, L.M. Mascarenhas, B.J.F. Silva, et al.<br />
A097 Effect of Treatment with GnRH Analogue at the Moment of Insemination on Pregnancy Rate in Primiparous<br />
Nelore (Bos Indicus) Cows Submitted to a Timed AI in the Brazilian South Pantanal ......................................... s385<br />
M.R. Silva, J.F. Massoneto, D.B. Marinho, I.C. Canavari, R.R. Raineri, et al.<br />
A098 Efficiency of Different Stimuli Gonadotropin in Resynchronization Nellore Lactating Cows Submitted to FTAI s385<br />
J.T. Campos, S.M. González, W. Blaschi, T.R.R. Barreiros & M.M. Seneda<br />
A099 Eficiency of Timed Artificial Insemination Protocol for Holstein Cows Reared under Semiarid Condition ........ s386<br />
S.I. Guido, A.S. Santos Filho, L.C. Pereira, J.C.O. Andrade, F.C.L. Guido, et al.<br />
A100 Luteolytic Effectiveness of Prostaglandina Tortuga® in Bovine Cattle ............................................................... s386<br />
M.S. Baruselli, R.M. Ferreira, M.F. Sá Filho, L.F.M. Tamassia, C.A. Nascimento, et al.<br />
A101 Establishment of a Synchronization of Ovulation Protocol for Fixed Time Embryo Transfer Applied on Dairy Cattle<br />
Genetic Improvement Program of Acre State ........................................................................................................ s387<br />
R. Satrapa, J.V.A. Diniz, F.L. Dantas, R.R. Marcelino, N.L. Vasconcelos, et al.<br />
A102 Use of the Estradiol Benzoate to Induction Follicular Regression in Mares Treated with Intravaginal Progesterone<br />
Device .................................................................................................................................................................... s387<br />
C. Meira, J.C. Ferreira & A.I.N. Boff<br />
A103 Influence of the Uterine Lavage to Endometrial Cytology on the Bovine Fertility ............................................... s388<br />
H.E. Thomé, R.P. Arruda, B.M.M. Oliveira, M. Maturana Filho, G.C. Oliveira, et al.<br />
A104 Artificial Insemination in Pheasants Lodged in Individual Cages ....................................................................... s388<br />
O.A. Resende, J. Almeida & E.P. Ponte<br />
A105 Early Embryo Mortality in Beef Cows after Fixed Time Artificial Insemination (FTAI) and Hormonal Treatmentss389<br />
R. Machado, M.J. Sudano, M.A. Bergamaschi, G.P. Nogueira, C.M.B. Membrive, et al.<br />
A106 Modified Method of Artificial Insemination with Cervical Retraction in Dorper Ewes ......................................... s389<br />
M.F. Cordeiro, T.V.C. Nascimento, C.H.S.C. Barros, L.C. Magalhães, A.P.O. Monte, et al.<br />
A107 Ovarian Parameters of Dairy Goats Undergoing Synchronization with PGF2α Associated or Not with the Use of<br />
hCG in the Beginning of Estrus ............................................................................................................................. s390<br />
L.V. Esteves, F. Zandonadi, A.F. Silva, S.S. Venturi & J.F. Fonseca<br />
A108 The Protocol for FTAI with Estradiol Cypionate does not Require Additional GnRH and Allows Insemination in<br />
Two Periods ........................................................................................................................................................... s390<br />
F.P. Torres, R.M. Ferreira, F.A. Lima, M.B. Veras, F.R.F. Silva, et al.<br />
A109 Synchronization and Resynchronization Using a Norgestomet Ear Implant in Nelore Heifers Timed Artificial<br />
Inseminated ........................................................................................................................................................... s391<br />
M.F. Sá Filho, R.A.I. Oliveira, R.V. Sala, G.G. Macedo, R.W. Girotto, et al.<br />
A110 Synchronization of Ovulation in Water-Buffalo Heifers with Estradiol Benzoate or Gonadorelin ...................... s391<br />
M. Nichi, N.A.T. Carvalho, J.G. Soares, D.C. Souza & P.S. Baruselli<br />
A111 Synchronization and Induction of Fertile Estrus of Paca (Cuniculus Paca L.) .................................................... s392<br />
V.M.F. Ribeiro, R. Satrapa, J.V.A. Diniz, R.A. Satrapa & R. Rumpf<br />
A112 Supplementation of Nelore Cows with Rumen-Protected Fat Before and/or After TAI ....................................... s392<br />
I.M. Sokoloski, M.M. Guardieiro, A.B. Prata, L.H.D. Carrijo, G.B. Mourão, et al.<br />
A113 Pregnancy Rates of Different Protocols for Fixed Time Transfer of In Vitro Produced Bovine Embryos ............ s393<br />
T.P. Coutinho, Y.F. Watanabe, L.M.C. Feitosa, M.V.G. Loiola, B.H.A. Andrade, et al.<br />
A114 Pregnancy Rate and Embryonic/Fetal Loss After eCG Administration Given 5 or 10 Days After Insemination in<br />
Cycling Ewes ......................................................................................................................................................... s393<br />
C. García-Pintos, P.C. Santos-Neto, A. Menchaca<br />
A115 Conception Rates in Beef Cattle Given a New or Previously Used Progesterone Device in Fixed-Time Artificial<br />
Insemination .......................................................................................................................................................... s394<br />
A. Kehrle, M. Pereira Neto, M. Maturana Filho, P.H.P. Miguez, G.C. Gomes, et al.<br />
A116 Use of DIB® and Crestar® to Synchronize Ovulation and FTAI in Buffalo Heifers during the Non-Breeding<br />
Season................................................................................................................................................................... s394<br />
N.A.T. Carvalho, J.G. Soares, E.L. Reis & P.S. Baruselli<br />
XIV
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A117 Reproductive Ultrasonography for Use of High Cost Semen in FTAI Programs ................................................ s395<br />
J.R.S. Torres Júnior, H.M.V.B. Aguiar & P.H. Cavalcanti<br />
A118 Pregnancy Rate Monthly Variation of the Nellore Cows Submitted to the FTAI in Bolivian Farm ....................... s395<br />
E. Nogueira, M.R. De Paula, H.R. Marques Junior & A.S. Silva<br />
OPU-IVP AND ET<br />
A119 Ultrasound-Guided Follicle Aspiration Do Not Reduce Blood Flow in the Follicular Wall - Preliminary Results ... s396<br />
A.M. Ghetti, F. Zandonadi, l.G.B. Siqueira, l.S.A. Camargo, E.K.N. Arashiro, et al.<br />
A120 Evaluation of Oocytes Recovered from Cows Undergoing Immunosuppressive Protocol with Dexamethasone .. s396<br />
A.P. Oliveira, P.M.P. Nascimento, J.M. Bicalho, G.G. Carvalho, R.S. Diniz, et al.<br />
A121 Evaluation of a Medium Based on Coconut Water (Cocos Nucifera) To In Vitro Embryo Production of Bovine s397<br />
M.S. Cordeiro, P.C.A. Ramos, N.N. Costa, T.V.G. Silva, P.P.B. Santana, et al.<br />
A122 Evaluation of a New Method for Identifying Endometritis in Anestrous Mares through Ultrassonography ........ s397<br />
G.M.Greco, L.M.P. Maia, M.M.C. Chaves & M.A. Alvarenga<br />
A123 Hemodinamic Evaluation of the Uterus in Mares Used as Recipient: Partial Results ........................................ s398<br />
F.S. Ignácio, J.C. Ferreira, R.P. Melo, N.C. Abreu & G.H.M. Araújo<br />
A124 Comparison of the Pregnancy Rate of Embryos Coming of Ovulation in Mares Single or Double Race Polo<br />
Argentino ............................................................................................................................................................... s398<br />
T.D. Reis, M.M. Higuti, J.T. Paula, S.M. González, E. Bernardi, et al.<br />
A125 Comparison between the Number of Aspiraded Follicles by Videolaparoscopy and the Oocyte Recovery in<br />
Dorper and Santa Inês Ewes ................................................................................................................................ s399<br />
A.S. Rodrigues, J.S. Chacon, A.L. Ribeiro Filho, B.H.A. Andrade, M.V.G. Loiola, et al.<br />
A126 Correlation Of Kinetics Of Development And Sex Of In Vitro Produced Bovine Embryos ................................. s399<br />
A.R. Pupulim, Á.R. Buzzo, J. Mazucheli, F.V. Meirelles, R.Z. Puelker, et al.<br />
A127 Different Volumes and Centrifugations Time of Percoll Gradient Do Not Affect the Sperm Quality and Embryo<br />
Development of In Vitro Produced Bovine Embryos ............................................................................................ s400<br />
L.L. Vianna, J. Pradieé, E.C.S. Santos, A.O. Gonçalves, L. Anghinoni, et al.<br />
A128 Cumulative Effect of Bovine Recombinant Somatotropin (Rbst) on the In Vitro Production of Embryos from<br />
Lactating Guzolando Females .............................................................................................................................. s400<br />
F.J. Moraes Junior, L.F. Schütz, C. Feltrin, L.T. Martins, S. Gaudencio Neto, et al.<br />
A129 Effect of High Energy Diet on Metabolic, Endocrine and Reproductive Parameters on Bos Indicus and Bos<br />
Taurus Cows .......................................................................................................................................................... s401<br />
J.N.S. Sales, L.T. Iguma, C.C.R. Quintão, M.A.S. Gama, C. Freitas, et al.<br />
A130 Effect of the Sperm Selection with Isolate ® or Percoll ® on Sperm Quality and In Vitro Bovine Embryo Development<br />
for Frozen-Thawed Sexed and Non-Sexed Semen .............................................................................................. s401<br />
P.R. Villamil, M.E.G. Gomez, M.F. Taranco, M. Caccia & G. Bó<br />
A131 The Effect of Follicle Wave Synchronization and eCG Treatment on Cumulus Oocyte Complex Recovery Rates<br />
and Quality in Brangus and Angus Donors .......................................................................................................... s402<br />
F.L. Ongaratto, P.R. Villamil, A. Tribulo & G. Bó<br />
A132 Effect of FSH Incorporated in a Polimeric Matrix of Pluronic F-127® about the Quality of Oocytes Collected by<br />
Follicular Aspiration in Zebu Heifers ..................................................................................................................... s402<br />
H.J. Narvaez, R.S. Fontes, G.M. Mican & C.R. Quirino<br />
A133 Effect of Corpus Luteum on the Recovery Rate and Number of Oocytes in Crossbred Zebu Heifers ............... s403<br />
B.P. Carvalho, F.Q. Costa, F.B. Rosa, A.M. Arrais, R.C. Maia, et al.<br />
A134 Effect of Stage of Development on the Sex Ratio of Pregnancies Originated from In Vitro Produced Embryos s403<br />
L.M. Gouvêa<br />
A135 Effect of the Method for the Retrieval of Cumulus-Oocyte Complexes on the In Vitro Production Efficiency of<br />
Sheep Embryos ..................................................................................................................................................... s404<br />
M.B. Salviano, C. Feltrin, J.L. Almeida, K.C.S. Tavares, F.J. Moraes Junior, et al.<br />
A136 Efficiency of OPU-IVP-Et of Fresh and Vitrified Embryos in Buffalo .................................................................... s404<br />
W.P. Saliba, R.M. Drumond, H.X. Bayão, M.T. Alvim, P.S. Baruselli, et al.<br />
XV
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A137 Efficacy of Short Protocols with CIDR® on Estrus Synchronization of Embryos Recipients Ewes .................... s405<br />
F.S. D´Angieri, J.C.B. Silva, W.K. Okabe, M. Maturana Filho & A.S. Traldi<br />
A138 Fertility of Recipients Transferred with Embryos in Oviduct by Two Days of Santa Inês and Dorper Sheep In Vitro<br />
Produced ............................................................................................................................................................... s405<br />
E.A.B. Araujo, S.N. Oliveira, A.C. Basso, J.S. Chacon, M. Chalhoub, et al.<br />
A139 Apoptosis in Fresh and Vitrified In Vitro and In Vivo Produced Bovine Embryos ............................................... s406<br />
M.J. Sudano, D.M. Paschoal, T.S. Rascado, M.D. Guastali, R.R.D. Maziero, et al.<br />
A140 Influence of Lineage of Oocyte Donor on the Yield and Morphology of Oocytes Recovered By Ultrasound-<br />
Guided Follicular Aspiration in Nellore Cows ....................................................................................................... s406<br />
A. Martins Júnior, R.S. Calegari, D.M. Paschoal, D.G. Souza & M.J. Sudano<br />
A141 Influence of the Sperm Pre-Incubation on Cleavage Rates, Blastocyst Production and Sex Distribution of In Vitro<br />
Produced Bovine Embryos .................................................................................................................................... s407<br />
A.G. Curcio, R.S. Fontes, S.R. Leal, C.R. Quirino, G.M. Mican, et al.<br />
A142 Influence of Semen on the Pregnancy Rate in Mares under Embryo Transfer in the Northeast of the State of Para ........ s407<br />
S.T. Rolim Filho, F.O. Barbosa Filho, H.F.L. Ribeiro, E.Y.E. Mesquita, K.C. Sousa, et al.<br />
A143 IGF-I (Insulin-Like Growth Factor I) Increases In Vitro Production, but Does Not Protect Embryos from Deleterious<br />
Effect of Heat Stress in Nelore (Bos indicus) and Holstein (Bos taurus) Breeds ................................................. s408<br />
A.C.S. Castilho, R.A. Satrapa, E.M. Razza, C.F. Silva, R. Simões, et al.<br />
A144 Treated Oocytes with Retinoids and with Retinoids and Growth Factor Improve the In Vitro Embryos Production<br />
and Inhibit the Apoptosis? ..................................................................................................................................... s408<br />
R.M. Chaves, J.C. Conceição, M.A.V. Silva, E.R. Santos Júnior, L.M. Freitas Neto, et al.<br />
A145 Gene Expression Profile of IGF System Members on In Vitro Produced Bovine Blastocysts: Comparison between<br />
Nelore (Bos Indicus) and Holstein (Bos taurus) ................................................................................................... s409<br />
R.A. Satrapa, A.C.S. Castilho, R. Simões, E.M. Razza, C.F. Silva, et al.<br />
A146 Potential of the Gyr (Bos taurus indicus) and Holstein (Bos taurus taurus) Donors for Embryo Production ...... s409<br />
J.H.M. Viana, T.M. Miyauchi, T.A. Miyauchi, E.R. Oliveira, J.A.D. Garcia, et al.<br />
A147 Preservation of Intrafollicular Oocytes at 4°C ...................................................................................................... s410<br />
R.S. Ferro & O.S. Garcia<br />
A148 Production of Ovine Oocytes from Slaughterhouse Ovaries Using Two Collection Procedures ........................ s410<br />
P.M. Rafaelli, P.Z.D. Pumará, P. Ortiz & J.I. Ernesto<br />
A149 In Vitro Embryo Production (IVEP) after the Laparoscopic Oocyte Recovery (LOR) In Canindé Goats ............ s411<br />
A.F. Pereira, R.R. Moura, R.I.T.P. Batista, J.M.G. Souza, A.S. Alcântara Neto, et al.<br />
A150 In Vitro Embryo Production Using Frozen Semen Previously Infected with Bovine Herpesvirus Type 5 ........... s411<br />
D.G. Souza, A. Martins Júnior, R.S. Calegari, J.O. Caldeira, C. Silva-Frade, et al.<br />
A151 In Vitro Production of Ovine Embryos of Dorper and Santa Ines Breed Aspirated by Videolaparoscopy and<br />
Transported over Long Distances ......................................................................................................................... s412<br />
M. Chalhoub, A.C. Basso, J.S. Chacon, A.L. Ribeiro Filho, M.V.G. Loiola, et al.<br />
A152 In Vivo Embryo Production in Cows Superovulated 1 or 2 Days after OPU ....................................................... s412<br />
R.S. Surjus, A.B. Prata, M. Borsato, F.C.S.Z. Mattos, M.C.M. Silveira, et al.<br />
A153 Hormonal Protocols for Oocyte Retrieval and In Vitro Production of Embryos in Bovine Donors ...................... s413<br />
T.M. Miyauchi, T.A. Miyauchi, M.P. Palhão, A.S. Camargos, C.A.C. Fernandes, et al.<br />
A154 Technical Report: Ovun Pick Up by Single for Recovery of Oocytes in Mares in Transition Period .................... s413<br />
A.G. Rigo, B.V. Sanches, J.H.F. Pontes, L.L. Moino, R.M. Untura, et al.<br />
A155 Ovulatory Response of Cycling Mares Stimulated with an Association of pFSH and EPE ................................ s414<br />
N.C. Abreu, C. Meira, F.S. Ignácio & G.H.M. Araújo<br />
A156 Pregnancy Rate in Equine Embryo Recipients Treated with Flunixin Meglumine ............................................. s414<br />
J.P. Oliveira, H.L. Resende & J.C.F, Jacob<br />
A157 Pregnancy Rate in Mangalarga Marchador Recipients at Different Ages with or without Progesterone<br />
Supplementation ................................................................................................................................................... s415<br />
S.O. Paiva, C.G.M. Oliveira, P.C. Almeida, M.A.F. Sá, R.G.C. Junqueira, et al.<br />
XVI
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A158 Conception Rates of In Vitro Produced Bovine Embryos with Conventional and Thawed-Sexed Sperm of Nellore<br />
Bulls ....................................................................................................................................................................... s415<br />
J.C.B. Silva, W.K. Okabe, M.C.C. Mattos, B.V. Sanches, J.H.F. Pontes, et al.<br />
A159 Use of Retinoids to Inhibit the In Vitro Apoptosis of Caprine Oocytes and Embryos ......................................... s416<br />
J.C. Conceição, R.M. Chaves, M.A.V. Silva, E.R. Santos Júnior, L.M. Freitas Neto, et al.<br />
A160 FSH in Protocols for Follicular Wave Synchronization and Embryo Production in Taurin and Zebu Donnors .. s416<br />
C.A.C. Fernandes, A.C.S. Figueiredo, B.F.L. Alves, E.R. Oliveira, T.C. Rossi, et al.<br />
EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
A161 Use of b-Mercaptoethanol for In Vitro Maturation and Culture of Ovine Embryos .............................................. s417<br />
J. Pradieé, L.L. Vianna, A.O. Gonçalves, E.C.S. Santos, R.G. Mondadori, et al.<br />
A162 Addition of Nerve Growth Factor on Maturation and Development Media for In Vitro Production of Sheep Embryos ...... s417<br />
M. Vilariño, M. Crispo, P.C. Santos Neto, R. Wijma, N. Barrera, et al.<br />
A163 Physiological and Endocrine Changes of Bovine Corpus Luteum after Challenges with Sub-Dose of Cloprotenol<br />
Sodium................................................................................................................................................................... s418<br />
E. Trevisol, W.C. Marques Filho, F.C. Destro, J.C. Ferreira, A.S. Camargos, et al.<br />
A164 Changes in Blood Chemistry Profiles and Association with Birthweights in IVF-Derived Newborn Calves ..... s418<br />
L.F. Schütz, F.C. Zago, L.H.Aguiar, P.C. Santos Neto, J. Machado Júnior, et al.<br />
A165 Breeding Season Anticipation in Mares Submitted to a Progesterone Intravaginal Implant or Artificial Lighting ... s419<br />
R.P. Melo, F.S. Ignácio, J.C. Ferreira, J.N.P. Puoli Filho & C. Meira<br />
A166 Evaluation of Hypothalamic-Pituitary Responsiveness during the Postpartum Nellore Cows .......................... s419<br />
C.M. Barros, J.R. Cury, R.A. Satrapa, M. Pegorer, L.A. Trinca, et al.<br />
A167 Immunohistochemical Evaluation of Collagen I and III in Cervix and Uterus from Bitches with Open or Closed<br />
Pyometra................................................................................................................................................................ s420<br />
R. Volpato, I. Martin, C.L. Ackermann, M.H. Tsunemi, R.L. Amorim, et al.<br />
A168 Ultrasonographic Evaluation of the Criollo Cloned Equine Placenta ................................................................. s420<br />
A. Gambini, J. Jarazo, F. Karlanian, A. Stefano, C. Bergadá, et al.<br />
A169 Chimeric Mouse Blastocysts Reconstructed by Inner Cell Mass and Trophectoderm Aggregation .................. s421<br />
I.P. Emanuelli, C.P. Godói, B.C.S. Campanha, P.D. Moço, B. Cazari, et al.<br />
A170 Characterization of Secondary Corpora Lutea Formation in Non-Cyclic and Cyclic Embryo Recipient Mares<br />
(Partial Results) ..................................................................................................................................................... s421<br />
E.S.M. Silva, S.F. Frade & C. Meira<br />
A171 Morphological Characterization of the Placenta of Curraleiro/Pé-Duro Cows ................................................... s422<br />
H.C.A. Teixeira, P.L.G. Souto, E.A. Barbosa, N.H. Moreira, A.S. Mariante, et al.<br />
A172 Sodium Cloprostenol Treatment and Postpartum Uterine Involution in Dairy Goats - Preliminary Results ....... s422<br />
L.B. Rizzoni, J.A.D. Garcia, M.P. Palhão, T.A. Miyauchi, S.D.A. Ribeiro, et al.<br />
A173 Sodium Cloprostenol and Postpartum Treatments of Dairy Cows ...................................................................... s423<br />
R.J. Carvalho, M.P. Palhão, M.M. Gioso, B.F.L. Alves, E.R. Oliveira, et al.<br />
A174 Plasma Leptin Concentration in Nelore Heifers Supplemented with Protected Fat or Carbohydrate Excess ... s423<br />
G.P. Nogueira, R.S. Cipriano, M.C.V. Miguel, H.F. Costa, L.M. Pavanello, et al.<br />
A175 Progesterone Concentration during Estrous Cycle in Two Seasonal Periods in Jennies .................................. s424<br />
J.V. Oliveira, E.S.M. Silva & C. Meira<br />
A176 Culture of Inner Cell Mass Cells Obtained from Bovine Embryos Produced In Vitro and In Vivo in the Presence of<br />
Leukemia Inhibitory Factor (LIF) ............................................................................................................................ s424<br />
M.D. Guastali, M.J. Sudano, D.M. Paschoal, T.S. Rascado, L.F. Crocomo, et al.<br />
A177 Reproductive Performance of Two Wistar Rats Generations Supplemented with the Omega-3 Fatty Acid ....... s425<br />
C.B. Jacometo, S. Halfen, F. Bado, F.T. Rosa, E. Schmitt, et al.<br />
A178 The Onset of Puberty in Ewe Lambs After Biostimulation and Medroxyprogesterone Acetate or Long-Acting<br />
Progesterone ......................................................................................................................................................... s425<br />
C.D.M. Toma, S.D. Bicudo, H.S. Toma, C.E.A. Biscarde, T.M. Oliveira, et al.<br />
XVII
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A179 Dimensions, Steroidogenesis and Vascularization of the Bovine Corpus Luteum in Response to Manipulation of<br />
the Pre-Ovulatory Follicle Growth ......................................................................................................................... s426<br />
F.S. Mesquita, M.R. França, S.C. Scolari, V. Pavanello Jr., F.F. Bressan, et al.<br />
A180 Effect of Dexamethasone and Triiodothyronine on In Vitro Bovine Embryo ....................................................... s426<br />
N.N. Costa, M.S. Cordeiro, T.V.G. Silva, P.D.P.B. Santana, R.V. Sampaio, et al.<br />
A181 Effect of Cell Quantity in Obtaining Mice Chimeras by Demi-Blastocysts Aggregation ...................................... s427<br />
C.P. Godói, P.D. Moço, B. Cazari, P.T. Mihara, P.V. Silva, et al.<br />
A182 Effect of Different Media on Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-<br />
9) mRNA Expression During Oocyte In Vitro Maturation (IVM) ............................................................................. s427<br />
P.F. Lima, M.F. Machado, E.S. Caixeta, F.M. Dalanezi, C. Price, et al.<br />
A183 Effect of Dry and Wet Seasons on the Ability of Oocytes Developing and In Vitro Production of Embryos in Sheep ....... s428<br />
M.T.C. Gonçalves, F.Q.G. Bezerra, R.M. Chaves, C.R. Aguiar Filho, F.F. Paula-Lopes, et al.<br />
A184 Uterine Hemodynamics Changes After Semen Infusion in Mares ..................................................................... s428<br />
J.C. Ferreira, F.S. Ignácio & C. Meira<br />
A185 Effects of Oxygen Tension and Glucose Concentration During In Vitro Maturation of Bovine Oocyte on Oocyte<br />
Quality and Maturation and Embryonic Development .......................................................................................... s429<br />
G.Z. Mingoti, F. Filion, L.C. Smith & P. Vincent<br />
A186 Florfenicol and Sodium Cloprostenol for the Treatment of Retained Placenta in Dairy Cows ........................... s429<br />
A. Pereira, M.P. Palhão, B.F.L. Alves, E.R. Oliveira, T.M. Miyauchi, et al.<br />
A187 Estrogen Sources in Female Sirio Hamsters (Mesocricetus auratus) After Ovarietomy ..................................... s430<br />
K. Annes, M.O.M. Chelini, N.L. Souza, S.R.G. Cortopassi & M.P. Milazzotto<br />
A188 Immunolocalization of the Epidermal Growth Factor (EGF), Transforming Growth Factor- Alpha (TGFA) and EGF<br />
Receptor (EGFR) in the Ovary of the Bitch ............................................................................................................ s430<br />
D.J. Cardilli, J.F. Pérez-Gutiérrez, K.S. Oliveira, C.F. João, F.A. Voorwald, et al.<br />
A189 Induction of Parturition with Aglepristone in Ewes .............................................................................................. s431<br />
F.M. Regazzi, L.G. Silva, G.A.L. Veiga, C.F. Lucio, G.C. Oliveira, et al.<br />
A190 Evidence of the Nitric Oxide Importance to In Vitro Development of Bovine Embryos ...................................... s431<br />
P.D.P.B. Santana, T.V.G. Silva, B.B. Silva, N.N. Costa, D.C.N. Santos, et al.<br />
A191 Influence of Follicle Stimulating Hormone on Genes Involved in the Regulation of Maturation after Culture of<br />
Bovine Cumulus-Oocyte Complexes .................................................................................................................... s432<br />
P.R.L. Pires, K.R.L. Schwarz, L.G. Mesquita, M.R. Chiaratti, L.V.F. Pessôa, et al.<br />
A192 Influence of the Bull and the Grade of Morphological Quality of Cumulus-Oocyte Complexes In Vitro Bovine<br />
Embryonic Developmental .................................................................................................................................... s432<br />
R.C. Lemes, F.P. Elias, A. Renzi, R.A. Vila, C.C. Paz, et al.<br />
A193 Bovine Embryo Modulates Gene Expression of Uterine Growth Factors ........................................................... s433<br />
A.D.B. Lira, A.E. Silva Júnior, L.A. Fátima, L.O. Barros, P.C. Papa, et al.<br />
A194 New Medium for the Use of Brilliant Cresyl Blue in Oocyte Selection for In Vitro Swine Embryos Production -<br />
Preliminary Data .................................................................................................................................................... s433<br />
E.C.S. Santos, J. Pradieé, A.O. Gonçalves, R.G. Mondadori, T. Lucia Jr, et al.<br />
A195 The WNT Signaling-Antagonist DKK1 does Not Impair Development of Bovine Embryos to the Blastocyst Stage s434<br />
K.B. Dobbs, A.C. Denicol & P.J. Hansen<br />
A196 The Effect of Insulin-Like Growth Factor-I on Mitochondrial Activity of ovine Oocytes Exposed to Heat Shock . s434<br />
J. Ispada, R.S. Lima, P.H.B. Risolia, M.E. Assumpção, J.A. Visintin, et al.<br />
A197 The Role of Insulin-Like Growth Factor-I on Developmental Competence of Bovine Oocytes Exposed to Heat<br />
Shock ..................................................................................................................................................................... s435<br />
P.H.B.Risolia, R.S. Lima, J. Ispada, M.E. Assumpção, J.A. Visintin, et al.<br />
A198 Expression Profile of Histone Deacetylase 2 (HDAC2) During Development of Bovine Embryos Produced In Vitro ..... s435<br />
A.R. Ferreira, G.M. Machado, I.R. Bessa, R.C. Nishimura, R. Sartori, et al.<br />
A199 Leptin Gene Polymorphisms and their Associations with Puberty in Nellore Cattle .......................................... s436<br />
L.L. Coutinho, G. Gasparin, F. Savalio, G.B. Mourão, J.B. Ferraz, et al.<br />
XVIII
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A200 Production of Bovine Embryonic Chimeras by Aggregation of Diploid (Bos taurus) and Tetraploid Embryos (Bos<br />
indicus) .................................................................................................................................................................. s436<br />
E.M. Razza, R.A. Satrapa, I.P. Emanuelli, C.M. Barros & M.F.G. Nogueira<br />
A201 Embryo Production by Parthenogenetic Activation and IVF in Capuchin Monkeys ........................................... s437<br />
S.F.S. Domingues, J.S. Lima, D.L. Leão, A.B. Brito, R.V. Sampaio, et al.<br />
A202 Selection for Post Weaning does Not Change Age at Puberty in Heifers ........................................................... s437<br />
J.M. Garcia, F.M. Monteiro & A.T. Arevalo<br />
A203 Supplementation of Media for In Vitro Maturation of Oocytes of Rabbits ........................................................... s438<br />
T.M. Gonçalves, J.R. Sangalli, M.R. Chiaratti & F.V. Meirelles<br />
A204 Use of C57BL/6/EGFP Mouse Testicular Cells to Validate the Technique of Microinjection in Embryonic Chimera<br />
Production ............................................................................................................................................................. s438<br />
D.M. Souza, H. Fernandes, P.V. Silva, B. Cazari, P.D. Moço, et al.<br />
A205 Alterations in Methylation Profiles of Lysine Residues 4 and 27 of Histone H3 in Bovine Blastocysts Following<br />
Cryopreservation ................................................................................................................................................... s439<br />
D.R. Arnold, B.M.C. Faccio, R. Vantini, C.V. Rocha Jr, C.A.P. Corrêa, et al.<br />
CLONING, TRANSGENESIS AND STEM CELLS<br />
A206 Mesenchymal Stem Cells Isolated from Subcutaneous Fat of a Mare Display Chondrogenic, Osteogenic and<br />
Adipogenic Potential .............................................................................................................................................. s439<br />
A.T. Arevalo, J.C. Salazar, L. Rodriguez & F.O. Castro<br />
A207 Cloned Embryos from Two Cell Types of Endangered Pig Breeds ..................................................................... s440<br />
L.U. Ohlweiler; J.C. Mezzalira, D.S. Brum, J. Cristani, A.N. Moraes, et al.<br />
A208 Establishment of a hG-CSF Transgenic Goat Line Originated from a Male Founder and Detection of Age at<br />
Puberty in F1.......................................................................................................................................................... s440<br />
R.I.T.P. Batista, J.M.G. Souza, C.R.G.S. Oliveira, T.M. Almeida, M.P. Vieira, et al.<br />
A209 In Vitro Culture and Osteogenic Differentiation of Cryopreserved Canine Adipose-Derived Stem Cell ........... s441<br />
I. Arruda, C.C. Macedo, A.J. Listoni, M.D. Guastali, M. Pardo, et al.<br />
A210 Effect of Fibroblast Cell Passage on Production of Gyr Embryos by Nuclear Transfer Technique ..................... s441<br />
C.C.R. Quintão, M.M. Pereira, N.C. Rabelo, J.R.T. Alonso, L.T. Iguma, et al.<br />
A211 Effect of Trichostain a on Production of Bovine Embryos Produced by Somatic Cell Nuclear Transfer and<br />
Subsequent Gestations ......................................................................................................................................... s442<br />
L.S.A. Camargo, C.C.R. Quintão, A.C. Denicol; L.T. Iguma; B.C. Caravalho, et al.<br />
A212 Efficiency of Nuclear Transfer in Equine: Direct Injection of the Donor Cell Nucleus into the Recipient Cytoplasts s442<br />
C.B. Fernandes, C.G.P. Gonçalves, L.R. Martins & F.C.L. Alvarenga<br />
A213 New Strategy to Induce Transgenesis in In Vitro Fertilization (IVF) Bovine Embryos ......................................... s443<br />
N.G. Canel, R.J. Bevacqua, M.I. Hiriart & D. Salamone<br />
A214 Expression of Xist, G6pd and Hspa1a Genes in Bovine Blastocysts Reconstituted by NT from Recipient Oocytes<br />
Produced by Chemically Assisted Enucleation .................................................................................................... s443<br />
N.Z. Saraiva, C.S. Oliveira, T.A.D. Tetzner, M.R. Lima, S.C. Méo & J.M. Garcia<br />
A215 Development of Bovine Clone Embryos Using Fibroblasts Cultured in SOF Supplemented with Conjugated<br />
Linoleic Acid (C-9,T-11) ......................................................................................................................................... s444<br />
N. Klein, L.U. Ohlweiler, F.G. Leivas, D.S. Brum, M.G.L.Pinto, et al.<br />
A216 Pregnancy of Cloned Embryos from a Freemartin Cow of an Endangered Breed ............................................. s444<br />
J.C. Mezzalira, M.G.L. Pinto, F.C. Zago, A.M. Silva, N. Albino, et al.<br />
A217 Age Influence of Cytoplst Donor in Bovine Clone Production by Nuclear Transfer ............................................ s445<br />
E. Siqueira Filho, T.O. Diesel, E. Ramiro Júnior, A.A. Fidelis & R. Rumpf<br />
A218 Isolation and Immunophenotyping of Multipotent Mesenchimal Stem Cells from Bovine Embryos Yolk Sac .... s445<br />
C.A.F. Mançanares, L.J. Oliveira, R.V. Sampaio, R.S.N. Barreto, A.C.F. Mançanares, et al.<br />
A219 The Expression of Pluripotence Markers in Donor Cells Increased Cloning Efficiency in Bovine ..................... s446<br />
J.R. Manriquez, A.E.Velasquez; F.O. Castro & L. Rodriguez<br />
XIX
Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A220 In Vitro Culture of Bovine Embryos in Murine Es Cell Conditioned Media Negatively Affects Expression of<br />
Pluripotency Related Markers Oct4, Sox2 and SSEA1 ........................................................................................ s446<br />
C.S. Oliveira, M.M. Souza, N.Z. Saraiva, T.A.D. Tetzner, M.R. de Lima, et al.<br />
A221 Technical Report: First Pregnancy of a Equine Clone in Brazil ........................................................................... s447<br />
B.V. Sanches; A.G. Rigo, J.H.F. Pontes; R.M. Untura, L.L. Moino, et al.<br />
A222 Toxicity of Carbon Nanotubes in Bovine Fibroblast Cells ................................................................................... s447<br />
M.M. Pereira, C.C.R. Quintão, H.M. Brandão, N.B.R. Raposo, N. Rabelo, et al.<br />
A223 Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed? ......... s448<br />
M.S. Miranda; F.F. Bressan; T.H.C. de Bem, G.K. Merighe4, W.A. King, et al.<br />
A224 Use of Embryo Transcervical Collection in a Founder Transgenic Goat for Human Granulocyte Colony-<br />
Stimulating Factor (Hg-CSF) .................................................................................................................................. s448<br />
R.R. Moura, J.M.G. de Souza, T.M. de Almeida, M.P. Vieira, C.H, S. de Melo, et al.<br />
A225 Success on Isolation, Immunophenotypical Characterization and Differentiation in Osteogenic, Adipogenic and<br />
Chondrogenic Lineages of Mesenchymal Stem Cells (MSCS) From the Equine Amniotic Liquid ...................... s449<br />
B. de Vita, I. Martin, L.L. Campos, A. J. Listoni, N.P.P. Freitas, et al.<br />
SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
A226 Altered Expression of Genes Related to Glucose Metabolism in Bovine Oocytes Maturated In Vitro or In Vivo s449<br />
S. Wohlres-Viana, M.M. Pereira, J.N.S. Sales, A.J.R. Camargo, C.C.R. Quintão, et al.<br />
A227 Comparative Analysis of Membrane Integrity Methods of Ovine Cryopreserved Semen .................................. s450<br />
A.M. Arrais, C.S.P. Carvalho, B.P. Carvalho, F.Q. Costa, E.M. Mogollon-Waltero, et al.<br />
A228 Peptide Fingerprinting Analysis of Bovine Embryos of Nelore (Bos indicus) and Red Angus (Bos taurus) Races<br />
Obtained Through In Vitro Fertilization ................................................................................................................. s450<br />
R.C. Rochetti, Y.F. Watanabe, F. Perecin, J. R. Sangalli, F.V. Meirelles, et al.<br />
A229 Proteomic Analysis of Primitive Gut in Bovine Embryos ..................................................................................... s451<br />
A.C.F. Mançanares, L.J. Oliveira, E. Zimberknopf, F. Perecin, G.H.M.F. Souza, et al.<br />
A230 Evaluation of Different Times of Equilibrium on Intensive Progressive Motility and Sperm Pathologies of<br />
Cryopreserved Ram Semen .................................................................................................................................. s451<br />
R.T. Pereira & O. L. Garszareck<br />
A231 Endometrial Biopsy in Mares in Heat Repeater: Immunohistochemical Analysis of T Lymphocytes and<br />
Macrophages ......................................................................................................................................................... s452<br />
F.B.F. Silva, J.F.E. Moreira, J.S. Leite & A.M.R. Ferreira<br />
A232 Micro-FTIR Biochemical Characterization of Bull Spermatozoa Irradiated by Low Power Laser ...................... s452<br />
T.R. Dreyer, A.F.Siqueira, T.D. Magrini, M.E. Assumpção, H.S. Martinho, et al.<br />
A233 Comparison of mRNA Expression of Stat3 and Akp2 in Bovine Embryos with 7 and 11 Days after In Vitro<br />
Fertilization ............................................................................................................................................................ s453<br />
T.S. Rascado, M.D. Guastali, L. E. Vergara, R.R.D. Maziero, M. J. Sudano, et al.<br />
A234 Correlation Between Stallion Fertility and DNA Fragmentation of Frozen Sperm Analysed by The Acridina Orange<br />
Test ......................................................................................................................................................................... s453<br />
C.P.F. Dell´aqua, J.A. Dell ´aqua Junior, M. A. Alvarenga & F.O. Papa<br />
A235 Cryopreservation of Somatic Cells in 0.5 Ml Or 0.25 Ml Straws and Propylene Glycol or Ethylene Glycol ....... s454<br />
M. Urio, C.R.A. Duarte, R.C. Fleith, M.L. Munhoz, F.C. Zago, et al.<br />
A236 Ovarian Tissue Cryopreservation from Bitches: Morphologic Evaluation of Pre-Antrals Follicles ..................... s454<br />
J.L.J.P. Rôlo, M.S. Araújo, F. Paulini & C.M. Lucci<br />
A237 Cryotolerance of Bovine In Vitro-Produced Embryos Subjected to Conjugated Linoleic Acid .......................... s455<br />
L.S.R. Marinho, J.C. Mezzalira, L.U. Ohlweiler, F. Forell, D.E. Oliveira, et al.<br />
A238 BVDV Detection by a Polymerase Chain Reaction-Based Assay in Bovine Follicular Fluid ............................. s455<br />
A.G. Galuppo, M.N. Weber, R.F. Budaszewski, A.O. Corbellini, L.S. Marques, et al.<br />
A239 Echogenicity of Testes of Buffalo Crossbreds Created in the State Of Para, Amazon, Brazil ............................. s456<br />
H.D.M. Ayala, S.T.R. Filho, H.F.L. Ribeiro, K.B.Nunes, E.Y.E. Mesquita, et al.<br />
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Acta Scientiae Veterinariae. 39 (Suppl 1): <strong>2011</strong>.<br />
A240 Effect of Different Cryoprotectant Solutions in the Bovine Spermatogonial Cell Viability Submitted to Two Cooling<br />
Curves ................................................................................................................................................................... s456<br />
A.M. Cunha, H.C.Bessler, E.R.Cunha, C.G. Silva, G.H.L. Martins, et al.<br />
A241 Effect of Antioxidant Substances in Cryopreserved Semen from Buffalos (Bubalus bubalis) ........................... s457<br />
A.A. Gonçalves, S.R.S. Castro, A.R. Garcia, A.X. Santos, G.R. Silva, et al.<br />
A242 Bull Effect in the Embryo Survival Produced In Vitro after Submission to Slow Freezing .................................. s457<br />
M.B. Fernandes, A. Mioranza, V.T.F. CiprianO, R.A. Vila, M.A. Galerani, et al.<br />
A243 Effect of Polyunsaturated Fatty Acids during In Vitro Culture of Bovine Embryos on the Success of Vitrification -<br />
Preliminary Results ............................................................................................................................................... s458<br />
N.A.S. Rocha, B.C.S. Leão, G.Z. Mingoti & M.F. Accorsi<br />
A244 Effects of Polyunsaturated Fatty Acids Omega 3 and 6 during In Vitro Maturation and Culture on Bovine Embryo<br />
Development and Cryotolerance .......................................................................................................................... s458<br />
B.C.S. Leão, N.A.S. Rocha, M.F.Accorsi, G.Z. Mingoti<br />
A245 Study of Folicular Vascularization Using Tridimensional Images: A New Approach ........................................... s459<br />
E.K.N Arashiro, M.P. Palhão, S. Wohlres-Viana, L.G.B. Siqueira, M.R.J.M. Henry, et al.<br />
A246 Qualitative Study of the Interaction of BSPS (Binder Of Sperm Proteins) With The Sperm Cells in Ruminants s459<br />
M.F.V. Tilburg, Í.C. Lima, V.G. Cadavid, A.A. Araújo, D.R. Rocha, et al.<br />
A247 Gene Expression of Embryos In Vitro Produced With Sorted Sperm by Density Gradient Centrifugation and Flow<br />
Cytometry ............................................................................................................................................................... s460<br />
A.C. Lucio<br />
A248 DNA Extraction by Alkaline Lysis for Molecular Diagnosis: A Fast, Simple and Efficient Method for the PCR-Based<br />
Sexing of In Vitro-Produced Sheep Embryos ....................................................................................................... s460<br />
K.C.S. Tavares, C. Feltrin, I.S. Carneiro, D.B. Riosa. R.K.Costa, et al.<br />
A249 Multiparous Buffaloes Submited to Exogenous Melatonin in Long Photoperiod ............................................... s461<br />
A.O. Sousa<br />
A250 Equine Epididymal Semen Fertility after the Addition Of Seminal Plasma ......................................................... s461<br />
C.M.M. Oña, L.C.O. Magalhães, F.O.Papa, F.C.L. Alvarenga, I. Martin, et al.<br />
A251 Embryo Genotyping from Biopsied Bovine Embryos Using A 50 K Snp Chip .................................................... s462<br />
D. Bourhis, E. Mullaart, P. Humblot, H. Knijn, B. Guienne, et al.<br />
A252 Acrosome Integrity of Ram Spermatozoa Submitted to Cryopreservation: Dynamics Related to Plasmatic<br />
Membrane Cryoresistence .................................................................................................................................... s462<br />
H.C. Azevedo, S.D. Bicudo, M.S. Maia, D.B. Sousa, L. Rodello, et al.<br />
A253 In Vitro Maturation of Oocytes Vitrified Immature in Different Conditions ........................................................... s463<br />
F. Forell, N. Albino, J.M. Junior, F.C.Zago & A. Mezzalira<br />
A254 DNA Methylation Pattern of the Igf2 and Xist Genes in Oocytes Obtained of the Preantral Follicles from Nellore<br />
Cows (Bos taurus indicus) ..................................................................................................................................... s463<br />
L.F.S. Gomes, I.R. Bessa, F.C. Rodrigues, P.J.S. Rua, O. Bravim, et al.<br />
A255 Ultrasonographic Profile of Embryonic/Fetal Growth of Transgenic Goats for hG-CSF ...................................... s464<br />
D.Í.A. Teixeira, C.R.G.S. Oliveira, C.H.S. Melo, A.C.A.T. Filho, R.I.T.P. Batista, et al.<br />
A256 Proteome-Based Insights on Oocyte Competence from a Retrospective Analysis of Bovine Follicular Fluid ... s464<br />
F. Perecin, L.J. Oliveira, J.R.Sangalli, T.H.C. De BEM & G.H.M.F. Souza<br />
A257 Relative Abundance of Maternal Transcripts in Oocytes with and Without Polar Body after In Vitro Maturation s465<br />
L.T. Iguma, M. M. Pereira, S. Wohlres-Viana, J.N.S. Sales, C.C.R. Quintão, et al.<br />
A258 Relationship between Acrossome Reaction, Post-Thaw Sperm Evaluation and Total Protein in Moxotó Goat<br />
Semen ................................................................................................................................................................... s465<br />
R.V. Valle, Â.M.X. Eloy, N. M. M. Silva, F.W.V. Silva & J.R. Furtado<br />
A259 Moura Pig Sperm Viability after Cryopreservation .............................................................................................. s466<br />
M.G. Marques, A. Dahmer, V. H. Grings & E.A. P. Figueiredo<br />
A260 Vitrification of In Vitro Produced Bovine Embryos after Chemical Lipolysis ....................................................... s466<br />
D.M. Paschoal, M.J. Sudano, T. S. Rascado, L.C.O. Magalhães, L.F.Crocomo, et al.<br />
A261 Vitrification of In Vitro Produced Bovine Embryos Supplemented With Triiodothyronine (T3) and Thyroxine (T4) . s467<br />
M.C.C. Mattos, E.R. Siqueira, F.S.P. Rochelle, A.C.F. Pereira, B.V. Sanches, et al.<br />
XXI
A262 Bovine Oocyte Vitrification: Effects on Fertilization Rates ................................................................................... s467<br />
J.F.W. Sprícigo, R. Rumpf & M.A.N. Dode<br />
www.ufrgs.br/actavet<br />
Acta Scientiae Veterinariae. 39(Suppl 1): s1-s467.<br />
<strong>2011</strong><br />
XXII
R.C. Uliani, L.A. Silv<br />
ilva,<br />
M.A. Alv<br />
lvar<br />
arenga.<br />
<strong>2011</strong>. Mare’s Folliculogenesis: Assessment of ovarian and perifollicular<br />
vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s1.<br />
ISSN 1679-9216 (Online)<br />
What is producing the dramatic improvement in reproductive efficiency in U.S.<br />
dairy herds from 2000 until now?<br />
Milo C. Wiltbank 1 , Alexandre H. Souza 1 , Jerry N. Guenther 1 , Mary M. Herlihy 1 & Roberto Sartori 2<br />
ABSTRACT<br />
Background: Efficient reproduction is essential for profitability on dairy farms. Nevertheless, reproductive efficiency is not<br />
optimal on many dairies in the United States (US) and Brazil.<br />
Review: Daughter pregnancy rate has been used to monitor changes in reproductive efficiency in US dairy herds using data on<br />
days open collected by the US Department of Agriculture. Between 1977 and 2000, this value decreased over 25%, from 27.78<br />
to 20.97, for an average decrease of about 1% per year. The decline in reproductive efficiency was due to declines in both<br />
fertility (pregnancies per AI; P/AI) and heat detection rate (percentage of eligible cows that receive AI every 21 days).<br />
Surprisingly, there has now been a sustained increase in daughter pregnancy rate from 20.97 to 25.48 between 2000 and 2008,<br />
representing an increase at about twice the rate (2%/year) as the previous decline. The increase in reproductive efficiency is<br />
due to improvements in heat detection rate without any consistent change in P/AI. Between 1998 and 2008, there was a<br />
dramatic change in the US dairy industry from less than 10% (1998) of dairy cows managed on dairies that used timed AI (TAI)<br />
to more than 50% (58%) of cows on dairies that use TAI by 2008. The improvements in reproductive efficiency were observed<br />
in herds using TAI and not in herds that were not synchronized. The P/AI are similar on herds that use TAI, compared to herds<br />
that breed after detection of estrus. Protocols, such as Ovsynch, that allow TAI but do not increase P/AI are termed “service rate<br />
programs” because they only improve the heat detection rate without a change in P/AI. An understanding of the physiology<br />
of dairy cows has allowed the development of TAI protocols that consistently improve P/AI, compared to AI after detection of<br />
N<br />
estrus. However, these “fertility programs” are just beginning to be effectively utilized on most commercial US dairies, leaving<br />
the possibility for further improvements in reproductive efficiency in US dairy herds during the next few years. Further, the TAI<br />
programs utilized in the US generally have a lack of an effect of milk production on either heat detection rate or P/AI.<br />
Conclusion: It is now possible to achieve high milk production and exceptional reproduction by effectively utilizing current<br />
nutritional, genetic, and reproductive management programs.<br />
Keywords: fertility, reproductive management, dairy cattle.<br />
1<br />
Department of Dairy Science, University of Wisconsin-Madison, USA. 2 Department of Animal Science, Superior School of Agriculture “Luiz<br />
de Queiroz” (ESALQ), University of São Paulo, Piracicaba, SP, Brazil. CORRESPONDENCE: M.C. Wiltbank [wiltbank@wisc.edu]. 1675<br />
Observatory Drive, Madison, WI 53706-1284, USA.<br />
s1
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s3- s13.<br />
ISSN 1679-9216 (Online)<br />
How FTAI and FTET Impact t Repr<br />
eproduc<br />
ductiv<br />
tive Efficienc<br />
fficiency of Brazilian Dair<br />
airy Her<br />
erds<br />
Carlos Alberto Rodrigues 1 , Roberta Machado Ferreira 2 , Lais Mendes Vieira 2 , Andressa L. Ranieri 3 ,<br />
Péricles R.L. Silva 1 & Pietro Sampaio Baruselli 2<br />
ABSTRACT<br />
Background: Throughout dairy cows evolution, milk production was always the key point to select the superior animal.<br />
Currently, several evidences has shown that high milk production have intensively contributed to the decline of dairy cattle<br />
fertility. Beyond milk production, dairy cows have their reproductive performance impaired by another factors, heat stress and<br />
repeat-breeding. Methods like fixed time artificial insemination and embryo transfer were developed to minimize the effects of<br />
these factors, and improve dairy herds profitability. This review aims to show some key-point experiments conducted to improve<br />
the efficiency of the self-appointed protocols for artificial insemination and embryo transfer in Brazil, overcoming several<br />
reproductive problems. Our goal is to develop cheap and easy self-appointed programs that facilitate animal handling and<br />
maximize their reproductive outcomes all over the year.<br />
Review: Failure in estrus detection is the mainly limiting factor for the use of artificial insemination in high-production dairy herd.<br />
An excellent alternative to overcome the need of estrus detection is fixed time artificial insemination. Many protocols with and<br />
without the use of estradiol have been developed to that end. Among the protocols for fixed time artificial insemination without<br />
estradiol, DoubleOvsynch has been extensively used recently in American dairy herds. In Brazil, similar pregnancy rate was<br />
obtained compared to progesterone-estradiol based protocols for fixed time artificial insemination. Particularities of progesteroneestradiol<br />
based protocols as (1) new progesterone device or devices previously used for eight days; (2) different doses of eCG;<br />
and (3) the use of estradiol cypionate for fixed time artificial insemination have been studied in Brazil. The use of self-appointed<br />
artificial insemination also enabled the reduction of the interval calving-conception compared to cows inseminated following the N<br />
standing estrus. Regarding the low fertility of repeat breeders and the effect of heat stress at early pregnancy, other methods like<br />
embryo transfer became important tools to enhance reproductive efficiency of Brazilian dairy herds. Protocols were also developed<br />
to allow fixed time embryo transfer, eliminating the need of estrus detection and improving the reproductive efficiency of lactating<br />
recipients. As well as described for fixed time artificial insemination treatments, there is a large variety of hormone combination<br />
for fixed time embryo transfer (with and without estradiol). An experiment conducted in Brazil demonstrated that protocols for<br />
fixed time embryo transfer without estradiol can be as good as with estradiol to synchronize high-producing Holstein recipients,<br />
essentially during summer. Particularities related to embryos cryopreservation, synchronization of the estrus cycle of donors<br />
and recipients and the site of embryo release into the uterine horn were also investigated. Greater conception rates were achieved<br />
when fresh embryos were transferred compared to frozen-thawed ones. Also, the tight synchronization between donor and<br />
recipient (same day of estrus) resulted more pregnancies than when recipients were one day later or in advantage in relation to<br />
donors. Moreover, the site of embryo release into the uterine horn (ipsilateral to the corpus luteum) had no effect on pregnancy<br />
rates after in vivo produced embryo transfer.<br />
Conclusion: Both fixed time artificial insemination and fixed time embryo transfer are important tools to improve reproductive<br />
efficiency of high-producing dairy cows. These biotechnologies help bypassing some of the greatest challenges of dairy cattle<br />
reproduction: the difficulties of estrus detection, and the low fertility associated to heat stress and repeat breeding.<br />
Keywords: FTAI, FTET, heat stress, repeat breeders, reproductive efficiency.<br />
Descritores: IATF, TETF, estresse térmico, repetidoras de serviço, eficiência reprodutiva.<br />
1<br />
Clínica SAMVET de São Carlos, São Carlos, SP, Brazil. 2 Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e<br />
Zootecnia (FMVZ) da Universidade de São Paulo (USP), São Paulo, SP, Brazil. 3 Departamento de Reprodução Animal e Medicina Veterinária<br />
Preventiva da Faculdade de Ciências Agrárias e Veterinária (FCAV) da Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil.<br />
CORRESPONDENCE: C.A. Rodrigues [carlos.samvet@terra.com.br/barusell@usp.br]. Clínica SAMVET de São Carlos, Avenida Getúlio<br />
Vargas, n. 300, Jardim São Paulo. CEP 13570-390, São Carlos, SP, Brazil<br />
s3
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
I. INTRODUCTION<br />
II. THE USE OF FTAI IN HIGH PRODUCTION HOLSTEIN<br />
CATTLE<br />
2.1 Protocols for FTAI without estradiol<br />
2.2 Protocols for FTAI with estradiol<br />
III. THE USE OF ET IN HIGH-PRODUCING HOLSTEIN<br />
CATTLE<br />
3.1 Benefits of applying embryo technology in Holstein cattle<br />
raised in tropical condition<br />
3.2 Advantages of using FTET<br />
3.3 FTET protocols with or without E2<br />
3.4 Particularities of ET process<br />
IV. CONCLUSION<br />
I. INTRODUCTION<br />
During the last decades, the selection of<br />
genetically superior cows for milk production has been<br />
correlated to the lower fertility of dairy herds. However,<br />
the decline in reproductive efficiency has become<br />
alarming [10,11,21]. Several evidences lead to a role of<br />
high milk production and heat stress (HS) in contributing<br />
with changes in reproductive physiology that may underlie<br />
the decline of cows profitability [10,27].<br />
Aiming to avoid the need for estrus detection<br />
and enhance pregnancy rates, different protocols for<br />
synchronization of follicular wave emergence and<br />
ovulation with self-appointed managements have been<br />
developed. Mainly, the hormonal treatments are based<br />
on a combination of GnRH/PGF 2α<br />
/GnRH – Ovsynch<br />
[14] or association of progesterone (P4) releasing devices<br />
and estradiol (E2) [3]. Currently, there are numerous<br />
protocols for fixed time artificial insemination (FTAI) and<br />
embryo transfer (FTET) with different combination.<br />
Based on the knowledge that oocytes and<br />
embryos at early stages of development are extremely<br />
sensitive to HS [5,15,7], embryo transfer (ET) was<br />
employed as a potential tool to improve summer fertility<br />
by bypassing the effects of HS on early embryonic<br />
development [1,9]. Moreover, FTET can be successfully<br />
employed to bypass the conception failure observed in<br />
repeat breeding (RB) cows, especially during HS<br />
[reviewed by 6, 17].<br />
This review aims to show some key-point<br />
experiments conducted to improve the efficiency of FTAI<br />
and FTET protocols in Brazil, overcoming several<br />
reproductive problems. Our goal is to develop cheap and<br />
easy self-appointed programs that facilitate animal<br />
handling and maximize their reproductive outcomes all<br />
over the year.<br />
II. THE USE OF FTAI IN HIGH PRODUCTION HOLSTEIN<br />
CATTLE<br />
The main limiting factors for the massive use<br />
of artificial insemination (AI) are the failures and<br />
difficulties to perform efficient estrus detection. Even<br />
in well-organized farms the rate of estrus detection<br />
barely achieves 50% [26], which in turn result in few<br />
number of AI. When FTAI is used the service rate<br />
achieves 100%, ending up with a larger number of<br />
cows been inseminated, increasing the percentage of<br />
P/AI. Protocols currently used combine GnRH and<br />
PGF 2α<br />
or P4 and E2. The choice for one or another<br />
mainly relies on the availability of the drugs, the<br />
permission to use them (federal laws) and their cost.<br />
2.1 Protocols for FTAI without estradiol<br />
A protocol developed to synchronize both<br />
cyclic and anovular cows was the Double-Ovsynch<br />
(DO) [23]. It consists of a pre-synchronization<br />
method using an Ovsynch 7d prior to the Ovsynchtimed<br />
AI protocol. In addition to treatment of anovular<br />
cows, it seems likely that DO more tightly<br />
synchronized the stage of the estrous cycle at<br />
initiation of Ovsynch compared to Presynch,<br />
increasing fertility to the FTAI [23]. Recently, DO<br />
was compared to a P4-E2 based protocol [P4 device<br />
and estradiol benzoate (EB) - 8d - device removal,<br />
eCG, PGF 2α<br />
and EC - 56h – GnRH and AI]<br />
commercially used in Brazilian Holstein cows<br />
(Ranieri & Baruselli, unpublished data). Both<br />
protocols resulted similar 30 (P = 0.15) and 60d<br />
pregnancy rate (P = 0.20) and pregnancy loss (P =<br />
0.83; Figure 1). Also, when cows had a corpus luteum<br />
(CL) at the beginning of the treatment greater 30 and<br />
60d pregnancy rates were observed, but pregnancy<br />
loss was similar (Figure 2).<br />
In conclusion, the P4-E2 based protocols require<br />
lesser managements (3 vs 7) and have shorter duration<br />
(10 vs 25d) than DO with similar 30 and 60d P/AI<br />
(although it reached 12.5 and 9.6 points over DO,<br />
respectively).<br />
2.2. Protocols for FTAI with estradiol<br />
Currently, numerous P4-E2 based protocols for<br />
FTAI [2,12] with different combinations of hormones,<br />
doses and duration are available in Brazil. The possibility<br />
of using P4 devices previously used for eight days and<br />
the benefits of enlarging the proestrus period [by giving<br />
estradiol cypionate (EC) at device withdrawal] during<br />
s4
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
Figure 1. Percentage of 30 and 60 d pregnancy and pregnancy loss of high-producing Holstein<br />
cows treated with different protocols for FTAI. P = 0.15, 0.20 and 0.83, respectively (Adapted<br />
from Ranieri & Baruselli, unpublished data).<br />
N<br />
Figure 2. Percentage of 30 and 60 d pregnancy and pregnancy loss of high-producing<br />
Holstein cows with or without a CL at the begging of the protocol for FTAI.* P = 0.01.<br />
Adapted from Souza et al.[23].<br />
protocols for FTAI was evaluated [22]. Neither the<br />
type of device (new or previously used) nor the<br />
administration of EC affected P/AI of cows receiving<br />
GnRH as ovulatory stimulus. Thus, the device<br />
previously used can be efficiently associated with EB<br />
in protocols for FTAI. Also, there was no additional<br />
effect of using EC to enlarge the proestrus duration<br />
when cows are treated with GnRH prior to AI (Figure<br />
3). However, cows with BCS < 2.75 EC improved<br />
P/AI compared to GnRH (21.7% vs. 6.1%; P <<br />
0.05)[22].<br />
Another goal was to verify if additional eCG<br />
(400IU) at device removal could improve P/AI. Also,<br />
the type of ovulatory stimulus (GnRH or EC) was<br />
s5
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
evaluated [22,24]. The average diameter of the<br />
ovulatory follicle (13.9 mm) and time to the LH peak<br />
(average 43.6 h after device removal) was unaffected<br />
by the type of ovulatory stimulus or the use of eCG.<br />
However, greater variability in time to the LH peak<br />
was found when cows where treated with EC than<br />
GnRH (S.E.M. 2.6 vs 1.8). Overall, time to ovulation<br />
averaged 73.2 h after device removal. The size of<br />
the CL, P4 concentrations in the subsequent diestrus,<br />
and P/AI (Figure 4) were unaffected by the use of<br />
eCG or type of ovulatory stimulus. However, thinner<br />
cows (body condition score below 2.75) receiving<br />
eCG had greater P/AI than those not treated with eCG<br />
(Figure 5).<br />
Another study was done to reevaluate the<br />
need of using eCG (at device removal) and the benefit<br />
Figure 3. P/AI for Holstein cows treated for FTAI with new progesterone device<br />
or device previously used for 8 days, and receiving or not estradiol cypionate at<br />
device removal. P > 0.05. Adapted from Souza et al. [23].<br />
Figure 4. P/AI for Holstein cows treated or not with eCG, and receiving or not<br />
estradiol cypionate at device removal for FTAI. P > 0.05. Adapted from Souza et<br />
al. [23].<br />
s6
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
Figure 5. P/AI for Holstein cows receiving or not eCG for FTAI and<br />
with BCS < 2.75 or = 2.75. P < 0.05. Adapted from Souza et al. [23].<br />
of increasing its dose (0, 400 or 600 IU) in protocols<br />
for FTAI. Although follicular growth rate tended to<br />
increase in cows treated with eCG (no eCG = 2.5 ±<br />
0.5 a vs 400IU eCG = 3.8 ± 0.6 b and 600IU eCG = 3.5<br />
± 0.5 b ; P = 0.06), follicular diameter, ovulation rate,<br />
time to ovulation, size of the CL and P4 concentration<br />
were not affected by eCG or its dose (unpublished<br />
data). P/AI was also similar among groups (Figure<br />
6), suggesting that eCG might not be essential to FTAI<br />
protocols in high-producing confined Holstein cows<br />
in Brazil.<br />
Recently, the interval calving-AI and calvingconception<br />
and P/AI at first, second, third services, and<br />
150 DIM were compared among FTAI and AI 12h after<br />
estrus detection [25]. Similar P/AI after first, second and<br />
third AI and at 150 DIM were achieved. However,<br />
pregnancy loss was greater with FTAI compared to<br />
AI following estrus (Figure 7). The interval calving-<br />
AI and calving-conception were reduced in FTAI<br />
cows, because they started the protocol around 57<br />
DIM regardless the observation of estrus, anticipating<br />
the first and second AI postpartum with similar P/AI<br />
(Figure 8). Thus, although FTAI protocols initiated<br />
around 60 DIM decreased the interval calvingconception,<br />
the percentage of pregnant cows at 150<br />
DIM was similar among cows subjected to FTAI and<br />
AI after estrus detection.<br />
N<br />
III. THE USE OF ET IN HIGH-PRODUCING HOLSTEIN<br />
CATTLE<br />
The main advantages of implementing the ET<br />
is (1) to accelerate the herd genetic gain by producing<br />
more calves per selected donors, (2) to increase<br />
reproductive efficiency of RB cows, and (3) to reduce<br />
the deleterious effects of HS.<br />
Figure 6. P/AI for high-producing Holstein cows receiving 0, 400 or<br />
600 IU of eCG for FTAI. P > 0.05. Adapted from Ferreira & Baruselli,<br />
unpublished data.<br />
s7
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
Figure 7. P/AI at first, second and third services and at 150 days in milk<br />
(DIM), and pregnancy loss (first to third services) of Holstein cows<br />
inseminated 12 h following estrus detection or treated for FTAI. P > 0.05.<br />
Adapted from Teixeira [25].<br />
Figure 8. Interval calving-insemination and calving-conception of Holstein cows<br />
inseminated 12 h following estrus detection or treated for FTAI. *P = 0.0001; **P<br />
= 0.02. Adapted from Teixeira [25].<br />
3.1 Benefits of applying embryo technology in Holstein<br />
cattle raised in tropical condition<br />
It is already well established that HS strongly<br />
impairs Holstein cows fertility, decreasing the herd<br />
reproductive efficiency [9]. Oocytes and embryos at early<br />
stages of development are extremely sensitive to heat<br />
stress [7,9]. A strategy that has been used to improve<br />
fertility during HS is ET.<br />
In attempt to elucidate the reproductive performance<br />
of Brazilian high-producing Holstein cows after<br />
AI or ET, a retrospective study was conducted [16].<br />
Cows receiving an embryo had higher P/AI (41.9%) than<br />
s8
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
those inseminated (28.5%) throughout the year (Figure<br />
9). Similar results were observed when RB were<br />
evaluated [17]; Figure 9. Therefore, ET came out as a<br />
valuable alternative to obtain satisfactory P/AI throughout<br />
the year, especially during HS and in RB.<br />
Although ET enhances P/AI compared to AI,<br />
Freitas et al. [2010] demonstrated that pregnancy loss<br />
between 30 and 60 d pregnancy was higher when<br />
cows received embryos (20.5%) than AI (17.3%; P =<br />
0.001). RB had similar (P = 0.39) embryonic losses<br />
compared to non RB after AI (17.1 vs 17.9%) or ET<br />
(19.4 vs 20.7%). Overall, HS increased (P = 0.001)<br />
pregnancy loss regardless the use of AI (16.1 vs<br />
19.1%) or ET (18.4 vs 23.1%).<br />
3.2 Advantages of using FTET<br />
As for FTAI, an alternative to avoid the need<br />
of estrus detection to transfer embryos is the use of<br />
FTET [2,4]. Rodrigues et al. [2009] compared the<br />
reproductive efficiency of high-producing RB<br />
recipients receiving embryo at fixed time or 7 d after<br />
estrus detection. The protocol (P4 implant and E2 -<br />
8d - implant removal, eCG and PGF2α - 9d – ET and<br />
GnRH) increased transferred-treated and pregnancy<br />
rates. When cows had a CL at the beginning of the<br />
treatment transferred-treated rate was enhance,<br />
however similar pregnancy was obtained compared<br />
to cows that started the protocol without a CL (Figure<br />
10). Thus, FTET increased the number of recipients<br />
suitable for ET compared to estrus detection, and<br />
allow the use of cows regardless the presence of a<br />
CL at the begging of the protocol, with same<br />
efficiency.<br />
3.3 FTET protocols with or without E2<br />
High-producing Holstein cows submitted to<br />
FTET using or not E2, and with or without eCG at<br />
device removal had similar 25 and 42 d pregnancy<br />
rates during winter. However, during summer the<br />
recipients treated with GnRH (without E2) had greater<br />
25 d pregnancy rates. But protocols with E2 seem to<br />
be more efficient during winter (Figure 11;<br />
unpublished data). Similarly to FTAI, the use of eCG<br />
had no effect in RB recipients subjected to FTET (Figure<br />
12). In conclusion, FTET without E2 can be an<br />
important strategy (need more studies) during HS in<br />
high-producing Holstein recipients<br />
3.4 Particularities of ET process<br />
A retrospective study with Holstein cows<br />
raised in tropical regions showed that greater<br />
conception rates were achieved when fresh embryos<br />
were transferred (43.9%; n = 2,634) compared to<br />
frozen-thawed ones (39.5%; n = 2,237) [18]. Further,<br />
the tight synchronization between donor and recipient<br />
N<br />
(same day of estrus) showed higher efficacy (43.6%;<br />
n = 2,648) than when recipients were one day later<br />
(41,5%; n = 1,259) or advantage (37.6%; n = 964) in<br />
Figure 9. Monthly conception rate of high-producing Holstein cows after AI or ET over six years: (A) overall (all services), AI = 18,568 and<br />
ET = 4,871 and (B) repeat-breeder cows (= 3 services), AI = 5,693 and ET = 3,858. Adapted from Rodrigues et al. [16,17].<br />
s9
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
Figure 10. Transferred-to-treated and 60 d pregnancy rates of recipient Holstein cows receiving an<br />
embryo at fixed time (FTET) or following estrus detection. *P < 0.0001; ** P = 0.003; ***P = 0.001.<br />
Adapted from Rodrigues et al. [20].<br />
Figure 11. In sequence, 25 and 42 d pregnancy rate of recipient Holstein cows receiving an embryo<br />
following a FTET protocol using GnRH or estradiol. Values within treatment (ab) and within<br />
seasons (*) differ (P < 0.05). Adapted from Rodrigues et al. [20].<br />
s10
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
Figure 12. Reproductive parameters of recipient Holstein cows receiving an embryo<br />
following a FTET protocol using or not eCG. P > 0.05. Adapted from Rodrigues<br />
et al. 2010.<br />
relation to donors, especially when cryopreserved<br />
embryos were used [19]. These results suggest that,<br />
in Holstein cows raised in tropical climates, the use<br />
of fresh embryos produce greater conception rates<br />
than frozen embryos. In general, the difference in<br />
conception rate between fresh and frozen embryos seems<br />
to be greater in later stages of embryo development.<br />
Another study investigated the effect of the site<br />
of embryo release (proximal, median or distal) into<br />
the uterine horn ipsilateral to the ovary with the CL<br />
on pregnancy rates after fresh and frozen-thawed<br />
embryo transfer [13]. It was verified that the site of<br />
embryo release did not influence (P = 0.84) 27 and<br />
50d pregnancy rates in recipients receiving fresh or<br />
frozen-thawed embryos. Also, pregnancy loss was<br />
similar among sites, regardless the type of embryo<br />
(Figure 13).<br />
In conclusion, results are suggestive that fresh<br />
N<br />
or frozen-thawed embryos can be transferred to any<br />
site of the uterine horn adjacent to the ovary<br />
Figure 13. Conception rate and pregnancy loss of recipient Holstein cows receiving<br />
fresh or frozen-thawed embryo following a FTET. Embryos were released in<br />
different sites (proximal, median or distal) of the uterine horn adjacent to the<br />
corpus luteum. P > 0.05. Adapted from Pieroni et al. [13].<br />
s11
C.A. Rodr<br />
drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
containing the CL, without interfering in pregnancy<br />
rates.<br />
IV. CONCLUSION<br />
Both FTAI and FTET are important tools to<br />
improve reproductive efficiency of high-producing dairy<br />
cows. These biotechnologies help bypassing some of the<br />
greatest challenges of dairy cattle reproduction: the<br />
difficulties of estrus detection, and the low fertility<br />
associated to HS and RB.<br />
Unpublished data. Ferreira & Baruselli (robertinhavet@yahoo.com.br);<br />
Ranieri & Baruselli (barusell@usp.br)<br />
- abstract sent to this meeting.<br />
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drigues<br />
igues, R.M. Fer<br />
erreir<br />
eira,<br />
L.M. Vieir<br />
ieira,<br />
et al. <strong>2011</strong>. How FTAI and FTET Impact Reproductive Efficiency of Brazilian Dairy<br />
Herds. Acta Scientiae Veterinariae. 39(Suppl 1): s3 - s13.<br />
16 Rodrigues C.A., Ayres H., Ferreira R.M., Teixeira A.A., Mancilha R.F., Oliveira M.E.F., Souza A.H. & Baruselli P.S.<br />
2007. Comparison of pregnancy rates after artificial insemination or embryo transfer in highproducing repeat breeder<br />
Holstein cows. In: <strong>Annual</strong> meeting Brazilian embryo technology society (Costa do Sauípe, Brasil). Acta Scientiae<br />
Veterinariae. 35: 1255.<br />
17 Rodrigues C.A., Ayres H., Ferreira R.M., Teixeira A.A., Mancilha R.F., Oliveira M.E.F., Souza A.H. & Baruselli P.S.<br />
2007. Comparison of pregnancy rates after artificial insemination or embryo transfer in high-producing repeat breeder<br />
Holstein cows. In: <strong>Annual</strong> meeting Brazilian embryo technology society (Costa do Sauípe, Brasil). Acta Scientiae<br />
Veterinariae. 35: 1255.<br />
18 Rodrigues C.A., Ayres H., Ferreira R.M., Teixeira A.A., Mancilha R.F., Oliveira M.E.F., Souza A.H. & Baruselli P.S.<br />
2007. Conception rate in fresh and cryopreserved embryos transfered in high producing holstein cows. In: <strong>Annual</strong> meeting<br />
Brazilian embryo technology society (Costa do Sauípe, Brasil). Acta Scientiae Veterinariae. 35: 1256.<br />
19 Rodrigues C.A., Ayres H., Ferreira R.M., Teixeira A.A., Mancilha R.F., Oliveira M.E.F., Souza A.H. & Baruselli P.S.<br />
2007. Effect of the synchrony between donor and recipient in conception rates after transfer of fresh or frozen embryos in<br />
high producing holstein cows. In: <strong>Annual</strong> meeting Brazilian embryo technology society (Costa do Sauípe, Brasil). Acta<br />
Scientiae Veterinariae. 35: 1257.<br />
20 Rodrigues C.A., Teixeira A.A., Ferreira R.M., Ayres H., Mancilha R.F., Souza A.H. & Baruselli P.S. 2010. Effect of fixedtime<br />
embryo transfer on reproductive efficiency in high-producing repeat-breeder Holstein cows. Animal Reproduction<br />
Science. 118(2-4): 110-117.<br />
21 Sartori R., Sartor-Bergfelt R., Mertens S.A., Guenther J.N., Parrish J.J. & Wiltbank M.C. 2002. Fertilization and early<br />
embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter. Journal of Dairy<br />
Science.85(11): 2803-12.<br />
22 Souza A.H. 2008. Inseminação artificial em tempo fixo em vacas Holandesas de alta produção.152p. São Paulo, SP.<br />
Dissertation (PhD in Animal Science) - Programa de pós-graduação em Reprodução animal, Universidade de São Paulo.<br />
23 Souza A.H., Ayres H., Ferreira R.M. & Wiltbank M.C. 2008. A new presynchronization system (Double-Ovsynch) increases<br />
fertility at first postpartum timed AI in lactating dairy cows. Theriogenology.70(2): 208 -215.<br />
24 Souza A.H, Viechnieski S., Lima F. A., Silva F. F., Araújo R., Bó G. A., Wiltbank M.C. & Baruselli P.S. 2009. Effects of<br />
equine chorionic gonadotropin and type of ovulatory stimulus in a timed-AI protocol on reproductive responses in dairy<br />
N<br />
cows. Theriogenology. 72(1): 10-21.<br />
25 Teixeira A.A. 2010. Impacto da inseminação artificial em tempo fixo na eficiência reprodutiva de vacas de leite de alta<br />
produção. 69p. São Paulo, SP. Dissertation (Master in Animal Science) - Programa de pós-graduação em Reprodução animal,<br />
Universidade de São Paulo.<br />
26 Van Eerdenburg F.J., Karthaus D., Taverne M.A., Merics I. & Szenci O. 2002. The relationship between estrous<br />
behavioral score and time of ovulation in dairy cattle. Journal of Dairy Science. 85(5): 1150-1156.<br />
27 Wiltbank M.C., Lopez H., Sartori R., Sangsritavong S. & Gümen A. 2006. Changes in reproductive physiology of lactating<br />
dairy cows due to elevated steroid metabolism. Theriogenology. 65(1): 17-19.<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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L.F. Nasser<br />
asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and Embryo<br />
Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s15 - s22.<br />
ISSN 1679-9216 (Online)<br />
Fix<br />
ixed time Artificial Insemination and Embryo Transf<br />
ansfer Progr<br />
grams in Brazil<br />
Luis Fer<br />
ernando Nasser<br />
1,2 , Luciano Pen<br />
enteado<br />
eado 1,2 , Car<br />
arlos R. Rezende<br />
ende 1 , Mano<br />
anoel F. Sá Filho<br />
3 & Pietr<br />
ietro S.<br />
Baruselli 3<br />
ABSTRACT<br />
Background: Currently, fixed-time protocols for either artificial insemination or embryo transfer can be routinely applied in<br />
the reproductive programs on commercial farms. The control of the follicular wave emergency and the induction of ovulation<br />
on a pre determined time without the need for heat detection facilitate the application of such biotechnologies on a large scale<br />
basis increasing the reproductive and productive efficiency. This article will discuss the development of reproductive programs<br />
that became practical to apply on Brazilian commercial farms as well as the factors that affect its efficiency.<br />
Review: Nowadays, Brazil is the world leader on bovine meat market and also in commercial application of biotechnology,<br />
such as fixed-time artificial insemination (FTAI) and fixed-time embryo transfer (TETF). In Brazil, there are several hormones<br />
commercially available that can be used for manipulation of follicular wave dynamics and induction of ovulation. The<br />
evolution of such protocols were also driven towards the decrease on the amount of time that the animals should came to the<br />
curral for hormones treatments in order to make it practical to be use on a large scale basis. Among the commercial established<br />
synchronization protocols, one of the most commonly used is with the insertion of a progesterone (P4) device associated with<br />
an injection of Estradiol Benzoate (EB) at the beginning of the synchronization protocol to induce an emergence of the new<br />
follicular wave. Eight days later, the P4 device is removed associated with the intramuscular administration of prostaglandin,<br />
equine chorionic gonadotropin (eCG) and Estradiol Cipionate (ECP). In spite of satisfactory follicular manipulation and<br />
precisely synchronization of the time of ovulation there are several factors that can affect the efficiency of FTAI or TETF<br />
programs. The FTAI pregnancy rate was influenced by the farm as well as by the body condition scores that the cows presented N<br />
at the beginning of the synchronization protocol. Other important factors that alter the programs results were the bull used and<br />
personal performance during artificial insemination. In general, treatments used for FTET are very similar to those applied for<br />
FTAI. In the FTET protocols, the main objective is to increase follicular growth and the diameter of the dominant follicle in<br />
order to increase P4 concentrations of the subsequent cycle. Some of the strategies used during the evolution of FTET protocol<br />
were to superstimulate the growing follicles of the induced wave after the injection of EB at the beginning of synchronization<br />
protocol with eCG or to decrease P4 blood concentration increasing LH pulse frequency promoting an increase on the growth<br />
of the dominant follicle during synchronization treatment by advancing the PGF treatment. The final result is to have a large<br />
pre ovulatory follicle or a pool of follicles in order to produce a single large or multiples functional CL at the time of embryo<br />
transfer. The FTET pregnancy rate was influenced by the diameter of the single CL; by recipient superovulation response by<br />
the time of year being lower during the months of autumn and winter in relation to the months of spring and summer. Besides,<br />
pregnancy rate at 30 days was also affected by the age of the embryo. Additionally, the correct application of cited biotechnologies<br />
enhances reproductive efficiency of livestock bringing sustainable and economic return, increasing the viability of the<br />
activity.<br />
Conclusion: Therefore, the standardization of the procedures is necessary for the commercial application of FTAI and FTET in<br />
Brazil being fundamental for obtaining expressive results, so that Brazil could also export such technologies for countries<br />
under the same management production system.<br />
Keywords: cattle, estrus synchronization, embryo, artificial insemination, biotechnology.<br />
1<br />
Firmasa Tecnologia para Pecuária, Campo Grande, MS, Brasil, 2 Born Animal Biotechnology Cuidad del Saber, Panama. 3 Departamento de<br />
Reprodução Animal (VRA), Universidade de São Paulo, São Paulo SP. CORRESPONDENCE: L.F. Nasser [nasser@firmasa.com.br/<br />
nasser@born.com.pa]. Rua Antonio Maria Coelho M.3443. Bairro V Lia. CEP 79020-210. Campo Grande, MS, Brazil.<br />
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L.F. Nasser<br />
asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and<br />
Embryo Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
I. INTRODUCTION<br />
II. THE EVOLUTION OF FTAI PROTOCOLS IN BRAZIL<br />
III. FACTORS THAT AFFECT THE EFFICIENCY OF<br />
FTAI PROGRAMS<br />
IV. THE EVOLUTION OF FTET PROTOCOLS IN<br />
BRAZIL<br />
V. FACTORS THAT AFFECT THE FTET PROGRAMS<br />
VI. CONCLUSIONS<br />
I. INTRODUCTION<br />
The animal protein production system is under<br />
constant pressure by the great demand for these<br />
products in a growing world market. Particularly in<br />
the case of beef, the decrease of available areas implies<br />
the need for greater production efficiency and<br />
therefore the use of reproductive biotechnology is<br />
the tool to achieve these goals.<br />
Nowadays, Brazil is the world leader on<br />
bovine meat market, in addition the country find itself<br />
in a prominent place in scientific development and<br />
commercial application of biotechnology, such as<br />
fixed-time artificial insemination (FTAI) and in vitro<br />
embryo production (IVP). Once these techniques are<br />
inserted into the herd reproductive management, it<br />
resulted on an increased of reproductive efficiency<br />
and a genetic improvement on its livestock, projecting<br />
the country as one of the largest beef producer and<br />
meat supplier for the coming years.<br />
The dimensions and the national productive<br />
system feature require the development of programs<br />
for FTAI and FTET that can be practical and easy to<br />
apply on a large scale basis decreasing the amount<br />
of hormonal treatments and cattle handling required<br />
for such protocols.<br />
The objective of this article will be discuss<br />
the main factors that can affect the FTAI and FTET<br />
programs, as well as the scientific lines of research<br />
commercially applied in Brazil.<br />
II. THE EVOLUTION OF THE FTAI PROTOCOLS IN<br />
BRAZIL<br />
In Brazil, there are several hormones<br />
commercially available that can be used for manipulation<br />
of follicular wave dynamics and induction<br />
of ovulation enabling large-scale employment of<br />
FTAI programs. The protocols are well established<br />
and without doubt one of the most commonly used<br />
is with the insertion of a device containing<br />
progesterone (P4) associated with an injection of 2<br />
mg of Estradiol Benzoate (EB) at the beginning of<br />
the synchronization protocol (D 0) to induce a new<br />
wave of follicular growth. Eight days later (D 8) along<br />
with the withdrawal of the device, an injection of<br />
prostaglandin (PGF) (to ensure luteolysis) and 400<br />
IU of the hormone equine chorionic gonadotropin<br />
(eCG) is performed along with 0.5 mg of Estradiol<br />
Cipionate for an induction of ovulation [1,4]. The<br />
benefits of the application of eCG at P4 device<br />
removal made it possible to obtain an average<br />
pregnancy rate around 50% [2,8] that drove the<br />
implementation of this biotechnology throughout the<br />
national herds.<br />
III. FACTORS THAT AFFECT THE EFFICIENCY OF FTAI<br />
PROGRAMS<br />
In spite of satisfactory follicular manipulation<br />
and precisely synchronization of the time of ovulation<br />
there are several factors that can affect the efficiency<br />
of FTAI programs. In a retrospective field study<br />
performed with information originated from Firmasa<br />
and Geraembryo, companies specialized in applied<br />
biotechnology; an average of 32.000 FTAI was<br />
analyzed in order to identify the factors affecting its<br />
efficiency when applied on a large scale. The FTAI<br />
pregnancy rate was influenced by the farm (P < 0.01;<br />
Figure 1) as well as by the body condition scores that<br />
the cows presented at the beginning of the<br />
synchronization protocol (P < 0.01; Figure 2). Other<br />
important factors that alter the programs results were<br />
the bull used (Figure 3) and personal performance<br />
during artificial insemination (Figures 4 and 5). An<br />
interesting point was that only 50% of the bulls<br />
achieved results above 50% of pregnancy rate. These<br />
results corroborate with those observed by other<br />
authors [8]. The knowledge of the factors that affect<br />
commercial FTAI programs in Brazil has fundamental<br />
importance for increasing reproductive and<br />
productive efficiency of the national herd. Currently,<br />
the FTAI was inserted into the routine management<br />
of commercial properties serving as example for other<br />
countries that rely on productive systems similar to<br />
those used in Brazil.<br />
IV. EVOLUTION OF FTET PROTOCOLS IN BRAZIL<br />
In general, treatments used for FTET are very<br />
similar to those applied for FTAI. In the FTET<br />
protocols, the main objective is to increase follicular<br />
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L.F. Nasser<br />
asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and Embryo<br />
Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
Figure 1. Effect of farm on the pregnancy per fixed time artificial insemination (FTAI; n=32.213<br />
FTAIs performed on 24 commercial farms). Proportion of farms that achieved low (= 40 %; n= 5),<br />
average (entre 40 a 50 %; n= 12) or high (= 50 %; n= 7) pregnancy results after FTAI.<br />
N<br />
Figure 2. Effect of body condition score (1 to 5 scale) at the first day of the synchronization<br />
protocol on the pregnancy per fixed time artificial insemination (FTAI; n=27.443 FTAIs).<br />
Figure 3. Effect of bull on the pregnancy per fixed time artificial insemination (FTAI; n=32.213 FTAIs<br />
performed on 24 commercial farms). Proportion of bull that promoted low (= 40 %; n= 13), average<br />
(entre 40 a 50 %; n= 23) or high (= 50 %; n= 37) pregnancy results after FTAI.<br />
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L.F. Nasser<br />
asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and<br />
Embryo Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
Figure 4. Pregnancy per fixed time artificial insemination (FTAI) according the AI technician (n=2.873<br />
FTAI performed on a single commercial farm).<br />
Figure 5. Effect of AI technician on the pregnancy per fixed time artificial insemination (FTAI; n=31.906<br />
FTAIs performed by 47 different AI technicians). Proportion of AI technician that achieved low (= 40 %;<br />
n= 12), average (entre 40 a 50 %; n= 25) or high (= 50 %; n= 10) pregnancy per AI.<br />
growth and the diameter of the dominant follicle in<br />
order to increase P4 concentrations of the subsequent<br />
cycle.<br />
Firstly, studies were done in order to compare<br />
the effect of PGF treatment on day 5 of the<br />
synchronization protocol with the traditional luteolytic<br />
treatment at the time of P4 device removal. The<br />
intention of advancing the PGF treatment is to<br />
decrease P4 blood concentration increasing LH pulse<br />
frequency promoting an increase on the growth<br />
of the dominant follicle during synchronization<br />
treatment [4].<br />
Another strategy used to improve the results<br />
of the FTET was the inclusion of hormone equine<br />
chorionic gonadotrophin (eCG) on day 5 of the<br />
protocol [3]. In an initial study carried out in Argentina<br />
with crossbred recipients [4], eCG treatment<br />
increased the corpus luteum (CL) diameter when<br />
compared to control group (18.5 ± 17.7 ± 0.4 vs 0.4<br />
mm, respectively, P < 0.05), as well as the conception<br />
rate [76/132 (57.6) v. 53/127 (41.7%), respectively,<br />
P < 0.05]. Although previous studies demonstrate the<br />
effectiveness of the protocol on achieving high rates<br />
of utilization and satisfactory conception rates, the<br />
recipients had to be brought to the curral at least five<br />
times (four times for drugs administration and one<br />
for embryo transfer). In this sense, with the aim of<br />
reducing the number of managements required to<br />
achieve the FTET, studies were conducted to simplify<br />
synchronization protocol [1].<br />
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asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and Embryo<br />
Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
In a first moment, it was studied the effect of<br />
the change of time of administration of eCG and PGF<br />
usually done on day 5 to day 8, coinciding with the<br />
removal of the P4 device in order to try to avoid the<br />
treatment on day 5. In addition, it was further<br />
investigated the effect of different doses of eCG (400<br />
IU 500 IU and 600 IU) on pregnancy rate on Bos<br />
indicus x Bos taurus recipients which received an in<br />
vitro embryo produced [7]. The results showed that<br />
the dose of eCG does not interfere with the<br />
effectiveness of the Protocol. However, administration<br />
of eCG on day 8 of the Protocol has reduced the<br />
pregnancy rate when compared to administration on<br />
day 5. Similar results were reported by Nasser et al.<br />
[5], who demonstrated that recipients receiving eCG<br />
on day 5 achieved higher pregnancy rates than those<br />
treated at day 8 of the protocol [(47.0% (71/151) and<br />
40.7% (61/150), respectively].<br />
It was also demonstrated by Nasser et al. [5]<br />
that the administration of 2 mg EB without 50 mg of<br />
progesterone at the beginning of synchronization<br />
protocol did not reduce the FTET protocol efficiency.<br />
Treatment with BE + P4 presented the same<br />
pregnancy rate than the treatments with only EB<br />
[(45.3% (68/150) to EB and 42.4% (64/151) EB +<br />
P4], eliminating the need for an application of<br />
injectable progesterone at the beginning of<br />
synchronization protocols for FTET.<br />
These studies have demonstrated that<br />
changing the day of administration of eCG (5 th to 8 th<br />
after the beginning of the synchronization protocol)<br />
had a negative influence on the results of FTET<br />
Protocol. This decrease can be related to the<br />
suppression of follicular growth between days 5 and<br />
8. Since the majority of recipients used in the<br />
experiment have a CL at the moment of P4 device<br />
insertion, the delay in the administration of PGF for<br />
day 8 of the protocol may have exposed the recipients<br />
to high circulating concentrations of P4 (CL more P4<br />
device), which may have reduced the LH pulse<br />
frequency [9]. This factor, associated with the absence<br />
of eCG treatment to stimulate follicular growth at the<br />
beginning of the follicular wave, compromised the<br />
dominant follicle growth.<br />
An alternative to reduce P4 concentrations<br />
during synchronization treatment with intravaginal<br />
P4 device without increasing the number cattle of<br />
management is to administrate PGF at the moment<br />
of P4 device insertion (D0). In a study using Bos<br />
indicus x Bos taurus cross heifers, heifers receive a<br />
half dose of PGF at the P4 device insertion and another<br />
half dose at P4 device removal or a single dose of<br />
PGF on P4 device removal [6]. All recipients received<br />
1 mg EC and 400 IU of eCG on P4 device removal<br />
(Day 8). Recipients treated with PGF on day 0 and 8<br />
have larger diameter of the dominant follicle (14.7 ±<br />
0.4 vs. 12.6 ± 0.5 mm, P < 0.05) and the CL on the<br />
day of embryo transfer (19.3 ± 0.5 vs. 18.1 ± 0.4<br />
mm) and presented higher pregnancy rate [50.5%<br />
(50/99) vs. 39.4% (39/99), respectively] than those<br />
receiving PGF treatment only on Day 8, demonstrating<br />
the possibility of a protocol with two managements<br />
for FTET programs.<br />
V. FACTORS THAT AFFECT THE EFFICIENCY OF FTET<br />
PROGRAMS<br />
Similar to previous described for FTAI<br />
programs, there are several factors that can influence<br />
the FTET results when applied in commercial farms.<br />
Recently we evaluate the possible factors that could<br />
influence the conception and embryo survival of the<br />
recipients receiving an in vitro produced embryo at<br />
fixed time. In this study, 10,195 embryo recipients<br />
Bos taurus x Bos indicus during the years 2004-2007 N<br />
were evaluated. In all FTET only females with CL<br />
and absence of uterine abnormalities were selected<br />
to synchronization. All females were fixed-time<br />
embryo transferred after synchronization of ovulation<br />
using intravaginal P4 device and EB at the beginning<br />
of treatment. The time of P4 device withdraw, a PGF<br />
injection were administered along with eCG (400 IU)<br />
and ECP (0.5 mg). All recipients received an in vitro<br />
produced embryo and were evaluated by<br />
ultrasonography at the time of embryo transfer to<br />
verify the presence of CL, diameter (16, 18, 20 or 22<br />
mm; Figure 6) and also to quantify the number of<br />
CL. Pregnancy diagnoses were done by ultrasound<br />
at 30 and 60 days after transfer, where it was also<br />
evaluated the occurrence of pregnancy loss.<br />
The conception rate varied depending on the<br />
time of year. The conception rate was lower during<br />
the months of autumn and winter (41.1%; 448/1090)<br />
in relation to the months of spring and summer<br />
(48.1%; 1760/3658). These results may be related to<br />
the dry climate and/or lower availability/quality of<br />
pasture supply typically observed during these<br />
periods in tropical regions (Figure 7).<br />
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L.F. Nasser<br />
asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and<br />
Embryo Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
Figura. 6. Pregnant-to-transferred rate according to the diameter of the corpus luteum (CL) in Bos indicus x Bos<br />
taurus recipients (n=8,034) receiving in vitro-produced embryos (P < 0.0001).<br />
Figure 7. Conception rates in bovine embryo recipients receiving an in vitro produced embryo according the<br />
month of the year (n=4748 FTET).<br />
Besides, pregnancy rate at 30 days was also<br />
affected by the diameter of the single CL [Figure 5;<br />
P < 0.0001]; by recipient superovulation response<br />
[superovulated=51.3 (328/639) or nonsuperovulated=45.8%<br />
(1880/4109); P = 0.01; Figure<br />
8] and by the age of the embryo [6 days=55.0% (132/<br />
240) a ; 7 days=46.5% (2046/4404) b ; 8 days=28.9%<br />
(30/104) b ; P < 0.0001].<br />
The average gestational loss rate (between 30<br />
to 60 days) was 11.7% (258/2208), and was not being<br />
influenced by the diameter of the single CL or by<br />
recipient superestimulation, by season or by stage of<br />
embryonic development. However, the gestational<br />
loss was influenced by the parity of the recipient.<br />
Heifers have higher rate of gestational loss (14.5%,<br />
42/269) than cows with calves (9.2%; 27/294; P =<br />
0.05; Figure 9).<br />
VI. CONCLUSIONS<br />
Currently, it is possible to manipulate the<br />
follicular dynamics and luteal phase, abolishing the<br />
need of the heat detection for FTAI or FTET. The<br />
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asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and Embryo<br />
Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
Figura 8. Conception rates after embryo transfer according to the number of corpus luteum (CL) at fixed<br />
time embryo transfer [>1CL (n= 639) vs 1 CL (n=4109); P = 0.01].<br />
N<br />
Figure 9. Conception rates at 30, at 60 and pregnancy loss occurred from 30 to 60 days in bovine in vitro<br />
embryo recipients after fixed time embryo transfers according the animal category (lactating cows or<br />
cyclic heifers). [Cyclic heifers (n=611) and Lactating cows (n=587)].<br />
correct application of such biotechnologies enhances<br />
reproductive efficiency of livestock bringing sustainable<br />
and economic return, increasing the viability<br />
of the activity. It is important to mention that other<br />
factors such as nutrition and sanitary management as<br />
well as availability of personal qualification or training<br />
can influence significantly on the successful use of<br />
these biotechnologies.<br />
Therefore, the standardization of the<br />
procedures necessary for the commercial application<br />
of FTAI and FTET in Brazil are fundamental for<br />
obtaining expressive results. This reality has placed<br />
the country in a prominent position as an exporter of<br />
biotechnologies for other countries that use similar<br />
production systems.<br />
REFERENCES<br />
1 Baruselli P.S., Ferreira R.M., Sá Filho M.F., Nasser L.F.T., Rodrigues C.A. & Bó G.A. 2010. Bovine embryo transfer<br />
recipient synchronization and management in tropical environments. Reproduction, Fertility and Development. 22: 67-<br />
74.<br />
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L.F. Nasser<br />
asser, L. Pen<br />
enteado<br />
eado, C.R. Rezende<br />
ende, M.F. Sá Filho & P.S.<br />
Bar<br />
aruselli.<br />
<strong>2011</strong>. Fixed time Artificial Insemination and<br />
Embryo Transfer Programs in Brazil. Acta Scientiae Veterinariae. 39(Suppl 1): s15 - s22.<br />
2 Baruselli P.S., Reis E.L., Marques M.O., Nasser L.F. & Bo GA. 2004. The use of hormonal treatments to improve reproductive<br />
performance of anestrous beef cattle in tropical climates. Animal Reproduction Science. 82: 479-486.<br />
3 Baruselli P.S., Marques M.O., Madureira E.H., Costa Neto W.P., Grandinetti R.R. & Bó GA. 2001. Increased pregnancy<br />
rates in embryo recipients treated with CIDR-B devices and eCG. Theriogenology. 55: 157.<br />
4 Bó G.A., Baruselli P.S., Moreno D., Cutaia L., Caccia M., Tríbulo R., Tríbulo H. & Mapletoft R.J. 2002. The control of<br />
follicular wave development for self-appointed embryo transfer programs in cattle. Theriogenology. 57: 53-72.<br />
5 Nasser L.F., Reis E.L., Oliveira M.A., Bó GA. & Baruselli PS. 2004. Comparison of four synchronization protocols for<br />
fixed-time bovine embryo transfer in Bos indicus X Bos taurus recipients. Theriogenology. 62: 1577–84.<br />
6 Reis P.O., Martins C.M., Ferreira R.M., Ayres H., Sales J.N.S., Crepaldi G.A. & Baruselli P.S. 2008. Effect of the<br />
administration of PGF at the beginning of the protocol for synchronization of ovulation on pregnancy rate in crossbred<br />
(Bos taurus x Bos indicus) heifers submitted to FTET. In: XXII Reunião Anual da Sociedade Brasileira de Tecnologia de<br />
Embriões. Acta Scientiae Veterinariae. 36: 637.<br />
7 Reis E.L., Nasser L.F.T., Menegatti J.A., Resende L.F., Mantovani A.P. & Baruselli P.S. 2004. Effect of time and dose of<br />
eCG treatment in Bos indicus x Bos taurus recipients treated with progesterone for timed embryo transfer. In: <strong>International</strong><br />
Congress on Animal Reproduction. 395 [abstract].<br />
8 Sá Filho O.J., Meneghetti M., Peres R., Lamb G. & Vasconcelos J.L.M. 2009. Fixed-time artificial insemination with<br />
estradiol and progesterone for Bos indicus cows II: Strategies and factors affecting fertility. Theriogenology. 72: 210-218.<br />
9 Savio J.D., Thatcher W.W., Morris G.R., Entwistle K., Drost M. & Mattiacci M.R. 1993. Effects of induction of low plasma<br />
progesterone concentrations with a progesterone-releasing device on follicular turnover and fertility in cattle. Journal of<br />
Reproduction and Fertility. 98: 77-84.<br />
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R.B. Lôbo, D. Nkruman,<br />
D.A.<br />
Grossi,<br />
P.S Bar<br />
arros<br />
os, L.A.F. Bezer<br />
erra,<br />
et al. <strong>2011</strong>. Implementation of DNA Markers to<br />
Produce Genomically – Enhanced EPDs in Nellore Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s23 - s27.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s23 - s27.<br />
ISSN 1679-9216 (Online)<br />
Implementation of DNA Markers to Produce Genomically - Enhanced EPDs in<br />
Nellore Cattle<br />
Raysildo Barbosa Lôbo 1 , Donald Nkrumah 2 , Daniela do Amaral Grossi 3 , Priscila Sales de Barros 4<br />
Pablo Paiva 5 , Luiz Antônio Framatino Bezerra 6 , Henrique Nunes de Oliveira 7 & Marcos Vinícius<br />
Barbosa da Silva 8<br />
ABSTRACT<br />
Background: The sequencing and publication of the cattle genome and the identification of single nucleotide polymorphism<br />
(SNP) molecular markers have provided new tools for animal genetic evaluation and genomic-enhanced selection. These new<br />
tools aim to increase the accuracy and scope of selection while decreasing generation interval. The objective of this study was<br />
to evaluate the enhancement of accuracy caused by the use of genomic information (Clarifide® - Pfizer) on genetic evaluation<br />
of Brazilian Nellore cattle.<br />
Review: The application of genome-wide association studies (GWAS) is recognized as one of the most practical approaches to<br />
modern genetic improvement. Genomic selection is perhaps most suited to the improvement of traits with low heritability in<br />
zebu cattle. The primary interest in livestock genomics has been to estimate the effects of all the markers on the chip, conduct<br />
cross-validation to determine accuracy, and apply the resulting information in GWAS either alone [9] or in combination with<br />
bull test and pedigree-based genetic evaluation data. The cost of SNP50K genotyping however limits the commercial application<br />
of GWAS based on all the SNPs on the chip. However, reasonable predictability and accuracy can be achieved in GWAS by<br />
using an assay that contains an optimally selected predictive subset of markers, as opposed to all the SNPs on the chip. The best<br />
way to integrate genomic information into genetic improvement programs is to have it included in traditional genetic N<br />
evaluations. This approach combines traditional expected progeny differences based on phenotype and pedigree with the<br />
genomic breeding values based on the markers. Including the different sources of information into a multiple trait genetic<br />
evaluation model, for within breed dairy cattle selection, is working with excellent results. However, given the wide genetic<br />
diversity of zebu breeds, the high-density panel used for genomic selection in dairy cattle (Ilumina Bovine SNP50 array)<br />
appears insufficient for across-breed genomic predictions and selection in beef cattle. Today there is only one breed-specific<br />
targeted SNP panel and genomic predictions developed using animals across the entire population of the Nellore breed<br />
(www.pfizersaudeanimal.com), which enables genomically - enhanced selection. Genomic profiles are a way to enhance our<br />
current selection tools to achieve more accurate predictions for younger animals.<br />
Material and Methods: We analyzed the age at first calving (AFC), accumulated productivity (ACP), stayability (STAY) and<br />
heifer pregnancy at 30 months (HP30) in Nellore cattle fitting two different animal models; 1) a traditional single trait model,<br />
and 2) a two-trait model where the genomic breeding value or molecular value prediction (MVP) was included as a correlated<br />
trait. All mixed model analyses were performed using the statistical software ASREML 3.0.<br />
Results: Genetic correlation estimates between AFC, ACP, STAY, HP30 and respective MVPs ranged from 0.29 to 0.46. Results<br />
also showed an increase of 56%, 36%, 62% and 19% in estimated accuracy of AFC, ACP, STAY and HP30 when MVP<br />
information was included in the animal model.<br />
Conclusion: Depending upon the trait, integration of MVP information into genetic evaluation resulted in increased accuracy<br />
of 19% to 62% as compared to accuracy from traditional genetic evaluation. GE-EPD will be an effective tool to enable faster<br />
genetic improvement through more dependable selection of young animals.<br />
Keywords: DNA Markers, Genomic Enhanced EPDs, Genetic Evaluation, Genomic Selection, Nellore Cattle, SNP.<br />
1<br />
Universidade de São Paulo (USP), Ribeirão Preto, SP e Associação Nacional de Criadores e Pesquisadores (ANCP). 2 Pfizer Animal Genetics<br />
- Kalamazoo, MI, USA. 3 Associação Nacional de Criadores e Pesquisadores (ANCP) – Ribeirão Preto, SP, Brazil. 4 Pfizer Animal Genetics, São<br />
Paulo, SP, Brazil. 5 Pfizer Saúde Animal, São Paulo, SP, Brazil. 6 Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil. 7 Universidade<br />
Estadual Paulista (UNESP), Jaboticabal, SP, Brazil. 8 Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA),Juiz de Fora, MG, Brazil.<br />
CORRESPONDENCE: R.B. LÔBO [raysildo@ancp.org.br - FAX: +55 (16) 3877-3260]. Associação Nacional de Criadores e Pesquisadores<br />
(ANCP), Rua João Godoy, 463 - Jardim América, CEP: 14020-230. Ribeirão Preto, SP, Brazil.<br />
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R.B. Lôbo, D. Nkr uman, D.A.<br />
Grossi,<br />
P.S Bar<br />
arros<br />
os, L.A.F. Bezer<br />
erra,<br />
et al. <strong>2011</strong>. Implementation of DNA Markers to<br />
Produce Genomically – Enhanced EPDs in Nellore Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s23 - s27.<br />
I. INTRODUCTION<br />
II. MATERIAL AND METHODS<br />
III. RESULTS<br />
IV. DISCUSSION<br />
V. CONCLUSIONS<br />
I. INTRODUCTION<br />
Genetic evaluation programs have<br />
significantly increased the productivity of animals and<br />
the quality as well as yield of beef products throughout<br />
the world. In Brazil, there was a significant positive<br />
genetic trend in traits of interest in beef cattle [10].<br />
However, this genetic progress can be maximized if<br />
the best animals are identified early in life and more<br />
aggressively propagated.<br />
After the discovery of single nucleotide<br />
polymorphism (SNP) molecular markers and the<br />
genome cattle publication, new approaches have been<br />
proposed for genetic evaluation in order to increase<br />
the accuracy of estimated breeding values and<br />
decrease the time needed for dependable evaluation<br />
of the animals (i.e. decrease generation interval and<br />
increase the genetic progress). The process of using<br />
genomic information to assist in animal selection is<br />
called genomic-enhanced selection.<br />
The genomic-enhanced selection in dairy<br />
cattle is working with excellent results. However,<br />
given the wide genetic diversity of the zebu breeds,<br />
and considering the influence of Taurus breeds, the<br />
panel used for genomic selection in dairy cattle (Ilumina<br />
Bovine SNP50 array) appears insufficient for<br />
across-breed genomic predictions and selection in<br />
zebu cattle [10]. Despite being less informative for<br />
zebu breeds, recent efforts have proven that it is<br />
possible to use the SNP50K for effective genomicenhanced<br />
predictions and selection. Therefore a breed<br />
specific targeted SNP panel and genomic predictions<br />
was developed by Pfizer which enables genomicallyenhanced<br />
selection on Nellore cattle.<br />
In the present study, we proposed an approach<br />
to evaluate the improvement in accuracy from<br />
integration of genomic information (Clarifide® -<br />
Pfizer) into the genetic evaluation of Brazilian Nellore<br />
cattle.<br />
II. MATERIALS AND METHODS<br />
Phenotypic data were collected from Nellore<br />
animals, belonging to farms participating in the<br />
Nellore Brazil Genetic Evaluation Program,<br />
coordinated by the National Association of Breeders<br />
and Researchers (“Associação Nacional de Criadores<br />
e Pesquisadores” - ANCP). Traits included in these<br />
analyses were: age at first calving (AFC), accumulated<br />
productivity (ACP), stayability (STAY), and heifer<br />
pregnancy at 30 months (HP30). Molecular value<br />
predictions (MVP) for each trait (MVP AFC<br />
, MVP ACP<br />
,<br />
MVP STAY<br />
, MVP HP30<br />
) were determined from Clarifide<br />
prediction equations (Clarifide is a registered<br />
trademark of Pfizer Animal Health) specifically<br />
developed for Nellore cattle.<br />
AFC is a measure of the age of entry of heifers<br />
into the beef cattle production system. This is an easily<br />
measured trait that can be used as a selection criterion<br />
for earlier expressed reproductive performance. ACP<br />
is an index that evaluates female productivity,<br />
considering progeny weight at weaning and number<br />
of offspring produced. The ACP depends directly on<br />
age at first calving, the calving intervals, and on the<br />
duration of time the cow remains in the herd. ACP<br />
expresses the cow’s ability to conceive and give birth<br />
regularly, to begin production early in life, and to<br />
wean heavier calves [6]. STAY is a trait that has a<br />
large impact on the costs of beef production because<br />
it is directly related to the cow’s ability to produce a<br />
number of calves over a given period of time [1],<br />
and the need for resources to be used for producing<br />
replacement females. HP30 quantifies the probability<br />
of successful conception and calving by 30 months<br />
of age.<br />
The best way to implement the genomic<br />
information into breeding programs is to simply<br />
integrate the genomic predictions into traditional<br />
genetic evaluation. Using this approach, traditional<br />
expected progeny differences based on phenotypic<br />
and pedigree information is combined with genomic<br />
predictions based on markers. Another approach is<br />
to include the two sources of information into a<br />
multiple trait genetic evaluation model [10].<br />
We analyzed reproductive traits in Nellore<br />
cattle with two different animal models. The<br />
traditional single trait model and a two-trait model<br />
where the MVP was fit as a correlated trait were<br />
analyzed. The linear mixed model used to estimate<br />
MVP genetic variances and covariances, breeding<br />
values and respective accuracy is described in [8].<br />
All mixed models analyses were performed using the<br />
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R.B. Lôbo, D. Nkruman,<br />
D.A.<br />
Grossi,<br />
P.S Bar<br />
arros<br />
os, L.A.F. Bezer<br />
erra,<br />
et al. <strong>2011</strong>. Implementation of DNA Markers to<br />
Produce Genomically – Enhanced EPDs in Nellore Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s23 - s27.<br />
statistical software ASREML 3.0 [3] fitting an animal<br />
model similar to those used in the Nellore Brazil<br />
Genetic Evaluation to all traits analyzed. MVP models<br />
included just the overall mean. We analyzed 813<br />
animals with MVP for all traits evaluated. The data<br />
available for each trait are described on Table 1.<br />
Estimated accuracies of EPD from analyses<br />
with and without MVP information were compared<br />
to calculate the increase in accuracy caused by the<br />
inclusion of MVP in the genetic evaluation of animals<br />
for these traits.<br />
III. RESULTS<br />
Mean values, standard deviations, minimums<br />
and maximums for AFC, ACP, STAY, HP30 and<br />
respective MVPs are provided in Table 2.<br />
The estimated heritabilities for AFC, ACP,<br />
STAY and HP30 were 0.11, 0.20, 0.12 and 0.24,<br />
respectively and for MVP AFC<br />
, MVP ACP<br />
, MVP STAY<br />
and<br />
MVP HP30<br />
the heritabilities ranged from 0.95 to 0.98.<br />
Genetic correlation estimates between AFC, ACP,<br />
STAY, HP30 and respective MVPs ranged from 0.29<br />
to 0.46.<br />
The difference between the average GE-EPD<br />
and the average of traditional EPD ranged from 1 to<br />
11%. This variation occurs because the MVP is new<br />
information that can move up or down the traditional<br />
EPD. However, the GE-EPD estimates are in average<br />
more accurate than traditional EPD (Table 3).<br />
Table 3. shows the accuracy increase for all<br />
traits when the MVP information was included in the<br />
genetic evaluation.<br />
Table 1. Description of data used to estimate the variance<br />
components and genomic-enhanced expected progeny<br />
difference for age at first calving (AFC), accumulated<br />
productivity (ACP), stayability (STAY) and heifer<br />
pregnancy at 30 months (HP30).<br />
Trait N Phenotype MVP<br />
AFC 18462 18457 813<br />
ACO 10325 9837 813<br />
N<br />
STAY 17218 16694 813<br />
HP3O 4014 3760 813<br />
Table 2. Means, standard deviations (SD), and minimum (Min) and maximum<br />
(Max) values for age at first calving (AFC), accumulated productivity (ACP),<br />
stayability (STAY) and heifer pregnancy at 30 months (HP30) and respective<br />
molecular value predictions (MVP).<br />
Trait N Min Max Mean+SD<br />
AFC (months)<br />
18457 21.00 49.00 35.80±5.59<br />
MVP AFC(months)<br />
813 -2.21 1.28 -0.33±0.55<br />
ACP (kilograms)<br />
9837 55.00 264.00 150.99±29.87<br />
MVP ACP(kilograms)<br />
813 -5.48 12.01 1.51±3.03<br />
STAY (probability)<br />
16694 0.00 1.00 0.43±0.50<br />
MVP STAY(probability)<br />
813 46.96 63.74 53.40±3.00<br />
HP30 (probability)<br />
3760 0.00 1.00 0.26±0.44<br />
MVP HP30(probability)<br />
813 43.81 58.68 49.42±2.59<br />
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R.B. Lôbo, D. Nkr uman, D.A.<br />
Grossi,<br />
P.S Bar<br />
arros<br />
os, L.A.F. Bezer<br />
erra,<br />
et al. <strong>2011</strong>. Implementation of DNA Markers to<br />
Produce Genomically – Enhanced EPDs in Nellore Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s23 - s27.<br />
Table 3. Mean accuracy of expected progeny difference estimated in the analysis without<br />
using Clarifide predictions (without MVP) and considering the molecular value predictions<br />
(with MVP) in the model for age at first calving (AFC), accumulated productivity<br />
(ACP), stayability (STAY) and heifer pregnancy at 30 months (HP30).<br />
Trait<br />
Accuracy Mean a<br />
Increase<br />
Without MVP With MVP Difference (%) b<br />
AFC 0.16 0.25 0.09 56<br />
ACP 0.14 0.19 0.05 36<br />
STAY 0.13 0.21 0.08 62<br />
HP30 0.16 0.19 0.03 19<br />
a<br />
Beef Improvement Federation (BIF) accuracy; b Increase observed by inclusion of predicted<br />
molecular value (MVP) in animal genetic evaluation.<br />
IV. DISCUSSION<br />
The observed means are similar to those<br />
reported for the Nellore breed [7], showing that the<br />
data file analyzed is representative of the breed in<br />
Brazil.<br />
Estimated heritabilities are consistent with<br />
those previously reported to Nellore cattle [5,14],<br />
except for HP30 where [2,12] reported large<br />
heritabilities. However, the data used was smaller than<br />
those considered by [2,12]. In despite of the large<br />
MVP heritabilities estimates, it was expected since<br />
MVPs are the sum of SNP additive genetic effects<br />
present on Clarifide® panel and it should have a<br />
smaller environment variance component.<br />
Considering that heritabilities account for<br />
correlation between estimated breeding values and<br />
phenotypes and that the MVP genetic correlation with<br />
the phenotype (from 0.29 to 0.46) are higher than<br />
the estimated heritabilities (from 0.11 to 0.24), we<br />
can summarize that GE-EPD are more correlated with<br />
phenotype than traditional EPD. It will result in greater<br />
response to selection consequently more genetic gain<br />
per generation when used GE-EPD as a selection<br />
criterion.<br />
The increase in accuracy observed in this<br />
research (Table 3) are similar with those reported by<br />
[8] in carcass marbling trait of Angus cattle that found<br />
an accuracy increase ranging from 36 to 85%. It<br />
should be recognized from this results that the use of<br />
Clarifide® information can provide more accurate<br />
EPDs for these traits.<br />
V. CONCLUSIONS<br />
Depending upon the trait, integration of MVP<br />
information into genetic evaluation resulted in<br />
increased accuracy of 19% to 62% as compared to<br />
accuracy from traditional genetic evaluation. GE-EPD<br />
will be an effective tool to enable faster genetic<br />
improvement through more dependable selection of<br />
young animals.<br />
Acknowledgements. The project for discovery and<br />
validation of molecular markers (Clarifide®) was developed<br />
by Brazilian and abroad researchers and Pfizer Animal<br />
Genetics using Nellore cattle from Brazilian breeders. We<br />
thank National Association of Breeders and Researchers<br />
(ANCP), Pfizer and breeders for providing the data used in<br />
this study, CTAG for analysis support.<br />
REFERENCES<br />
1 Buzanskas M.E., Grossi D.A., Baldi F., Barrozo D., Silva L.O.C., Torres Júnior R.A.A., Munari D.P. & Alencar M.M.<br />
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erra,<br />
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2 Eler J.P., Silva J.A., Ferraz J.B., Dias F., Oliveira H.N., Evans J.L. & Golden B.L. 2002.Genetic evaluation of the<br />
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3 Gilmour A.R., Gogel B.J., Cullis B.R. & Thompson R. 2009. ASReml User Guide Release 3.0 VSN <strong>International</strong> Ltd, Hemel<br />
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4 Goddard M.E., & Hayes B.J. 2007. Genomic selection. Journal of Animal Breeding and Genetics. 124(6): 323-330.<br />
5 Grossi D.A., Frizzas O.G., Paz C.C.P., Bezerra L.A.F., Lôbo R.B., Oliveira J.A. & Munari D.P. 2008. Genetic associations<br />
between accumulated productivity, and reproductive and growth traits in Nelore cattle. Livestock Science. 117: 139-146.<br />
6 Lôbo R.B., Bezerra L.A.F., Oliveira H.N., Garnero A.V., Schwengber E.B. & Marcondes C.R. 2000. Avaliação genética de<br />
animais jovens, touros e matrizes: Sumário ANCP. Ribeirão Preto, SP, Brasil. p.90.<br />
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& Oliveira H.N. <strong>2011</strong>. Avaliação genética de touros das raças Nelore, Guzerá, Brahman e Tabapuã: Sumário ANCP<br />
(Ribeirão Preto, Brasil) p.136.<br />
8 MacNeil, M.D., Nkrumah J.D., Woodward B.W. & Northcutt S.L. 2010. Genetic evaluation of Angus cattle for carcass<br />
marbling using ultrasound and genomic indicators. Journal of Animal Science. 88 (2):517-22.<br />
9 Meuwissen T.H., Hayes B.J. & Goddard M. E. 2001. Prediction of total genetic value using genome-wide dense marker<br />
maps. Genetics. 157(4): 1819-1829.<br />
10 Miller S. 2010. Genetic improvement of beef cattle through opportunities in genomics. Revista Brasileira de Zootecnia. 39<br />
(Supp l) 1: 247-255.<br />
11 Northcutt S.L. 2010. Implementation and deployment of genomically enhanced EPDS: challenges and opportunities. In:<br />
<strong>Proceedings</strong> of the Beef improvement federation: Research Symposium & <strong>Annual</strong> <strong>Meeting</strong> (Columbia, Missouri). [Fonte:<br />
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12 Shiotsuki L., Silva J.A.II V. & Albuquerque L.G. 2009. Associação genética da prenhez aos 16 meses com o peso à<br />
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www.ufrgs.br/actavet<br />
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M.E.F<br />
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liveir<br />
eira.<br />
<strong>2011</strong>. Estado da arte da superovulação em ovelhas. aaaaaa aaaaaaaaaaaa<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s29 - s35.<br />
ISSN 1679-9216 (Online)<br />
State-of-the-art in the superovulation of ewes<br />
Maria Emilia ia Franco Oliveira<br />
RESUMO<br />
Background: Os programas de múltipla ovulação e transferência de embriões (MOTE) em ovinos têm sido implantados com<br />
sucesso em todo mundo. O impacto é evidente nos programas de melhoramento genético, zootécnicos e sanitários, bem como,<br />
no resgate e conservação de raças ameaçadas de extinção e no apoio a outras biotécnicas relacionadas. A simplificação da<br />
técnica, incremento da eficiência dos resultados, assim como, o aumento no número de técnicos capacitados, pode acelerar<br />
ainda mais este desenvolvimento. De suas etapas, os protocolos superovulatórios são os responsáveis por induzir ampla<br />
variabilidade das respostas ovulatórias e produção de embriões. Tal efeito é, indubitavelmente, o principal limitante da MOTE<br />
nesta espécie.<br />
Revisão: Tradicionalmente os protocolos utilizados consideram fundamentalmente a duração do ciclo estral, e não o fenômeno<br />
biológico da dinâmica folicular, desconhecendo-se as condições prejudiciais ao desenvolvimento folicular adequado e<br />
produção de oócitos/embriões de qualidade. Alguns pontos críticos têm sido apontados como potenciais responsáveis pelos<br />
efeitos negativos, destacando-se: (i) o perfil de progesterona induzido pelos dispositivos utilizados no tratamento; (ii) a<br />
condição folicular presente ao início do protocolo superestimulatório e; (iii) a deficiência ou inexistência do pico préovulatório<br />
de LH após tratamento com gonadotrofinas. As respostas são acompanhadas pela ampla variação nas taxas de<br />
ovulação e fecundação dos oócitos, bem como, no número e qualidade dos embriões recuperados. Neste contexto, diversos<br />
estudos têm buscado desenvolver novas estratégias para o controle da dinâmica folicular. Acredita-se que há um efeito<br />
prejudicial da dominância folicular na resposta superovulatória em pequenos ruminantes. Nos tratamentos tradicionais, 70-<br />
85% das doadoras apresentam grandes folículos dominantes ao início dos tratamentos com FSH. Considerando Na<br />
imprevisibilidade do dia da emergência de cada onda folicular em ovinos, a verdadeira questão é como sincronizá-la. O<br />
emprego da pré-sincronização do estro e superestimulação da primeira onda emergente têm promovido maior eficiência. Outra<br />
estratégia, comumente empregada em bovinos, é a indução de uma nova onda folicular pelo emprego de estrógenos associado<br />
ao progestágeno, entretanto, sua eficiência em ovinos ainda é incipiente. Paralelamente, a indução do pico de LH ao final do<br />
tratamento superestimulatório tem sido investigada quanto aos benefícios em promover incremento da taxa ovulatória e<br />
número de embriões viáveis, bem como, melhoria da sincronia entre as ovulações visando aumentar a taxa de fecundação dos<br />
oócitos. A regressão luteal precoce é outra problemática que afeta os resultados dos programas de MOTE em pequenos<br />
ruminantes. Este fenômeno parece estar associado a elevadas concentrações plasmáticas de estrógenos durante a fase luteal<br />
inicial, resultando em decréscimo na resposta superovulatória e diminuição do número e qualidade dos embriões. A administração<br />
de progesterona exógena, agentes anti-luteolíticos ou luteotróficos pode prevenir ou reduzir os efeitos deletérios da<br />
regressão luteal precoce.<br />
Conclusão: A indústria da múltipla ovulação e transferência de embrião tem se tornado um negócio de escala internacional.<br />
As inúmeras vantagens relacionadas à biotécnica, a crescente exigência mundial por produção de alimentos seguros e sustentáveis<br />
têm demandado o incremento da eficiência reprodutiva e produtiva dos animais. Neste contexto, esta revisão abordará<br />
o estado da arte da superovulação em ovinos, bem como, as perspectivas voltadas a melhoria de seus resultados e dos<br />
programas de MOTE.<br />
Descritores: múltipla ovulação, produção de embrião, ovino.<br />
Universidade Estadual Paulista “Julio de Mesquita Filho”, Faculdade de Ciências Agrárias e Veterinárias (UNESP/FCAV - Jaboticabal).<br />
CORRESPONDÊNCIA: M.E.F. Oliveira [m_emiliafraoli@yahoo.com.br - TEL: +55 (16) 3209-2633]. Faculdade de Ciências Agrárias e<br />
Veterinárias (UNESP/FCAV - Jaboticabal). Via de acesso Prof. Paulo Donato Castellane. CEP 14.884-900 Jaboticabal, SP, Brasil.<br />
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I. INTRODUÇÃO<br />
II. PROTOCOLOS SUPEROVULATÓRIOS<br />
2.1 Superestimulação gonadotrófica<br />
2.2 Protocolos base para superovulação<br />
2.3 Indução da ovulação<br />
2.4 Controle da regressão luteal precoce<br />
III. CONCLUSÕES<br />
I. INTRODUÇÃO<br />
O processo superovulatório tem como princípio<br />
o fornecimento de preparações hormonais que<br />
estimulam o crescimento e, subsequente, ovulação<br />
de uma série de folículos. Tal procedimento dá início<br />
ao programa de múltipla ovulação e transferência de<br />
embriões (MOTE); biotecnologia que alavanca grandes<br />
avanços na multiplicação dos rebanhos, programas<br />
de melhoramento genético, trâmites comerciais<br />
(nacional ou internacionalmente) e conservação de<br />
germoplasmas animais em todo mundo.<br />
Os registros da movimentação mundial na<br />
área de transferência de embriões em pequenos ruminantes<br />
[14], embora instável e, de certo modo,<br />
imprecisa, podem demonstrar o sucesso desta prática.<br />
Entretanto, há grande demanda por melhorar a<br />
eficiência em resposta aos tratamentos superovulatórios<br />
e obter uma consistente produção de embriões<br />
viáveis.<br />
Dentre uma série de fatores intrínsecos e<br />
extrínsecos aos animais, a alta variabilidade das respostas<br />
à estimulação ovariana é destacadamente o<br />
maior desafio frente ao aumento da eficiência dos<br />
programas de MOTE em ovinos. O mesmo comportamento<br />
é registrado em outras espécies, a exemplo<br />
dos bovinos. Acredita-se que esta heterogeneidade<br />
nos resultados estimulatórios, em ovinos, decorra do<br />
uso de protocolos que consideram fundamentalmente<br />
a duração do ciclo estral, e não o fenômeno biológico<br />
da dinâmica folicular. Alguns pontos críticos têm<br />
sido apontados como potenciais responsáveis pelos<br />
efeitos negativos, destacando-se: (i) o perfil de<br />
progesterona induzido pelos dispositivos utilizados<br />
no tratamento [6]; (ii) a condição folicular presente<br />
ao início do protocolo superestimulatório [19,39,40]<br />
e; (iii) a deficiência ou inexistência do pico préovulatório<br />
de LH após tratamento com gonadotrofinas<br />
[18]. As respostas são acompanhadas pela ampla<br />
variação nas taxas de ovulação e fecundação dos<br />
oócitos, bem como, no número e qualidade dos embriões<br />
recuperados. Neste contexto, esta revisão abordará<br />
o estado da arte da superovulação em ovinos e<br />
as perspectivas voltadas a melhoria de seus resultados.<br />
II. PROTOCOLOS SUPEROVULATÓRIOS<br />
2.1 Superestimulação gonadotrófica<br />
Durante o intervalo interovulatório (i.e. ciclo<br />
estral) nas ovelhas, há tipicamente três ou quatro<br />
ondas de crescimento folicular. A emergência de cada<br />
onda é primariamente controlada pelo incremento das<br />
concentrações do hormônio folículo estimulante<br />
(FSH). Com o desenvolvimento desse pool de folículos,<br />
as concentrações do FSH diminuem pelo estabelecimento<br />
da dominância [3]. Em outras palavras,<br />
um a quatro folículos podem alcançar a diâmetro<br />
ovulatório em um ciclo normal [12], dentre outros<br />
aspectos, privando os demais folículos (i.e. subordinados)<br />
do FSH [17]. Com base neste princípio, o<br />
tratamento superovulatório é realizado mediante<br />
aporte de altas doses de gonadotrofinas exógenas,<br />
como FSH, gonadotrofina coriônica equina (eCG) e<br />
gonadotrofina menopausal humana (hMG). Em resposta<br />
a este procedimento há incremento da taxa<br />
ovulatória e, consequentemente, do número de embriões<br />
obtidos por fêmea tratada.<br />
Diversas preparações hormonais estão disponíveis<br />
comercialmente. O número e a frequência de<br />
administrações, bem como, a dose empregada pode<br />
variar de acordo com o protocolo. Como relato histórico,<br />
os tratamentos estimulatórios iniciaram-se com<br />
o uso da eCG em dose única, normalmente, de 1000-<br />
1500 UI administrada 48 h antes da retirada do<br />
progestágeno [36]. Atualmente, a eCG não é utilizada<br />
isoladamente em protocolos de superovulação por<br />
promover alta taxa de folículos anovulatórios, que<br />
se luteinizam; efeito dependente da dose, momento<br />
de aplicação e vida média do preparado comercial<br />
[26]. A produção de anticorpo anti-eCG, observada<br />
em fêmeas que recebem o tratamento repetidas vezes,<br />
tem ação biológica de inibir a atividade<br />
estimulatória da gonadotrofina administrada, levando<br />
a um atraso ou ausência da ovulação, podendo<br />
afetar a fertilidade dos animais [37].<br />
Grande melhoria na eficiência dos programas<br />
de superovulação para produção in vivo de embriões<br />
tem sido observada nas últimas três décadas pela<br />
substituição da eCG por preparados de FSH de origem<br />
porcina, ovina ou caprina. Com este hormônio<br />
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se obtêm taxas de ovulações superiores e menor incidência<br />
de folículos anovulatórios, além de resposta<br />
individual mais uniforme quando comparado aos tratamentos<br />
com eCG [1]. No entanto, para estimular<br />
adequadamente os folículos é necessário administrálo<br />
a intervalo de 12 h, devido suas concentrações na<br />
circulação periférica decrescerem a concentrações<br />
basais em 10 h [11]. Tal fato gera a necessidade de<br />
repetir as administrações em um curto intervalo de<br />
tempo, o que intensifica o manejo, tornando-o menos<br />
prático.<br />
Usualmente, o tratamento superestimulatório<br />
é iniciado 48 h antes da retirada do progestágeno,<br />
administrado em doses decrescentes, durante 2 - 4<br />
dias [8]. Recomenda-se o emprego de FSH em 6 a 8<br />
aplicações. Em geral, o FSH exógeno começa a atuar<br />
sobre a população de folículos presente nos ovários<br />
das ovelhas entre 12 e 24 h após o início do tratamento.<br />
Decorridas 48 h, se observa crescimento de<br />
folículos de 2 a 5 mm. Os folículos em crescimento<br />
alcançam o tamanho pré-ovulatório entre 36 e 60 h,<br />
concentrando-se entre 48 e 60 h o processo ovulatório<br />
[21]. A dose total utilizada é relativamente elevada<br />
[13]. No Brasil, emprega-se, comumente, protocolos<br />
com doses totais de 256 mg (Folltropin ® ) [23,32] ou<br />
200 UI (Pluset ® ) [9] por ovelha superestimulada.<br />
Novas pesquisas estão sendo desenvolvidas por nosso<br />
grupo de pesquisa utilizando-se dose total de 200<br />
mg (Folltropin ® ), as quais tem obtido desempenho<br />
(taxa de ovulação e produção de embriões viáveis)<br />
similar aos relatados na literatura e resultados de campo<br />
(M.E.F. Oliveira, dados não publicados).<br />
Muitas vezes, uma pequena dose da eCG (200<br />
- 400 UI) é associada ao tratamento, sendo administrada<br />
no momento da retirada do dispositivo [29].<br />
Esta prática baseia-se na hipótese de que a suplementação<br />
com LH é importante para a maturação final<br />
dos folículos, entretanto, sua eficácia é discutível<br />
[31]. Devido a longa meia-vida da eCG, há produção<br />
de estradiol após a ovulação [4]. Consequentemente<br />
têm sido reportados efeitos deletérios no oviduto<br />
(resultando em falhas de fecundação dos oócitos)<br />
e, sobre a frequência de regressão luteal prematura.<br />
Vale ressaltar que o momento da inseminação artificial<br />
deve ser antecipado quando se fizer uso desta<br />
associação.<br />
Em decorrência dos tratamentos com FSH<br />
envolverem várias administrações durante a fase<br />
folicular (i.e. intensa manipulação dos animais), há<br />
uma grande demanda por simplificação dos tratamentos<br />
superovulatórios. Nesta perspectiva, a redução do<br />
número de administrações de FSH pode ser obtida<br />
pela associação do fármaco a moléculas que propiciem<br />
sua liberação lenta. A associação de FSH com<br />
polivinilpirrolidina (PVP) obteve taxas de recuperação<br />
embrionárias idênticas aos protocolos de múltiplas<br />
administrações [10].<br />
2.2 Protocolos base para superovulação<br />
O procedimento superestimulatório pode ser<br />
realizado com base na observação de estro natural<br />
ou sincronização de estro. Tradicionalmente, utilizamse<br />
progesterona ou progestágenos impregnados em<br />
dispositivos/pessários vaginais ou implantes<br />
auriculares, os quais permanecem por um tempo de<br />
exposição superior a 10 dias para a indução e sincronização<br />
do estro [13]. O mais comum é que em ovelhas<br />
o progestágeno permaneça por 14 dias (i. e. período<br />
de duração da fase lútea. Nesses protocolos as<br />
administrações de gonadotrofinas exógenas iniciamse<br />
dois dias antes do término do tratamento.<br />
Apesar dos benefícios de sincronização, atribuí-se<br />
aos progestágenos efeito negativo ao número<br />
de ovulações e embriões transferíveis [6]. O perfil de<br />
N<br />
progesterona induzido pelo tratamento não é constante,<br />
havendo diminuição a concentrações abaixo<br />
do fisiológico, particularmente, ao final dos protocolos<br />
longos (10 a 14 dias). Esse evento é associado à<br />
alteração do padrão de crescimento folicular e persistência<br />
folicular [24], bem como a interferência no<br />
processo de fecundação e desenvolvimento de embriões<br />
de boa qualidade [22]. Estratégias usadas, até<br />
o momento, para superar o efeito negativo dos<br />
tratamentos com progestágenos incluem: (i) inserção<br />
de um segundo dispositivo de progesterona (do Dia<br />
7 ao Dia 14) para evitar concentrações subluteais do<br />
hormônio durante o tratamento [23,19]; (ii) suplementação<br />
de gonadotrofinas logo após a remoção<br />
do dispositivo até a ovulação para evitar efeito nocivo<br />
do folículo dominante [28,30]; (iii) administrar<br />
tratamento superovulatório baseando-se na detecção<br />
do estro natural (início das administrações de<br />
gonadotrofinas quatro dias após o estro), assim, fazse<br />
uso de níveis fisiológicos de progesterona produzidos<br />
pelo corpo lúteo cíclico [27] ou; (iv) encurtar o<br />
período do tratamento com progestágenos para 5 - 7<br />
dias [31].<br />
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Outra problemática que tem intensificado as<br />
pesquisas é referente à ampla variabilidade das respostas<br />
superovulatórias aos tratamentos<br />
gonadotróficos. Essa é, sem dúvida, o principal fator<br />
limitante da transferência de embriões em ovinos e<br />
caprinos. Na produção in vivo de embriões, independentemente<br />
dos hormônios utilizados no tratamento<br />
superovulatório, protocolo de administração e método<br />
de sincronização das ovulações, as maiores limitações<br />
responsáveis pela variabilidade das respostas<br />
estão, possivelmente, relacionadas à dinâmica<br />
folicular, equilíbrio estímulo-inibição que determinam<br />
à taxa de ovulação em cada espécie, e aos mecanismos<br />
intra-ovarianos que controlam o crescimento<br />
folicular [26]. Os tratamentos tradicionais de<br />
superestimulação ovariana em pequenos ruminantes<br />
foram definidos há vários anos e não consideravam<br />
os conhecimentos atuais do desenvolvimento<br />
folicular [29].<br />
Neste contexto, diversos estudos apontam que<br />
a condição folicular presente no início do protocolo<br />
superovulatório interfere na resposta ao tratamento.<br />
Acredita-se que há um efeito prejudicial da<br />
dominância folicular na resposta superovulatória em<br />
pequenos ruminantes. Em outras palavras, tratamentos<br />
superovulatórios iniciados na ausência de um<br />
folículo dominante têm resultado em melhores taxas<br />
de recrutamento folicular, ovulação e produção de<br />
embriões [19,39,40]. A presença de folículos dominantes<br />
(maiores que 5-6 mm) no início do tratamento<br />
com FSH parece afetar a maturação e a ovulação<br />
[39,40] dos folículos menores. Esta seria uma das<br />
possíveis justificativas para a alta frequência de<br />
folículos anovulatórios em tratamentos<br />
superovulatórios destes animais. A evidência de que<br />
o folículo dominante possa exercer domínio, não só<br />
por vias sistêmicas, mas também por fatores locais,<br />
reforça a necessidade de garantir a ausência de<br />
folículos grandes no início da superestimulação [29].<br />
Nos tratamentos tradicionais, 70-85% das doadoras<br />
apresentam grandes folículos dominantes ao início<br />
dos tratamentos com FSH [30]. Considerando a<br />
imprevisibilidade do dia da emergência de cada onda<br />
folicular em ovinos, a verdadeira questão é como<br />
sincronizá-la [29].<br />
Já existem algumas estratégias focadas em<br />
começar o tratamento superovulatório na ausência<br />
de um folículo dominante (i. e. próximo a emergência<br />
da onda folicular) em pequenos ruminantes, destacando-se<br />
o: (i) protocolo “Dia 0”, no qual se emprega<br />
a pré-sincronização do estro e superestimulação<br />
da primeira onda emergente [30,38] e; (ii) tratamentos<br />
com antagonistas do GnRH antes da superestimulação<br />
gonadotrófica, o qual pode ser uma alternativa<br />
para eliminar o folículo dominante [20].<br />
Outra estratégia, comumente empregada em bovinos,<br />
é a indução de uma nova onda folicular pelo<br />
emprego de estrógenos associado ao progestágeno<br />
[7]. Entretanto, poucos estudos têm avaliado este tratamento<br />
em ovinos e caprinos [29]. Os dois fármacos<br />
conjuntamente promovem uma elevação transitória<br />
de FSH e emergência da nova onda folicular, aproximadamente,<br />
três a cinco dias após [2,5]. Nestes estudos,<br />
o 17â-estradiol foi administrado por volta da<br />
metade do tratamento (14 dias) com progestágeno,<br />
resultando em uma redução da variabilidade da resposta<br />
ovariana, sem, no entanto, afetar a produção<br />
embrionária.<br />
Embora alguns avanços estejam sendo conquistados,<br />
roga-se por mais estudos na busca por tratamentos<br />
que efetivamente sincronizem uma nova<br />
onda folicular em ovinos.<br />
A ampliação de conhecimentos nesta perspectiva<br />
terá grande importância e relevância quando<br />
associados à superovulação. Acredita-se que haverá<br />
melhora na resposta superovulatória e produção de<br />
embriões viáveis em ovinos.<br />
2.3 Indução da ovulação<br />
Outra possível causa relacionada à alta variabilidade<br />
da resposta superovulatória e produção de<br />
embriões em pequenos ruminantes é associada à deficiência<br />
ou inexistência do pico pré-ovulatório de<br />
LH após tratamento com gonadotrofina exógena [18]<br />
ou devido à presença de folículos não responsivos, o<br />
que é relacionado a uma baixa regulação dos receptores<br />
de LH na granulosa e teca [25]. Paralelamente,<br />
há registro de falhas de fecundação em ovelhas<br />
superestimuladas, o que poderia ser reduzido melhorando<br />
a sincronia entre as ovulações e os procedimentos<br />
de inseminação artificial [29].<br />
Neste contexto, alguns estudos em MOTE<br />
[31,32,35] têm focado no incremento da taxa<br />
ovulatória e número de embriões viáveis por modificar<br />
a taxa FSH:LH no final do tratamento superestimulatório,<br />
administrando-se LH ou GnRH exógeno.<br />
É importante ressaltar ainda que, nesses protocolos<br />
há modificação no momento ovulatório, informação<br />
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que deve ser considerada quando for utilizar a<br />
inseminação artificial em tempo fixo [34,33].<br />
2.4 Controle da regressão luteal precoce<br />
A regressão luteal precoce é outra problemática<br />
que afeta os resultados dos programas de MOTE<br />
em pequenos ruminantes. Este fenômeno é exacerbado<br />
em ovelhas superovuladas e parece estar associado<br />
a elevadas concentrações plasmáticas de<br />
estrógenos durante a fase luteal inicial, notando-se<br />
como consequência, um decréscimo na resposta<br />
superovulatória e diminuição do número e qualidade<br />
dos embriões [13]. A regressão luteal precoce é<br />
evidente cerca de quatro dias após o estro, mas as<br />
concentrações plasmáticas de progesterona incompatíveis<br />
com atividade luteal normal já são detectadas<br />
aos três dias após o estro em cabras acometidas<br />
[41]. Em ovelhas superovuladas, a frequência de regressão<br />
luteal precoce pode variar de 6 a 75% [16], e<br />
acometer a formação de todos ou parte dos corpos<br />
lúteos de um mesmo animal [32]. A administração<br />
de progesterona exógena, agentes anti-luteolíticos<br />
(antiinflamatório; i. e. inibidores da prostaglandina)<br />
ou luteotróficos (hCG, GnRH, LH) pode prevenir ou<br />
reduzir os efeitos deletérios da regressão luteal precoce<br />
[15]. Inibidores da prostaglandina-sintetase<br />
como a Flunixin meglumine administrado de uma a<br />
duas vezes ao dia, entre o 2º e 4º dia após a detecção<br />
do estro (período crítico), auxiliam no bloqueio do<br />
processo de regressão prematura dos corpos lúteos.<br />
Neste sentido, tem-se observado incremento na recuperação<br />
de embriões viáveis [42].<br />
III. CONCLUSÃO<br />
Por muitos anos os protocolos empregados<br />
como base para o processo de superovulação, desconsideraram<br />
os eventos relacionados à dinâmica<br />
folicular. Com o emprego da ultrassonografia e da<br />
avaliação dos perfis hormonais, as investigações mais<br />
recentes têm-se voltado para elaboração de protocolos<br />
que efetivamente controlem a atividade ovariana,<br />
evitando os efeitos negativos dos hormônios<br />
exógenos. A meta principal é atingir o equilíbrio estímulo-inibição<br />
das respostas aos protocolos hormonais,<br />
visando incrementar o sucesso desta etapa<br />
dos programas de MOTE.<br />
REFERÊNCIAS<br />
N<br />
1 Armstrong D.T. & Evans G. 1983. Factors affecting success of embryo transfer in sheep and goats. Theriogenology. 19: 31-<br />
42.<br />
2 Bartlewski P.M., Alexander B.D., Rawlings N.C., Barrett D.M.W. & King W.A. 2008. Ovarian responses, hormonal profiles<br />
and embryo yields in anoestrous ewes superovulated with Folltropin ® -V after pretreatment with medroxyprogesterone<br />
acetate-releasing vaginal sponges and a single dose of oestradiol-17β. Reproduction in Domestic Animals. 43(3): 299-307.<br />
3 Bartlewski P.M., Baby T.E. & Giffin J.L. <strong>2011</strong>. Reproductive cycles in sheep. Animal Reproduction Science. 124(3-4):<br />
259-268.<br />
4 Barrett D.M.W., Bartlewski P.M., Batista-Arteaga M., Symington A. & Rawlings N.C. 2004. Ultrasound and endocrine<br />
evaluation of the ovarian response to a single injection of 500 IU of PMSG following a 12-day treatment with progestogenreleasing<br />
intravaginal sponges in and out of the breeding season in ewes. Theriogenology. 61(2-3): 311-327.<br />
5 Barrett D.M.W., Bartlewski P.M., Duggavathi R., Davies K.L., Huchkowsky S.L., Epp T. & Rawlings N.C. 2008.<br />
Synchronization of follicular wave emergence in the seasonally anestrous ewe: The effects of estradiol with or without<br />
medroxyprogesterone acetate. Theriogenology. 69(7): 827-836.<br />
6 Berlinguer F., González-Bulnes A., Succu S., Leoni G., Mossa F., Bebbere D., Ariznavarreta C., Tresguerres J.A.F., Veiga-<br />
Lopez A. & Naitana S. 2007. Effects of progestagens on follicular growth and oocyte developmental competence in FSHtreated<br />
ewes. Domestic Animal Endocrinology. 32(Suppl 4): 303-314.<br />
7 Bó G.A., Adams G.P.; Caccia M., Martinez M., Pierson R.A. & Mapletoft R.J. 1995. Ovarian follicular wave emergence<br />
after treatment with progestogen and estradiol in cattle. Animal Reproduction Science. 39: 193-204.<br />
8 Cognié Y. 1999. State of the art in sheep-goat embryo transfer. Theriogenology. 51(1): 105-116.<br />
9 Cordeiro M.F., Lima-Verde J.B., Lopes-Júnior E.S., Teixeira D.I.A., Farias L.N., Sales H.O., Simplício A.A, Rondina D.<br />
& Freitas V.J.F. 2003. Embryo recovery rate in Santa Inês ewes subjeted to successive superovulatory treatments with<br />
pFSH. Small Ruminant Research. 49: 19-23.<br />
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10 D’Alessandro A.G., Martemucci G., Colonna M.A., Borghese A., Terzano M.G. & Bellitti A. 2001. Superovulation in<br />
ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration<br />
and schedule of treatment. Animal Reproduction Science. 65(3-4): 255-264.<br />
11 Demoustier M.M., Beckers J.F., Van Der Zwalmen P., Closset J., Gillard J.L. & Ector F.R. 1988. Determination of<br />
porcine plasma levels during superovulation treatment in cows. Theriogenology. 30(2): 379-386.<br />
12 Evans A.C.O. 2003. Ovarian follicle growth and consequences for fertility in sheep. Animal Reproduction Science.78(3-4):<br />
289-306.<br />
13 Fonseca J.F., Souza J.M.G. & Bruschi J.H. 2007. Sincronização de estro e Superovulação em Caprinos e Ovinos. In: II<br />
Simpósio de Caprinos e Ovinos da UFMG (Belo Horizonte, Brasil). pp.167-195.<br />
14 Fonseca J.F., Souza J.M.G. & Camargo L.S.A. 2010. Estado da Arte de ovócitos e embriões de caprinos e ovinos: passado,<br />
presente, futuro. Acta Scientiae Veterinariae. 38: 353-369.<br />
15 Fonseca J.F., Viana J.H.M., Bruschi J.H., Zambrini F.N., Palhão M.P. & Santos A.F.A. 2005. Resposta superovulatória<br />
em cabras Saanen lactantes utilizando curtos protocolos de exposição à progesterona e somatotropina bovina recombinante<br />
(rbST). Acta Scientiae Veterinariae. 32: 243.<br />
16 Fukui Y., Okada M. & Ishida M. 1998. Incidence of premature luteal regression in ewes superovulated with a single<br />
injection of follicle-stimulating hormone combined with equine chorionic gonadotropin. Journal of Reproduction and<br />
Development. 44: 407-412.<br />
17 Ginther O.J., Wiltbank M.C., Friche P.M., Gibbons J.R. & Kot K. 1996. Selection of the dominant follicle in cattle.<br />
Biology of Reproduction. 55(6): 1187-1194.<br />
18 González-Bulnes A., Carrizosa J.A., Díaz-Delfa C., Garcia-Garcia R.M., Urrutia B., Santiago-Moreno J., Cocero M.J.<br />
& López-Sebastián A. 2003. Effects of ovarian follicular status on superovulatory response of dairy goats to FSH treatment.<br />
Small Ruminant Research. 48: 9-14.<br />
19 González-Bulnes A., Santiago-Moreno J., Cocero M.J., Souza C.J.H., Groome N.P., Garcia-Garcia R.M., Lopez-<br />
Sebastián A. & Baird D.T. 2002. Measurement of inhibin A predicts the superovulatory response to exogenous FSH in<br />
sheep. Theriogenology. 57: 1263-1272.<br />
20 González-Bulnes A., Santiago-Moreno J., Garcia-Garcia M.R. & Souza C.J.H. 2004. Effect of GnRH antagonist treatment<br />
on gonadotropin secretion, follicular development and inhibin A secretion in goat. Theriogenology. 61(5): 977-985.<br />
21 González-Bulnes A., Santiago-Moreno J. & Lopez-Sebastián A. 1997. Effect of follicular development and superovulatory<br />
protocol on ovulation rate in ewes. In: I Congresso Ibérico de Reprodução Animal (Estoril, Portugal). pp. 40-41.<br />
22 González-Bulnes A., Veiga-Lopez A., Garcia P., Garcia-Garcia R.M., Ariznavarreta C. & Sanchez M.A. 2005. Effects of<br />
progestagens and prostaglandin analogues on ovarian function and embryo viability in sheep. Theriogenology. 63(9):<br />
2523-2534.<br />
23 Gusmão A.L. 2006. Transferência de embriões em pequenos ruminantes. O Embrião. 25: 6-9.<br />
24 Letelier C.A., Contreras-Solis I., García-Fernández R.A., Ariznavarreta C., Tresguerres J.A.F., Flores J.M. & González-<br />
Bulnes A. 2009. Ovarian follicular dynamics and plasma steroid concentrations are not significantly different in ewes<br />
given intravaginal sponges containing either 20 or 40 mg of fluorogestone acetate. Theriogenology. 71(4): 676-682.<br />
25 Lopez-Diaz M.C. & Bosu W.T.K. 1992. A review and an update of cystic ovarian degeneration in ruminants. Theriogenology.<br />
37: 1163-1183.<br />
26 López-Sebastián A., Gonzáles-Bulnes A. & Moreno J. S. 2006. Control y manejo reproductivo en pequeños rumiantes. In:<br />
XXIX Curso Internacional de Reproducción animal (Madrid, Espanha). pp.43-52.<br />
27 Mayorga I., Mara L., Sanna D., Stelletta C., Morgante M., Casu S. & Dattena M. <strong>2011</strong>. Good quality sheep embryos<br />
produced by superovulation treatment without the use of progesterone devices. Theriogenology. [in press].<br />
28 Menchaca A. & Rubianes E. 2002. Relation between progesterone concentrations during the early luteal phase and<br />
follicular dynamics in goats. Theriogenology. 57(5): 1411-1419.<br />
29 Menchaca A., Vilariño M., Crispo M., Castro T. & Rubianes E. 2010. New approaches to superovulation and embryo<br />
transfer in small ruminants. Reproduction, Fertility and Development. 22(1): 113-118.<br />
30 Menchaca A., Vilariño M., Crispo M., Pinczak A. & Rubianes E. 2007. Day 0 Protocol: Superstimulatory treatment<br />
initiated in the absence of a large follicle improves ovarian response and embryo yield in goats. Theriogenology. 68(8):<br />
1111-1117.<br />
31 Menchaca A., Vilariño M., Pinczak A., Kmaid S. & Saldaña J.M. 2009. Progesterone treatment, FSH plus eCG, GnRH<br />
administration, and Day 0 protocol for MOET programs in sheep. Theriogenology. 72(4): 477-483.<br />
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32 Oliveira M.E.F. 2008. Efeito da administração do LH ao final do tratamento superestimulatório na taxa de ovulação e<br />
produção de embriões em ovelhas da raça Santa Inês. 67f. Jaboticabal, SP. Dissertação (Mestrado em Medicina Veterinária)<br />
- Universidade Estadual Paulista “Júlio de Mesquita Filho”, Faculdade de Ciências Agrárias e Veterinárias, Campus de<br />
Jaboticabal.<br />
33 Oliveira M.E.F., Ferreira R.M., Cordeiro M.F., Pieroni J.S.P., Souza S.F., Santos, I.C.C., Rodrigues, L.F.S., Fonseca J.<br />
F. & Vicente W.R.R. 2008. Efeito da administração do LH ao final do tratamento superovulatório sobre as taxas de<br />
ovulação e produção de embriões em ovelhas Santa Inês. Acta Scientiae Veterinariae. 36: 598.<br />
34 Oliveira M.E.F., Vicente W.R.R., Costa D.A.C.P., Cordeiro M.F., Ferreira R.M., Sousa S.F. & Rodrigues L.F.S. 2008.<br />
Effects of LH administration at end of the FSH superovulatory regimen on ovulatory period in Santa Inês sheep [abstract].<br />
In: XXV Jubilee World Buiatrics Congress (Budapest, Hungary). Hungarian Veterinary Journal. 130: 129.<br />
35 Picazo R.A., Cocero M.J., Barragan M.L. & López-Sebastián A. 1996. Effects of LH administration at the end of an FSH<br />
superovulatory regimen on ovulation rate and embryo production in three breeds of sheep. Theriogenology. 45(5): 1065-<br />
1073.<br />
36 Ranio V. 1991. PMSG-dose in Finn-sheep embryo production. In: <strong>Annual</strong> Conference of the <strong>International</strong> Embryo Transfer<br />
Society. Theriogenology. 35: 261. [abstract].<br />
37 Roy F., Combes B., Vaiman D., Cuibiu E.P., Pobel T., Deletang F., Combarnous Y., Guillou F. & Maurel M.C. 1999.<br />
Humoral immune response to equine chorionic gonadotropin in ewes: Association with major histocompatibility complex<br />
and interference with subsequent fertility. Biology of Reproduction. 61(1): 209-218.<br />
38 Rubianes E. & Menchaca A. 2006. Dinâmica folicular, sincronização de estro e superovulação em ovinos. Acta Scientiae<br />
Veterinariae. 34: 251-261.<br />
39 Rubianes E., Ibarra D., Ungerfeld R., Carbajal B. & De Castro T. 1995. Superovulatory response in anestrous ewes is<br />
affected by the presence of a large follicle. Theriogenology. 43(2): 465-472.<br />
40 Rubianes E., Ungerfeld R., Viñoles C., Rivero A. & Andadams G.P. 1997. Ovarian response to gonadotropin treatment<br />
initiated relative to wave emergence in ultrasonographically monitored ewes. Theriogenology. 47(8): 1479-1488.<br />
41 Saharrea A., Valencia J., Balcázar A., Medja O., Cerbón J.L., Caballero V. & Zarco L. 1998. Premature luteal regression<br />
in goats superovulated with PMSG: effect of hCG or GnRH administration during the early luteal phase. Theriogenology.<br />
50(7): 1039-1052.<br />
N<br />
42 Traldi A.S. 2002. Biotecnologia da Reprodução de Pequenos Ruminantes. Laboratório de Biotecnologia de Ovinos e<br />
Caprinos. VRA – Faculdade de Medicina Veterinária e Zootecnia - USP, 85p.<br />
www.ufrgs.br/actavet<br />
39 (Suppl 1)<br />
s35
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usmão. <strong>2011</strong>. Estado da Arte da Coleta de Embriões pela via Transcervical em Cabras e Ovelhas. ssssss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s37 - s42.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s37 - s42.<br />
ISSN 1679-9216 (Online)<br />
State-of-the-art in the transcervical embryo collection in goats and sheep<br />
Alberto Lopes Gusmão<br />
ABSTRACT<br />
Background: The Embryo Transfer (ET) in small ruminants raised in the last ten years, the interest of business people and<br />
veterinarians, because of the great movement of frozen embryos that entered Brazil, from South Africa and Australia. The heat<br />
generated from the market demands have stimulated our technicians to look to develop skills to meet these demands. The<br />
immediate need to produce results and profits, made the TE was used in animals with a genetic quality and undervalued using<br />
with greater frequency, methods that can jeopardize the future reproductive donor, as is the case of harvested by laparotomy or<br />
better known as surgical harvesting. In seeking to develop an alternative, less aggressive and given to animals appeals to the<br />
modern animal welfare, many researchers have developed techniques to overcome the barriers imposed by the anatomy of the<br />
female genital tract of small ruminants and managed to collect embryos of sheep and goats or via transcervical non-surgical.<br />
This review aims to gather information about the current availability of this technique of embryo collection and propose a new<br />
approach for this purpose.<br />
Review: In goats, the distribution of the cervical rings allows transposition to be easily obtained, and it is quite possible to get<br />
goat embryos using urethral catheters devoid of balloons. In sheep, the obstacle is presented by the great difficulty of<br />
transposing the cervical canal, as it is long and winding, with excentric openings of the rings and small diameter. Studies have<br />
tested a wide variety of drugs with the purpose of promoting cervical relaxation in sheep, in order to overcome the barrier of<br />
the cervical canal. The administration of substances such as oxytocin, estradiol, and interleukin 8, produced results incompatible<br />
with the requirements for utilization of the method on a commercial scale. Promising results were obtained however by<br />
inserting a vaginal device containing prostaglandin E (PGE 2<br />
). The natural softening of the cervix has been described as the N<br />
result of intrinsic changes of the extracellular components of the stroma. The most striking change is caused by a reduction in<br />
the concentration of collagen and glycosaminoglycans (GAGs), associated with a significant elevation of one of the primary<br />
GAGs, hyaluronic acid (HA), which has high affinity for water molecules. The relaxation of the cervix observed during estrus<br />
is associated with a high content of HA and water. Positive results were obtained when HA was injected into the cervix of ewes<br />
to be inseminated cervically. Using Posi tweezers for traction and fixation of the cervix, is essential to achieving success in the<br />
transcervical collection, for both goat and sheep.<br />
Conclusion: The growing awareness around animal welfare issues seems to be one of the factors that favor the use of transcervical<br />
sampling in small ruminants, however further studies are necessary in order to make this technique fully viable in the field,<br />
especially in sheep. Injecting HA into the cervix before the transcervical collection, in my opinion, represents a new alternative<br />
to be investigated in the pursuit of full success in this method in sheep.<br />
Keywords: embryos,goat, sheep, transcervical.<br />
Escola de Medicina Veterinária e Zootecnia (EMEVZ), Universidade Federal da Bahia (UFBA), Salvador, Bahia, Brasil. CORRESPONDÊN-<br />
CIA: A.L. Gusmão [gusmao@ufba.br]. Escola de Medicina Veterinária e Zootecnia, Av. Ademar de Barros n.500, Ondina. CEP 41940-250,<br />
Salvador, Bahia, Brasil.<br />
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ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s37 - s42.<br />
I. INTRODUÇÃO<br />
II. COLETA TRANSCERVICAL NA CABRA<br />
III. COLETA TRANSCERVICAL NA OVELHA<br />
IV. CONCLUSÕES<br />
V. REFERÊNCIAS<br />
I. INTRODUÇÃO<br />
Com a crescente demanda por produtos destas<br />
espécies, e um grande número de empresários<br />
dispostos a investirem nessa atividade, a agroindústria<br />
instalada e as tecnologias já disponibilizadas pela<br />
pesquisa, capazes de atender aos diversos segmentos<br />
da cadeia produtiva, a ovinocaprinocultura tem<br />
se destacado no cenário brasileiro como atividade<br />
de grande impacto sócioeconômico.<br />
Apesar das limitações existentes, tem havido<br />
uma crescente demanda por parte da iniciativa<br />
privada, por biotécnicas da reprodução que visem o<br />
incremento da produtividade e da rentabilidade dos<br />
rebanhos e das unidades produtivas. Dentre estas<br />
biotécnicas podemos ressaltar a sincronização de<br />
estros, a inseminação artificial, o diagnóstico precoce<br />
de prenhez, Transferência de Embriões (TE) e mais<br />
recentemente, coleta de oócitos e produção de embriões<br />
em laboratório através da fertilização in vitro.<br />
A principal limitação da TE em pequenos<br />
ruminantes sempre esteve relacionada a dois fatores:<br />
o custo e a metodologia cirúrgica de coleta de<br />
embriões [10]. Assim como no bovino, que até<br />
meados dos anos 70 a colheita cirúrgica de embriões<br />
também era um dos principais fatores limitantes,<br />
sendo esta técnica, substituída com sucesso pela colheita<br />
não cirúrgica [7].<br />
II. COLETA TRANSCERVICAL NA CABRA<br />
A coleta de embriões em cabras, que até recentemente<br />
era realizada predominantemente pelo<br />
método cirúrgico ou laparoscópico [9], limitando o<br />
número de vezes em que uma doadora pudesse ser<br />
utilizada, devido ao estresse e a formação de aderências,<br />
obteve um significativo avanço após a introdução<br />
da técnica não cirúrgica (transcervical). No<br />
Brasil, em nosso ponto de vista, os trabalhos desenvolvidos<br />
por Pereira et al. [25] e Suyadi et al. [36],<br />
que utilizaram cateter Nelaton - Robinson (Ruesch,<br />
Nº Ref. 220500) desprovido de balão com algumas<br />
adaptações, foram de fundamental importância para<br />
a popularização da TE em cabras. Atualmente, não<br />
se justifica mais a coleta de embriões em cabras por<br />
um método que não seja o transcervical.<br />
III. COLETA TRANSCERVICAL NA OVELHA<br />
Na ovelha, um fator que limita a utilização<br />
da TE em escala comercial, é a dificuldade de serem<br />
realizadas coletas pelo método transcervical, devido<br />
ao obstáculo representado pela grande dificuldade<br />
de transposição do canal cervical, já que este é longo,<br />
sinuoso, com abertura dos anéis excêntricos e<br />
com diâmetro reduzido. O orifício externo apresenta<br />
diferentes formatos em bico de pato, flap, roseta e<br />
espiral [8,15,33], criando um obstáculo à passagem<br />
de instrumentos que permitam o acesso ao útero pela<br />
via transcervical. Sendo assim, a utilização comercial<br />
destas biotécnicas fica limitada aos procedimentos<br />
cirúrgicos e laparoscópicos que trazem como grande<br />
desvantagem a predisposição a formação de aderências<br />
do sistema genital das doadoras, reduzindo o<br />
número de colheitas efetuadas em uma mesma fêmea,<br />
e em algumas vezes até comprometendo a vida<br />
reprodutiva futura do animal [1,11,12,15,16,20,23,<br />
24,34].<br />
O sucesso na transposição da barreira cervical<br />
parece depender de vários fatores como a raça, o<br />
número de partos até o momento da coleta e fatores<br />
inerentes ao indivíduo [2,34].<br />
O relaxamento do cérvix em ovelhas em cio<br />
foi descrito como sendo o resultado da ação préovulatória<br />
dos hormônios progesterona, estradiol e<br />
oxitocina atuando sobre o cérvix. Foi possível detectar<br />
uma elevação da expressão de receptores para<br />
oxitocina nas células do epitélio luminal do cérvix<br />
durante o estro. Na vaca, o efeito potencial da<br />
oxitocina sobre o relaxamento cervical é mediado<br />
pela elevação local da ciclooxigenase-2(COX2), com<br />
subsequente elevação na síntese de prostaglandina<br />
E 2<br />
(PGE 2<br />
) [3,32].<br />
O amolecimento natural do cérvix tem sido<br />
descrito como resultado de alterações intrínsecas dos<br />
componentes extracelulares do estroma. A mais<br />
marcante das mudanças é provocada por uma redução<br />
na concentração de colágeno e glicosaminoglicans<br />
(GAGS), associado a um significativa elevação<br />
de um dos GAGS primários, o ácido hialurônico<br />
(HA), que tem elevada afinidade por moléculas de<br />
água. O relaxamento do cérvix observado durante o<br />
estro está associado a um elevado conteúdo de HA e<br />
água [17,18,35,37,40].<br />
Perry et. al. [26] investigaram o efeito da aplicação<br />
local de HA sobre a profundidade de penetração<br />
da pipeta para inseminação no cérvix de ovelhas<br />
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ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s37 - s42.<br />
em cio, e sugeriram que a aplicação de HA com baixo<br />
peso molecular é um passo adiante para tornar<br />
inseminação transcervical mais acessível na ovelha.<br />
O influxo de neutrófilos no cérvix parece ser<br />
um importante componente para o amolecimento<br />
cervical. Postula-se que a colagenase liberada por<br />
neutrófilos recrutados para o interior do cérvix é muito<br />
importante para o rompimento das fibras colágenas<br />
que são os principais elementos estruturais do cérvix.<br />
Em cobaias, coelhos e humanos a interleucina 8 têm<br />
sido associada com o recrutamento de leucócitos e a<br />
elevação da colagenase cervical no final da gestação<br />
[28,29].<br />
Alguns estudos testaram uma grande variedade<br />
de fármacos com o propósito de promover o<br />
relaxamento cervical em ovelhas, com o objetivo de<br />
ultrapassar a barreira do canal cervical. A administração<br />
de ocitocina isoladamente ou em combinação<br />
com estradiol produziu resultados incompatíveis com<br />
as necessidades para adoção do método em escala<br />
comercial [19,31,41]. Efeito semelhante foi descrito<br />
quando da utilização da interleucina 8, que produziu<br />
efeito insignificante sobre o relaxamento e abertura<br />
do cérvix [6]. Resultados promissores foram obtidos<br />
através da introdução vaginal de um dispositivo contendo<br />
prostaglandina E (PGE 2<br />
), que possibilitou a<br />
penetração do cérvix 12 h após o tratamento [4,5,14,<br />
22,30].<br />
Leethongdee et al. [20], verificaram o efeito<br />
da administração do análogo da PGE 2<br />
, misoprostol e<br />
do hormônio folículo estimulante (FSH) sobre a<br />
penetrabilidade do cérvix de ovelhas durante o período<br />
peri-ovulatório e concluíram que FSH ou<br />
misoprostol foram capazes de aumentar o relaxamento<br />
natural do cérvix ao ponto de permitir uma efetiva<br />
penetração intra-uterina no momento da inseminação<br />
artificial.<br />
Durante a prenhez, a PGE 2<br />
é liberada continuamente<br />
pela placenta e desempenha um importante<br />
papel nos eventos que conduzem ao parto. É<br />
conhecido que PGE 2<br />
estimula a produção de PGF 2<br />
á,<br />
que por sua vez sensibiliza o miométrio à ocitocina<br />
endógena ou a administração exógena [26], entretanto,<br />
as evidências disponíveis indicam que a PGE 2<br />
desempenham seu principal papel, na preparação e<br />
amadurecimento do cérvix para o parto sem afetar a<br />
contratilidade uterina [4,25,26], promovendo um<br />
complexo conjunto de alterações estruturais e bioquímicas<br />
envolvidas no processo.<br />
O amadurecimento e abertura do cérvix envolvem<br />
um marcado relaxamento das fibras da musculatura<br />
lisa cervical, que necessitam serem transformadas<br />
de uma estrutura rígida em uma estrutura<br />
amolecida, complacente e com configuração dilatada.<br />
Esse processo envolve a ativação da enzima<br />
colagenase que é responsável pela digestão de parte<br />
da cadeia estrutural de colágeno do cérvix [21,27].<br />
Este mecanismo está associado a uma concomitante<br />
elevação nos níveis cervicais de glicosaminoglicans<br />
e ácido hialurônico [27]. Em pacientes humanos, o<br />
efeito local da administração da PGE 2<br />
é manifesto<br />
por mudanças na consistência, traduzido por um<br />
amolecimento e abertura do cérvix.<br />
Com base nas informações acima descritas,<br />
estudos desenvolvidos por Gusmão et al. [13], obtiveram<br />
promissores resultados quando depositaram<br />
200 µg Misoprostol (Cytotec®) no fundo de saco<br />
vaginal de ovelhas Santa Inês e Dorper. Foi observado<br />
que a utilização do misoprostol cinco horas antes<br />
das coletas, associado à tração e dilatação mecânica<br />
do cérvix, foi efetiva, possibilitando a transposição<br />
cervical. Os autores relatam que, independente da<br />
utilização do Misoprostol, ovelhas da raça Santa Inês<br />
impuseram maior dificuldade para a transposição do<br />
cérvix, quando comparadas com ovelhas da raça N<br />
Dorper.<br />
Além de utilizar a PGE 2<br />
, a tração cervical com<br />
pinças de Pozzi e o emprego de velas tipo Hegar<br />
para promover a dilatação mecânica do canal cervical<br />
foram indispensáveis para a execução da técnica com<br />
sucesso. O calibre da vela (N o 2 ou 3) variou de acordo<br />
com o diâmetro do canal cervical do animal trabalhado.<br />
Outro fator importante na rotina de coleta<br />
trascervical, tanto em ovelhas quanto em cabras, é o<br />
tipo de cateter utilizado. Sondas nasoesofágicas utilizadas<br />
na medicina humana, com calibres que variam<br />
de oito a dez, podem ser utilizadas, entretanto,<br />
em função de sua consistência amolecida, frequentemente<br />
dobram no interior do útero inviabilizando a<br />
coleta após a injeção do meio coletor, uma vez que<br />
ocorrendo o dobramento, faz-se necessário a retirada<br />
completa da sonda e com isso ocorrerá o<br />
extravasamento do meio com perda de embriões.<br />
Apesar da dificuldade em se obter no mercado nacional,<br />
a sonda modelo Nelaton-Robinson (Ruschâref.<br />
220500 nº 10) com via única fabricada pela Rusch<br />
produziu bons resultados e raros episódios de dobramento<br />
[13].<br />
s39
A.L. Gusmão<br />
usmão. <strong>2011</strong>. Estado da Arte da Coleta de Embriões pela via Transcervical em Cabras e Ovelhas. ssssss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s37 - s42.<br />
Transposta a barreira cervical, o prosseguimento<br />
da lavagem uterina pode ser executado em<br />
circuito fechado ou não. Importante salientar que,<br />
em função dos cateteres utilizados serem desprovidos<br />
de balão, uma vez que necessitam de manipulação<br />
durante a lavagem, o volume de meio a ser injetado<br />
no útero da doadora depende do seu porte. Em<br />
ovelhas é seguro utilizar-se porções de 20 mL, podendo<br />
chegar a 30 mL em ovelhas maiores, no final<br />
das lavagens [13].<br />
IV. CONCLUSÕES<br />
A evolução da coleta transcervical tanto na<br />
cabra quanto na ovelha, em escala comercial vai<br />
depender muito do mercado e da mentalidade dos<br />
criadores. Atualmente, a transferência de embriões<br />
em pequenos ruminantes é utilizada, menos como<br />
ferramenta de melhoramento genético e mais como<br />
uma forma de multiplicação rápida do rebanho. Isso<br />
faz com que os criadores busquem a biotécnica para<br />
qualquer tipo de animal, não levando em consideração<br />
o método utilizado nem suas consequências.<br />
Acreditamos que no futuro, com um mercado mais<br />
exigente em qualidade, não será tolerado a adoção<br />
de métodos que coloquem em risco o futuro<br />
reprodutivo de doadoras com comprovada qualidade<br />
genética.<br />
Outro ponto favorável à técnica transcervical,<br />
é a crescente conscientização da sociedade para o<br />
bem estar animal.<br />
A utilização de HA, injetado no cérvix antes<br />
da coleta transcervical, no meu ponto de vista, representa<br />
uma nova alternativa a ser investigada com o<br />
objetivo de alcançar pleno sucesso na coleta<br />
transcervical na ovelha.<br />
REFERÊNCIAS<br />
1 Almeida V.M., Câmara D.R., Salles H.O., Oliveira D.P.F., Medeiros J.N. & Alves O.M.M. 2002. Colheita de embriões via<br />
transcervical em ovinos. Revista Brasileira de Reprodução Animal (Supl 5): 82-84.<br />
2 Andrioli-Pinheiro A. 1993. Métodos de Colheita e de Inovulação de Embriões Caprinos (Capra hircus, Linnaeus, 1758) e<br />
os Efeitos de Repetidas Colheitas na Vida Reprodutiva de Doadoras. São Paulo. 100f. Dissertação (Mestrado) - Faculdade<br />
de Medicina Veterinária e Zootecnia, Universidade de São Paulo.<br />
3 Ayad V.J., Leung S.T., Parkinson T.J. & Wathes DC. 2004. Coincident increases in Oxytocin receptor expression and EMG<br />
responsiveness to Oxytocin in the ovine cervix at oestrus. Animal Reproduction Science. 80: 237–250.<br />
4 Barry D.M., Van Niekerk C.H., Rust J. & Van Der Walt T. 1990. Cervical embryo collection in sheep after ripening of the<br />
cervix with prostaglandin E 2<br />
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5 Candappa I.B., Bainbridge H.C., Price N.T., Hourigan K.R. & Bartlewski P.M. 2009. A preliminary study on the<br />
suitability of Cervidil to induce cervical dilation for artificial insemination in ewes. Research in Veterinary Science.<br />
87(2): 204-206.<br />
6 Croy B., Prudencio J., Minhas K., Ashkar A., Galligan C., Foster R., Buckrell B. & Coomber B.A. 1999. A preliminary<br />
study on the usefulness of huil-8 in cervical relaxation of the ewe for artificial insemination and for embryo transfer.<br />
Theriogenology. 52(2): 271-287.<br />
7 Elsden R.P., Hasler J.F. & Seidel G.E. 1976. Nonsurgical recovery of bovine eggs. Theriogenology. 6(5): 523-532.<br />
8 Evans G. & Maxwell W.M.C. 1987. Salamon’s artificial insemination of sheep and goats. Sydney: Butterworths,194p.<br />
9 Flores-Foxworth G., Mac Bride B.M., Kraemer D.C. & Nuti L.C.A. 1992. Comparison between laparoscopic and transcervical<br />
collection and transfer in goats. In: Theriogenology. 37(1): 213. [Abstract].<br />
10 Fonseca J.F., Souza J.M.G. & Camargo L.S.A. 2010. Produção de oócitos e embriões de pequenos ruminantes: passado,<br />
presente e futuro. Acta Scientiae Veterinariae. 38 (Supl 2): 337-369.<br />
11 Gonzales C.I.M. & Oliveira V.S. 1991. Técnicas para incrementar a eficiência reprodutiva de caprinos e Ovinos. Revista<br />
Sociedade Brasileira de Zootecnia. 28: 71-102.<br />
12 González-Bulnez A., Baird D.T., Campbell B.K., Cocero M.J., García-Garcia R.M., Inskeep E.K., López-Sebastián A.,<br />
McNeilly A.S., Santiago-Moreno J., Souzac J.H. & Veiga-López A. 2004. Multiple factors affecting the efficiency of<br />
multiple ovulation and embryo transfer in sheep and goats. Reproduction, Fertility and Development. 16: 421-435.<br />
13 Gusmão A.L., Silva J.C, Bittencourt T.C.C., Martins L.E.P., Gordiano H.D. & Barbosa L.P. 2009. Coleta transcervical<br />
de embriões em ovinos da raça Dorper no semiárido do Nordeste Brasileiro. Arquivos Brasileiros de Medicina Veterinária<br />
e Zootecnia. 61(2): 313-318.<br />
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usmão. <strong>2011</strong>. Estado da Arte da Coleta de Embriões pela via Transcervical em Cabras e Ovelhas. ssssss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s37 - s42.<br />
14 Halbert G.W., Dobson H., Walton J.S. & Buckrell B.C. 1990. A technique for transcervical intrauterine insemination of<br />
ewes. Theriogenology. 33(5): 993-1010.<br />
15 Halbert G.W., Dobson H., Walton J.S. & Buckrell B.C. 1990. The structure of the cervical canal of the ewe. Theriogenology.<br />
33(5): 977-992.<br />
16 Ishwar A.K. & Memon M.A. 1996. Embryo Transfer in Sheep and Goats: a Review. Small Ruminant Research. 19(1): 35-<br />
43.<br />
17 Kershaw C.M., Khalid M., McGowan M.R., Ingram K., Leethongdee S., Wax G. & Scaramuzzi R.J. 2005. The anatomy<br />
of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen.<br />
Theriogenology. 64(5): 1225-1235.<br />
18 Kershaw C.M. 2006. Mechanisms of cervical relaxation in the ewe. Ph.D. thesis. University of London.<br />
19 Khalifa R., Sayre B. & Lewis G. 1992. Exogenous oxytocin dilates the cervix in ewes. Journal of Animal Science. 70(1):<br />
38.<br />
20 Leethongdee S., Khalid M., Bhatti A., Ponglowhapan S., Kershaw C.M. & Scaramuzzi R.J. 2007. The effects of the<br />
prostaglandin E analogue Misoprostol and follicle-stimulating hormone on cervical penetrability in ewes during the periovulatory<br />
period. Theriogenology. 67(4): 767-777.<br />
21 MacLennan A.H., Katz M. & Creasey, R. 1985. The morphological characteristics of cervical ripening induced by the<br />
hormones relaxin and prostaglandins F 2<br />
in a rabbit model. American Journal of Obstetrice and Gynecology. 152: 91-96.<br />
22 McKelvey W.A.C., McEvoy T.G., Dingwall W.S., Robinson J.J., Lindsay E., King M.E., Fitzsimons J. & Mylene M.J.A.<br />
1997. The use of exogenous hormones to facilitate transcervical embryo recovery in sheep and their effect on embryo<br />
development. Theriogenology. 47(1): 369.<br />
23 More J. 1984. Anatomy and histology of the cervix uteri of the ewe: new insights. Acta Anatomica. 120: 156-159.<br />
24 Naqvi S.M., Pandey G.K., Gautam K.K., Geethalakshmi V. & Mittal J.P. 2005. Evaluation of gross anatomical features of<br />
cervix of tropical sheep using cervical silicone moulds. Animal Reproduction Science. 85(3-4): 337-344.<br />
25 Pereira R.J.T.A., Sohrey B. & Holtz W. 1998. Nonsurgical embryo collection in goats treated with prostaglandin F2α and<br />
oxytocin. Journal of Animal Science. 76: 360-363.<br />
26 Perry K., Haresign W., Wathes D.C. & Khalid M. 2010. Intracervical application of hyaluronan improves cervical<br />
relaxation in the ewe. Theriogenology. 74(9): 1685-1690.<br />
N<br />
27 Pritchard J.A., MacDonald P.C. & Gant N.F. 1985. Physiology of Labor. In: Williams Obstetrics. Aplenton Century-Crofts,<br />
pp.295-321.<br />
28 Rajabi M.R., Dean D.D., Beydoun S.N. & Woessner J.R. 1988. Elevated tissue levels of collagenase during dilation of the<br />
uterine cervix in human parturition. American Journal of Obstetric and Gynecology. 159: 971-976.<br />
29 Rajabi M.R. 1991. Biochemical evidence of collagenase-mediated collagenolysis as a mechanism of cervical dilation at<br />
parturition in the guinea-pig. Biology of Reproduction. 45: 764-772.<br />
30 Rickords L.F. & White KL. 1988. Dinoprostone induced cervical dilation in the ewe Theriogenology. 29(1): 296.<br />
31 Sayre B. & Lewis G. 1996. Cervical dilation with exogenous oxytocin does not affect sperm movement into the oviducts<br />
in ewes. Theriogenology. 45(8): 1523-1533.<br />
32 Shemesh M., Dommbrovski L., Gurevich M., Shore L.S., Fuchs A.R. & Fields M.J. 1997. Regulation of bovine cervical<br />
secretion of prostaglandins and synthesis of cyclooxygenase by oxytocin. Reproduction Fertility and Development. 9:<br />
525-530.<br />
33 Silva J.C., Quintela A., Andrade Moura J.C., Resende J., Gordiano H.D., Martins L.E.P., Chalhoub M., Ribeiro Filho<br />
A.L. & Gusmão A.L. 2004. Avaliação da colheita transcervical de embriões ovinos da raça Santa Inês. Acta Scientiae<br />
Veterinariae. 32: 90.<br />
34 Sousa J.H.M. 1999. Coleta de embriões e resposta superovulatória utilizando diferentes preparações de FSH em ovelhas<br />
deslanadas na região semi-árida da Paraíba. 57f. Paraíba. Dissertação (Mestrado) - Universidade Federal da Paraíba.<br />
35 Stern R., Asari A. & Sugahara K.N. 2006. Hyaluronan fragments: an information-rich system. European Journal of<br />
Cellular Biology. 85(8): 699-715.<br />
36 Suyadi B. Sohnrey & Holtz, W. 2000. Transcervical embryo collection in Boer goats. Small Ruminat Research. 36(2):<br />
195-200.<br />
37 Timmons B., Akins M. & Mahendroo M. 2010. Cervical remodeling during pregnancy and parturition. Trends on<br />
Endocrinology and Metabolism. 21(6): 353-361.<br />
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usmão. <strong>2011</strong>. Estado da Arte da Coleta de Embriões pela via Transcervical em Cabras e Ovelhas. ssssss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s37 - s42.<br />
38 Tsiligianni T.H., Karagiannidis A., Brikas P. & Saratsis P.H. 2001. Chemical properties of bovine cervical mucus during<br />
normal estrus and estrus induced by progesterone and/or PGF2alpha. Theriogenology. 56(1): 41-50.<br />
39 Weiss G. 2000. Endocrinology of parturition. Journal of Clinical Endocrinology and Metabolism. 85(12): 4421.<br />
40 Wulster-Radcliffe M.C., Wang S. & Lewis G.S. 2004. Transcervical artificial insemination in sheep: Effect of new transcervical<br />
artificial insemination instrument and traversing the cervix on pregnancy and lambing rate. Theriogenology.<br />
62(6): 990-1002.<br />
41 Wulster-Radcliffe M.C., Costine B.A. & Lewis G.S. 1999. Estradiol-17β-oxytocin – induced cervical dilation in sheep:<br />
application to transcervical embryo transfer. Journal Animal Science. 77(10): 2587-2593.<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
s42
J. R. Figueir<br />
igueiredo<br />
edo, A.P.R.<br />
.R. Rodr<br />
drigues & V.R.<br />
Araújo<br />
aújo. <strong>2011</strong>. In vitro culture of goat preantral follicles. ssssss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s43 - s44.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s43 - s44.<br />
ISSN 1679-9216 (Online)<br />
In vitro culture of goat preantral follicles<br />
José Ricardo de Figueiredo, Ana Paula Ribeiro Rodrigues & Valdevane Rocha Araújo<br />
ABSTRACT<br />
Background: The majority of thousands of oocytes in the ovaries are small, non-growing and reside in preantral follicles (PFs).<br />
The development of a culture system for preantral follicle may be very useful for understanding the complex mechanism in<br />
folliculogenesis at early stages of development as wells as could offer a significant way for the propagation of livestock,<br />
including goats. This paper describes the results of a number of studies aimed to evaluate the effects of several substances on<br />
in vitro culture of caprine preantral follicles highlighting the many advances, limitations and prospects.<br />
Review: Caprine PFs are usually cultured either in ovarian cortical slices or after isolation. Although IVC (in vitro culture) of PFs<br />
enclosed in cortical slices is practical, non-time-consuming, maintains three dimensional follicle architecture and preserves<br />
interactions between follicles and surrounding stroma cells, the cortical tissue may act as a barrier to IVC medium perfusion.<br />
Conversely, IVC of isolated PFs allows monitoring of individual follicles throughout the growing period, but is time-consuming,<br />
may be affected by the isolation procedure, demands a more sophisticated IVC system and is often applied to secondary and not<br />
to primordial and primary follicles. In general, several studies with farm animals and primates have successfully shown the<br />
activation and transition of primordial follicles to primary stages during in vitro culture of ovarian cortical slices. However, using<br />
these mammalian models primary follicles do not grow to secondary stages. Despite that, we succeeded to overcome this<br />
problem using an appropriate concentration of growth differentiation factor-9 (GDF- 9). Indeed, Martins et al. [2] demonstrated<br />
that GDF-9 (200 ng/mL) maintained the survival of PF and promoted activation of primordial follicles. Furthermore, GDF-9<br />
stimulated the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles. Despite a small<br />
number of publications on IVC of isolated caprine PFs, promising results such as maintenance of follicular survival, follicle and N<br />
oocyte growth, antrum formation, meiosis resumption and development of embryos after IVC of oocytes enclosed in caprine PFs<br />
were achieved [3]. The main results obtained by our research group (for review see [1]) after 18-day in vitro culture of isolated<br />
goat preantral follicles are as follows: follicular survival (89.74%), antrum formation (100%), Growth rate (25.52 µm/day), recovery<br />
rate of oocyte = 110 µm (77.10%), meiosis resumption (78.13%), nuclear maturation-MII (29.41%) and embryo production (n = 2).<br />
Conclusions: The basic culture system for the in vitro culture of caprine PF which maintains follicular survival is well established.<br />
Primordial follicle activation and their further growth up to secondary stage in vitro were achieved. Isolated primary follicles can<br />
growth up to antral stage although this rate is still low. Antrum formation and fully grown oocytes were obtained from in vitro<br />
culture of large secondary follicles even yielding a few mature oocytes and embryos. At present, the key challenge for researchers<br />
is to increase the rates of maturation and in vitro production of embryos from goat oocytes enclosed in PF grown in vitro,<br />
specially from very early stages, in order to produce, in the future, large number of offsprings.<br />
Keywords: preantral follicles, culture, goat.<br />
Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), Faculty of Veterinary, State University of Ceara, Fortaleza,<br />
Ceara, Brazil. CORRESPONDENCE: J.R. Figueiredo [jrfig@pesquisador.cnpq.br - Fax: + 55 (85) 3101-9860]. Av. Paranjana n. 1700, Campus<br />
do Itaperi, CEP 60740-000 Fortaleza, CE, Brazil.<br />
s43
J. R. Figueir<br />
igueiredo<br />
edo, A.P.R.<br />
.R. Rodr<br />
drigues & V.R.<br />
Araújo<br />
aújo. <strong>2011</strong>. In vitro culture of goat preantral follicles. ssssss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s43 - s44.<br />
REFERENCES<br />
1 Figueiredo J.R., Rodrigues A.P.R., Silva J.R.V. & Santos R.R. 2010. Cryopreservation and in vitro culture of caprine preantral<br />
follicles. Reproduction, Fertility and Development. 23(1): 40-47.<br />
2 Martins F.S., Celestino J.J., Saraiva M.V., Matos M.H.T., Bruno J.B., Rocha-Junior C.M., Lima-Verde I.B., Lucci C.M., Báo<br />
S.N., & Figueiredo J.R. 2008. Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro<br />
and their progression to secondary follicles. Reproduction, Fertility and Development. 20(8): 916-924.<br />
3 Saraiva M.V., Rossetto R., Brito I.R., Celestino J.J.H., Silva C.M., Faustino L.R., Almeida A.P., Bruno J.B., Magalhães D.M.,<br />
Matos M.H., Campello C.C. & Figueiredo J.R. 2010. Dynamic medium containing FSH, LH and EGF produces caprine<br />
embryo from preantral follicles grown in vitro. Reproductive Sciences. 17(12): 1135-1143.<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
s44
M.E.F<br />
.F. Oliv<br />
liveir<br />
eira.<br />
<strong>2011</strong>. Estado da arte da superovulação em ovelhas. aaaaaa aaaaaaaaaaaa<br />
aaaaaaaaaaaa Acta Scientiae Veterinariae. 39(Supl 1): s1 - s7.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s45.<br />
ISSN 1679-9216 (Online)<br />
Use of Embryo Transf<br />
ansfer for the Preser<br />
eservation of Small Ruminan<br />
uminants in Risk of<br />
Extinction<br />
Edilson Soares Lopes Júnior 1 & Vicente José de Figueirêdo Freitas 2<br />
Background: For about 9000 years, farmers have been managing sheep and goats in a sustainable way. From the 1800’s the<br />
situation started to change dramatically, with the rise of the concept of breed. With the development of industrial breeds came<br />
economic pressure on farmers to abandon their traditional breeds. This means that genetic resources in sheep and goats are<br />
highly endangered. It is therefore important to take measures that promote a sustainable management of these genetic resources;<br />
first, by in situ preservation of endangered breeds; second, by using modern reproductive techniques, particularly the embryo<br />
transfer to restore the genetic diversity of breeds. The objective of this paper is to present the results obtained with indigenous<br />
breeds of small ruminants, raised in the Northeastern Brazil, which are with a reduced number of individuals.<br />
Review: Small ruminant production in Northeastern Brazil is of great socio-economic importance to the region. Among others,<br />
this activity stands out regarding the size of the herds. That herd is made up of undefined breeds together with imported breeds<br />
of high production levels and the indigenous breeds that are highly adapted to the region’s prevailing semi-arid conditions.<br />
The indigenous breeds play an important role in subsistence stock farming, although the uncontrolled crossbreeding of these<br />
breeds with exotic breeds has caused genetic degeneration, placing the indigenous breeds at risk of extinction. The main<br />
examples of this condition are Canindé and Moxotó (goats) and Morada Nova var. white (sheep) breeds. Modern reproductive<br />
techniques, such as artificial insemination (AI), multiple ovulation/embryo transfer (MOET) and in vitro embryo production<br />
(IVEP) have exhibited great potential to preserve endangered breeds. Our group used these techniques, especially the MOET,<br />
in order to form an embryo bank of these indigenous breeds. Estrus synchronization was performed using vaginal sponge<br />
containing 60 mg MAP inserted for 11 (goats) or 14 (sheep) days. Forty-eight hours prior to progestagen removal, superovulatory<br />
treatment was started. Females received injections of pFSH at 12h intervals in decreasing doses. The sponges were removed Nat<br />
the time of the fifth pFSH injection. In goats, cloprostenol injection was used at the same time of the first pFSH injection. The<br />
natural mating or AI was performed at the onset of estrus and 24h later and the embryo recovery at six (sheep) or seven (goats)<br />
days after estrus. Two methods of embryo recovery are used: laparotomy or surgical (sheep and goats) and transcervical or non<br />
surgical (goats) and the embryo transfer was performed by semi-laparoscopy in synchronized recipients. The non transferred<br />
embryos were cryopreserved by slow freezing. The ovulation rate varied widely, which is a major problem in the process as a<br />
whole. However, the use of nonsurgical technique in goats was very interesting, resulting in a recovery rate greater than 80%.<br />
In sheep, it was observed an effect of age of donor, because young animals (< 2 years), when compared to those older (3-4 years),<br />
showed a higher ovulation rate (10.2 ± 1.2 vs 5.0 ± 0.8, P < 0.05).<br />
Conclusion: Indigenous breeds of small ruminants responded satisfactorily to the MOET procedure, which leaves an excellent<br />
perspective of its use in preservation of these breeds. In addition, the embryo recovery by transcervical way will greatly<br />
improve the MOET procedure, since the same donor can be used several times without the inconvenience of the occurrence of<br />
postoperative adhesions.<br />
Keywords: goat, sheep, endangered, embryo transfer.<br />
1<br />
Universidade Federal do Vale do São Francisco (UNIVASF), Petrolina, PE. Brazil. 2 Faculdade de Veterinária, Universidade Estadual do Ceará<br />
(UECE). V.J.F. Freitas [vjff@pq.cnpq.br. - Fax: +55 (85) 3101-9840]. Av. Dedé Brasil n. 1700. Fortaleza CEP 60.740-903 Fortaleza, CE,<br />
Brazil.<br />
s45
E.L.A.<br />
Motta,<br />
M. Nichi & P.C.<br />
Ser<br />
erafini.<br />
<strong>2011</strong>. State of the Art of Assisted Human Reproduction. sssssssssssss<br />
aaaaa<br />
Acta Scientiae Veterinariae. 39(Suppl 1): s47 - s55<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s47- s55.<br />
ISSN 1679-9216 (Online)<br />
State of the Art of Assisted Human Reproduction<br />
Eduardo Leme Alves da Motta 1,2 , Marcilio Nichi 1 & Paulo Cesar Serafini 1,3<br />
ABSTRACT<br />
Background: Infertility is a disease observed in approximately 10% of the reproductive age population (20-44 years old), and<br />
is defined as the failure to conceive after twelve months of regular sexual intercourse, without contraception; in women older<br />
than 35 years old, this period is reduced to 6 months. The main causes of infertility are tubal, ovarian and uterine and sperm<br />
abnormalities, endometriosis, and those with undetermined causes. Over the past 30 years, several techniques were developed<br />
to overcome these factors including gamete cryopreservation, controlled ovarian stimulation, intra-uterine insemination, in<br />
vitro fertilization, intracytoplasmatic sperm injection).<br />
Review: Despite advances in assisted reproductive technologies (ART), treatment success is still strongly dependent on<br />
oocyte and sperm quality, and resulting embryo viability. The most promising advance on oocyte quality assessment is the<br />
evaluation of the ovarian reserve by the quantification of the anti-müllerian hormone (AMH). Since ovarian reserve is closely<br />
related to oocyte quality, AMH levels could be an indicator of both oocyte production capacity and the potential of these<br />
oocytes to generate a viable embryo. On the other hand, despite the development of techniques to overcome male factor<br />
infertility, attention has been paid on the semen evaluation, since routine sperm evaluation techniques are known to be<br />
ineffective, especially in those cases of unexplained infertility. Therefore, techniques were developed to assess acrosome and<br />
membrane integrity, mitochondrial potential, DNA integrity, and fertilizing capacity of sperm. However, further studies are<br />
necessary to evaluate sperm DNA integrity without damaging the cell, allowing the injection of a spermatozoon with an intact<br />
DNA when using ICSI. Regarding embryo quality, even with a good quality oocyte (as assessed by the current techniques) and<br />
an apparently normal sperm, there are still chances of generating an embryo with genetic abnormalities. In such cases, and Nin<br />
cases of recurrent failures, women over 35 years of age, and couples with a pre-existing genetic risk, the preimplantation<br />
genetic diagnosis (PGD) appears to be an important tool to improve the odds of pregnancy and avoid abortions or the<br />
conception of fetuses with genetic abnormalities. The technique of PGD, usually performed with PCR or FISH, has gained a<br />
powerful tool with the development of the Comparative Genomic Hybridization (CGH). However, recent studies aiming to<br />
identify markers of oocyte and sperm quality and embryo viability are in course using mass spectroscopy. With this sensitive<br />
technique applied to body fluids (i.e., blood, follicular fluid, seminal plasma), granulosa cells, sperm, and culture media,<br />
researches are being conducted to non-invasively identify biomarkers that will help understand reproductive mechanisms and<br />
to efficiently predict the outcome of ARTs.<br />
Conclusion: Significant advances in ART have been observed in the last few years, yet, failures still occur with high frequency.<br />
This review will focus on techniques to assess oocyte quality, sperm function and embryo viability, aiming to provide tools for<br />
a precise prognosis when treating infertile couples.<br />
Keywords: Assisted reproductive technologies, anti-müllerian hormone, sperm function tests, preimplantation genetic<br />
diagnosis, comparative genomic hybridization, omics.<br />
1<br />
Huntington Medicina Reprodutiva, São Paulo, SP, Brazil. 2 Disciplina de Ginecologia, Universidade Federal de São Paulo, São Paulo, SP,<br />
Brazil. 3 Departamento de Ginecologia, Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP, Brazil. CORRESPONDENCE:<br />
E.L.A. Motta [emotta@huntington.com.br]. Huntington Medicina Reprodutiva, Av. República do Líbano n. 529, CEP 04501000 São Paulo,<br />
SP, Brazil.<br />
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I. INTRODUCTION<br />
II. OOCYTE<br />
III. SPERMATOZOA<br />
3.1.1 Sperm functionality tests<br />
3.1.1 Membrane integrity (vitality)<br />
3.1.2 Acrosome status<br />
3.1.3 Mitochondrial potential<br />
3.1.4 DNA integrity<br />
3.1.5 Penetration assays<br />
IV. EMBRYO<br />
V. PERSPECTIVES<br />
I. INTRODUCTION<br />
Infertility was defined as a disease for the first<br />
time in 2008 by the Practice Committee of the<br />
American Society for Reproductive Medicine [48].<br />
Soon after, the World Health Organization (WHO)<br />
also defined infertility as a disease, publishing<br />
simultaneously in Human Reproduction [78] and<br />
Fertility and Sterility [79]. The modern societal trend<br />
of women trying to conceive after 35 years of age,<br />
combined with the dramatic and irreversible decrease<br />
in oocyte potential with increased age, has lead to<br />
more couples facing infertility issues. Therefore, in<br />
the near future, medical insurance companies will<br />
likely be obliged to cover the costs of assisted<br />
reproduction techniques (ART).<br />
Although considerable advances have been<br />
achieved in recent years, many new technologies to<br />
improve the fertility rate will likely be introduced in<br />
the near future. A successful human pregnancy<br />
depends on several factors, such as ovarian function,<br />
sperm quality, embryo development, and a receptive<br />
endometrium; these factors, alone or combined,<br />
strongly influence ART results [5]. The purpose of<br />
this review is to briefly describe some of the most<br />
recent advances in the infertility field.<br />
II. OOCYTE<br />
The oocyte is well known to be the key player<br />
in reproduction outcome, however, techniques to<br />
evaluate fertility of the female gamete are still poor.<br />
Assessment of the ovarian reserve prior to ART is<br />
essential to predict outcome, select the best<br />
stimulation protocol, and retrieve the most viable<br />
oocytes in order to increase the success rate of the<br />
treatments employed. Furthermore, studies indicate<br />
that poor ovarian reserve is also accompanied by a<br />
decrease in oocyte quality [32]. Antral follicle count<br />
by ultrasound [17], early (basal) measures of follicle<br />
stimulating hormone (FSH) [1]), estradiol (E 2<br />
, [77]),<br />
and inhibin B [24] levels, as well as the clomiphene<br />
citrate challenge test [49] remain the standard<br />
evaluations. Also, increased female age correlates<br />
with diminished ovarian function [16], and may be<br />
used as an indicator of ovarian reserve. However,<br />
when performed separately, the above mentioned tests<br />
are not efficient to reflect ovarian status [23]. A<br />
promising tool to evaluate ovarian reserve is<br />
assessment of anti-Müllerian hormone (AMH) levels.<br />
AMH is produced in Sertoli cells and was previously<br />
believed to function only in the differentiation of males<br />
by inducing regression of the Müllerian ducts, the<br />
primordial anlage of the female reproductive tract<br />
[44]. AMH is a dimeric glycoprotein of the<br />
transforming growth factor family (TGF) known to<br />
regulate cell growth and differentiation [50]. This<br />
hormone appears to play an inhibitory role in the early<br />
recruitment, selection, and growth of primordial<br />
follicles and in cyclic FSH-induced antral and preantral<br />
follicular growth [75]. Granulosa cells of preantral<br />
and antral follicles are known to produce AMH.<br />
Collectively, these data indicate that this hormone may<br />
be a potential marker to predict the state of the ovarian<br />
primordial follicle reserve. In fact, several studies have<br />
shown the efficacy of AMH level testing, alone or<br />
combined with ultrasound antral follicle count, on<br />
predicting ovarian responsiveness to stimulation<br />
protocols when compared to traditional procedures<br />
[26,58,71].<br />
III. SPERMATOZOA<br />
Semen analysis (SA) is used worldwide to<br />
assess male fertility and is mainly focused on sperm<br />
concentration, progressive motility, and morphology;<br />
WHO recently published a new reference value for<br />
fertile men based on these variables (Table 2) [18].<br />
These values are based on the fifth centile found in a<br />
population of fertile men (men with a formerly<br />
pregnant partner with a time-to-pregnancy < 12<br />
months). However, despite studies indicating the<br />
efficiency of sperm count and morphology<br />
assessment as predictors of pregnancy likelihood [14],<br />
SA has some well known limitations since it<br />
represents the ability to fertilize an egg, not the ability<br />
to produce a viable embryo. In fact, the current WHO<br />
standard for normozoospermia does not accurately<br />
predict subfertile men [4], which was found to<br />
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Table 1. Ovarian reserve markers.<br />
Marker Advantages Limitations<br />
Age<br />
Antral follicle count<br />
FSH on day 3<br />
Inhibin B<br />
Clomiphene challenge test<br />
AMH<br />
Correlates with several reproductive outcomes<br />
Gold standard, performed by ultrasound (non-invasive)<br />
Routinely used, may reflect the ability of the particular<br />
month and not the overall reproductive ability<br />
Correlates with response to gonadotropin<br />
Good predictor of ovarian response, not IVF outcome<br />
Low variation between cycles and individuals, not affected<br />
by GnRH agonists<br />
High variability between women with the<br />
same age<br />
Subjective, intra-observer variations,<br />
response dependent on LH levels<br />
Variation between immunoassays, inter- and<br />
intra-cycle variations, feedback influence<br />
High false-positive rate, not widely available,<br />
feedback influence<br />
Time consuming, depends on FSH<br />
immunoassay<br />
Not widely available<br />
Table 2. Reference values for semen characteristics – WHO 2010 [18].<br />
Semen Variable<br />
Reference values<br />
Volume<br />
Sperm concentration<br />
Total sperm count<br />
Progressive motility<br />
Strict Morphology<br />
> 1.5 mL<br />
> 15 million spermatozoa/mL<br />
At least 39 million spermatozoa/ejaculate<br />
> 32% of progressive spermatozoa<br />
> 4.0% of normal spermatozoa<br />
N<br />
contribute to almost 40% of couples presenting with<br />
unexplained infertility [70]. Furthermore, with the<br />
advent of intracytoplasmic sperm injection (ICSI), the<br />
assessment of sperm motility, concentration, and<br />
morphology may be obsolete. In the last decade, new<br />
techniques for semen analysis have been developed<br />
based on routine methods with promising results, such<br />
as computer-assisted sperm analysis (CASA; [47]) and<br />
SuperICSI (ICSI using sperm specifically selected by<br />
high magnification microscopy; [35]). However, new<br />
techniques are necessary to not only determine sperm<br />
fertilization capacity, but also functional abilities, and<br />
are essential for diagnostic and prognostic purposes<br />
in helping to determine the most efficient treatment<br />
and to select the ideal gamete to be used.<br />
3.1 Sperm functionality tests<br />
Studies indicate that sperm functional competence<br />
rather than the number of motile and morphologically<br />
normal cells is determinant to predict sperm<br />
fertility (22). Therefore, techniques to evaluate sperm<br />
membrane integrity, acrosome status, mitochondrial<br />
activity, DNA integrity and sperm fertilizing capacity<br />
have been developed (Table 3).<br />
3.1.1 Membrane integrity (vitality)<br />
The integrity of the sperm plasma membrane<br />
is essential to protect the DNA from injury during<br />
fertilization. Also, reported data indicates that a<br />
number of motile cells have a disrupted membrane;<br />
the identification of such cells would be extremely<br />
beneficial in increasing the efficiency of ART.<br />
Several techniques to evaluate sperm<br />
membrane integrity are available. Most dye-exclusion<br />
techniques can accurately identify sperm with a<br />
damaged membrane in the ejaculate, including<br />
techniques developed more than 60 years ago (eosin<br />
and nigrosin [11]) to the more recently developed<br />
fluorescent probes (propidium iodide [25] and<br />
Hoechst 33258 [56]). This information is critical<br />
when analyzing the semen sample for IVF. However,<br />
most of the established techniques kill the<br />
spermatozoa, preventing its use for ICSI. Therefore,<br />
the ideal technique to test sperm membrane integrity<br />
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Table 3. Sperm function tests.<br />
Sperm function<br />
Commercially available tests<br />
Plasma Membrane Hyposmotic swelling test, eosin-nigrosin, Propidium iodide, Hoechst 33258<br />
Acrosome Arachis hypogaea agglutinin, Pisum sativum agglutinin, Trypan Blue/Bengal rose, Hoechst 33258<br />
Mitochondria<br />
DNA fragmentation<br />
Penetration assays<br />
JC1, Rhodamine 123, Mito tracker, 3,3'-diaminobenzidine<br />
Comet assay, TUNEL, Sperm Chromatin Structure Assay, CMA3<br />
Zona-free hamster oocyte, binding to egg yolk membrane, hyaluronic acid assay<br />
is the hypo-osmotic swelling test, which results in<br />
tail swelling of spermatozoa with damaged<br />
membranes while leaving normal spermatozoa intact.<br />
3.1.2 Acrosome status<br />
The acrosome, an organelle formed from the<br />
Golgi complex that develops over the anterior head<br />
of sperm, is required for binding of sperm to the<br />
oocyte and contains proteolytic enzymes that digest<br />
through the zona pellucida (acrosome reaction) [73].<br />
The effect of the acrosome reaction on the fertilization<br />
process is well established. Therefore, assessment of<br />
acrosome integrity is important in determining<br />
subfertility, particularly after failure of multiple IVF<br />
cycles. Furthermore, since acrosome and membrane<br />
integrity are closely related, the assessment of<br />
acrosome integrity is usually accompanied by a<br />
vitality test.<br />
Acrosome integrity can be assessed by<br />
fluorescent lectins that bind to the acrosomal<br />
membrane (Arachis hypogaea agglutinin) or to the<br />
acrosomal contents (Pisum sativum agglutinin) [45].<br />
Trypan Blue [68] and Hoechst 33258 [56] staining<br />
have also been successfully used to accurately detect<br />
acrosome integrity. A technique that is frequently used<br />
to evaluate the potential of the acrosome to react is to<br />
induce the acrosome reaction with ionophore A23187<br />
[19], progesterone [15], human zona pellucida [57],<br />
or zona-free hamster oocytes [76].<br />
3.1.3 Mitochondrial potential<br />
A number of enzymes responsible for<br />
anaerobic glycolysis to generate ATP for sperm<br />
motility were identified in the sperm tail [54].<br />
However, mitochondrial ATP produced by oxidative<br />
phosphorylation has been shown to play an essential<br />
role in flagellar movement [6]. Additionally, sperm<br />
mitochondria are important in the regulation of<br />
oxidative stress and apoptosis [8,46]. Therefore, the<br />
functional properties of the mitochondria likely affect<br />
the fertility potential of the sperm.<br />
The assessment of mitochondrial status is<br />
usually performed using fluorescent probes, such as<br />
Rhodamine 123, JC-1, and MitoTracker red, green,<br />
or orange, each having their own advantages and<br />
limitations [33]. However, all tests require a fluorescent<br />
microscope or a flow cytometer (sometimes with more<br />
than one color detector) for the assay, which improves<br />
the efficiency of the technique, but limits the use for<br />
most ART centers. An alternative method is the use<br />
of 3,3'-diaminobenzidine (DAB); the oxidation of<br />
DAB by cytochrome c oxidase forms a brown<br />
complex and may reflect sperm mitochondria activity<br />
[39]. The grade of staining in the sperm midpiece<br />
can be visualized by contrast microscopy and correlated<br />
with the level of mitochondrial activity [12].<br />
3.1.4 DNA integrity<br />
The spermatozoa should carry an intact male<br />
genome to properly activate an oocyte during the<br />
fertilization process [65]. Studies indicate that even<br />
with fragmented DNA the spermatozoa may correctly<br />
fertilize the egg [5,52], if such abnormalities are below<br />
a critical threshold [2]. However, depending on the<br />
extent of the DNA fragmentation, embryo potential<br />
may be affected [3], leading to impaired development,<br />
low implantation rates, miscarriages, and likely<br />
contribute to abnormalities in the offspring [9, 53].<br />
Several tests have been developed to assess<br />
sperm DNA fragmentation, and the results are<br />
reported, generally, as “DNA damage”. However, the<br />
different tests can distinguish distinct properties of<br />
the DNA, determining several aspects of the DNA<br />
abnormality. While the Terminal Transferase dUTP<br />
Nick End Labeling assay (TUNEL) assesses the ‘real’<br />
DNA damage, the Sperm Chromatin Structure Assay<br />
(SCSA) evaluates the ‘potential’ DNA damage in<br />
terms of susceptibility to DNA denaturation [36]. On<br />
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the other hand, the Comet assay, when performed at<br />
neutral pH, is able to detect double-strand breaks [64],<br />
and when performed at acidic or alkaline pH, detects<br />
single-stranded breaks [61]. This distinction is<br />
important since single-stranded breaks are easier to<br />
repair than double-stranded breaks. Therefore, care<br />
should be taken when counseling patients and making<br />
recommendations based on the results of a sperm<br />
DNA test. According to Sakkas and Alverez [61], only<br />
a combination of tests can properly evaluate different<br />
aspects of the DNA damage (i.e., single versus<br />
double-stranded breaks, real versus induced<br />
fragmentation).<br />
3.1.5 Penetration assays<br />
The sperm penetration assays is probably the<br />
most effective technique to evaluate sperm fertilizing<br />
capacity, since it simultaneously evaluates the sperms’<br />
ability to capacitate, undergo the acrosome reaction,<br />
and fuse with the vitelinic membrane of an oocyte.<br />
Studies performed with animal and artificial<br />
models, such as the zona-free hamster oocyte [38],<br />
egg yolk membrane [10], and the hyaluronic acid<br />
binding assay [41], suggest that these tests are<br />
accurate in determining the ability of sperm to bind<br />
to the oocyte. Furthermore, the number of sperm with<br />
chromosomal disomy is decreased in hyaluronic acid<br />
treated sperm, which allows for selection of healthy,<br />
mature sperm for ICSI.<br />
IV. EMBRYO<br />
The embryonic potential to invade and attach<br />
at the endometrial cavity still remains the critical event<br />
to achieve a viable pregnancy. Previous studies<br />
indicate that genetic abnormalities are involved in 50-<br />
80% of first-trimester miscarriages [31,62], 20-30%<br />
of neonatal deaths [40], [20], and 30-50% of postnatal<br />
deaths [37]. Furthermore, genetic abnormalities<br />
were observed in 50% of mentally retarded patients<br />
[42], 10% of cancer patients [28], and are linked with<br />
many other diseases. Until recently, the diagnosis of<br />
genetic abnormalities was based exclusively on results<br />
from nonspecific ultrasonographic findings [67],<br />
maternal blood tests [22,63], amniocentesis [63], and<br />
chorionic villus sampling [29], which are performed<br />
after implantation occurs. Since termination of the<br />
pregnancy is complicated by ethical and legal<br />
concerns [43], the antenatal diagnosis of a genetically<br />
abnormal fetus would be a better choice to select the<br />
most viable embryo. The pre-implantation genetic<br />
diagnosis (PGD) procedure was developed more than<br />
20 years ago, which allowed for the genetic testing<br />
of embryos before implantation [34], selecting only<br />
healthy embryos for transfer.<br />
PGD analyzes the polar body in the oocyte,<br />
the cleavage stage embryo, or the blastocyst stage<br />
embryo by genetic analysis, which can be performed<br />
by polymerase chain reaction (PCR), fluorescent in<br />
situ hybridization (FISH), and, more recently, by<br />
comparative genomic hybridization (CGH). PCR<br />
analysis is used to test for monogenic diseases, such<br />
as cystic fibrosis [51], Huntington disease [66], fragile<br />
X syndrome [7], and Duchenne muscular dystrophy<br />
[51]. FISH analysis is used to detect structural<br />
(deletion, duplication, and translocation) and<br />
numerical (trysomy and monosomy) chromosomal<br />
abnormalities which result in diseases, such as Down<br />
syndrome [80], Turner’s syndrome [55], and Klinefelter’s<br />
syndrome [59]. However, despite continuous<br />
advances, these techniques can only identify a limited<br />
number of genetic abnormalities. With the<br />
development of CGH, it is now possible to perform a<br />
screening of the whole genome to identify an<br />
unbalanced number of chromosomal copies [74].<br />
V. PERSPECTIVES<br />
Over the past couple decades, several studies<br />
have been performed aiming to identify and quantify<br />
“substances” that could elucidate biological<br />
mechanisms and explain differences between<br />
individuals, races, and species. The term “biomarker”<br />
was then created to refer to such substances that<br />
indicate normal biological processes, pathogenic processes,<br />
or responses to the environment or therapeutic<br />
intervention. Since the sequencing of the human<br />
genome [72], several studies were performed to better<br />
characterize the function and interaction between key<br />
biomolecules, such as proteins (proteome), DNA<br />
transcripts (transcriptome), lipids (lipidome), and<br />
metabolites (metabolome), the so-called “Omics”<br />
approach. However, care should be taken when<br />
correlating these results with the biological process<br />
since the state of the genome may not reflect the<br />
transcripts generated [27], changes in mRNA do not<br />
reflect absolute or relative changes in protein levels<br />
[69], and the proteins produced may not reflect the<br />
biological process and phonotypical characteristics<br />
[60]. A revolution has been observed in the fields of<br />
proteome, lipidome, and metabolome research with<br />
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the development of more sensitive and selective<br />
techniques, including mass spectrometry, which, in<br />
contrast to previously used techniques, allows the<br />
screening of thousands of proteins, peptides, and<br />
small molecules (metabolites and lipids) [30]. In<br />
reproduction, an Omics approach applied to follicular<br />
fluid, granulosa cells, embryo culture media, semi-<br />
nal plasma, and other biological fluids may lead to<br />
the identification of biomarkers that elucidate several<br />
still unknown mechanisms related to the oocyte,<br />
spermatozoa, and/or embryo, and is a non-invasive<br />
test that may identify couples with higher odds of<br />
achieving pregnancy.<br />
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www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s57 - s70.<br />
ISSN 1679-9216 (Online)<br />
The Equine Model to Study the Influence of Obesity and Insulin Resistance in<br />
Human Ovar<br />
arian Func<br />
unction<br />
Eduardo Leite Gastal 1 , Melba de Oliveira Gastal 1 , Áurea Wischral 2 & Jeremy Davis 1<br />
ABSTRACT<br />
Background: Understanding ovarian folliculogenesis is critical to the study of fertility and in the development of fertility<br />
techniques as well as contraception. Mares and women share striking similarities in ovarian folliculogenesis, and in insulin<br />
resistance and obesity syndromes. The effects of insulin resistance and obesity on follicular development and the surrounding<br />
endocrinology and genes in mares may shed light on the causes and effects of metabolic and reproductive disorders such as<br />
polycystic ovarian syndrome (PCOS) in women using an appropriate research model.<br />
Review: Studies in laboratory animals (e.g. mice and rats) have demonstrated that, in general, these animals are not good<br />
research models to study ovarian function in women because of the remarkable physiological differences in ovarian<br />
folliculogenesis and luteogenesis/luteolysis. Therefore, there is an urgent need for the development of in vivo (whole animal)<br />
research models using species (e.g. mare and cow) that have a similar physiological ovarian function to the woman. The use of<br />
such models will allow for an understanding of the causes and effects of different pathological reproductive processes involved<br />
in diseases. Several studies from our group and others have shown that nowadays the mare seems to be one, if not the best,<br />
animal model to study ovarian function in women. In addition, the recent elucidation of the equine genome has provided<br />
evidence of the high gene conservation and similar chromosomal order of this species to humans, reinforcing the importance<br />
of this species for comparative studies with humans.<br />
Conclusions: The use of farm animal models is also relevant for agricultural and biomedical research because this might help<br />
N<br />
to improve reproductive efficiency and health in animals and humans, as well as the quality of products (e.g. oocytes, embryos,<br />
etc.). This review will focus on the potential use of the mare as a model to study the effects of obesity and insulin resistance<br />
syndromes on ovarian function in women.<br />
Keywords: insulin resistance, obesity, ovary, mares, women.<br />
1<br />
Department of Animal Science, Food and Nutrition, Southern Illinois University Carbondale. 2 Department of Veterinary Medicine, Federal<br />
Rural University of Pernambuco (UFRPE), Recife, PE, Brazil. CORRESPONDENCE: E.L. Gastal [egastal@siu.edu]. Department of Animal<br />
Science, Food and Nutrition, Southern Illinois University Carbondale, 1205 Lincoln Drive, MC 4417, Carbondale, IL 62901, USA.<br />
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E.L.Gastal, M.O. Gastal, A. Wischral & J. Davis. <strong>2011</strong>. The Equine Model to Study the Influence of Obesity and<br />
Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
I. INTRODUCTION<br />
II. GENERAL ASPECTS OF FOLLICLE DEVE-<br />
LOPMENT IN THE MARE<br />
III. SIMILARITIES IN FOLLICLE DEVELOPMENT<br />
AND SYSTEMIC HORMONES BETWEEN MARES<br />
AND WOMEN<br />
IV. OBESITY<br />
V. INSULIN RESISTANCE<br />
5.1 Molecular mechanisms and defects of insulin activity<br />
in target tissues<br />
5.2 Insulin resistance detection<br />
VI. CONCLUSIONS<br />
I. INTRODUCTION<br />
The growing interest in research in insulin<br />
resistance and obesity is centered on the recognition<br />
that both conditions play a role in the pathogenesis<br />
of several adverse metabolic disturbances in humans<br />
and domestic animals, including horses. Reproductive<br />
disorders such as anovulation and Polycystic<br />
Ovarian Syndrome have been associated with the<br />
action of insulin on its target tissues. The striking<br />
similarities between mares and women in follicular<br />
dynamics and hormonal changes during the interovulatory<br />
interval and the ovulatory follicular wave<br />
[7,37,49,50,52,77], in ultrasonographic changes of<br />
the preovulatory follicle before ovulation [36,40-<br />
42,75,82], in reproductive aging processes<br />
[15,54,55], and in reproductive dysfunction,<br />
including development of HAFs (hemorrhagic<br />
anovulatory follicles) or LUFs (luteinized unruptured<br />
follicles [17,22,23,65,74,101]), demonstrate the<br />
importance of the mare as a relevant experimental<br />
model for the study of ovarian function in women.<br />
Among the agriculturally important domestic<br />
species studied so far, the equine is the one with the<br />
highest incidence of metabolic syndrome (i.e. Equine<br />
Metabolic Syndrome). In addition, similar to what<br />
has been found in women, insulin resistance and<br />
obesity syndromes in mares have become serious<br />
and frequent problems for today’s farm operations,<br />
associated or not with metabolic syndromes.<br />
Reproductive disorders at the ovarian level in women<br />
with these syndromes are very common and are<br />
usually a serious infertility problem (i.e. Polycystic<br />
Ovarian Syndrome). Few reproductive studies in<br />
mares with these syndromes have been reported.<br />
However, the few results available demonstrate that<br />
mares’ ovarian function and estrous cycle also seem<br />
to be altered. The importance of the mare over other<br />
domestic species in the study of obesity and insulin<br />
resistance is warranted because of its monovular<br />
condition, significant similarity in ovarian<br />
folliculogenesis and metabolic disturbs (e.g. Equine<br />
Metabolic Syndrome) with humans, and the relative<br />
ease with which follicles are imaged, tracked, and<br />
targeted (e.g. follicle sampling, aspiration, injection)<br />
using transrectal and transvaginal ultrasonography<br />
(reviewed in [36,37]). Moreover, the similarities<br />
existing between mares and women in insulin<br />
resistance and obesity syndromes associated with<br />
reproductive disturbances during the estrous/menstrual<br />
cycle encourage the use of the equine model to<br />
test hypotheses using invasive technologies and may<br />
provide additional information that can also be<br />
considered for other farm animal species and in<br />
human clinical medicine. Additionally, studies in this<br />
area shall have beneficial impacts in agriculture<br />
allowing: (1) a better understanding of Equine<br />
Metabolic Syndrome; (2) improvement in the control<br />
and treatment of obese horses (males and females),<br />
cows, and other farm animals that might suffer from<br />
these syndromes; and (3) improvement on ovarian<br />
cyclicity, oocyte and embryo production, and fertility<br />
rates in farm animals with metabolic syndrome, insulin<br />
resistance and obesity. This review will focus on the<br />
similarities existing between mares and women in<br />
insulin resistance and obesity syndromes associated<br />
with reproductive disturbances during the estrous/<br />
menstrual cycle.<br />
II. GENERAL ASPECTS OF FOLLICLE DEVELOPMENT IN<br />
THE MARE<br />
Ovarian follicle development in the mare is<br />
characterized by waves of several follicles that emerge<br />
and initially grow in synchrony (reviewed in [35,36]).<br />
Various numbers and types of follicular waves<br />
develop during an equine interovulatory interval (IOI)<br />
[47]. In a major wave, the largest follicle attains the<br />
diameter of a dominant follicle (=28-30 mm), whereas<br />
in minor waves, the largest follicle does not become<br />
dominant. The ovulatory wave emerges midway<br />
during an IOI of 21-24 days. After emergence at 6<br />
mm [39], the follicles of a wave develop in a<br />
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E.L.Gastal, M.O. Gastal, A. Wischral & J. Davis. <strong>2011</strong>. The Equine Model to Study the Influence of Obesity and<br />
Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
common-growth phase for several days [38]. At the<br />
end of the common-growth phase, a distinctive<br />
change in growth rates begins. This process is called<br />
deviation, and in mares it begins when the diameter<br />
of the two largest follicles are, on average, 22.5 mm<br />
and 19.0 mm [39,43,50]. The deviation in growth<br />
rates between the future dominant and subordinate<br />
follicles is a key event during the selection of the<br />
ovulatory follicle. After deviation (“follicle selection”),<br />
the developing dominant follicle maintains a constant<br />
growth rate until 1 or 2 days before ovulation [41]<br />
and the remaining follicles (subordinate follicles)<br />
grow at a reduced rate or establish a plateau phase<br />
and then regress.<br />
The ovulatory waves, as well as major<br />
anovulatory waves and minor waves, originate from<br />
the stimulation of a follicle-stimulating hormone (FSH)<br />
surge, which reaches a peak when the largest follicle<br />
is about 13 mm [39]. The initial decline in the FSH<br />
surge appears to be a function of inhibin. Circulating<br />
estradiol does not begin to increase until about 2 days<br />
after the FSH peak or about 1 day before the beginning<br />
of deviation. Concentrations of luteinizing hormone<br />
(LH) during the ovulatory LH surge reach a transient<br />
plateau encompassing deviation of the ovulatory<br />
wave. The intrafollicular concentrations of estradiol,<br />
insulin-like growth factor 1 (IGF1), inhibin-A, and<br />
activin-A increase differentially in the future dominant<br />
follicle versus the future subordinate follicles about<br />
1 day before the beginning of diameter deviation.<br />
These factors may be enablers for differentially<br />
enhancing the FSH and LH responsiveness of the<br />
future dominant follicle.<br />
The characteristics and the intrafollicular and<br />
systemic hormonal events associated with the<br />
beginning of deviation have been reviewed for mares<br />
[50], mares and heifers [12,48], and have been<br />
compared between mares and women [49,52].<br />
Briefly, dramatic changes in the IGF system lead to<br />
increased free IGF1 in the most developed follicle<br />
before the beginning of diameter deviation and play<br />
a crucial role in the events that lead to deviation in<br />
both horses and cattle. Estradiol and LH receptors<br />
also play a role, at least in cattle. The intrafollicular<br />
events prepare the selected follicle for the decreasing<br />
availability of FSH from the wave stimulating FSH<br />
surge and increasing availability of LH. Other follicles<br />
of the wave have a capability for future dominance<br />
similar to that of the largest follicle but do not have<br />
adequate time to attain the required preparatory stage.<br />
In this regard, the essence of deviation is a close twoway<br />
functional coupling between FSH and products<br />
of the follicles (inhibin, estradiol).<br />
III. SIMILARITIES IN FOLLICLE DEVELOPMENT AND<br />
SYSTEMIC HORMONES BETWEEN MARES AND WOMEN<br />
As mentioned above, mares and women<br />
share striking similarities regarding ovarian folliculo<br />
genesis. Some of the similarities between the two<br />
species are: (1) emergence of the ovulatory follicle<br />
before the first subordinate follicle; (2) length of<br />
intervals between sequential emergence of follicles<br />
within a wave; (3) interval from emergence to<br />
deviation of the ovulatory follicle; (4) percentage<br />
growth of follicles during the common growth phase;<br />
(5) deviation in growth rates between the two largest<br />
follicles of a major wave (Figure 1); (6) percentage<br />
of a predeviation follicle that regressed an average of<br />
1 day before deviation; (7) consistency of a<br />
mare:woman diameter ratio (approximately 2:1) of<br />
the ovulatory follicle; (8) prevalence of major<br />
anovulatory follicular waves during an interovulatory<br />
interval; (9) presence of major and minor follicular<br />
waves (Figure 2); (10) peak of the wave-stimulating N<br />
FSH surge on the day of emergence of the follicular<br />
wave; (11) occurrence of follicle deviation during a<br />
decline in FSH concentrations; (12) estradiol and<br />
progesterone profiles during the periovulatory period;<br />
(13) long duration of the follicular phase; (14) increase<br />
in the length of the follicular phase and IOI with aging;<br />
(15) changes in concentrations of gonadotropins with<br />
aging; (16) cessation of follicular activity with aging;<br />
(17) preovulatory follicle characteristics; (18)<br />
anovulatory disturbs such as HAFs and LUFs<br />
[7,15,23,35-37,40,41,49,52,55]. Similar follicle<br />
dynamics between mares and women indicate the<br />
mare may be a useful experimental model for the<br />
study of folliculogenesis in women, with the<br />
advantage of larger follicle size.<br />
It is still not clear whether or not obesity,<br />
insulin resistance, abnormal ovarian morphology and<br />
function, and/or altered endocrinology are causes or<br />
merely symptoms of ovarian disturbs in women with<br />
PCOS and in mares. The scientific literature pertaining<br />
to PCOS is vast and commonly based on clinical<br />
research, although some studies have focused on in<br />
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Figure 1. Means (±SEM) for diameters of four follicle types during ovulatory waves in 27 mares and 25 women. Numbers<br />
of waves for mares and women, respectively, with various follicle types were: dominant and first subordinate follicles (27 and<br />
25), second subordinate follicles (14 and 9), and predeviation follicles (10 and 12). Dominant and subordinate follicles are<br />
centralized among individuals to the beginning of deviation (Day 0), and the predeviation follicle is centralized to the mean day<br />
at the beginning of its regression (Day 21). Adapted from [52].<br />
Figure 2. Comparative ovarian follicular wave patterns between mares and women. Presented on the left panel are individual<br />
follicle profiles in mares for three ovulatory waves (M1, M3, M5) and three interovulatory intervals (M2, M4, M6). Emerging<br />
follicles were not detected earlier in the interovulatory interval in M1, M3, and M5. Follicles of the ovulatory wave intermingled<br />
with regressing follicles from a previous ovulatory wave (M3, M6), a major anovulatory wave (M4), and static follicles from<br />
a previous wave (M2). A predeviation follicle (M3, M5) and a late regressing follicle (M4) are shown. Presented on the right<br />
panel are individual follicle profiles in women for three interovulatory intervals (W1, W3, W5) and three ovulatory waves (W2,<br />
W4, W6). Emerging follicles were not detected before 12 days postovulation in W2, W4, and W6. Note the predeviation follicle<br />
(W3, W6), a second-largest subordinate follicle (W1, W3), and the transiently arrested growth of the follicles during 13–18<br />
days postovulation (W5). MAW = major anovulatory wave. mW = minor wave. OV = ovulation. An arrow indicates the<br />
beginning of deviation. Adapted from [52].<br />
vitro culture and experimentation with follicular cells<br />
from PCOS-affected women. Using an animal model<br />
for controlled experiments to investigate the<br />
interrelationships between ovarian follicular development,<br />
reproductive endocrinology, and metabolic<br />
status at the whole-animal level and down to the<br />
cellular level may shed new light on some of the causes<br />
and effects of PCOS in women and ovarian disturbs<br />
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Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
in farm animals. In this regard, there is strong<br />
evidence that an appropriate animal model for this<br />
purpose can be the mare.<br />
IV. OBESITY<br />
In domestic farm animals, research with an<br />
emphasis on nutrition was mainly related to the effects<br />
of poor nutrition on productivity and reproductive<br />
performances for a very long time. However, recent<br />
investigations have demonstrated an increasing<br />
awareness of obesity as a main detrimental factor that<br />
affects the health of companion [60,108] and some<br />
domestic animals, including horses [33,86,112].<br />
Obesity is defined as an accumulation of excessive<br />
amounts of adipose tissue in the body. Human obesity<br />
or overweight diagnosis is relatively simple, requiring<br />
only the determination of the body mass index (BMI)<br />
by measurement of height and weight; however the<br />
measuring of the circumference of the individual’s<br />
waist has been suggested as an excellent indicator of<br />
visceral adiposity [72]. In the equine field, various<br />
methods have been recommended for the purpose<br />
of assessing the relative adiposity of equine patients,<br />
including the body condition score (BCS; [58]) the<br />
equine BMI [66], and the use of ultrasonography to<br />
assess subcutaneous fat thickness near the tail head<br />
[28].<br />
Every year, the scientific community and<br />
media dedicate increasing amounts of time and<br />
resources to the health complications caused by<br />
obesity in humans, which is currently considered one<br />
of the fastest-growing epidemics throughout the<br />
world. Obesity in humans is associated with an<br />
increased risk of mortality by a number of different<br />
diseases, and usually precedes many of the<br />
acknowledged metabolic disturbances including<br />
prediabetes, diabetes and cardiovascular disease.<br />
Obesity-related deaths are expected to exceed the<br />
number of all other causes of death for preventable<br />
conditions in the near future [14]. The occurrence of<br />
obesity-related diseases has been mainly linked to<br />
the visceral (abdominal) adiposity [3,72]. In horses<br />
and ponies, the association between regional adiposity<br />
and disease risk seems to be similar to the human<br />
[16]. Obese animals usually have elevated body<br />
condition scores and enlarged fat deposits on the neck<br />
(“cresty neck”), thoracic and/or tailhead regions [16].<br />
In parallel with human obesity, there is a<br />
growing concern about the occurrence of obesity and<br />
insulin resistance in animals. Studies have shown a<br />
prevalence of obesity of 20 to 25% in dogs<br />
[31,92,108] and 26% in cats [4]. Few studies have<br />
examined the prevalence of obesity in horse<br />
populations. An estimate of only 4.5% overweight or<br />
obese horses was reported in 1998 in the U.S.<br />
population (NAHMS). However, the accuracy of this<br />
prevalence may be questioned because it was based<br />
on owner reporting, and not from results of physical<br />
measurements. More recent studies have shown that<br />
the prevalence of overweight and obese horses has<br />
increased to 45% and 51%, in a subpopulation of<br />
pleasure riding horses in North-West Glasgow in 2005<br />
and Virginia in 2006, respectively [100,112]. Hence,<br />
the number of obese horses has shown a strong incline<br />
over time, and the reasons by which domesticated<br />
animals develop obesity are broadly similar to those<br />
reasons that have been attributed to obesity in<br />
humans [62]. In domesticated horses, for instance,<br />
obesity develops in light of the fact that they tend to<br />
be physically inactive and are provided with rations<br />
that are excessive in terms of energy [63].<br />
Although health problems such as heart<br />
diseases and diabetes do not occur in horses as<br />
frequently as in humans, the clinical implications of<br />
obesity are just as serious. In human obesity, the N<br />
underlying mechanism contributing to metabolic<br />
dysfunction appears to be the presence of low grade<br />
systemic inflammation (initiated in adipose tissue).<br />
Obese horses and ponies are known to have a higher<br />
risk of developing laminitis, an inflammation of the<br />
sensitive laminae of the hooves [70], and a lower<br />
fertility rate with a greater risk of dystocia [105].<br />
Furthermore, higher fat mass leads to hyperthermia<br />
and an increased incidence of recurrent exertional<br />
rhabdomyolysis (RER), or tying-up [103]. Laminitis,<br />
with a prevalence of 7% [57], and RER, with a<br />
prevalence of 5 to 10% in Thoroughbreds [73], are<br />
considered two of the most common reasons for<br />
presenting a horse for veterinary consultation [57]<br />
and for exclusion of horses from competitions [73].<br />
All these numbers stress the impact of obesity and<br />
associated disorders on equine health and welfare.<br />
Obesity in women is often inextricably linked<br />
with ovarian function leading to clinical reproductive<br />
manifestations such as menarche onset, subfertility<br />
and Polycystic Ovarian Syndrome (PCOS; reviewed<br />
in [89]). PCOS is one of the most common endocrine<br />
disorders in women of reproductive age [29,34]. It is<br />
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Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
characterized by hyperandrogenic chronic<br />
anovulation, and has a mean prevalence of 7-8%<br />
(range, 4-12%) in the general population [5,6,25].<br />
Insulin resistance has been recognized as the major<br />
factor related to PCOS and also a significant<br />
contributor to its reproductive and metabolic<br />
complications [26]. The potential mechanism related<br />
to reproductive disturbs seems to involve<br />
compensatory hyperinsulinemia, which stimulates<br />
ovarian androgen production, and also increases<br />
peripheral aromatization of androgens to estrogens;<br />
these factors may alter gonadotropin secretion,<br />
subsequently altering follicular development [24].<br />
However, the molecular mechanism involving insulin<br />
resistance in the pathophysiology of PCOS remains<br />
obscure. PCOS is characterized by a group of features<br />
with no specific diagnostic and a large diversity in its<br />
clinical presentation [61]. As the name implies, PCOS<br />
is in part identifiable by abnormal ovarian<br />
morphology [98]. In the last 30 years, the<br />
development of ultrasound as a noninvasive<br />
diagnostic tool has resulted in numerous studies that<br />
have characterized abnormal ovarian morphology<br />
associated with PCOS. The most pertinent and<br />
clinically-relevant ultrasound diagnostic criteria<br />
include the presence of =12 follicles, 2-9 mm in<br />
diameter (small- to medium-sized follicles), and an<br />
ovarian volume >10 cm 3 that is approximately 2-fold<br />
greater than normal ovaries [10]. In a large study of<br />
1,741 women diagnosed with PCOS by the presence<br />
of abnormal ovarian morphology, 38% were obese<br />
and 29% and 40% had elevated serum concentrations<br />
of LH and testosterone, respectively [9]. Currently,<br />
endocrinological abnormalities such as elevated<br />
systemic LH concentrations and hyperandrogenism<br />
are used for the diagnosis of PCOS [8].<br />
The abnormal endocrinology associated with<br />
PCOS occurs locally at the level of the ovary and<br />
systemically at the hypothalamic-pituitary-adrenal<br />
axis. The gonadotropins are differentially altered in<br />
PCOS women. Elevated serum concentrations of LH<br />
[83,99] and normal [81,99] to low [83] concentrations<br />
of FSH were reported in women suffering from PCOS.<br />
The increase in systemic LH concentrations likely<br />
augment androgen production in the thecal cells of<br />
the follicle by activation of the LH receptor, and<br />
subsequent cAMP/Protein Kinase-A signaling<br />
increases the activation of enzymes responsible for<br />
the conversion of cholesterol to androstenedione [27].<br />
In addition, increased insulin concentrations, which<br />
are associated with insulin resistance, may stimulate<br />
the enzyme 3âHSD in follicle and adrenal cells. This<br />
enzyme, mediated by IGF and paracrine factors,<br />
converts dehydroepiandrosterone (DHEA) to<br />
androstenedione [27]. Keeping in mind that<br />
androstenedione is a precursor of testosterone in the<br />
steroidogenic pathway, the increase in steroidogenic<br />
enzyme activity is a plausible reason for the reported<br />
increased serum concentrations of both<br />
androstenedione and testosterone in women with<br />
PCOS compared to normal women [81,83,99].<br />
Despite increases in concentrations of<br />
androstenedione [83,99] and testosterone [80,83,99],<br />
serum estradiol concentrations in PCOS women were<br />
not different from normal women. A regulator of<br />
testosterone and estradiol activity and transport, the<br />
sex hormone binding globulin (SHBG), was reduced<br />
in blood samples of PCOS women compared to normal<br />
women [81].<br />
In humans and rodents, some of the metabolic<br />
changes associated with obesity are the increase in<br />
free fatty acids (FFAs), altered adipokine production<br />
by adipose tissues, and elevated inflammatory<br />
cytokine concentration within the blood [91]. One<br />
explanation of the link between obesity and insulin<br />
resistance is the higher FFA concentrations in insulinsensitive<br />
tissues that can result in a process referred<br />
to as lipotoxicity that can cause insulin resistance [97].<br />
Adipokines are hormones produced by adipocytes<br />
that have local (paracrine) and remote (endocrine)<br />
effects on tissues. Leptin and adiponectin are the best<br />
known adipokines, and obesity has been associated<br />
with higher plasma leptin concentrations and lower<br />
plasma adiponectin concentrations in horses [67].<br />
Recent studies have focused on leptin, which acts on<br />
the hypothalamus as a mediator of nutritional effects<br />
on reproductive function (reviewed in [20,110]. Low<br />
systemic leptin concentrations are associated with<br />
poor body condition in humans, ruminants, and<br />
horses [18,32,46,87]. In mares, systemic<br />
concentrations of leptin were lower in younger (10 yr of age) animals<br />
[13,45]. However, lower circulating concentrations<br />
were also observed during the winter, independent<br />
of age or nutritional status and body condition<br />
[32,45]. Long-term dietary restriction resulted in both<br />
lower body condition scores and lower systemic<br />
leptin concentrations [45]. A recent study [44]<br />
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Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
demonstrated that mares submitted to a dietary shortterm<br />
feed restriction presented decreased systemic<br />
and intrafollicular concentrations of leptin and tended<br />
to have greater intrafollicular concentrations of<br />
inhibin-A and VEGF. In follicular fluid, the<br />
concentration of leptin was positively correlated with<br />
free IGF1, and both intrafollicular leptin and free IGF1<br />
were positively correlated with body condition score.<br />
Blood flow of the preovulatory follicular wall was<br />
greater in the restricted group and combined for both<br />
groups (control and treated) blood flow was negatively<br />
correlated with intrafollicular concentrations of leptin.<br />
Therefore, studies are necessary on the relationship<br />
of obesity and insulin resistance with the systemic<br />
and intrafollicular concentrations of insulin and IGF<br />
and its binding proteins as well as angiogenic factors<br />
(e.g. VEGF, NO).<br />
V. INSULIN RESISTANCE<br />
Besides being involved in the process of<br />
carbohydrate metabolism, insulin takes part in an<br />
extensive range of physiologic processes, including<br />
stimulation of fatty acid and triglyceride synthesis in<br />
the liver and adipose tissue, inhibition of lipolysis,<br />
enhancement of protein anabolism, cell growth and<br />
survival, regulation of vascular endothelial function,<br />
and anti-inflammatory effects [56,109]. In ovarian<br />
function, insulin has been shown to have a major<br />
role, including the regulation of ovarian<br />
steroidogenesis, follicular development, and<br />
granulosa cell proliferation [1,84,111]. The reduction<br />
in sensitivity to the biological actions of insulin affects<br />
not only glucose metabolism, but also all aspects of<br />
insulin action. In this regard, the insulin-like growth<br />
factor system (e.g. IGF1 and IGF binding proteins)<br />
plays a crucial role in the ovary in processes of follicle<br />
selection and dominance in mares [12,51,53] as<br />
discussed before.<br />
Insulin resistance or decreased insulin<br />
sensitivity is defined as the decreased biological<br />
response of cells to the action of insulin in transporting<br />
glucose from the bloodstream into the target tissue<br />
(e.g. liver, muscle and adipose tissue). It is important<br />
to emphasize that insulin resistance is not a disease<br />
itself, but a physiological status. The reduced insulin<br />
sensitivity and resulting hyperglycemia are usually<br />
compensated by increased insulin production by the<br />
pancreatic β-cells or by an increase in the glucosemediated<br />
glucose disposal [69]. Insulin resistance has<br />
been well documented in humans [26,30] and horses<br />
[59,102]. In humans, obesity has been related to the<br />
development of insulin resistance. The link between<br />
obesity and insulin resistance has been suggested to<br />
be a consequence of inflammation in adipose tissue<br />
and the liver and the accumulation of by-products of<br />
nutritional overload (e.g. diacyglycerol) in insulinsensitive<br />
tissues [78]. In horses, Vick et al. [104]<br />
reported associations between obesity and blood<br />
mRNA expression of tumor necrosis factor alpha<br />
(TNFα) and interleukin (IL-1β), suggesting that<br />
systemic inflammation may be involved in the<br />
development of insulin resistance.<br />
Insulin resistance has been shown to play a<br />
central role in some health problems in humans [94]<br />
and also in horses [21,106]. In horses, insulin<br />
resistance has been associated with pathogenesis of<br />
diseases such as laminitis, pituitary adenoma,<br />
hyperlipidemia, osteochondritis, and also seems to<br />
influence negatively reproductive efficiency [105]<br />
and probably exercise [64].<br />
Original research pertaining to the effects of<br />
obesity and insulin resistance on reproductive<br />
cyclicity and fertility in the mare is sparse, and the<br />
data from this limited amount of research have<br />
yielded no solid conclusions. Kubiak et al. [71] N<br />
reported that there was no difference in the intervals<br />
from parturition to first ovulation and first to second<br />
ovulation and first cycle conception rate between<br />
postpartum mares fed to obesity and control mares.<br />
However, the results of the previous data are<br />
questionable considering the low numbers of mares<br />
used per experimental group. A few studies have<br />
reported on the effects of obesity and/or insulin<br />
resistance on the estrous cycle. Induction of transient<br />
insulin resistance via infusion of a heparinized lipid<br />
emulsion lengthened the interovulatory interval and<br />
reduced peak plasma progesterone concentrations<br />
during the luteal phase, but did not affect mean LH<br />
concentrations compared to control mares [96]. Vick<br />
et al. [105] reported that obese mares (BCS =7) with<br />
reduced insulin sensitivity had longer interovulatory<br />
intervals and luteal phases compared to feed-restricted<br />
mares; however, Metformin (anti-diabetic drug)<br />
treatment of obese mares did not alter the<br />
interovulatory interval or luteal phase. Daily<br />
intravenous infusion of insulin during the mid to late<br />
luteal phase (7-17 days after ovulation) did not alter<br />
the length of the estrous cycle, corpus luteum area,<br />
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Insulin Resistance in Human Ovarian Function. Acta Scientiae Veterinariae. 39(Suppl 1): s57 - s70.<br />
or plasma LH concentrations, but tended to lower<br />
plasma progesterone concentrations [90].<br />
Surprisingly, to our knowledge, the effects of insulin<br />
resistance and obesity on the preovulatory follicular<br />
wave have not been studied in detail in mares or<br />
women.<br />
5.1 Molecular mechanisms and defects of insulin<br />
activity in target tissues<br />
Insulin signaling involves a wide number of<br />
substances and affects both transport and subsequent<br />
utilization of glucose, especially in relation to<br />
hexokinase, glycogen synthase, and other key<br />
enzymes in glucose and lipid metabolism [95]. A<br />
cascade of intracellular events is initiated when insulin<br />
binds and activates its receptor at the cell-surface,<br />
resulting in the tyrosine phosphorylation of several<br />
substrates, including the insulin receptor substrate.<br />
Insulin resistance may result from a reduction in the<br />
density of receptors, malfunction of insulin receptors,<br />
defective internal signaling pathways, and interference<br />
with the translocation or function of glucose<br />
transporter type 4 (GLUT-4) proteins [68]. Decreased<br />
levels of insulin receptors -1 and -2 tyrosine<br />
phosphorylation [19,88,107] and reduced numbers<br />
of insulin receptors [19] in adipose tissue from insulin<br />
resistant PCOS patients have been shown. Also, the<br />
expression of GLUT-4 was significantly decreased<br />
in PCOS adipocytes [93].<br />
The presence of insulin receptors in both stromal<br />
and follicular compartments of the human ovary<br />
[85] and the ability of insulin to stimulate<br />
steroidogenesis in ovarian cells in vitro [11] has<br />
established the ovary as a target organ for insulin<br />
activity. Hyperinsulinemia can potentiate<br />
gonadotropin-stimulated steroidogenesis in granulosa<br />
and theca cells by increasing the low-density<br />
lipoprotein (LDL) receptor, 3β-hydroxysteroid<br />
dehydrogenase, 17α-hydroxylase, and 17,20 lyase<br />
expression [76,79,113]. Recently, the insulin<br />
signaling pathways on ovaries from obese and insulin<br />
resistant rats were studied [2]. The ovaries from obese<br />
and insulin resistant rats showed a reduction in the<br />
insulin receptor substract/phosphatidylinositol 3-<br />
kinase/AKT intracellular pathway, associated with an<br />
increase in FOXO3a, IL1B, and TNFα protein<br />
expression [2]. Similarly TNFα and IL-1â were<br />
elevated in obese insulin resistant mares [104].<br />
5.2 Insulin resistance detection<br />
A variety of tests, each with their own pros<br />
and cons, are available to test insulin resistance in<br />
human and veterinary medicine. There seems to be a<br />
consensus that the hyperinsulinemic – euglycemic<br />
clamp procedure is considered the “gold standard”<br />
for assessment of insulin sensitivity. The clamp<br />
procedure involves maintaining a constant<br />
supraphysiological insulin concentration while<br />
differentially controlling glucose infusions to maintain<br />
a steady state of glycemia. The greater the rate of<br />
glucose infusion, the more sensitive the subject is to<br />
insulin. A lower infusion rate indicates decreased<br />
insulin sensitivity (or higher insulin resistance). Singlepoint<br />
measurements of insulin and glucose during<br />
fasting states are convenient and practical; however,<br />
they do not offer the amount of information provided<br />
by the clamp or minimal model analysis [102]. The<br />
minimal model employs a frequently-sampled i.v.<br />
glucose tolerance test (FSIGT) with an i.v. insulin dose<br />
20 min after the glucose bolus. Based on the<br />
differential effects of glucose on insulin and vice<br />
versa, the following endpoints are calculated [102]:<br />
1) insulin sensitivity of tissue (SI); 2) difference<br />
between insulin-stimulated and insulin-independent<br />
glucose clearance (Sg); 3) pancreatic â-cell response<br />
to glucose (AIRg); and 4) comparison of insulin<br />
sensitivity to insulin response (DI).<br />
VI. CONCLUSIONS<br />
Mares and women share striking similarities<br />
in ovarian folliculogenesis, and in insulin resistance<br />
and obesity syndromes. The effects of insulin<br />
resistance and obesity on follicular development and<br />
the surrounding endocrinology and genes in mares<br />
may shed light on the causes and effects of metabolic<br />
and reproductive disorders such as polycystic ovarian<br />
syndrome (PCOS) and anovulatory processes in<br />
women. Understanding the mechanisms related to<br />
this effect might help to improve reproductive<br />
efficiency and health in animals and humans, as well<br />
as the quality of products (e.g. oocytes, embryos,<br />
etc.). Moreover, the similarities existing between<br />
mares and women in insulin resistance and obesity<br />
syndromes associated to reproductive disturbances<br />
during the estrous/menstrual cycle encourage the use<br />
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of the equine model to test hypotheses using invasive<br />
technologies and may provide additional information<br />
that can also be considered for other farm animal<br />
species and in human clinical medicine.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s71.<br />
ISSN 1679-9216 (Online)<br />
Using Animals to Develop Assisted Human Reproduction<br />
Carlos Gilberto Almodin 1,2<br />
ABSTRACT<br />
Background: There is no denying the importance of animal experimentation in the development of biological sciences, the<br />
importance due to the extraordinary benefits for human and animal health. Without the use of laboratory animals in experiments,<br />
it would be impossible to achieve the knowledge gained in the last two centuries. Hippocrates (450 BC) has related the<br />
appearance of ill human organs patients with those of animals, with clear didactic purpose. It was also through the use of<br />
animals that human reproduction has advanced in scientific terms in recent decades.<br />
Review: Bioassays with mouse embryos have been used as a means of controlling water quality, culture media, solutions,<br />
materials and standardization of techniques and training in assisted reproduction techniques. Most of these bioassays use<br />
embryos in early stages of development and evaluate development to the blastocyst stage. Changes in substrate utilization<br />
follow similar patterns in mice and humans. This similarity in metabolic parameters between the two species has facilitated the<br />
use of mice as a model for studying the formulation of culture media for use in different stages of preimplantation period, from<br />
fertilization to the blastocyst stage. There are aspects of the physiology of both species that still require more studies, such as<br />
the requirement of inorganic phosphate in the culture medium, the specific requirement of amino acids for optimal development<br />
before and after compaction, and the importance of including EDTA in the culture medium. The evaluation of chemically<br />
defined media for the generation of viable cleaved embryos is of fundamental importance. Several cryopreservation studies<br />
have been performed with gametes and embryos from cattle and pigs, because such species have greater difficulty in survival<br />
after cryopreservation compared to the same cell types in the mouse and in humans, due to the increased amount of lipids they<br />
have in their constitution. That is, if the cryopreservation protocol under test meets the requirements for cryopreservation Nof<br />
bovine and swine oocytes and embryos, it will bring great benefits to the cryopreservation of such cells in humans. More than<br />
50 years ago, Parrot (1960) showed that frozen ovarian fragments can restore fertility in mice when re-implanted. Such study<br />
was later extended to other species: rats (Aubard et al.), rabbits and sheep (Gosden et al., Baird, Almodin et al.). The latter model<br />
is more relevant to human physiology because sheep, as well as women, are single ovulators. In addition, ovaries from sheep<br />
ovaries resemble humans in terms of size and tissue composition. Thus, cryopreservation of ovarian tissue was consolidated<br />
after experiments in sheep.<br />
Conclusion: Presently not only are animals used in experimentation on human reproduction, but are also used as quality<br />
control in human IVF laboratories. Quality control is no longer just a necessity to minimize possible flaws in the process, but<br />
a requirement. However, it is very important to stress that it should only be carried out under absolutely defined conditions,<br />
with complete safety, ethical rigor and respect for the animals used. Moreover, it is scientifically proven that the genetic<br />
quality, the standard of health and the environment affect the animal’s behavior, thus modifying experimental results. It is<br />
therefore important to outline the “big picture” of animal experimentation, in terms of objectives, types of procedures and<br />
numbers of animals and procedures involved.<br />
Keywords: assisted reproduction, bioassays, human IVF, animal experimentation, fertility.<br />
1<br />
Materbaby-Reprodução Humana e Genética, Maringá, PR, Brazil. 2 Universidade Federal de São Paulo, Departamento de Tocoginecologia,<br />
São Paulo, SP, Brazil. CORRESPONDENCE: C.G. Almodin [almodin@materbaby.com.br - FAX: +55 (11) 22722605]. Universidade Federal<br />
de São Paulo, Departamento de Tocoginecologia, Rua Napoleão de Barros, n. 715 - 8º andar, Vila Clementino. CEP 04024-002 São Paulo, SP,<br />
Brazil.<br />
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orges Jr. <strong>2011</strong>. The Role of Veterinarians in Human In Vitro Embryo Production. SSSSSSSSSSS<br />
ssssssssssssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s73 - s80.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s73 - s80.<br />
ISSN 1679-9216 (Online)<br />
The Role of Vet<br />
eter<br />
erinar<br />
inarians ians in Human In Vitr<br />
itro Embryo Produc<br />
duction<br />
Daniela Paes de Almeida Fer<br />
erreir<br />
eira Braga & Edson Bor<br />
orges Jr.<br />
ABSTRACT<br />
Background: Infertility affects approximately 15% of couples of childbearing age, and there is evidence that fertility has been<br />
declining at an accelerated pace in recent years. Recent advances in assisted reproductive technologies (ART) have made<br />
biological parenthood possible for couples who previously had no hope of childbearing. On the other hand, the primary use<br />
of embryo transfer in cattle has been amplification of the reproductive rates of valuable females. In 2007, 245,257 bovine<br />
embryos were produced globally; Brazil accounted for 86.6% of this output. The popularity of this technology has contributed<br />
to the growing knowledge of embryology among veterinarians. Because of ethical concerns regarding the use of human<br />
subjects, bovines have been used as a model for the study of reproductive biology in women. Therefore, this manuscript will<br />
discuss approaches for oocyte selection and sperm selection that were developed in animal models and adapted for ART in<br />
humans.<br />
Review: One of the most important factors determining the outcome of embryo development is gamete quality. Numerous<br />
attempts have been made to identify prognostic factors for oocyte development potential. One promising approach uses<br />
spindle imaging as a predictor of oocyte quality. The development of a polarised light microscope that evaluates the birefringence<br />
of living cells enabled the evaluation of highly birefringent oocyte spindles without damage to the cell. Polarised light<br />
microscopy also enables the evaluation of other birefringent, sub-cellular oocytesstructures, such as the zona pellucida. The<br />
birefringence of the meiotic spindle and the zona pellucida are valuable tools in predicting the developmental potential of<br />
oocytes and embryos. The post-genomic era has facilitated the growth of non-invasive approaches to the study of embryonic<br />
physiology and to predicting oocyte developmental competence and viability. These approaches include the assessment Nof<br />
proteomic profiling and, most recently, of the oocyte and embryonic metabolome. Although intracytoplasmic sperm injection<br />
ICSI outcomes are apparently unrelated to basic sperm parameters, recent reports have suggested a paternal contribution to the<br />
success of embryonic development and implantation, and a new method for sperm evaluation has been proposed. It is now<br />
possible to examine the nuclear morphology of spermatozoa at 6600x magnification. This technique has allowed the<br />
introduction of a modified ICSI procedure called “intracytoplasmic morphologically selected sperm injection” (IMSI). As a<br />
consequence, real-time, detailed morphological sperm examination using motile sperm organelle morphology examination<br />
(MSOME) enables the best available spermatozoa to be selected prior to oocyte injection. Recent reports have suggested that<br />
normal nuclear morphology, as viewed under high magnification, may be useful for selecting spermatozoa with higher<br />
developmental capacity.<br />
Conclusion: Studies in animal reproduction have allowed the development of non-invasive methods of evaluating sperm and<br />
oocytes prior to their use in ART. Gamete quality biomarkers, once identified, may allow embryos with the highest implantation<br />
potential to be selected with greater accuracy. The use of such biomarkers may thus yield an increased chance of pregnancy,<br />
which is the primary objective of ART.<br />
Keywords: assisted reproduction, non-invasive biomarkers, oocyte quality, sperm morphology.<br />
Fertility - Assisted Fertilization Center, São Paulo, SP, Brazil. CORRESPONDENCE: D.P.A.F. Braga [dani@webbraga.com/dbraga@fertility.com.br<br />
- FAX: +55 (11) 3885-9858]. Av. Brigadeiro Luis Antonio, n. 4545, CEP:01401-002, São Paulo, SP, Brazil.<br />
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orges Jr. <strong>2011</strong>. The Role of Veterinarians in Human In Vitro Embryo Production. SSSSSSSSSSS<br />
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I. INTRODUCTION<br />
II. EVALUATION OF HUMAN OOCYTES<br />
2.1 Meiotic spindle image<br />
2.2 Zona Pellucida Birefringence<br />
2.3 Assessment of metabolites and proteins in the oocyte<br />
culture media<br />
III. EVALUATION OF HUMAN SPERMATOZOA<br />
IV. CONCLUSION<br />
I. INTRODUCTION<br />
Human infertility is defined as the failure to<br />
conceive after one year of unprotected intercourse<br />
[4] and affects approximately 15% of couples at<br />
childbearing age, with substantial clinical and social<br />
impacts [7].<br />
There is some evidence that fertility has been<br />
decreasing at an accelerated rate in recent years. One<br />
prominent cause of reduced fertility is age-related<br />
decline in the number and quality of eggs; women<br />
now wait longer to have their first child than they did<br />
in previous decades [35]. In the so-called natural<br />
fertility populations in which no birth control is<br />
practised, fertility starts to decline at age 30 or earlier<br />
[21]. Based on such historical cohorts, it is known<br />
that the time required to conceive a child increases<br />
with age; the risk of infertility is also higher for older<br />
women [40].<br />
Moreover, lifestyle factors and nutritional<br />
status are known to be critical determinants of normal<br />
reproductive function. A combination of reduced<br />
exercise, changes in dietary composition and social<br />
habits have contributed to a growing worldwide<br />
epidemic of infertility [45].<br />
Recent advances in assisted reproductive<br />
technologies (ART) have made biological parenthood<br />
possible for couples who previously had no hope of<br />
childbearing. Since the first healthy child was<br />
conceived via in vitro fertilisation (IVF) [54], the use<br />
of ART has increased dramatically; as many as 1%<br />
of all children are now conceived in vitro [2]. More<br />
recently, intracytoplasmic sperm injection (ICSI) has<br />
become the treatment of choice for severe male factor<br />
infertility [48].<br />
On the other hand, the primary application<br />
of embryo transfer in cattle has been to amplify the<br />
reproductive rates of valuable females. Ideally,<br />
embryo transfer can be used to enhance stock<br />
improvements and to increase marketing<br />
opportunities for purebred cattle. After transferable<br />
embryos are collected from a donor cow, it is decided<br />
which of the available recipients should receive<br />
embryos, with the goal being to achieve the greatest<br />
number of pregnancies [61]. In 2007, 245,257 bovine<br />
embryos were produced globally; Brazil accounted<br />
for 86.6% of this output [52]. The popularity of these<br />
technologies has contributed to the growing<br />
knowledge of embryology among veterinarians.<br />
Moreover, follicular development in cattle has<br />
been characterised in detail over the last two decades<br />
[26], and the bovine model [1] was the foundation<br />
for the recent discovery of follicular wave<br />
development in women [6,7]. Indeed, the<br />
development of ovarian follicular waves in women<br />
is comparable to the follicular wave phenomenon<br />
described in cattle. In both species, the menstrual [7]<br />
and oestrous [16,26] cycles are characterised by two<br />
or three follicular waves.<br />
The main challenge for successful IVF is the<br />
production of viable embryos with high implantation<br />
potential. Although high-quality human embryos may<br />
be available for transfer, only a small proportion of<br />
patients undergoing IVF will ever achieve a pregnancy.<br />
In fact, previous studies most of the embryos<br />
transferred into the uterus fail to implant [19]. Oocyte<br />
quality influences the implantation potential of IVFderived<br />
embryos [18,60] and recent reports have also<br />
suggested a paternal contribution to the success of<br />
embryonic development and implantation [31,47].<br />
Because of ethical concerns regarding the use<br />
of human subjects, and human embryos in particular,<br />
bovines have been used as a model for the study<br />
of reproductive biology in women [16,38]. Therefore,<br />
this manuscript will describe approaches for oocyte<br />
selection and sperm selection that were developed in<br />
animal models and adapted for human ART.<br />
II. EVALUATION OF HUMAN OOCYTES<br />
Advances in reproductive medicine have<br />
made clear that one of the most important factors<br />
determining the outcome of embryonic development<br />
is oocyte quality; in particular, success depends on<br />
optimal nuclear and cytoplasmic oocyte maturity<br />
[15,17].<br />
There are many published reports on the<br />
impact of oocyte morphology on embryonic<br />
development [18,60]. Numerous attempts have been<br />
made to identify the characteristics of oocyte<br />
morphology that can be used to predict oocyte quality,<br />
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fertilisation rates and embryonic development;<br />
however, the predictive value of the criteria used in<br />
these studies is still controversial.<br />
2.1 Meiotic spindle image<br />
During very early transition from Metaphase<br />
I to Metaphase II of meiosis, a highly dynamic<br />
microtubule structure called the meiotic spindle is<br />
formed. The meiotic spindle controls the movement<br />
of chromosomes throughout meiosis; the spindle is<br />
also involved in various functions that are essential<br />
for fertilisation and in early post-fertilisation events,<br />
such as chromosome segregation and genomic<br />
stability after oocyte activation [20,51].<br />
Over the years, transmission electron microscopy<br />
and immunofluorescence have been used to<br />
study oocytes from both humans and experimental<br />
animals; these techniques are used to detect spindle<br />
aberrations and to identify conditions that adversely<br />
affect spindle function and chromosome segregation,<br />
for instance, at IVF. Errors in meiotic division may<br />
be one of the most common causes of aneuploidy<br />
[15].<br />
One promising approach uses spindle<br />
imaging as a predictor of oocyte quality [15,59]. The<br />
development of a polarised light microscope that<br />
evaluates the birefringence of living cells enabled the<br />
evaluation of the highly birefringent oocyte spindles<br />
(Figure 1) without damage to the cell [33,46].<br />
Previous reports have demonstrated that<br />
oocyte developmental competence is lower when<br />
meiotic spindles are not detected under polarised light<br />
microscopy. It has been suggested that the absence<br />
of meiotic spindles detection may compromise not<br />
only fertilisation but also embryonic development<br />
[30,33,59]. Moreover, a previous study presented by<br />
our group demonstrates that when only embryos<br />
derived from oocytes with detectable spindles were<br />
used, higher pregnancy and implantation rates were<br />
achieved [36]. These data suggest that spindle<br />
visualisation can be an important tool in predicting<br />
oocyte and embryo competence.<br />
2.2 Zona Pellucida Birefringence<br />
Polarised light microscopy also enables the<br />
evaluation of other birefringent sub-cellular oocyte<br />
structures, such as the zona pellucida [29,49]. The<br />
zona pellucida is a unique extracellular coat that<br />
surrounds the maturing oocyte during ovulation,<br />
fertilisation, and early embryonic development [22].<br />
One possible role of zona birefringence in<br />
predicting embryonic implantation potential has been<br />
previously discussed [50]. Shen et al. [29] reported a<br />
higher zona retardance in oocytes contributing to<br />
conception cycles when compared to oocytes in nonconception<br />
cycles. Embryonic development was also<br />
reported to be better in embryos derived from oocytes N<br />
with high zona birefringence [41,50]. Higher rates<br />
of implantation, pregnancy and live birth have also<br />
been reported when transferred embryos were derived<br />
from oocytes with high birefringence zona pellucida<br />
[37,41]. Moreover, when only embryos derived from<br />
Figure 1. Meiotic spindle in a human oocyte under polarised light<br />
microscopy.<br />
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orges Jr. <strong>2011</strong>. The Role of Veterinarians in Human In Vitro Embryo Production. SSSSSSSSSSS<br />
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oocytes with high zona birefringence were transferred,<br />
the miscarriage rate was considerably lower [37].<br />
2.3 Assessment of metabolites and proteins in the<br />
oocyte culture media<br />
In the post-genomic era, research efforts have<br />
deepened our understanding of the relationships<br />
among genomes, DNA transcripts, proteins,<br />
metabolites and the phenotypes expressed in cells<br />
and organisms. Microarray studies have provided a<br />
great deal of information regarding gene expression<br />
and the changing transcriptome. Nonetheless, gene<br />
expression is only one aspect of the complex<br />
regulatory network that allows cells to respond to<br />
intracellular and extracellular signals [24].<br />
Although transcript analysis of granulosa or<br />
cumulus cells can provide information regarding<br />
oocyte quality, oocyte or embryo transcriptomics is<br />
invasive [14,27]. This limitation has prompted the<br />
development of non-invasive adjunctive technologies<br />
for use in the study of embryonic physiology and<br />
for predicting oocyte and embryonic developmental<br />
competence and viability [27]. These approaches<br />
include the assessment of glucose, lactate, pyruvate,<br />
or amino acid levels in the embryonic culture media,<br />
evaluation of oxygen consumption, proteomic<br />
profiling, and most recently, examination of the<br />
oocyte and embryo metabolome [5,12].<br />
Proteomics alone involves several sophisticated<br />
techniques, including imaging, mass spectrometry<br />
and bioinformatics to identify, quantify and<br />
characterise a proteome. It has been suggested, that<br />
both metabolomic and proteomic tests may be strong<br />
predictor for implantation potential of human oocytes<br />
[43,53].<br />
III. EVALUATION OF HUMAN SPERMATOZOA<br />
Although ICSI outcomes are apparently not<br />
related to basic sperm parameters [32,55], recent<br />
reports have suggested a paternal contribution to<br />
successful embryonic development and implantation<br />
[31,47].<br />
Intracytoplasmic sperm injection is usually<br />
performed under an optical magnification of 400x.<br />
This magnification makes it possible to detect, in<br />
living cells, most of the sperm anomalies that are<br />
recognised in fixed and stained sperm samples in<br />
conventional basic sperm analyses. This system,<br />
however, has severe limitations, as only major<br />
morphological defects are detectable. The more minor<br />
morphologic defects related to ICSI outcome [11] are<br />
often overlooked.<br />
One new technique, “motile sperm organelle<br />
morphology examination” (MSOME), uses unstained,<br />
real-time observation of spermatozoa. It is now<br />
possible to examine the nuclear morphology of<br />
spermatozoa at a magnification of 6600x (Figure 2)<br />
using Nomarski differential interference contrast [13].<br />
Because MSOME is an unstained cytological<br />
technique, its incorporation, together with a<br />
micromanipulation system, has allowed the<br />
Figure 2. A: A human sperm under 400X magnification. B: Human sperm under 6600X magnification.<br />
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orges Jr. <strong>2011</strong>. The Role of Veterinarians in Human In Vitro Embryo Production. SSSSSSSSSSS<br />
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introduction of a modified ICSI procedure called<br />
“intracytoplasmic morphologically selected sperm<br />
injection” (IMSI). In this procedure, real-time, detailed<br />
morphological sperm examination at magnifications<br />
ranging from 6600x to 13000x [25] enables the<br />
selection of the best available spermatozoa prior to<br />
oocyte injection.<br />
A positive correlation between sperm<br />
morphological normality as determined through<br />
MSOME and the fertilisation rate has previously been<br />
demonstrated [13]. Moreover, it has been suggested<br />
that high magnification sperm morphology has a<br />
major impact on the percentage of high quality<br />
embryos [9,10,13], as well as on the implantation<br />
rate [10,13,60], pregnancy rate [8,10,13] and<br />
miscarriage rate [8,10,28]. Recently, Figueira et al.<br />
[23] demonstrated that morphological normality of<br />
the sperm nucleus also has a significant impact on<br />
the occurrence of aneuploidy in the developing<br />
embryo.<br />
In summary, these data suggest that the<br />
selection of sperm for intracytoplasmic injection<br />
based on normal nuclear morphology as seen under<br />
high magnification may be a useful tool in selecting<br />
euploid spermatozoa with higher developmental<br />
capacity.<br />
IV. CONCLUSION<br />
Ethical concerns surrounding experiments on<br />
human embryos and gametes have largely restricted<br />
studies on the molecular biology of human gametes<br />
to non-human animal experiments.<br />
Studies in non-human animal reproduction<br />
have allowed the development of non-invasive<br />
methods of evaluating sperm quality and oocyte<br />
quality prior to their use in ART.<br />
The evaluation of human gamete quality has<br />
important benefits when applied to ART. These<br />
benefits are especially important in countries with legal<br />
restrictions, where oocyte selection prior to IVF<br />
has particular importance. Moreover, the identification<br />
of gamete quality biomarkers may allow more<br />
accuracy in the selection of the best embryo for<br />
transfer with the highest implantation potential. The<br />
use of such biomarkers may also yield an increased<br />
chance of pregnancy, which is the primary objective<br />
of ART.<br />
N<br />
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27 Hamel M., Dufort I., Robert C., Gravel C., Leveille M. C., Leader A. & Sirard M. A. 2008. Identification of differentially<br />
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28 Hazout A., Dumont-Hassan M., Junca A. M., Cohen Bacrie P. & Tesarik J. 2006. High-magnification ICSI overcomes<br />
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29 Hermann B.P., Sukhwani M., Lin C.C., Sheng Y., Tomko J., Rodriguez M., Shuttleworth J.J., McFarland D., Hobbs<br />
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30 Hu Y., Betzendahl I., Cortvrindt R., Smitz J. & Eichenlaub-Ritter U. 2001. Effects of low O2 and ageing on spindles and<br />
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31 Krawetz S.A. 2005. Paternal contribution: new insights and future challenges. Nature Review Genetics. 6(8): 633-642.<br />
32 Kupker W., al-Hasani S., Schulze W., Kuhnel W., Schill T., Felberbaum R. & Diedrich K. 1995. Morphology in<br />
intracytoplasmic sperm injection: preliminary results. Journal of Assisted Reproduction and Genetics. 12(9): 620-626.<br />
33 Liu L., Oldenbourg R., Trimarchi J.R. & Keefe D.L. 2000. A reliable, noninvasive technique for spindle imaging and<br />
enucleation of mammalian oocytes. Nature Biotechnology. 18(2): 223-225.<br />
34 Lundin K., Soderlund B. & Hamberger L. 1997. The relationship between sperm morphology and rates of fertilization,<br />
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35 Lutz W. 2006. Fertility rates and future population trends: will Europe’s birth rate recover or continue to decline? <strong>International</strong><br />
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ssssssssssssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s73 - s80.<br />
36 Madaschi C., Aoki T., Braga D.P.A.F., de Cassia Savio Figueira R., Semiao Francisco L., Iaconelli A. & Borges E. 2009.<br />
Zona pellucida birefringence score and meiotic spindle visualization in relation to embryo development and ICSI outcomes.<br />
Reproductive Biomedicine Online. 18(5): 681-686.<br />
37 Madaschi C., Bonetti T.C.S., Braga D.P.A.F., Pasqualotto F. F., Iaconelli A. & Borges E.2008. Spindle imaging: a marker<br />
for embryo development and implantation. Fertility and Sterility. 90(1): 194-198<br />
38 Malhi P.S., Adams G.P. & Singh J. 2005. Bovine model for the study of reproductive aging in women: follicular, luteal, and<br />
endocrine characteristics. Biology of Reproduction. 73(1): 45-53.<br />
39 Mansour R.T., Aboulghar M.A., Serour G.I., Amin Y.M. & Ramzi A.M. 1995. The effect of sperm parameters on the<br />
outcome of intracytoplasmic sperm injection. Fertility and Sterility. 64(5): 982-986.<br />
40 Menken J., Trussell J. & Larsen U. 1986. Age and infertility. Science. 233(4771): 1389-1394.<br />
41 Montag M., Schimming T., Koster M., Zhou C., Dorn C., Rosing B., van der Ven H. & Ven der Ven K. 2008. Oocyte zona<br />
birefringence intensity is associated with embryonic implantation potential in ICSI cycles. Reproductive Biomedicine<br />
Online. 16(2): 239-244.<br />
42 Moon J.H., Hyun C.S., Lee S.W., Son W.Y., Yoon S.H. & Lim J.H. 2003. Visualization of the metaphase II meiotic spindle<br />
in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI.<br />
Human Reproduction. 18(4): 817-820.<br />
43 Nagy Z.P., Sakkas D. & Behr B. 2008. Symposium: innovative techniques in human embryo viability assessment. Noninvasive<br />
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44 Nagy Z.P., Liu J., Joris H., Verheyen G., Tournaye H., Camus M., Derde M.C., Devroey P. & Van Steirteghem A.C. 1995.<br />
The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Human Reproduction.<br />
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45 Norman R.J., Noakes M., Wu R., Davies M.J., Moran L. & Wang J.X. 2004. Improving reproductive performance in<br />
overweight/obese women with effective weight management. Human Reproduction Update. 10(3): 267-280.<br />
46 Oldenbourg R. 1996. A new view on polarization microscopy. Nature. 381(6585): 811-812.<br />
47 Ostermeier G.C., Goodrich R.J., Moldenhauer J.S., Diamond M.P. & Krawetz S.A. 2005. A suite of novel human<br />
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48 Palermo G., Joris H., Devroey P. & Van Steirteghem A.C. 1992. Pregnancies after intracytoplasmic injection of single<br />
spermatozoon into an oocyte. The Lancet. 340(8810): 17-18.<br />
49 Pelletier C., Keefe D.L. & Trimarchi J.R. 2004. Noninvasive polarized light microscopy quantitatively distinguishes the<br />
multilaminar structure of the zona pellucida of living human eggs and embryos. Fertility and Sterility. 81(Suppl 1): 850-<br />
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50 Rama Raju G.A., Prakash G.J., Krishna K.M. & Madan K. 2007. Meiotic spindle and zona pellucida characteristics as<br />
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14(2): 166-174.<br />
51 Rienzi L., Ubaldi F., Iacobelli M., Minasi M.G., Romano S. & Greco E. 2005. Meiotic spindle visualization in living<br />
human oocytes. Reproduction Biomedicine Online. 10(2): 192-198.<br />
52 <strong>SBTE</strong>. 2008. Mudanças e tendências no mercado de embriões bovinos no Brasil. O Embrião. 5-7.<br />
53 Scott R., Seli E., Miller K., Sakkas D., Scott K. & Burns D.H. 2008. Noninvasive metabolomic profiling of human embryo<br />
culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study.<br />
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54 Steptoe P.C. & Edwards R.G. 1978. Birth after the reimplantation of a human embryo. The Lancet. 2(8085): 366.<br />
55 Sukcharoen N., Sithipravej T., Promviengchai S., Chinpilas V. & Boonkasemsanti W. 1998. Sperm morphology evaluated<br />
by computer (IVOS) cannot predict the fertilization rate in vitro after intracytoplasmic sperm injection. Fertility and<br />
Sterility. 69(3): 564-568.<br />
56 Svalander P., Jakobsson A.H., Forsberg A.S., Bengtsson A.C. & Wikland M. 1996. The outcome of intracytoplasmic<br />
sperm injection is unrelated to ‘strict criteria’ sperm morphology. Human Reproduction. 11(5): 1019-1022.<br />
57 Wang W.H., Meng L., Hackett R.J., Odenbourg R. & Keefe D.L. 2001. Limited recovery of meiotic spindles in living<br />
human oocytes after cooling-rewarming observed using polarized light microscopy. Human Reproduction. 16(11): 2374-<br />
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ssssssssssssssssssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s73 - s80.<br />
58 Wang W.H., Meng L., Hackett R.J., Odenbourg R. & Keefe D.L. 2001. The spindle observation and its relationship with<br />
fertilization after intracytoplasmic sperm injection in living human oocytes. Fertility and Sterility. 75(2): 348-353.<br />
59 Wang W.H., Meng L., Hackett R.J. & Keefe D.L. 2001. Developmental ability of human oocytes with or without<br />
birefringent spindles imaged by Polscope before insemination. Human Reproduction. 16(7): 1464-1468.<br />
60 Wilding M., Di Matteo L., D’Andretti S., Montanaro N., Capobianco C. & Dale B. 2007. An oocyte score for use in<br />
assisted reproduction. Journal of Assisted Reproduction and Genetics. 24(8): 350-358.<br />
61 Wright J.M. 1981. Non-surgical embryo transfer in cattle embryo-recipient interactions. Theriogenology. 15(1): 43-56.<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
s80
M.E.F<br />
.F. Oliv<br />
liveir<br />
eira.<br />
<strong>2011</strong>. Estado da arte da superovulação em ovelhas. aaaaaa aaaaaaaaaaaa<br />
aaaaaaaaaaaa Acta Scientiae Veterinariae. 39(Supl 1): s1 - s7.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s81.<br />
ISSN 1679-9216 (Online)<br />
Vet<br />
eter<br />
erinar<br />
inary applications in regener<br />
egenerativ<br />
tive medicine; developmen<br />
elopment t of induced<br />
pluripotential stem cells (iPSC) in dogs<br />
Sehw<br />
ehwon Koh,<br />
Steve e Bischoff<br />
ischoff, Shengdar<br />
Tsai & Jor<br />
orge A. Piedr<br />
iedrahita<br />
ABSTRACT<br />
Background: Pluripotent stem cells such as embryonic stem cells can give rise to derivatives of all three germ layers and thus<br />
have great potential for clinical applications related to regenerative medicine. By analyzing totipotent or pluripotent gene<br />
expression signatures from eggs, embryonic blastomeres and embryonic stem cells, Takahashi & Yamanaka (2006) developed<br />
a method to directly rewire the circuitry of adult somatic cells to pluripotency by transfection with a range of transcription<br />
factors. Ectopic expression of four transcription factors Oct4, Klf4, Sox2 and c-Myc (OKSM) was capable of resetting the adult<br />
somatic cell to pluripotency. This approach has now been applied to a range of mammalian species including mice, humans,<br />
horses and pigs. Our interest is the application of this technology to the dog both for clinical application in veterinary<br />
medicine and as a way to understanding some of the limitations and strengths of this technology when applied to humans.<br />
Review: Here we report the derivation of iPS cells from adult canine fibroblast by retroviral OSKM transduction. The isolated<br />
canine iPS cells were expanded in three different iPS culture media (FGF2, LIF and FGF2 plus LIF) and only the cells cultured<br />
in FGF2 plus LIF showed strong AP activity and to express pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28<br />
as well as ES cells-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1). To determine the ability of the cell to differentiate<br />
into derivatives of all tree germ layers; endoderm, mesoderm and ectoderm, we utilized both embryoid body formation and<br />
directed differentiation using chemical inducers. In vitro differentiation by formation of embryoid bodies (EBs) and directed<br />
differentiation showed cell derivatives of all three germ layers as confirmed by expression for AFP, CXCR4 and SOX17 N<br />
(endoderm), desmin (DES), vimentin (VIM), MSX1 and BMP2 (mesoderm) and glial fibrillary acidic proten (GFAP), TUJ1,<br />
NCAM and bIII-tubulin (TUBB, ectoderm). In vivo differentiation was tested by development of teratomas after injection into<br />
immunodeficient mice. Results indicated that the putative canine iPS cells were capable of creating solid tumors that expressed<br />
markers for all three germ layers.<br />
Conclusion: Embryonic stem cells have tremendous potential in the area of regenerative medicine but the difficulties in their<br />
isolation, and the inability to rapidly and efficiently develop lines that can be genetically matched to the recipient, reduces<br />
their clinical usefulness. In contrast, pluripotential stem cells (iPS) developed through direct reprogramming with transcription<br />
factors allow the derivation of patient-derived stem cells. This will allow the development of cell lines that can be used in the<br />
area of regenerative medicine in both human and veterinary medicine. However, there are still some issues of stability and<br />
culture requirements that must be elucidated before this technology can be fully applied in the clinics.<br />
Keywords: induced pluripotent stem cells, stem cells, dogs, regenerative medicine.<br />
Center for Comparative Medicine and Translational Research and Department of Molecular Biomedical Sciences, College of Veterinary<br />
Medicine, North Carolina State University. CORRESPONDENCE: J.A. Piedrahita [jorge_piedrahita@ncsu.edu]. Center for Comparative<br />
Medicine and Translational Research and Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State<br />
University, 4700 Hillsborough St., ZIP CODE: 27606, Raleigh, NC, U.S.A.<br />
s81
F.F<br />
.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s83 - s95.<br />
ISSN 1679-9216 (Online)<br />
Reprogramming somatic cells: pluripotency through gene induction and<br />
nuclear transfer<br />
Fabiana Fer<br />
ernandes Bressan,<br />
Felip<br />
elipe e Per<br />
erecin,<br />
ecin, Juliano Rodr<br />
drigues Sangalli & Flávio<br />
Vieir<br />
ieira Meir<br />
eirelles<br />
elles<br />
ABSTRACT<br />
Background: The understanding of nuclear reprogramming pathways provides important contributions to applied and basic<br />
sciences such as the development of autologous cellular therapies for the treatment of numerous diseases, the improved<br />
efficiency of animal-based biotechnology or the generation of functional gametes in vitro. Strategies such as nuclear transfer<br />
and induced reprogramming have been used to induce somatic cells into an embryonic-like pluripotent state. Both techniques<br />
have been routinely performed worldwide, and live offspring have been successfully derived from them, resulting in a proof of<br />
efficacy of both techniques. Detailed studies on cellular and molecular mechanisms that mediate reprogramming, however,<br />
still require further investigation to develop practical applications in veterinary and human medicine.<br />
Review: Studies on cell reprogramming, differentiation and proliferation have revealed that a core of transcription factors, as<br />
for example, OCT4, SOX2 and NANOG, act together promoting cell commitment or pluripotency. Mechanisms of induced<br />
reprogramming by pluripotency-related transcription factors forced expression or nuclear transfer seems to be mediated by the<br />
same pathways observed in fertilization, eliciting nuclear remodeling and modulating gene expression. However, abnormal<br />
chromatin conformation, often leading to disrupted imprinting and atypical gene expression patterns are frequently observed<br />
on in vitro reprogramming. Strategies used to facilitate nuclear remodeling, such as chromatin modifying agents, as for<br />
example, histone deacetilases inhibitors or DNA methyltransferases; or chemicals responsible for the inhibition of developmentrelated<br />
pathways, as for example, MEK and GSK3 inhibitors, when used in the in vitro culture of cells or embryos, have proved<br />
N<br />
to favors transcriptional regulation and improve reprogramming. Such alternatives are highly prone to enable the routine use<br />
of in vitro reprogramming in animal production and medical sciences, for example, by promoting the generation of functional<br />
male and female functional gametes capable of producing viable offspring. Thus, the properties, deficiencies and implications<br />
of induced reprogramming and nuclear transfer techniques in somatic cells were discussed in this review, as well as its probable<br />
outcomes.<br />
Conclusions: The combination of both reprogramming techniques - induced reprogramming and nuclear transfer, may be<br />
essential to clarify the mechanisms of gene expression that are responsible for induced pluripotency. As discussed here, the<br />
mechanisms responsible for triggering the pluripotency status of a somatic cell are probably closely related to the epigenetic<br />
changes and gene expression profiles present in early development following fertilization. We report here that the nuclear<br />
transfer of SOX2 expressing donor cells resulted in similar rates of embryo production when compared to control cells. A better<br />
understanding of the contribution of each reprogramming factor used in induced reprogramming may result in the establishment<br />
of strategies aiming to enhance in vitro reprogramming performance. Such knowledge will contribute to in vitro animal<br />
production by increasing the cloning efficiency and regenerative medicine through the derivation and adequate culture of<br />
reprogrammed embryonic stem cells.<br />
Keywords: bovine, pluripotency, reprogramming, nuclear transfer.<br />
Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo (USP), Pirassununga, SP, Brazil. CORRESPONDENCE: F.F.<br />
Bressan or F.V. Meirelles [fabianabressan@usp.br - meirellf@usp.br]. Faculty of Animal Sciences and Food Engineering - FZEA, University<br />
of São Paulo (USP), CEP 13635-900 Pirassununga, SP, Brazil.<br />
s83
F.F<br />
.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
I. Introduction<br />
II. Reprogramming and pluripotency<br />
III. Reprogramming through somatic cell nuclear transfer<br />
IV. Facilitating nuclear remodeling in SCNT<br />
V. Genetically induced pluripotency – new possibilities<br />
VI. Facilitating induced pluripotency<br />
VII. When reprogramming closes the cycle – back to the<br />
gametes<br />
VIII. SCNT and iPS cells: conclusions and future challenges<br />
I. INTRODUCTION<br />
Increased efficiency and control of nuclear<br />
reprogramming is desirable for developing<br />
therapeutics to allow the derivation of autologous<br />
stem cells in cellular therapies and for animal<br />
production because increased efficiency and control<br />
are indispensible for the success of several assisted<br />
reproductive technologies (ARTs). Several studies<br />
and reviews have attempted to elucidate the molecular<br />
mechanisms involved in cellular pluripotency.<br />
However, the exact mechanism of cellular<br />
reprogramming is not completely understood.<br />
The process of reprogramming into an<br />
undifferentiated stage similar to the embryonic<br />
pluripotency has been performed through the transfer<br />
of a somatic nucleus into an oocyte (somatic cell<br />
nuclear transfer (SCNT, or cloning [126]). Another<br />
process that is capable of reprogramming a cell is<br />
cellular fusion. In this technique, one somatic cell is<br />
fused with an embryonic cell, which reprograms the<br />
somatic cell into a pluripotent stage similar to the<br />
embryonic pluripotent stage [22]. More recently, the<br />
direct reprogramming of the somatic cell has been<br />
achieved through a revolutionary experiment that was<br />
based on the introduction and expression of known<br />
transcription factors, which have been designated as<br />
pluripotency induction factors [112].<br />
II. REPROGRAMMING AND PLURIPOTENCY<br />
In mammals, early embryonic development<br />
is characterized by the induction of totipotency, which<br />
reprograms male and female nuclei into an<br />
undifferentiated state, resulting in the formation of<br />
the zygote and the initiation of embryonic<br />
development. Consecutively, the process of<br />
embryonic development leads to the differentiation<br />
of cells into two different cell lineages, the inner cell<br />
mass and the trophectoderm, which are visually<br />
distinguishable at the blastocyst stage. The trophoblast<br />
originates part of extra-embryonic tissues, whereas<br />
cells from inner cell mass, which are classified as<br />
pluripotent cells, exhibit the ability to differentiate into<br />
one of the three following germ layers: endoderm,<br />
mesoderm and ectoderm. These cells will give rise to<br />
more than 200 different cellular types that constitute<br />
an organism [85]. Because no genetic modifications<br />
are involved in the developmental determination and<br />
cellular differentiation, these events depend on<br />
epigenetic modifications to drive the expression of<br />
specific genes [5].<br />
Initial experiments on the mechanisms of cell<br />
differentiation and reprogramming have revealed that<br />
pluripotent cells express a group of genes that are<br />
responsible for the dedifferentiation or pluripotency<br />
status. OCT4 and NANOG were first identified as fundamental<br />
transcription factors for early embryonic<br />
development and the maintenance of stem cell<br />
pluripotency. SOX2, which is another transcription<br />
factor, heterodimerizes with OCT4 and regulates<br />
several genes in pluripotent cells [12]. OCT4, SOX2<br />
and NANOG are essential but not unique transcription<br />
factors to regulate the pluripotent status in<br />
embryonic cells. Aside from the triadOCT4, SOX2<br />
and NANOG, many pluripotent factors have been<br />
identified such as SALL4, DAX1, ESSRB, TBX3, TCL1,<br />
RIF1 and NAC1 [51]. A connected network of<br />
regulatory genes is responsible for the development<br />
or maintenance of the pluripotency status of embryos<br />
in a complex and probably species-dependent<br />
manner [6]. Even in primordial germ cells, several<br />
pluripotent genes, including OCT4, SOX2<br />
and NANOG, are expressed in embryonic cells. The<br />
expression of these genes is reduced with the<br />
progression of gonadal cell differentiation into<br />
gametes [2].<br />
Not only the transcription factors bind their<br />
DNA target sites, the proteins interact with each other<br />
and also with other chromatin remodeling factors and<br />
enzymes that regulate histone modifications to<br />
modulate chromatin structure and, consequently, gene<br />
expression [17,124].<br />
Transcriptional regulation is an essential<br />
mechanism for the differentiation, dedifferentiation<br />
or maintenance of pluripotency and acts on normal<br />
or induced processes of reprogramming such as those<br />
s84
F.F<br />
.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
mediated by the fertilization of the oocyte by<br />
spermatozoa, the cytoplasm in SCNT or the fusion<br />
of somatic and stem cells and in directly induced<br />
pluripotency.<br />
III. REPROGRAMMING THROUGH SOMATIC CELL<br />
NUCLEAR TRANSFER<br />
The factors that determine the ability of the<br />
oocyte cytoplasm to reprogram the somatic cell<br />
nucleus have been under investigation for a long time.<br />
The first experiment on nuclear transfer was<br />
performed over 50 years ago and demonstrated that<br />
the development of embryos or adult individuals may<br />
be obtained after the injection of cells in the<br />
embryonic stage or the injection of differentiated cells<br />
into enucleated oocytes of amphibians, respectively<br />
[14].<br />
Notably, the reprogramming ability of the<br />
oocytes is not restricted to the embryonic nucleus.<br />
The birth of Dolly, which was the first viable mammal<br />
that was produced by SCNT, has shown that the<br />
oocyte can reprogram a highly differentiated nucleus<br />
into a pluripotent state and facilitate the development<br />
of viable animals.<br />
The reprogramming of differentiated cells into<br />
pluripotent embryonic cells using SCNT has been<br />
successfully applied in several species, including<br />
humans [31]. Moreover, several farm, pet and<br />
laboratory animals have already been cloned, e.g.,<br />
bovine, caprine, ovine, swine, equine, rabbits, mice,<br />
ferrets, camels, cats, dogs and wolves [3,53].<br />
Although SCNT reprograms a differentiated<br />
nucleus, it remains an inefficient technique.<br />
Commonly, less than 5% of produced embryos<br />
generate healthy adult animals [19,125]. Several<br />
studies have demonstrated nuclear reprogramming<br />
deficiencies in cloned embryos [11,25] leading to<br />
problems such as placental dysfunctions, large<br />
offspring syndrome, and hepatic and respiratory<br />
complications [39,72].<br />
Upon the formation of pluripotent cells after<br />
fertilization, chromatin that is derived from male and<br />
female gametes is independently demethylated. The<br />
paternal genome is actively demethylated, probably<br />
through the oxidation of 5-methylcitosine followed<br />
by the substitution for non-methylated cytosine [50],<br />
whereas the maternal genome is passively<br />
demethylated during first embryonic cleavages. For<br />
normal embryonic development, these processes are<br />
followed by de novomethylation, which is essential<br />
for establishing embryonic patterns of gene<br />
expression, X chromosome inactivation in females<br />
and the maintenance of genomic imprinting, which<br />
is established in germinal lineages [83].<br />
Remodeled global patterns of chromatin<br />
methylation are designated as nuclear remodeling and<br />
are incomplete in SCNT-derived embryos. The<br />
nucleus frequently fails to reestablish an embryonic<br />
pattern of chromatin modifications, resulting in<br />
abnormal expression patterns of genes that are related<br />
to early development [7]. Cloned bovine embryos<br />
show an incomplete wave of demethylation followed<br />
by a precocious de novo methylation [128].<br />
Therefore, the reprogramming of global methylation<br />
in cloned embryos is different from in vivo-derived<br />
embryos. The methylation patterns of cloned embryos<br />
are similar to somatic cells indicating that cloned<br />
embryos undergo only a partial remodeling of the<br />
somatic genome [9]. These abnormal patterns are<br />
frequently associated with the loss of monoallelic<br />
expression of imprinted genes, thus compromising<br />
the normal development, growth and placental<br />
function of these embryos.<br />
N<br />
IV. FACILITATING NUCLEAR REMODELING IN SCNT<br />
Numerous factors play a role in SCNT<br />
efficiency, and the failure of the donor nucleus to<br />
promote epigenetic reprogramming seems to be a<br />
central cause of low SCNT efficiency [25,]. Because<br />
nuclear remodeling is not complete, strategies that<br />
facilitate the relaxation of chromatin may be useful<br />
to enable nuclear reprogramming. Several<br />
approaches that are mainly based on the effects of<br />
proteins and transcription factors on chromatin have<br />
been described. Such approaches include the potential<br />
transfer of chromosomes and chromatin by increasing<br />
the probability of cytoplasmic proteins that are freely<br />
exchanged from the oocyte to the introduced genetic<br />
material, reducing the amount of proteins from<br />
somatic origin. It is also highlighted the relaxation of<br />
chromatin chromatin-modifying agents (CMAs).<br />
CMAs such as histone deacetylases inhibitors<br />
(HDACi) and DNA methyltransferases have emerged<br />
as important tools to study nuclear reprogramming<br />
mechanisms, to correct epigenetic failures and<br />
s85
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.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
improve the production of cloned animals [48].<br />
The inhibition of histone deacetylases in<br />
nuclei donor cells or reconstructed embryos with<br />
CMAs such as trichostatin A (TSA) or Scriptaid 6-<br />
(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-<br />
hexanoic acid hydroxyamide) have been effective<br />
to increase the global acetylation of histones, which<br />
facilitates transcription. In addition, modifying DNA<br />
methylation using chemicals such as 5-aza-2βdeoxycytidine<br />
is a possible approach to facilitate<br />
reprogramming in cultured donor cells or reconstructed<br />
embryos.<br />
Numerous studies have reported the use of<br />
CMAs in cultured donor cells before SCNT or cloned<br />
embryos after activation. Some groups have reported<br />
increased blastocyst production [1,21,59], quality<br />
and in vivo development [120]. Adverse effects have<br />
been observed with high doses of CMAs.<br />
The effects of these drugs on pregnancy rates<br />
and the development to term are rare in bovine.<br />
Reasons include the extended length of gestation and<br />
the high cost of recipients, which hinder further studies<br />
that evaluate the impact of these drugs on<br />
development in bovine. To our knowledge, one<br />
unique study has described a positive effect of CMAs<br />
on the development of viable cloned calves.<br />
Similarly, it is postulated that cloning<br />
efficiency is inversely correlated to the differentiation<br />
status of the donor cell, suggesting that an<br />
undifferentiated nucleus is more likely to be<br />
remodeled and reprogrammed [36]. SCNT<br />
procedures using embryonic cells [47] or<br />
differentiated somatic cells such as lymphocytes and<br />
fibroblasts [35] have demonstrated that the use of<br />
undifferentiated cells results in higher efficiency [40],<br />
suggesting that using a compromised nucleus reduces<br />
success rates [8,47].<br />
Data from our laboratory has indicated that<br />
efficient cloning is required for the production of<br />
transgenic animals with high and homogeneous<br />
transgene expression and it is improved by a second<br />
round of nuclear transfer. These data corroborated<br />
earlier studies, and we speculated that this effect was<br />
caused by the use of a cell lineage that has previously<br />
been successfully reprogrammed by SCNT [32,57].<br />
Therefore, the selection of cell populations<br />
that are, somehow, amenable to reprogramming using<br />
the ooplasm may be important to increase the cloning<br />
efficiency [105].<br />
V. GENETICALLY INDUCED PLURIPOTENCY – NEW<br />
POSSIBILITIES<br />
In 2006, directly induced dedifferentiation<br />
was established through the incorporation of known<br />
transcription factors into the mouse and human<br />
genomes. Takahashi and Yamanaka have shown that<br />
the expression of four transcription factors, OCT3/<br />
4, SOX2, KLF4 and C-MYC (represented by OSKM),<br />
is sufficient for the induction of fibroblasts into<br />
pluripotency. Induced pluripotent stem cells (iPSCs)<br />
retain most of the characteristics of embryonic stem<br />
cells, including a high proliferating rate and the ability<br />
to form many tissues from the three germinal layers in<br />
vitro and in vivo.<br />
Hundreds of studies have been published<br />
since 2006 to confirm the consistency of induced<br />
pluripotency in mice and human somatic cells and,<br />
more recently, in rats, rabbits, dogs, pigs, non-human<br />
primates, sheep and cattle through the expression of<br />
the OSKM factors in several combinations, in addition<br />
to NANOG, LIN28 and TCL-1A, which are genes that<br />
belong to a connected network of embryonic pluripotency<br />
as previously described [38,44,45,64,86,<br />
108,130].<br />
Interestingly, iPSCs have been characterized<br />
in some species such as bovine, ovine and equine, in<br />
which “true” embryonic stem cells had not been<br />
derived. The lack of defined characteristics in these<br />
species has been reported by several studies. Because<br />
these species do not show the same characteristics<br />
that are used to classify embryonic stem cells [114],<br />
the pluripotent cells are designated “stem cell-like”.<br />
After the derivation of iPSCs, seems that the main<br />
mechanisms of pluripotency in mammals use a<br />
constitutive pathway independent of peculiarities<br />
within each species.<br />
The iPS cells are extremely promising for the<br />
study and therapeutic development for human<br />
diseases [89] because their similarities to embryonic<br />
stem cells, regarding self-renewal and the<br />
development of originating tissues from the three<br />
germ layers. However, its clinical usage is limited for<br />
several reasons, including a low reprogramming<br />
efficiency and genomic alterations arising from viral<br />
integration. The derivation of these cells must be<br />
improved to allow its therapeutic use without<br />
restrictions [65]. On the other hand, iPSCs are a<br />
unique model to elucidate the mechanisms of the<br />
genetic induction into pluripotency and<br />
s86
F.F<br />
.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
differentiation as well as cellular commitment, which<br />
is usually represented by C. H. Waddington’s<br />
developmental potential model (epigenetic landscape<br />
model; Waddington, 1957; [43]).<br />
The induction of pluripotency without<br />
transgene integration in human and mouse genomes<br />
has been obtained through the continuous transfection<br />
of non-integrating OSKM cDNA in the genome of<br />
the cell, e.g., utilizing non-integrative adenovirus<br />
[107] or expression plasmids and episomes [87].<br />
Recent strategies such as mRNA and protein<br />
approaches have also been effective [62] and<br />
represent an important step toward its use in cell<br />
therapy. Nonetheless, these strategies are less efficient<br />
when compared with lentiviral transduction.<br />
VI. FACILITATING INDUCED PLURIPOTENCY<br />
The generation efficiency of cells that are<br />
completely pluripotent after genetic induction is<br />
usually lower at 0.01% to 0.1% than that related to<br />
SCNT at less than 5% (reviewed by [43]). Microarraybased<br />
gene expression analysis has revealed that the<br />
pattern of the global gene expression of iPS cells is<br />
similar to those of embryonic stem cells (ESCs) and<br />
not those from fibroblasts. However, differences<br />
between iPSCs and ESCs are evident [112,134].<br />
SCNT has a low efficiency that is mostly<br />
credited to the failure of donor cell epigenetic<br />
reprogramming. Unsurprisingly, the epigenetic<br />
reprogramming of iPSCs is considered abnormal.<br />
Methylation abnormalities of genes such as OCT3/<br />
4 and NANOG have been reported. Studies on the<br />
imprinting of these cells have shown that, similarly<br />
to cloned embryos, a significant number of lineages<br />
exhibits an abnormal expression of imprinted genes,<br />
including H19, IGF2R, PEG3 and MEG3[93].<br />
However, similar to SCNT, the ability of these cells<br />
to generate mice to term has been demonstrated.<br />
The “partially reprogrammed” status has been<br />
reported and characterized in iPSCs. In 2009, Chan<br />
and collaborators reported that the expression of<br />
commonly used stem-cell markers as follows: alkaline<br />
phosphatase, SSEA-4, GDF3, hTERT and NANOG.<br />
These markers do not distinguish between partially<br />
and fully reprogrammed colonies. In contrast,<br />
transgene silencing, TRA-1-60, DNMT3B and REX1<br />
expression, are found in colonies that are considered<br />
“true” (completely reprogrammed colonies [16]).<br />
The treatment of these partially reprogrammed<br />
cells with signaling cascade inhibitors, e.g., inhibitors<br />
against extracellular signal-related kinase (ERK) and<br />
glycogen synthase kinase 3 (GSK3) signaling<br />
pathways, have enabled the conversion of partially<br />
reprogrammed colonies into fully reprogrammed<br />
cells [104,129].<br />
Interestingly, the utilization of inhibitors and<br />
CMAs seems to favors the derivation of iPSCs in<br />
species with ESCs that have not been reported as<br />
“true” ESCs. Two interesting examples occur in rat<br />
and bovine, in which probably the same factors that<br />
support induced pluripotency may exert favorable<br />
roles in nuclear transfer by becoming collaborators<br />
in reprogramming events or by increasing the success<br />
rates. These factors, then, could enable the routine<br />
use of inhibitors and CMAs on animal production. In<br />
rats, the derivation of “true” ESCs was not reported<br />
until 2008 [15], when the use of inhibitors against<br />
the FGF receptor, MEK (MEKi) and GSK3 pathways<br />
(GSK3i, designated 3i) or only MEKi and<br />
GSK3i (designated 2i) enabled the derivation.<br />
Therefore, the use of these and other chemicals has<br />
been tested on the derivation of iPSCs from<br />
unconventional species [79].<br />
N<br />
VII. WHEN REPROGRAMMING CLOSES THE CYCLE –<br />
BACK TO THE GAMETES<br />
Male and female gametes are required for the<br />
continuation and evolution of species and are the<br />
main resource for a vast number of reproductive<br />
biotechnologies. Therefore, they are largely studied<br />
and manipulated directly in in vitro fertilization and<br />
culture or indirectly through hormonal stimulation in<br />
infertility treatments.<br />
The specialization of the pluripotent inner cell<br />
mass occurs during cellular differentiation at postblastocyst<br />
embryonic stage to form the epiblast and<br />
the hypoblast. A part of the epiblast is comprised of<br />
the cells that are highly related to the extra-embryonic<br />
ectoderm and acquires the property of contribution<br />
to the germ line [71]. This process is regulated by<br />
factors that are secreted by extra-embryonic cells<br />
(e.g., bone morphogenetic protein or BMP) which<br />
participate in a regulatory gene pathway, which will<br />
induce the development of primordial germ cells<br />
(PGCs) that are responsible for gamete generation.<br />
Structures that are similar to male or female<br />
s87
F.F<br />
.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
gametes have been derived in vitro from human,<br />
mice and swine or from human and mouse stem cell<br />
populations, respectively (for a review, see).<br />
Embryonic stem cells, when cultured in monolayers<br />
or in suspension (hanging drops) without<br />
pluripotency-maintaining factors for days or weeks<br />
tend to spontaneously differentiate into cells that are<br />
compromised with one of the three germ layers.<br />
Amongst these structures, cells that are similar to<br />
PGCs may be observed [46,123]. PGCs have been<br />
derived from mouse ESC populations and the culture<br />
of swine fetal skin-derived cells that have been<br />
supplemented with follicular liquid, probably derived<br />
from a subpopulation that may be similar to other<br />
undifferentiated cells [26,].<br />
These ESC-derived PGCs develop into<br />
structures that are similar to oogonia, which may<br />
undergo meiosis, recruit adjacent cells to form<br />
follicle-like structures, and mediate the embryonic<br />
development into blastocyst from spontaneous<br />
parthenogenesis [27]. Similar to female gametes, male<br />
germ cells have been reported to form blastocysts<br />
and viable adult animals after intracytoplasmic<br />
injections (ICSI) [33,115]. Despite the difficulties<br />
associated with the generation of human PGCs in<br />
vitro compared with that of mouse PGCs (revised by<br />
[90]), the possibility of using these cells as a tool for<br />
male and female infertilities and to provide autologous<br />
oocytes for many embryonic biotechnological<br />
techniques such as SCNT is invaluable.<br />
The induction into germinal lineages may be<br />
a rare event, and the similarities between this process<br />
and the natural process requires further investigation.<br />
Interestingly, these cells seem to lack Igf2r and -<br />
H19 imprints, which are representative characteristics<br />
of germ cells [33]. However, similar to studies on<br />
nuclear transfer reprogramming and the direct<br />
reprogramming by transcription factors, Nayernie and<br />
collaborators in 2006 identified remodeling events<br />
in DNA methylation with disruptions that lead to an<br />
incomplete epigenetic reprogramming of male germ<br />
cells in vitro.<br />
Obtaining functional gametes in vitro may<br />
become as intriguing as the success of IVF babies 40<br />
years ago. Using epigenetic studies of induced and<br />
cloned models, the ability of iPSCs to produce<br />
functional gametes may be used for the autologous<br />
treatment of several infertilities and avoid the ethical<br />
problems that are related to embryos that are produced<br />
using SCNT.<br />
VIII. SCNT AND IPS CELLS: CONCLUSIONS AND FUTURE<br />
CHALLENGES<br />
It is known that the reprogramming events<br />
are mediated by known transcription factors, which<br />
leads to chromatin remodeling and alter gene<br />
expression to create an ESC-like status and pluripotent<br />
cells. Induced reprogramming has raised questions<br />
concerning the manner by which reprogramming<br />
factors execute alterations in gene expression. These<br />
questions apply to reprogramming events that are<br />
induced by the oocyte cytoplasm during SCNT or<br />
the cytoplasm of ESCs after cellular fusion [22].<br />
A better understanding of the contribution of<br />
each reprogramming factor and the detailed<br />
characterization of each step of reprogramming in<br />
cloned embryos and iPSCs are required for the<br />
elucidation of the molecular basis of nuclear<br />
reprogramming.<br />
The bovine model provides great benefits to<br />
analyze reprogramming. The characterization of ESClike<br />
cells in these species requires further investigation<br />
because several research groups have reported<br />
conflicting characterizations [34,119]. The production<br />
of bovine iPSCs has been recently reported. In<br />
addition, bovine species shows a reasonable rate of in<br />
vivo development after nuclear transfer, which is<br />
indicated by the thousands of animals that are<br />
produced to term. Therefore, the bovine model is<br />
useful for understand the reprogramming events that<br />
are induced by the cytoplasm or specific<br />
reprogramming factors.<br />
The strategies for induced pluripotency and<br />
nuclear transfer should be studied together unearthing<br />
the deficiencies of both techniques. If the mechanisms<br />
of nuclear reprogramming through oocytes are<br />
understood, this information will be used to augment<br />
the conversion efficiency of fibroblasts into iPSCs.<br />
Conversely, if the mechanisms of gene expression<br />
that are responsible for induced pluripotency are<br />
elucidated, these mechanisms can be applied in nuclear<br />
transfer experiments. Moreover, iPSCs may be<br />
the next logical step for cloning if the concept of facility<br />
in reprogramming cells more amenable to<br />
reprogramming remains valid.<br />
s88
F.F<br />
.F. Bressan,<br />
F. Per<br />
erecin,<br />
ecin, J.R. Sangalli & F.V<br />
.V. Meir<br />
eirelles<br />
elles. <strong>2011</strong>. Reprogramming somatic cells: pluripotency through<br />
gene induction and nuclear transfer. Acta Scientiae Veterinariae. 39(Suppl 1): s83 - s95.<br />
In fibroblast-derived iPSCs, the integration of<br />
10 to 20 copies of each gene in the cellular genome<br />
and the varying proportions of each OKSM factor<br />
have been reported [68,112]. These studies have<br />
suggested that different relative quantities of each<br />
factor are important for reprogramming. Such<br />
assumptions have been confirmed by Papapetrou<br />
and collaborators in 2009, which have reported that<br />
a precise stoichiometry between each factor is critical<br />
for the efficient reprogramming of iPSCs through a<br />
mechanism that involved the use of reporter genes<br />
for each of the for pluripotency-inducing factors. This<br />
system has enabled the detailed study of the kinetics<br />
of the four transgenes silencing during reprogramming<br />
[88].<br />
Based on such mechanisms, our group studied<br />
the effect of exogenous SOX2 expression on nuclear<br />
reprogramming trough nuclear transfer (Bressan<br />
and collaborators, unpublished data). We observed<br />
that donor cells expressing exogenous SOX2 did not<br />
differ from non-genetically modified cells in the<br />
production of cloned embryos in vitro. The effects<br />
of each pluripotency-related transcription factor in<br />
SCNT-produced embryos should reveal unique or<br />
common molecular mechanisms for both SCNT and<br />
iPS techniques.<br />
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134 Zhao R. & Daley G.Q. 2008. From fibroblasts to iPS cells: induced pluripotency by defined factors. Journal of Cellular<br />
Biochemistry. 105(4): 949-955.<br />
135 Zhao X.Y., Li W., Lv Z., Liu L., Tong M., Hai T., Hao J., Guo C.L., Ma Q.W., Wang L., Zeng F. & Zhou Q. 2009. iPS cells<br />
produce viable mice through tetraploid complementation. Nature. 461(7260): 86-90.<br />
N<br />
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enceslau<br />
eslau, J.L. Nogueir<br />
gueira,<br />
et al. <strong>2011</strong>. Células-tronco de membranas fetais para aplicações em<br />
pequenos animais. Acta Scientiae Veterinariae. 39(Suppl 1): s97 - s101.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s97 - s101.<br />
ISSN 1679-9216 (Online)<br />
Fetal membranes stem cells application in pets<br />
Carlos Eduardo Ambrósio 1 , Cristiane Valverde Wenceslau 2 , José Luiz Nogueira 4 , Dilayla Kelly de<br />
Abr<br />
breu<br />
4 , Elaine Apar<br />
parecida Fer<br />
ernandes Rodr<br />
drigues<br />
4 , Thais Bor<br />
orges Lessa<br />
4 , Daniele dos San<br />
antos Mar<br />
artins<br />
1 ,<br />
Luciana Relly Bertolini 3 & Maria Angelica Miglino 4<br />
ABSTRACT<br />
Background:Stem cells are precursor cells that have the capacity for self-renewal and could generate cells with characteristics<br />
similar to cells and differentiation, generating varied cell lines. Considering the plasticity of cells can be classified into<br />
totipotent, pluripotent or multipotent.According to the isolation period, the stem cells can be classified as embryonic, fetal and<br />
adult. In the embryo stage are considered totipotent because they can rebuild any tissue in the body and adulthood are<br />
considered multipotent, since they have a more limited plasticity. The fetal tissues and the fetus is a potential source for stem<br />
cells, since they expand more rapidly compared to the cells after birth. Stem cells of fetal membranes are derived from extraembryonic<br />
tissues with high capacity to differentiate into various tissues. The cord blood stem cells have mesenchymal and<br />
hematopoietic, and mesenchymal cells have the potential to proliferate and differentiate into multiple cell lineages. The yolk<br />
sac in dogs is morphologically composed of three layers: a single layer of endoderm, a simple mesothelium, and intermediate<br />
to them, the vascular mesenchyme. Work identified a population of pluripotent cells in the yolk sac can differentiate into<br />
hematopoietic cells, however, can be isolated mesenchymal stem cells. In this review we aim to focus new isolations of cells<br />
from umbilical cord blood and yolk sac of dogs, reviewing the main literature on this species. The importance of using dogs<br />
out of work has intensified in recent years, since many diseases can manifest itself in a similar way to humans. Additionally, the<br />
dog is a pet, and interest in the treatment of diseases and improved quality of life of this species has been accentuated in<br />
veterinary medicine. Thus, identifying the cellular sources in the dog opens new horizons for preclinical studies and new N<br />
therapies for veterinary medicine.<br />
Review: This study is related to morphological biology multipotent stem cells, focusing its expansion and use in cell therapy<br />
in animal models that have different pathologies. A widely studied model for muscular dystrophy is the GRMD (Golden<br />
Retriever Muscular Dystrophy), which is homologous to DMD (Duchenne Muscular Dystrophy) that affects humans. It is a<br />
recessive genetic disease, X chromosome which affects approximately 1 in every 3500 boys. It is characterized by a progressive<br />
muscle degeneration, resulting from the absence or reduction in the production of dystrophin protein present in the sarcoplasmic<br />
membrane of muscle fibers.<br />
Conclusion: The use of cells derived from fetal tissue are strong candidates for veterinary regenerative medicine, since they<br />
have high capacity for cellular differentiation. The use of fetuses and fetal tissues of humans still has limitations, so the dog is<br />
a viable alternative for studies of fetal stem cells. Thus, it is extremely important to know the characteristics of morphology and<br />
proliferation of cells derived from fetuses and fetal annexes canines, including yolk sac and umbilical cord as well as know the<br />
feasibility of clinical application of these cells in preclinical testing in animal models and eventually in human medicine, thus<br />
contributing to regenerative medicine.<br />
Keywords: stem cells, fetal membranes, dogs, therapy.<br />
CORRESPONDÊNCIA: C.E Ambrósio [ceambrosio@usp.br- Fone: + 55 (11) 3565-4112]. Faculdade de Zootecnia e Engenharia de Alimentos<br />
(FZEA), USP. Av. Duque de Caxias Norte n. 225, ZAB. CEP 13635-900, Pirassununga, SP, Brazil. 1 Faculdade de Zootecnia e Engenharia<br />
de Alimentos (FZEA), Universidade de São Paulo (USP)-Pirassununga, SP, Brazil. 2 Instituto Butantan, São Paulo, SP, Brazil. 3 Universidade de<br />
Fortaleza (UNIFOR), Fortaleza, CE, Brazil. 4 Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, SP, Brazil.<br />
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et al. <strong>2011</strong>. Células-tronco de membranas fetais para aplicações em<br />
pequenos animais. Acta Scientiae Veterinariae. 39(Suppl 1): s97 - s101.<br />
I. INTRODUCTION<br />
II. MATERIALS AND METHODS<br />
III. STEM CELL POTENTIAL AND THEIR APPLI-<br />
CATIONS<br />
I. INTRODUCTION<br />
Transplantation of hematopoietic stem cells<br />
derived from umbilical cord has been widely used in<br />
various types of pathologies, including metabolic<br />
disorders, acquired immunodeficiencies, and<br />
hematological disorders [1].<br />
Ontogeny, hematopoiesis begins in the ventral<br />
aorta and the fetal yolk sac, and a second time<br />
they reach the bone marrow at the end of the second<br />
trimester. It is known that stem cells from umbilical<br />
cord blood contains hematopoietic progenitor cells<br />
in large numbers [1]. Already, some authors stated<br />
that the number of nucleated cells and progenitor cells<br />
in cord blood is lower than expected [2] showed that<br />
these umbilical cord cells have a greater capacity for<br />
expansion compared to the bone marrow in a particular<br />
medium. Several researchers said that the CD34<br />
+ cells from cord blood have a greater proliferative<br />
potential and require different growth factors of those<br />
CD34+ bone marrow [1]. The number of Colony<br />
Forming Units related to granulocytes, erythrocytes,<br />
monocytes and megakaryocytes (CFU - GEMM) as<br />
well as the proliferative capacity of these cells appear<br />
to be reasonably high in cord blood of newborns<br />
compared to peripheral blood of adults [10].<br />
Studies indicate that progenitor cells derived<br />
from umbilical cord blood that express the membrane<br />
antigen CD34+ does not have the same phenotypic<br />
features of cells extracted from bone marrow of<br />
adults, and also indicate that the CD34+ cells extracted<br />
from umbilical cord blood have a high proliferative<br />
capacity [1]. But these authors also claim that little<br />
has been studied on the morphofunctional<br />
characteristics of these cells.<br />
Some authors state that the amount of these<br />
progenitor cells derived from umbilical cord blood is<br />
insufficient for a successful transplant, and suggest<br />
the holding of an expansion of these cells “in vitro”<br />
in a culture medium with specific cytokines [2]. This<br />
process aims to increase the number of hematopoietic<br />
progenitor cells, reducing the time of cell<br />
reconstitution after transplantation. Spherical clusters<br />
of cells expressing several markers of HSCs were<br />
observed inside the major arteries within the assets<br />
(AGM) and extra-embryonic (vitelline and umbilical)<br />
in mouse embryos [4,5,13] and human [7,15,16],<br />
and it is believed that stem cells represent the definitive<br />
hematopoietic lineages. Definitive HSCs are also<br />
formed in the yolk sac. Although it was concluded in<br />
recent studies that correlated the hematopoiesis of the<br />
yolk sac to the AGM that definitive HSCs formed in<br />
the yolk sac only contribute to primitive hematopoiesis<br />
[12], and these cells have a definitive hematopoietic<br />
potential.<br />
Cells isolated from the yolk sac at day 9 may<br />
be grafted, and have the ability to repopulate the<br />
recipient animals after transplantation, the liver of<br />
newborn mice [19].<br />
The correlation of morphological synthesis,<br />
uptake, transport and erythropoiesis are found in the<br />
yolk sac of the dog at the end of pregnancy and<br />
decrease very close to delivery. In the developing<br />
embryo, hematopoiesis and early vascular structure<br />
are identified as blood islands of the yolk sac. Blood<br />
islands are formed of mesoderm aggregates that have<br />
migrated in the early training [9].<br />
The outer cells are differentiated into<br />
endothelial cells and the internal, primitive blood. The<br />
termination of the association between developmental<br />
hematopoiesis and endothelial cells suggests that they<br />
depart from a common progenitor, the hemangioblasts<br />
[4] and also shown in dogs by our group [9].<br />
Research focused on the canine model and<br />
its tissues, to describe new sources of stem cells and<br />
innovative use in therapy for treating genetic and<br />
acquired diseases were evaluated extensively by our<br />
laboratory, focusing so thorough and appropriate<br />
description of new sources of stem cells canine, as<br />
well as new treatments for diseases in veterinary<br />
medicine and to extrapolate future called translational<br />
medicine [6,8,17].<br />
II. MATERIALS AND METHODS<br />
For the extraction and analysis of<br />
morphological stem cells were used 20 newborn<br />
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et al. <strong>2011</strong>. Células-tronco de membranas fetais para aplicações em<br />
pequenos animais. Acta Scientiae Veterinariae. 39(Suppl 1): s97 - s101.<br />
animals affected by muscular dystrophy or normal<br />
GRMD obtained from the kennel (Golden Retriever<br />
Muscular Dystrophy - Brazil, Department of Surgery,<br />
Faculty of Veterinary Medicine, University of São<br />
Paulo.<br />
The newborn animals are monitored, and soon<br />
after birth proceeded to collect the cord blood, by<br />
puncturing the umbilical vein. In this act, the samples<br />
were divided into two aliquots, one was placed in<br />
vacountainers 9 mL EDTA, and the second in<br />
eppendorfs, with the same anticoagulant solution for<br />
genotyping of newborn dogs and quantification of<br />
the amounts of the enzyme creatine kinase (CK).<br />
Samples collected before proceeding to the<br />
separation of “pellet” of white blood cells, blood<br />
smears were made on slides and stained for cell<br />
overview and estimate the percentage of populations<br />
of white blood cells (trypan blue). In addition, the cells<br />
were analyzed by flow cytometry to quantify apoptosis<br />
and percentage of distinct populations of progenitor<br />
cells with different surface epitopes of the CD lines.<br />
Fragments of the placenta, the yolk sac and amnion<br />
were processed for cell culture. The culture of these<br />
cell lines was performed at 37°C and 5% CO2, with<br />
renewal of medium every 48 or 72 h, and tested<br />
different culture media to obtain the best solution for<br />
development, growth, maintenance and expansion<br />
these cells. Test curve of cell growth and differentiation<br />
were performed, and morphological analysis of the<br />
general cultivation.<br />
III. STEM CELL POTENTIAL AND THEIR APPLICATIONS<br />
The results so far have been very satisfactory,<br />
showing that these cellular sources can generate<br />
important information in the field of cellular and gene<br />
therapy with mesenchymal stem cells.<br />
The immunocytochemistry was performed<br />
using the protein vimentin, a cytoplasmic protein of<br />
the cytoskeleton that supports cellular organelles, and<br />
is present in large quantities in mesenchymal cells<br />
[11,14]. Despite the positive labeling will require<br />
further experiments with other markers of stem cells,<br />
since vimentin can also be found in high amounts in<br />
tumor cells [18].<br />
The structure of the yolk sac was marked by<br />
vimentin and demarcated the potential of their blood<br />
islets (Figure 1A) and its products, in case the<br />
hemangioblasts. Further details of this cell type was<br />
described by our group [17], and its potential for<br />
tissue production of classical mesenchymal lineages<br />
(Figure 1B) or hematopoietic lineages.<br />
The growth curve of cells from amniotic<br />
membrane showed an initial drop in the number of<br />
cells, stabilizing that amount in subsequent counts.<br />
As the count is done every 72h, it is possible that<br />
this is not the appropriate time for new trypsinization<br />
of cells, many do not resist the process and die. This<br />
could explain why that number is apparently<br />
stabilized, as if there were no cell multiplication.<br />
During the daily observation of the optical<br />
microscope used for counting cells we noticed that<br />
many cells are killed in the days following N<br />
trypsinization. The placenta was difficult as canine<br />
cell culture conditions and maintains (Figure 1C),<br />
however, the canine tissues and their cell culture and<br />
this process seems to be more fragile. However, it<br />
was possible to establish this simple line amniotic<br />
membrane (Figure 1D), with unique characteristics<br />
and its potential (Figure 1E-F).<br />
The use of these cells from fetal tissue is<br />
strong candidates for new possibilities for<br />
regenerative therapy in veterinary medicine.<br />
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enceslau<br />
eslau, J.L. Nogueir<br />
gueira,<br />
et al. <strong>2011</strong>. Células-tronco de membranas fetais para aplicações em<br />
pequenos animais. Acta Scientiae Veterinariae. 39(Suppl 1): s97 - s101.<br />
Figure 1. A: Photomicrography of canine yolk sac at middle gestation. Vimentin stain showing the mesechymal progenitor cells at<br />
epithelium layer. Arrows indicate normal erythrocytes inside blood island of this tissue. B: Typical stem cells from canine yolk sac. C:<br />
Placental cell culture at term pregnancy canine placenta. D: Semi thin section of dog amnion showing simple layers of tissue. E-F:<br />
Mesenchymal stem cell from canine amnion.<br />
REFERENCES<br />
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Review Oncology Hematology. 48(1): 35-43.<br />
2 Broxmeyer H.E., Gluckman E., Auerbach A., Douglas G.W., Friedman H., Cooper S., Hangoc G., Kurtzberg J., Bard J.<br />
& Boyse E.A. 1990. Human umbilical cord blood: a clinically useful source of transplantable hematopoietic stem/<br />
progenitor cells. <strong>International</strong> Journal Cell Cloning. 8(1): 76-91.<br />
3 Bruijn M.F., Speck N.A., Peeters M.C. & Dzierzak E. 2000. Definitive hematopoietic stem cells first develop within the<br />
major arterial regions of the mouse embryo. EMBO Journal. 19: 2465-2474.<br />
4 Choi K. 2002. The hemangioblast : a common progenitor of hematopoietic and endothelial cells. Journal of Hematotherapy<br />
and Stem Cell Research. 11: 91-101.<br />
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mbrósio, C.V. Wenc<br />
enceslau<br />
eslau, J.L. Nogueir<br />
gueira,<br />
et al. <strong>2011</strong>. Células-tronco de membranas fetais para aplicações em<br />
pequenos animais. Acta Scientiae Veterinariae. 39(Suppl 1): s97 - s101.<br />
5 Garcia-Porrero J.A., Godin I.E. & Dieterlen-Liévre F. 1995. Potential intraembryonic hemogenic sites at pre-liver stages<br />
in the mouse. Anatomy and Embryology. 192: 425-435.<br />
6 Kerkis I., Ambrosio C.E., Kerkis A., Martins D.S., Zucconi E., Fonseca S.A., Cabral R.M., Maranduba C.M., Gaiad T.P.,<br />
Morini A.C., Vieira N.M., Brolio M.P., Sant’Anna O.A., Miglino M.A. & Zatz M. 2008. Early transplantation of human<br />
immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?<br />
Journal of Translational Medicine. 6(3): 1-13.<br />
7 Labastie M.C., Cortes F.P., Romeo H., Dulac C. & Peault B. 1998. Molecular precursor cells emerging in the human<br />
embryo. Blood. 92: 3624-3635.<br />
8 Martins D.S., Ambrósio C.E., Saraiva N.Z., Wenceslau C.V., Morini A.C., Kerkis I., Garcia J.M. & Miglino M.A. <strong>2011</strong>.<br />
Early development and putative primordial germ cells characterization in dogs. Reproduction in Domestical Animals.<br />
46(1): 62-66. [doi: 10.1111/j.1439-0531.2010.01631.x.].<br />
9 Miglino M.A., Ambrosio C.E., dos Santos Martins D., Wenceslau C.V., Pfarrer C. & Leiser R. 2006. The carnivore<br />
pregnancy: the development of the embryo and fetal membranes. Theriogenology. 66: 1699-702.<br />
10 Mitchell P. 1997. European researchers condemn US firm’s cord-blood-storage patent. Lancet. 349(26): 1232.<br />
11 Moon H.K., Vinckier S., Smets E., Huysmans S. 2008. Comparative pollen morphology and ultrastructure of Mentheae<br />
subtribe Nepetinae (Lamiaceae). Review of Palaeobotany and Palynology. 149: 174-198.<br />
12 Muller A.M., Medvinsky A., Strouboulis J., Grosveld F. & Dzierzak E. 1994. Development of hematopoietic stem cell<br />
activity in the mouse embryo. Immunity. 1(4): 291-301.<br />
13 North T.L. & Stacy T. 1999. Cbfa2 is required for the formation of intra-aortic hematopoietic clusters. Development. 126:<br />
2563-2575.<br />
14 Rooney N.J., Gaines S.A., Denham H.D.C., Bradshaw J.W.S. 2008. Response to a standardized psychogenic stressor as an<br />
indicator of welfare status. Journal of Veterinary Behavior. [in press].<br />
15 Tavian M., Coulobel C., Luton D., San Clemente E., Dieterlen-Lievre F. & Peault B. 1996. Aorta-associated CD34+<br />
hematopoietic cells in the early human embryo. Blood. 87: 67-72.<br />
16 Tavian M., Hallais M.F. & Peault B. 1999. Emergence of intraembryonic hematopoietic precursors in the pre-liver human<br />
embryo. Development. 126: 793-803.<br />
17 Wenceslau C.V., Miglino M.A., Martins D.S., Ambrósio C.E., Lizier N.F., Pignatari G.C. & Kerkis I. <strong>2011</strong>. Mesenchymal<br />
N<br />
progenitor cells from canine fetal tissues: yolk sac, liver and bone marrow. Tissue Engineering Part A. [doi:10.1089/tem].<br />
18 Whipple R.A., Balzer E.M., Cho E.H., Matrone M.A., Yoon J.R. & Martin S.S. 2008. Vimentin Filaments Support<br />
Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells. Cancer Research. 68(14): 5678-5688.<br />
19 Yoder M.C., Hiatt K., Dutt P., Mukherjee P., Bodine D.M. & Orlic D. 1997. Characterization of definitive<br />
lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity. 7: 335-344.<br />
www.ufrgs.br/actavet<br />
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M.E.F<br />
.F. Oliv<br />
liveir<br />
eira.<br />
<strong>2011</strong>. Estado da arte da superovulação em ovelhas. aaaaaa aaaaaaaaaaaa<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s103.<br />
ISSN 1679-9216 (Online)<br />
Reprogramming of genomic imprints by in vitro culture and cloning<br />
procedures in cattle<br />
Lawr<br />
wrenc<br />
ence e Char<br />
harles Smith<br />
1,2 , João Suzuki i Jr. 1 , Felip<br />
elipe e Per<br />
erecin<br />
ecin 2 , Flávio<br />
Vieir<br />
ieira Meir<br />
eirelles<br />
elles 2<br />
ABSTRACT<br />
Background: Procedures for cloning cattle by somatic cell nuclear transfer (SCNT) have been widely used in cattle due to<br />
commercial interests in traditional animal breeding and to produce transgenic animals for the pharmaceutical industry. Even<br />
though cloning has proven to be more efficient in cattle than other species studied, certain problems are common, such as low<br />
efficiency, pregnancy failure and gross placental structural and functional abnormalities. Most of the developmental<br />
abnormalities observed in cloned animals are related to the proliferation of the fetus and placenta, a phenomenon known as<br />
“large offspring syndrome (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, i.e.<br />
genes that are expressed in a parental-specific manner, is at the root of LOS.<br />
Review: Our recent research has focused on understanding the epigenetic alterations associated with different technologies<br />
used for assisted reproduction. We compared samples from naturally derived individuals produced by artificial insemination<br />
(AI) to samples of individuals obtained from in vitro embryo culture (IVC) and SCNT. In order to perform parental-specific<br />
analysis of genetic imprinting we identified single nucleotide polymorphisms in DNA of Bos indicus that were used for<br />
analysis of parental alleles and their respective transcripts in the tissues of hybrid individuals obtained by crossing Bos indicus<br />
and Bos taurus. To investigate the epigenetic abnormalities, we performed bisulfate sequencing to analyze the differentially<br />
methylated domains (DMD) of a set of genes that are subjected to genomic imprinting in cattle, that is to say, SNRPN, H19 and<br />
N<br />
IGF2R. Since the expression of imprinted genes varies significantly in different tissues during development, we examined<br />
embryonic and extra-embryonic membranes at different periods, i.e. preimplantation (day 17), post-implantation (day 40- 60),<br />
and after birth. For SNRPN, day 40 fetuses in the IVF group showed significantly less methylation when compared to the AI<br />
group and SNRPN expression was mostly paternal in all fetal tissues studied, except in placenta. However, the SCNT group<br />
presented severe loss of DMR methylation in both day 17 embryos and 40 fetuses and biallelic expression was observed in all<br />
stages and tissues analyzed. For H19, biallelic expression was tightly associated with a severe demethylation of the paternal<br />
H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the<br />
reduced size and low implantation rates of cloned. Preliminary analysis of the paternally imprinted IGF2R gene, indicates that,<br />
although the methylation patterns of the DMD are reduced in the SCNT day-17 vesicles, expression is consistently bi-allelic,<br />
regardless of whether the embryos were derived in vivo, in vitro or by SCNT.<br />
Conclusion: Therefore, these results indicate a generalized hypomethylation of DMD in all three imprinted genes analyzed<br />
which, with the exception of IGF2R, seems to lead to the bi-allelic expression of individuals produced by SCNT. Together,<br />
these results suggest that epigenetic marks of imprinted genes are erased during the reprogramming of somatic cell nuclei<br />
during development, indicating that such epigenetic defects may play a key role in mortality and morbidity of cloned animals.<br />
1<br />
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada, and<br />
2<br />
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, Brazil.<br />
CORRESPONDENCE: L.C. Smith [smithl@medvet.umontreal.ca – TEL: +1 (450) 7738521, ext.: 8463]. Centre de recherche en reproduction<br />
animale (CRRA), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada J2S 7C6. O.E. Smith is supported by<br />
a scholarship from the Morris Animal Foundation.<br />
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J.C. Fer<br />
erreir<br />
eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Supl 1): s105 - s111.<br />
ISSN 1679-9216 (Online)<br />
Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares<br />
Jair Camargo Ferreira 1 , Fernanda Saules Ignácio 2 & Cezinande de Meira 2<br />
ABSTRACT<br />
Background: Doppler ultrasonography is a non-invasive real time pulse-wave technique recently used for the transrectal<br />
study of the reproductive system hemodynamics in large animals. This technic is based in the Doppler Effect Principle that<br />
proposes the change in frequency of a wave for an observer (red blood cells) moving relative to the source of the respective<br />
wave (ultrasonic transducer). This method had showed to be effective and useful for the evaluation of the in vivo equine<br />
reproductive tract increasing the diagnostic, monitoring, and predictive capabilities of theriogenology in mares. However, an<br />
accurate and truthful ultrasonic exam requires the previous knowledge of the Doppler ultrasonography principles.<br />
Review: In recent years, the capabilities of ultrasound flow imaging have increased enormously. The current Doppler ultrasound<br />
machines offer three methods of evaluation that may be used simultaneously (triplex mode). In B-mode ultrasound, a linear<br />
array of transducers simultaneously scans a plane through the tissue that can be viewed as a two-dimensional gray-scale image<br />
on screen. This mode is primarily used to identify anatomically a structure for its posterior evaluation using colored ultrasound<br />
modes (Color or Spectral modes). Colored ultrasound images of flow, whether Color or Spectral modes, are essentially<br />
obtained from measurements of moving red cells. In Color mode, velocity information is presented as a color coded overlay on<br />
top of a B-mode image, while Pulsed Wave Doppler provides a measure of the changing velocity throughout the cardiac cycle<br />
and the distribution of velocities in the sample volume represented by a spectral graphic. Color images conception varies<br />
according to the Doppler Frequency that is the difference between the frequency of received echoes by moving blood red cells<br />
N<br />
and wave frequency transmitted by the transducer. To produce an adequate spectral graphic it is important determine the<br />
position and size of the simple gate. Furthermore, blood flow velocity measurement is influence by the intersection angle<br />
between ultrasonic pulses and the direction of moving blood-red cells (Doppler angle). Objectively colored ultrasound exam<br />
may be done on large arteries of the reproductive tract, as uterine and ovary arteries, or directly on the target tissue (follicle, for<br />
example). Mesovarium and mesometrium attachment arteries also can be used for spectral evaluation of the equine reproductive<br />
system. Subjectively analysis of the ovarian and uterine vascular perfusion must be done directly on the corpus luteum,<br />
follicular wall and uterus (endometrium and myometrium associated), respectively. Power-flow imaging has greater sensitivity<br />
to weak blood flow and independent of the Doppler angle, improving the evaluation of vessels with small diameters and slow<br />
blood flow.<br />
Conclusion: Doppler ultrasonography principles, methods of evaluation and reproductive system anatomy have been described.<br />
This knowledge is essential for the competent equipment acquisition and precise collection and analysis of colored ultrasound<br />
images. Otherwise, the reporting of inconsistent and not reproducible findings may result in the discredit of Doppler technology<br />
ahead of the scientific veterinary community.<br />
Keywords: Doppler ultrasound imaging, hemodynamics, blood-red cells, spectrum, color-flow, power-flow.<br />
1<br />
Programa de Pós-graduação em Medicina Veterinária, FMVZ, Departamento de Reprodução Animal e Radiologia Veterinária, Universidade<br />
Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil. 2 Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista<br />
Júlio de Mesquita Filho, Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, SP, Brasil. CORRESPONDÊNCIA: J.C.<br />
Ferreira [jaircfvet@yahoo.com.br.]. Departamento de Reprodução Animal e Radiologia Veterinária, Universidade Estadual Paulista Júlio de<br />
Mesquita Filho. CEP18608-210 Botucatu, SP, Brasil.<br />
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J.C. Fer<br />
erreir<br />
eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
I. INTRODUCTION<br />
II. COLORED ULTRASONIC IMAGING<br />
2.1 Spectral mode<br />
2.2 Color-flow mode<br />
III. ARTERIAL SYSTEM OF THE REPRODUCTIVE<br />
TRACT<br />
IV. CONCLUSION<br />
I. INTRODUCTION<br />
In large animals, Doppler ultrasonography is<br />
a non-invasive real time pulse-wave technique recently<br />
used for the transrectal study of the reproductive<br />
system hemodynamics. The introduction of this<br />
technology in current researches has allowed<br />
reevaluating previously conceptions considered<br />
definitive regarding the physiology of reproduction.<br />
This method had showed to be effective and useful<br />
for the evaluation of the in vivo equine reproductive<br />
tract increasing the diagnostic, monitoring, and<br />
predictive capabilities of theriogenology in mares [4].<br />
Color-Doppler ultrasonic imaging is based in<br />
the Doppler effect principle that proposes the change<br />
in frequency of a wave for an observer moving<br />
relative to the source of the respective wave (Figure<br />
1). The frequency is constant when the wave source<br />
and observer are stationary. However, if there is a<br />
moving toward or away from each other, the returning<br />
echoes frequency increase and decrease, respectively<br />
[7]. At the moment, this principle is used for different<br />
applications as temperature, velocity and vibration<br />
measurements. In Doppler sonography, the wave<br />
source and stationary object are, respectively, the redblood<br />
cells and the transducer.<br />
During an ultrasonic doppler exam is possible<br />
to perform a qualitatively and/or quantitatively<br />
evaluation of specific vessels and tissues. The<br />
direction and velocity of red-blood cells can be<br />
represented by different types and intensities of colors<br />
pixels or as a velocity spectral graphic. However, to<br />
select the most appropriated mode of exam first it is<br />
necessary to have a complete knowledge of the<br />
Doppler ultrasonography principles and methods of<br />
evaluation.<br />
II. COLORED ULTRASONIC IMAGING<br />
There are two specifically approaches for<br />
colored ultrasonic imaging evaluation: Color-flow and<br />
Spectral modes (Figure 2). Color-flow mode uses<br />
Color-Doppler signals superimposes on a B-mode<br />
Figure 1. Collection of images representing the Doppler Effect. Wave frequency emitted by a static<br />
object (Vsource = 0) relative to the observer is constant, concentric and equally spaced. When the pulse<br />
is emitted during relative motion between source and observer, the wave fronts are no longer concentric.<br />
Wave frequency of echoes increases and decreases, respectively, during approach and away movements<br />
(Vsource < Vsound). When the velocity of the object is similar to the sound speed (Vsource =<br />
Vsound), the crests of waves emitted overlap forming a single ridge that reaches the observer<br />
simultaneously with the source. Emitted wave reaches the observer after passing the sound source at<br />
supersonic speeds (Vsource > Vsom). Font: Personal file.<br />
Figure 2. Ultrasonic images collection of female reproductive tract in B, Power, Color and Spectral modes (A, B, C and D, respectively). (A and<br />
B) Cross section image, using B-mode and Power-flow Doppler, of a non-pregnant uterine horn in diestrus. (C) Color Doppler evaluation of<br />
a pre-ovulatory follicle. (B and C) Presence of colored pixels within uterine tissue (endometrium and myometrium) and follicular<br />
wall indicate the red blood cells displacement and have been used to estimate the tissue vascularity. (D) Spectral graph<br />
consisting of maximum speeds values of blood flow in mesometrial artery. Source: Personal file.<br />
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J.C. Fer<br />
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eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
image of a structure to estimate its vascularity, while<br />
pulsed-Doppler spectral analysis of blood velocities<br />
of a specific artery is done on Spectral mode. Both<br />
methods are based on Doppler-shift frequencies, also<br />
called Doppler frequency [7].<br />
Doppler frequency is the difference between<br />
the frequency of received echoes and frequency<br />
transmitted by the transducer (Figure 3). The<br />
transducer transmitted frequency is constant while<br />
the frequency of the returning echoes varies according<br />
to the Doppler Effect principle. Therefore, when the<br />
wave source (blood-red cells) is stationary or moving<br />
parallel to the transducer there is no difference<br />
between transmitted and returning frequencies and<br />
colored Doppler signals are not detected. If the blood<br />
flow moves toward to the transducer, the returning<br />
frequency is greater than the transmitted frequency<br />
resulting in a positive Doppler frequency. A negative<br />
Doppler signal is produced when the returning<br />
frequency is lower than the transmitted frequency or,<br />
in other words, when the blood-red cells moves away<br />
to the transducer [11].<br />
1.1 Spectral mode<br />
In the spectral mode, blood flow velocity<br />
variations are represented as a graphic wave form<br />
called spectrum (Figure 4). The spectrum provides<br />
maximum velocities values as Peak systolic (PSV),<br />
End diastolic (EDV) and Time-average maximum<br />
(TAMV) velocities. PSV is the maximum point along<br />
the length of the spectrum, while EDV is the ending<br />
point of the cardiac cycle. TAMV is the maximum<br />
velocity values average. Additionally, the position of<br />
the spectrum in relation to the baseline of the monitor<br />
indicates the blood flow direction. By convection,<br />
wave forms above and under the baseline indicate,<br />
respectively, blood-red cells moving toward and away<br />
fromthe transducer [4].<br />
To measure blood velocities is necessary to<br />
determine the Doppler angle. Doppler angle or angle<br />
of insonation is the angle of intersection of the ultrasonic<br />
pulse with the direction of moving blood-red<br />
cells (Figure 5). To produce an accurate spectrum is<br />
required a doppler angle of 30° to 60°. However,<br />
determine the angle of insonation from uterine<br />
N<br />
Figure 3. Illustration of frequency echoes changes during a colored Doppler ultrasonic<br />
exam. Doppler frequency varies according to erythrocytes movement in relation to<br />
the transducer. (A and B) Frequency of echoes sent and received is similar in cases<br />
of stacionary sources or perpendicular motion, resulting in no production of colored<br />
pixels. (C) During an approach movement, the received frequency is greater than the<br />
emitted, generating positive Doppler signals. (D) Received frequency is lower than<br />
emitted frequency when red cells move away from the transducer, producing negative<br />
Doppler signals. Fontt: Personal file.<br />
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J.C. Fer<br />
erreir<br />
eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
Figure 4. Spectral Doppler evaluation of mesometrial attachment artery. The gate (yellow)<br />
is placed on an artery of the mesometrial attachment (red) to capture velocities data of<br />
uterine blood flow and, posteriorly, produce a spectral graph. Peak systolic (PSV) and<br />
end diastolic (EDV) velocities are, respectively, the maximum and ending blood flow<br />
velocities of a cardiac cycle. TAMV is the average of an espectral maximum blood flow<br />
velocities. Doppler device automatically calculates Pulsatility and Resistance indices<br />
(RI and PI, respectively). Spectral graph located above the baseline indicates erythrocytes<br />
moving toward to the transducer. Font: Personal file.<br />
Figure 5. Cosine values of the Doppler angle (cosO) and relationship between insonation angle and accuracy of<br />
measurement of blood flow velocities. Determination of blood flow velocity varies with the Doppler frequency (DF).<br />
DF = 2 ft. v. cosθ /c. While the movement of erythrocytes in relation to the transducer is perpendicular (90 °), cosθ is<br />
zero and the device do not produce Doppler signals. Value of cosθ is close to 1 when 0 °
J.C. Fer<br />
erreir<br />
eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
consuming to collect data with precision [4]. To<br />
produce a trustful spectral graphic is crucial to place<br />
the sample gate Doppler into an artery. Usually, this<br />
may be problematic considering the vessels dimensions<br />
and the animal and visceral mobility. It is<br />
recommended to perform the spectral-doppler exam<br />
in a closed dark room with temperature control to<br />
minimize animal movement and breathless. Evaluations<br />
of animals without feeding and exercising also<br />
can be helpful for avoid excessive visceral mobility.<br />
Additionally, according to Araujo & Ginther [1] sedation<br />
of pony mares do not affect the hemodynamics<br />
of the reproductive organs and are beneficial for the<br />
evaluation of the corpus luteum and endometrium.<br />
1.2 Color-flow mode<br />
Color-flow Doppler provides an immediately<br />
qualitative evaluation of the blood flow of different<br />
organs and tissues. The vascularity of a structure is<br />
estimated subjectively considering the extension of a<br />
tissue with colored pixels during a continuous realtime<br />
exam. Different than Spectral mode, Color-flow<br />
mode is a simple, rapid and functional method of<br />
evaluation, which has been applied for farm practical<br />
purposes [4].<br />
The current Doppler equipment offer two<br />
different modes of color-flow imaging: Color-flow<br />
and Power-flow modes. The classic color-flow mode<br />
use two distinct colors, usually variations of red and<br />
blue colors, to represent the vascular blood perfusion<br />
of a structure. Also, colored pixels indicate the bloodred<br />
cells direction in relation to the transducer. By<br />
convection, red colored spots indicate blood flow<br />
moving toward to the transducer, while blue colored<br />
spots represent blood-red cells moving away from<br />
the probe. Additionally, the intensity of the colored<br />
pixels suggests the velocity of blood flow ranging<br />
from dark to bright tonalities for slower and faster<br />
velocities, respectively.<br />
Recently, Power-flow imaging has been used<br />
to evaluate uterine and ovarian vascularity of mares<br />
and cattle [2,3,6,10]. In power-Doppler mode, the<br />
blood flow movement is graduated using a single<br />
color and the color pixels intensity vary according to<br />
the power of the Doppler signals (number of bloodred<br />
cells moving at a specific velocity). Power mode<br />
Doppler has advantages over conventional color-flow<br />
imaging for the evaluation of reproductive system<br />
hemodynamics in mares. According to Ginther [4],<br />
power-flow imaging has greater sensitivity to weak<br />
blood flow and is independent of the doppler angle.<br />
Also, its color display is not affected by aliasing and<br />
reduced blooming artifacts are observed when<br />
compared to the classic color-mode Doppler (Figure<br />
6). Therefore, power mode Doppler could improve<br />
the evaluation of vessels with small diameters and<br />
slow blood flow, as are present in the uterus<br />
(mesometrium attachment, endometrium and<br />
myometrium) and ovaries (mesovarium, corpus<br />
luteum and follicular wall) of mares [2,3].<br />
III. ARTERIAL SYSTEM OF THE REPRODUCTIVE TRACT<br />
The use of color and power Doppler ultrasonography<br />
has become one of the best available<br />
Figure 6. Selection of conventional and colored (Color and Powerflow)<br />
ultrasonic images of an equine corpus luteum. Sonograms<br />
within a same line were obtained simultaneously. Day of ovulation<br />
was considered day zero (D=0) of the estrous cycle. Number and<br />
intensity of colored pixels present inside and around the CL indicate<br />
luteal vascular perfusion. At a same time, extention of luteal tissue<br />
with color pixels using color-flow mode is lower than that displayed<br />
by Power-flow evaluation, showing the higher sensitivity of the second<br />
mode to weak and slow blood flows, as those present in the<br />
reproductive tract of mares. Font: Adapted from Ginther [4].<br />
N<br />
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J.C. Fer<br />
erreir<br />
eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
and reliable techniques for the diagnoses and studies<br />
of reproductive hemodynamics [2,4]. Considering<br />
the presence of a substantial number of small vessels<br />
with slow blood flow that do not appear on a<br />
conventional color-flow image, it is suggested the<br />
use of power-flow mode for the vascular perfusion<br />
evaluation of follicles, corpus luteum and uterus [2,<br />
6, 8].<br />
The uterine vascular perfusion evaluation,<br />
using color-flow imaging, must be done considering<br />
all uterine layers [2,3]. The vascularity of the uterus<br />
is estimated subjectively using the percentage of uterine<br />
tissue with color signals Doppler during a realtime<br />
cross-sectioning exam of the uterus in a continuous<br />
span of 1 minute. Multiple cross sections must<br />
be viewed, as consequence of animal and uterine<br />
movements [9].<br />
The mesometrium attachment is an important<br />
reference point in equine transrectal Doppler<br />
ultrasound for indicating the area of entry of the blood<br />
vessels into the uterus [4]. According to Silva et al.<br />
[9], color Doppler signals within the endometrium<br />
are commonly inadequate for the production of<br />
spectral waveforms. However, early studies have<br />
reported the successfully use of mesometrial arteries<br />
as an alternative assessment for the spectral<br />
evaluation of uterine blood flow velocities of pregnant<br />
and non-pregnant mares [3,9,10].<br />
Luteal vascularity is estimated while viewing,<br />
in a slow and continuous motion, of the entire CL<br />
real-time scan [6]. Color spots at the periphery and<br />
within the CL must be included in the luteal vascular<br />
perfusion estimation. Spectral mode from CL is<br />
considered too time consuming and, usually, it is not<br />
used for objectively evaluation [5]. An alternative to<br />
validate the subjectively exam of CL is determining<br />
the number of colored pixels in a captured selected<br />
image representing the largest cross-section of the<br />
CL with distinctive colored areas, as described<br />
previously by Ginther & Utt [7].<br />
Vascular blood perfusion of follicles is<br />
estimated considering the colored Doppler signals<br />
from the vessels of the follicular wall [8]. The entire<br />
follicle must be scanned several times, in a slow and<br />
continuous motion. However, the complete real-time<br />
exam of preovulatory follicles is, commonly, unable<br />
due to their dimensions. In these cases, the operator<br />
must scan parts of the follicle and use their average<br />
to estimate the vascularity. Similar to the reported for<br />
CL, the spectral mode is not recommended for<br />
objectively evaluation of follicular hemodynamics<br />
been suggested the validation of the subjectively<br />
exam using the colored pixels count.<br />
IV. CONCLUSION<br />
The advent of the Doppler ultrasonography<br />
had enabled to re-evaluate reproductive physiologic<br />
conceptions before considering them absolutes.<br />
However, the complete knowledge of Doppler<br />
principles, methods of evaluation and arterial<br />
anatomy of the reproductive system are essential to<br />
execute an exam with accuracy and excellence.<br />
Special care must be taken in order to enhance studies<br />
in this area. Otherwise, inconsistent and not<br />
reproducible findings may result in the discredit of<br />
Doppler technology ahead of the society and scientific<br />
veterinary community.<br />
REFERENCES<br />
1 Araujo R.R. & Ginther O.J. 2009. Vascular perfusion of reproductive organs in pony mares and heifers during sedation with<br />
detomidine or xylazine. American Journal of Veterinary Research. 70(1): 141-148.<br />
2 Ferreira J.C., Gastal E.L. & Ginther O.J. 2008. Uterine blood flow and perfusion in mares with uterine cysts: effect of the<br />
size of the cystic area and age. Reproduction. 135(4): 541-50.<br />
3 Ferreira J.C., Ignácio F.S. & Meira C. 2010. Uterine vascular perfusion and spectral-Doppler measurements during early<br />
gestation in mares: New concepts of evaluation. Animal Reproduction Science. 121(S): 281-283.<br />
4 Ginther O.J. 2007. Ultrasonic Imaging and Animal Reproduction: Color-Doppler Ultrasonography, Ginther O.J. (ed). Cross<br />
Plains: Equiservices Publishing, 258p.<br />
5 Ginther O.J., Gastal E.L., Gastal M.O., Utt M.D. & Beg M.A. 2007. Luteal blood flow and progesterone production in<br />
mares. Animal Reproduction Science. 99(1-2): 213-220.<br />
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J.C. Fer<br />
erreir<br />
eira,<br />
F.S.<br />
Ignácio & C Meir<br />
eira.<br />
<strong>2011</strong>. Doppler ultrasonography principles and methods of evaluation of the<br />
reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111.<br />
6 Ginther O.J., Rodrigues B.L., Ferreira J.C., Araujo R.R. & Beg M.A. 2008. Characterization of pulses of 13,14-dihydro-<br />
15-keto-PGF2alpha (PGFM) and relationships between PGFM pulses and luteal blood flow before, during, and after<br />
luteolysis in mares. Reproduction, Fertility and Development. 20(6): 684-693.<br />
7 Ginther O.J. & Utt M.D. 2004. Doppler Ultrasound in Equine Reproduction: Principles, Techniques, and Potential. Journal<br />
of Equine Veterinary Science. 24: 516-526.<br />
8 Siddiqui M.A., Ferreira J.C., Gastal E.L., Beg, M.A., Cooper D.A. & Ginther O.J. 2010. Temporal relationships of the LH<br />
surge and ovulation to echotexture and power Doppler signals of blood flow in the wall of the preovulatory follicle in<br />
heifers. Reproduction, Fertility and Development. 22 (7): 1110-1117.<br />
9 Silva L.A., Gastal E.L., Beg M.A. & Ginther O.J. 2005. Changes in vascular perfusion of the endometrium in association<br />
with changes in location of the embryonic vesicle in mares. Biology of Reproduction. 72(3): 755-761.<br />
10 Silva L.A., Ginther O.J. 2006. An early endometrial vascular indicator of completed orientation of the embryo and the role<br />
of dorsal endometrial encroachment in mares. Biology of Reproduction. 74(2): 337-343.<br />
11 Zagzebski J.A. 2005. Physics and instrumentation in Doppler and B-Mode ultrasonography. Philadelphia: Elsiever<br />
Saunders, 752p.<br />
N<br />
www.ufrgs.br/actavet<br />
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R.C. Uliani, L.A. Silv<br />
ilva,<br />
M.A. Alv<br />
lvar<br />
arenga.<br />
<strong>2011</strong>. Mare’s Folliculogenesis: Assessment of ovarian and perifollicular<br />
vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s113 - s116.<br />
ISSN 1679-9216 (Online)<br />
Mar<br />
are’s s Folliculo<br />
olliculogenesis:<br />
Assessmen<br />
ssessment t of ovar<br />
arian and per<br />
erif<br />
ifollicular<br />
vascular per<br />
erfusion by Doppler ultrasound<br />
Renata Cristina Uliani 1 , Luciano Andrade Silva 2 & Marco Antonio Alvarenga 1<br />
ABSTRACT<br />
Background: Ultrasound Doppler is a new technology that has recently been used in large animal reproduction. As the<br />
conventional ultrasound (B-mode) it is a noninvasive technique, but with the advantage of allows the assessment of the<br />
hemodynamic of reproductive tract in real time. The observation of important features of the vascularization and changes that<br />
occur during physiological processes that were not previously seen on B-mode encourage a reassessment of the concepts<br />
already established on the events of the reproductive physiology of animals and their applications.<br />
Review: In attempt to re-understand the equine reproductive physiology and finding practical uses to this new technique,<br />
authors showed that, during the follicular deviation, features are observed by Doppler before being observed under B-mode<br />
ultrasound like changes in the speed of the blood flow two days before deviation of the follicle size and one day before the<br />
increase in blood flow area of the follicular wall. According to another study ovulation is characterized by a decreased blood<br />
flow of the follicular wall in the last four hours preceding it, as well as the serration of the granulosa layer and formation of a<br />
non vascularized apex, but in our ongoing study, the ability to decrease the vascularity was not found. Very vascularized<br />
follicles are associated with higher rates of oocyte maturation and pregnant that does less vascularized follicles in the<br />
preovulatory phase. Those follicles that have septated evacuation (or prolonged) showed more vascularization and serration<br />
of the granulosa one hour before ovulation than follicles that ovulate normally, and this vascularization includes the apex of<br />
the follicle, the follicular wall portion that is not vascularized in normal ovulation. Another study reported that hemorrhagic<br />
N<br />
follicles have better vascularization of the follicular wall on the days preceding ovulation than follicles destined to ovulate.<br />
Some authors also showed that anovulatory follicles grow in size at the same rate as ovulatory follicles, but the percentage of<br />
vascularization of its wall is much smaller at 35 mm. Another study characterized that the vascular wall of the follicle that<br />
results in the first ovulation of the year is much smaller on the day before ovulation than the number of vessels present in a<br />
follicle that will ovulate in the middle of the breeding season. In these cases, the use of Doppler ultrasound can help to prevent<br />
economic losses as insemination of mares in cycles that are not able of resulting in pregnancy. This review aims to gather the<br />
information found in the literature about the characteristics of follicular hemodynamic of mares taking into account moments<br />
of deviation in follicle size, ovulation, ovulation failure and follicular viability.<br />
Conclusion: The Doppler technology has the potential to provide important information about the follicular environment and<br />
thus be used in practice in search of the perfect equine reproductive management, achieving better utilization of genetic<br />
material and increasing the financial return. The use of this new tool opens a large area for several interesting studies that will<br />
contribute to the knowledge of the physiological events of the mare for that this technique can soon be effectively applied.<br />
Keywords: mare, follicle, doppler, blood flow, follicular wall, ovulation.<br />
1<br />
Departamento de Reprodução Animal. Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista (UNESP) Botucatu, SP,<br />
Brazil. 2 Curso de Medicina Veterinária FZEA, Universidade de São Paulo (USP), Pirassununga, SP, Brazil. CORRESPONDENCE: R.C. Uliani<br />
[renatauliani@fmvz.unesp.br – PHONE: +55 (14) 3811-6249]. Distrito de Rubião Jr. s/n. CEP 18618-970 Botucatu, SP, Brazil.<br />
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R.C. Uliani, L.A. Silv<br />
ilva,<br />
M.A. Alv<br />
lvar<br />
arenga.<br />
<strong>2011</strong>. Mare’s Folliculogenesis: Assessment of ovarian and perifollicular<br />
vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
I. INTRODUCTION<br />
II. DOPPLER U.S. TO STUDY DEVIATION AND<br />
FOLLICULAR DOMINANCE<br />
III. DOPPLER US TO DETECT THE TIME OF OVU-<br />
LATION<br />
IV. CHARACTERISTICS OF FOLLICULAR DYNAMICS<br />
OBSERVED BY DOPPLER U.S. IN THE SPRING<br />
TRANSITION<br />
V. CONCLUSION<br />
I. INTRODUCTION<br />
Few, however exciting, are studies in the<br />
literature with use of Doppler in equine reproduction,<br />
this has been used for studying vascularization of<br />
the preovulatory follicle [5,13], predict oocyte<br />
maturation [13], follicular and oocyte quality check<br />
[14] and establishment of a pregnancy [15] and more.<br />
The characteristics of the ovarian antral<br />
follicles of non pregnant mares have relation with<br />
changing in their reproductive stages. Studies<br />
involving the hypothalamus, pineal gland, and<br />
concentrations of circulating hormones have been<br />
given productive consideration for many years.<br />
Recently, several productive ultrasound adaptations<br />
have been made to better consider the follicles as<br />
targets or end points in seasonality studies. These<br />
innovations include: 1) transvaginal ultrasoundguided<br />
ablation of follicles appropriate to a given<br />
hypothesis, 2) transvaginal sampling and experimental<br />
treatment of targeted follicles, and 3) color Doppler<br />
ultrasound for assessing the changing vascularity of<br />
the follicle wall. These advances in technology have<br />
produced results that encourage a review of the current<br />
status of follicle dynamics and seasonality [9].<br />
II. DOPPLER U.S. TO STUDY DEVIATION AND<br />
FOLLICULAR DOMINANCE<br />
Deviation is preceded by a common growth<br />
phase of several days. During this phase, the follicles<br />
grow at an approximately similar rate and each follicle<br />
has the capacity for future dominance [4]. During<br />
follicle growth, an extensive vascular plexus develops<br />
in the thecal layer surrounding the avascular basement<br />
membrane and granulosa layer. It has been suggested<br />
that the preferential delivery of gonadotropins and<br />
nutrients via a more highly developed vascular system<br />
in individual follicles plays a role in the selection and<br />
growth of the dominant follicle and that insufficient<br />
vascular support contributes to follicle atresia [16].<br />
In this regard deviation in mares is indicated<br />
morphologically not only by differential growth rate<br />
between the developing dominant and subordinate<br />
follicles, but also by an apparent expansion of the<br />
anechoic ultrasonic layer, as expressed subjectively,<br />
surrounding the granulosa of the dominant follicle<br />
[4]. This echotexture change distinguished the future<br />
dominant follicle from the future largest subordinate<br />
follicle about one day earlier than the beginning of<br />
diameter deviation and was attributed to increased<br />
vascularization. In another study, Acosta et al. [2]<br />
observed that even before the change in blood flow<br />
area can be viewed in color mode, the spectral analysis<br />
showed that the peak systolic velocity and timeaverage<br />
maximum velocity of blood flow begins to<br />
be higher in the dominant follicle in 6mm average<br />
(equivalent to 2 days) before deviation in follicle<br />
diameter. Although the concentrations of vascular<br />
endothelial growth factor in follicular fluid were higher<br />
in the largest follicle one day after the expected<br />
beginning of deviation [10], the original ratio of<br />
VEGF with the deviation is not known.<br />
III. DOPPLER US TO DETECT THE TIME OF OVULATION<br />
Currently, the imminence of ovulation in<br />
mares is estimated on B-mode ultrasound by the<br />
combination of thickness and echogenicity of the<br />
granulosa cell layer, decreased turgidity, loss of<br />
spherical shape of the follicle, granulosa detaching<br />
and echogenic spots in antrum, associated with<br />
decreased uterine edema, and can predict ovulation<br />
24 h before [3]. Likewise, an anechoic band seen on<br />
B-mode gradually increases in the theca layer during<br />
the days preceding ovulation representing the blood<br />
flow of the follicle [5].<br />
In a recent study, Gastal et al. [5] observed in<br />
ultrasound Doppler that in the last 4 h before ovulation<br />
occurs a decrease in the percentage of follicle<br />
circumference with color display and a decrease in<br />
the intensity of colors. The two surfaces of the<br />
granulosa (facing the antrum and the theca interna)<br />
become irregular during the last few hours prior to<br />
ovulation [6]. This phenomenon has been termed<br />
serration of the granulosa. Serration seemed most<br />
prominent at the base of the follicle opposite to the<br />
apex (future ovulation site). In this study, the authors<br />
noted also a formation of a not vascularized apical<br />
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M.A. Alv<br />
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arenga.<br />
<strong>2011</strong>. Mare’s Folliculogenesis: Assessment of ovarian and perifollicular<br />
vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
area recognized by a alteration on the formerly<br />
spherical follicle a few hours before ovulation,<br />
indicating the future rupture point. However, in<br />
ongoing study in our laboratory (Uliani et al,<br />
unpublished data) there was no decrease in the<br />
amount of vascularization at the time immediately<br />
preceding ovulation.<br />
In mares, increased blood flow and increase<br />
in diameter of the preovulatory follicle between the<br />
time of ovulation induction and 30 h after HCG<br />
injection was associated with higher rates of<br />
pregnancy [15]. Moreover, signs of more intense<br />
intraovarians signals of color indicated a decrease in<br />
rates of resistance (increased vascular perfusion) and<br />
increase in blood velocity in mares that became<br />
pregnant [15]. However the author showed the<br />
necessity to use more number of animals to confirm<br />
these findings. Ginther et al. [13] conducted a study<br />
in which follicles were aspirated 30 h after treatment<br />
with hCG and oocyte maturation rate was assessed.<br />
They observed that blood flow was better for the group<br />
of oocytes not recovered than for recovered oocytes<br />
and better for the group of mature oocyte than for<br />
immature oocyte. Spectral analysis revealed no<br />
differences between groups. These results are<br />
inconsistent with previous studies and therefore were<br />
considered inconclusive. In another study, Siddiqui<br />
et al. [14] evaluated blood flow in the follicular wall<br />
after treatment with hCG of mares whom had<br />
antibodies against hCG and mares that did not have<br />
antibodies. Follicles were aspirated 30 h after and<br />
were not differences between groups regarding the<br />
recovery rate, but showed significantly less blood flow<br />
in the follicular wall of mares antibody-negative than<br />
antibody-positive mares. Spectral analysis showed no<br />
differences between groups.<br />
Disturbances in ovulation may be submitted<br />
in the form of septed evacuations with prolonged<br />
discharge and no ovulation with the subsequent<br />
formation of a hemorrhagic anovulatory follicle<br />
(HAF). Septated evacuations are associated with<br />
better serration and vascularization of the follicular<br />
wall one hour prior to ovulation when compared to<br />
normal evacuation, also including the apical area [12].<br />
Serration and vascularization in mares with normal<br />
ovulation were not found in the apical pole. If the<br />
oocyte was included in the extended discharge is<br />
unknown [12]. Studying the HAFs, Ginter et al. [11]<br />
noted that follicular diameter was not different on the<br />
day before ovulation between the follicles that<br />
ovulated and those that formed one HAF. The results<br />
also indicated that high concentrations of systemic<br />
estradiol few days before the expected ovulation and<br />
better vascularization of the follicle on the day before<br />
ovulation are involved in the conversion of a viable<br />
preovulatory follicle into a HAF, but results are<br />
inconsistent because assessments were conducted<br />
with long intervals of 24 hours.<br />
IV. CHARACTERISTICS OF FOLLICULAR DYNAMICS<br />
OBSERVED BY DOPPLER U.S. IN THE SPRING<br />
TRANSITION<br />
The transition between the anovulatory and<br />
ovulatory seasons occurs in the spring and often is<br />
characterized by the formation of one or several<br />
anovulatory dominant follicles until a dominant<br />
ovulatory follicle terminates the anovulatory season<br />
[1]. The characteristics of the deviation are similar<br />
between both the phases. During the growth of<br />
follicles from 20 to 30 mm, the anovulatory follicles<br />
expanded at the same rate as the ovulatory follicles,<br />
being size of the dominant follicles unable to identify<br />
their health. The color Doppler ultrasound technology<br />
distinguished between future anovulatory and N<br />
ovulatory dominant follicles with reasonable accuracy.<br />
On the day that the follicle reached 35mm, vascularized<br />
area was 0.48 to 0.81 cm 2 in the ovulatory<br />
group and from 0.12 to 0.28 cm 2 in anovulatory<br />
group [1].<br />
The diameter of the preovulatory follicle on<br />
the day before ovulation is about 5 mm greater before<br />
the first than before the second ovulation of the year<br />
[8]. However, in one study, Gastal et al. [7] observed<br />
a much reduced rate of increase in vascularity<br />
preceding the first ovulation (day 0) of the year, The<br />
vascularized area (cm 2 ) of the follicle wall was similar<br />
between the preovulatory groups on Day -6, but<br />
the area was much smaller on Day -1 preceding the<br />
first ovulation, this characteristic may be used to<br />
identify the health of the follicle and thus prevent<br />
economic losses.<br />
V. CONCLUSION<br />
Doppler technology is a new tool for<br />
assessment of follicular characteristics and better<br />
understand the physiological changes of the estrous<br />
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arenga.<br />
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vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
cycle. However a wider range of studies is needed<br />
looking for the real applications of this technique<br />
aiming its use by veterinarians to increase fertility<br />
and improve the efficiency of biotechnologies such<br />
as artificial insemination.<br />
REFERENCES<br />
1 Acosta T.J., Beg M.A. & Ginther O.J. 2004. Aberrant Blood Flow Area and Plasma Gonadotropin Concentrations During the<br />
Development of Dominant-Sized Transitional Anovulatory Follicles in Mares. Biology of Reproduction. 71: 637-642.<br />
2 Acosta T.J., Gastal E.L., Gastal M.O., Beg M.A. & Ginther O.J. 2004. Differential Blood Flow Changes Between the Future<br />
Dominant and Subordinate Follicles Precede Diameter Changes During Follicle Selection in Mares. Biology of Reproduction.<br />
71: 502-507.<br />
3 Carnevale E.M., McKinnon A.O., Squires E.L. & Voss J.L. 1988. Ultrasonographic characteristics of the preovulatory<br />
follicle preceding and during ovulation in mares. Journal of Equine Veterinary Science. 8: 428-431.<br />
4 Gastal E.L., Gastal M.O., Beg M.A. & Ginther O.J. 2004. Interrelationships among follicles during the common-growth<br />
phase of a follicular wave and capacity of individual follicles for dominance in mares. Reproduction. 128: 417-422.<br />
5 Gastal E.L., Gastal M.O. & Ginther O.J. 2006. Relationships of changes in B-mode echotexture and colour-Doppler<br />
signals in the wall of the preovulatory follicle to changes in systemic oestradiol concentrations and the effects of human<br />
chorionic gonadotrophin in mares. Reproduction. 131: 699-709.<br />
6 Gastal E.L., Gastal M.O. & Ginther O.J. 2006. Serrated granulosa and other discrete ultrasound indicators of impending<br />
ovulation in mares. Journal of Equine Veterinary Science. 26: 67-73.<br />
7 Gastal E.L., Gastal M.O., Donadeu F.X., Acosta T.J., Beg M.A. & Ginther O.J. 2007. Temporal relationships among LH,<br />
estradiol, and follicle vascularization preceding the first compared with later ovulations during the year in mares. Animal<br />
Reproduction. 102: 314-321.<br />
8 Ginther O.J. 1990. Folliculogenesis during the transitional period and early ovulatory season in mares. Journal of Equine<br />
Veterinary Science. 90: 311-320.<br />
9 Ginther O.J., Gastal E.L., Gastal M.O. & Beg M.A. 2004. Seasonal influence on equine follicle dynamics. Anim. Reprod.<br />
1(1): 31-44.<br />
10 Ginther O.J., Gastal E.L., Gastal M.O., Checura, C.M. & Beg M.A. 2004. Dose response study of intrafollicular injection<br />
of insulin-like growth factor-1 on follicular-fluid factors and follicle dominance in mares. Biology of Reproduction. 70:<br />
1063-1069.<br />
11 Ginther O.J., Gastal E.L., Gastal M.O. & Beg M.A. 2006. Conversion of a viable preovulatory follicle into a hemorrhagic<br />
anovulatory follicle in mares. Animal Reproduction. 3(1): 29-40.<br />
12 Ginther O.J., Gastal E.L. & Gastal M.O. 2007. Spatial Relationships between Serrated Granulosa and Vascularity of the<br />
Preovulatory Follicle and Developing Corpus Luteum. Journal of Equine Veterinary Science. 27(1): 20-27.<br />
13 Ginther O.J., Gastal E.L., Gastal M.O., Siddiqui, M.A.R. & Beg M.A. 2007. Relationships of Follicle Versus Oocyte<br />
Maturity to Ultrasound Morphology, Blood Flow, and Hormone Concentrations of the Preovulatory Follicle in Mare.<br />
Biology of Reproduction. 77: 202-208.<br />
14 Siddiqui, M.A.R., Gastal E.L., Gastal M.O., Beg M.A. & Ginther O.J. 2008. Effect of HCG in the presence of HCG<br />
antibodies on the follicle, hormone concentrations, and the oocyte in mares. Reproduction in Domestic Animals. 44(3):<br />
474-479.<br />
15 Silva, L.A., Gastal E.L., Gastal M.O., Beg M.A & Ginther O.J. 2006. Relationship between vascularity of the preovulatory<br />
follicle and establishment of pregnancy in mares. Animmal Reproduction. 3: 339-346.<br />
16 Wulff C., Wilson H., Wiegand S.J., Rudge J.S. & Fraser H.M. 2002. Prevention of thecal angiogenesis, antral follicular<br />
growth, and ovulation in the primate by treatment with vascular endothelial growth factor Trap R1R2. Endocrinology.<br />
143: 2797-2807.<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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Ignácio, J.C. Fer<br />
erreir<br />
eira & C. Meir<br />
eira.<br />
<strong>2011</strong>. Uterine and Luteal hemodynamic evaluation of the non pregnant mare.<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s117 - s121.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s117 - s121.<br />
ISSN 1679-9216 (Online)<br />
Uterine and Luteal hemodynamic evaluation of the non pregnant mare<br />
Fernanda Saules Ignácio, Jair Camargo Ferreira & Cezinande de Meira<br />
ABSTRACT<br />
Background: Studies with Doppler ultrassonography started at the end of the 90s for the determination of physiological and<br />
pathological alterations in the reproductive tract of the mare. Uterine alterations caused by inflammation, response from<br />
seminal plasma infusion, hormonal variations during estrous and diestrus, pregnancy and action of various vasoactive factors<br />
influence on the vascular perfusion detected by Doppler ultrasound. The development of efficient methods for uterine quality<br />
evaluation is of big importance for field equine reproduction veterinarians, once uterine environment is responsible for<br />
pregnancy maintenance.<br />
Review: Nowadays, the most used methods of uterine evaluation are the mode B ultrassonography, cytology, culture and<br />
biopsy. Hemodynamic evaluation of the uterus can be done by spectral data collected from large vessels, as A. uterine and its<br />
ramifications, or from subjective or objective evaluations from endometrium, miometrium and mesometrium attachment,<br />
which provide data referent to local and specific alterations of the evaluated area. Alterations in uterine vascular perfusion has<br />
been detected during estrous cycle, during pregnancy and in cases of infusion of inflammatory substances. These alterations<br />
happen because of vasoactive substances that act in the uterus during these events, however, most of these vasoactive<br />
substances are probably not even known. Also, important hemodynamic alterations in old mares, as an increase in vascular<br />
resistance, have been described. This increase might result from fibrosis of the uterus and in women it is considered to be a<br />
cause of infertility. In mares, periglandular fibrosis of the endometrium is considered to be the major diagnosable cause of<br />
embryonic and fetal loss in older mares. For the CL, ovarian artery of the mare supplies the ovary as well as the oviduct and<br />
therefore can be used for evaluation of these areas. The CL evaluation can also be done by the percentage of luteum area with N<br />
colored signals as an indicator of the extent of blood flow. The percentage of the CL area with colored signals is determined<br />
subjectively by images observations in real time and/or by a freezing Power Doppler cross-section image with the maximum<br />
number of color pixels taped and the total number of color pixels is assessed by a computer analysis system. Therefore, a high<br />
correlation between plasmatic progesterone and CL vascularization also allows the CL evalution by this technique. In a first<br />
report, CL circulation reached its maximum on D5, the progesterone concentration in peripheral blood increased until D7 and<br />
in a posterior report, maximum perfusion was achieved two days after the maximum progesterone concentration (D8). Blood<br />
flow reduced between D10 - D14 some days before the plasma progesterone decrease and, during the luteolytic period (D15 -<br />
D17), the decline in CL blood-flow area was greater than blood flow decrease.<br />
Conclusion: Doppler ultrassonography add knowledge about uterine viability and CL functionality can be easily used by<br />
veterinarians in the field. It is a noninvasive method that provides real time results. However, because of the short time studies<br />
in this area have been done, many other answers still need to be found until normal and pathological patterns will be<br />
established.<br />
Keywords: Doppler ultrassonography, vascular perfusion, uterine hemodynamic, CL hemodynamic, mare, reproduction.<br />
Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, UNESP, Botucatu, SP, Brazil.<br />
CORRESPONDENCE: F.S. Ignácio [nandasaules@gmail.com - TEL: + 55 (14) 38116249]. Faculdade de Medicina e Veterinária, UNESP.<br />
CEP 18610-000 Bocatu, SP, Brazil<br />
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sss Acta Scientiae Veterinariae. 39(Suppl 1): s117 - s121.<br />
I. INTRODUCTION<br />
II. LITERATURE REVIEW<br />
2.1 Uterine evaluation<br />
2.2 Corpus luteum (CL) evaluation<br />
III. FINAL CONSIDERATIONS<br />
I. INTRODUCTION<br />
In the end of the 90s, studies utilizing Doppler<br />
ultrassonography started to be developed aiming to<br />
determine physiologic and pathologic alterations in<br />
the reproductive tract of the mare.<br />
Uterine, follicle and corpus luteum vascular<br />
perfusion is the focus of the most studies. The objective<br />
of this review is to show the Doppler findings<br />
so far detected in the uterus and in the corpus luteum<br />
of non pregnant mares.<br />
II. LITERATURE REVIEW<br />
2.1 Uterine evaluation<br />
The development of efficient methods for<br />
uterine quality evaluation is of big importance for<br />
field equine reproduction veterinarians, once uterine<br />
environment is responsible for pregnancy<br />
maintenance. Nowadays, the most used methods of<br />
uterine evaluation are the mode B ultrassonography,<br />
cytology, culture and biopsy.<br />
Doppler ultrassonography is a noninvasive<br />
method that brings information about vascular perfusion<br />
in real time. By this reason, it is noticed an<br />
increasing interest on the development of techniques<br />
applied to field for selection and detection of important<br />
uterine alterations about mare fertility.<br />
Hemodynamic evaluation of the uterus can<br />
be done by spectral data collected from large vessels,<br />
as A. uterine and its ramifications [4], or from<br />
subjective or objective evaluations from endometrium,<br />
miometrium and mesometrium attachment, which<br />
provide data referent to local and specific alterations<br />
of the evaluated area [16].<br />
In 1998 [4], a study aiming to develop a<br />
technique for measurement of the blood flow from<br />
spectral data (resistence index) from A. uterine in<br />
mares was done. The authors also evaluated changes<br />
in blood flow between mares, during the estrous cycle<br />
and between subsequent cycles.<br />
The observation that left-right differences in<br />
RI were not related to the side of ovulation [4]. Later<br />
studies confirmed these findings, there is no<br />
difference of perfusion between large uterine arteries<br />
[3] neither between vessels of the mesometrium<br />
attachment [16] of the horns ipsi and contralateral to<br />
ovulation (Figure 1). However, differences in uterine<br />
blood flow are seen during the estrous cycle [3,4].<br />
Not inseminated mares showed increased vascular<br />
perfusion [reduction in the resistence index (RI)] at<br />
D5 (D0 = ovulation) that was related to the importance<br />
of a high blood supply at the time of entry of embryo<br />
into the uterus. Around two or three days before<br />
ovulation and at D0, an increase in perfusion (redu-<br />
Figure 1. Values of uterine vascular perfusion from real time subjective evaluation graduated from 1 to 4 by<br />
Power Doppler mode of the left (CE) and right (CD) horns during estrous cycle days in mares used as embryo<br />
recipients.<br />
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sss Acta Scientiae Veterinariae. 39(Suppl 1): s117 - s121.<br />
ction on RI) followed by reduction (increase on RI)<br />
until ovulation were detected, probably because of<br />
the increase and decrease in estrogen (vasodilatory<br />
effect on uterine vessels) during these days. A<br />
perfusion reduction were also detected at D15 (end<br />
of diestrus) not related to estrogen levels but by other<br />
vasoative compounds that might be acting [4].<br />
Therefore, alterations on the uterine perfusion are<br />
correlated to plasmatic concentrations of estrogen<br />
during estrous but not during diestrus [3].<br />
In 2005, Silva et al. [16] described a subjective<br />
evaluation of the endometrium and myometrium and<br />
a objective evaluation from spectral data collected<br />
from vessels of the mesometrium attachment to<br />
evaluate uterine vascular perfusion alterations [16],<br />
different of the until now used [2,4]. In this experiment<br />
all mares were inseminated and the uterine<br />
perfusion of pregnant and non pregnant was<br />
compared. There was no detected difference in vascular<br />
perfusion between pregnant and non pregnant<br />
mares until D11, but from this day pregnant mares<br />
showed a gradual increase in vascular perfusion.<br />
A maximum value of mares with inadequate<br />
Doppler signals occurred on Days 4-6, which was<br />
attributed to a low RI. However, the presence or absence<br />
of an embryo on Days 1-8 cannot be considered,<br />
because embryonic loss could have occurred in the<br />
group with no embryo detected by D12 [16].<br />
It is important to point that hemodynamic<br />
alterations that indicate an increase on vascular perfusion<br />
during the beginning of diestrus (D4 – D6)<br />
have been described [3,9,14]. Non pregnant and no<br />
inseminated mares have shown a reduced RI from<br />
the spectral evaluation of A. uterine [3]. Using the<br />
same technique as Silva et al. [16], recent studies<br />
showed a gradual increase on RI and PI from D3 in<br />
pregnant mares [9] and reduction on PI at D5 (Figure<br />
2) in mares used as embryo recipients [14]. In this<br />
later report, mares receiving embryos at D4 - D6<br />
demonstrated uterine blood flow increase on both<br />
N<br />
Figure 2. Values of uterine vascular perfusion from real time subjective analysis graduated from 1 to 4 by<br />
Power Doppler (uterine perfusion, means of both horns) and objective analysis by pulsatility index values (PI)<br />
from spectral mode of mesometrial attachment vessels during the estrous cycle days in mares used as embryo<br />
recipients.<br />
uterine horns during these days [14]. All the results<br />
show a vascular perfusion increase during the<br />
beginning of diestrus, however, results are still note<br />
conclusive if the increase is a result of entry of the<br />
embryo into the uterus or if it is part of a uterine preparation<br />
for a possible entry of the embryo.<br />
Important hemodynamic alterations in old<br />
mares, as an increase in vascular resistance [4,9], have<br />
been described. This increase might result from<br />
fibrosis of the uterus [4] and in women it is considered<br />
to be a cause of infertility [5]. In mares, periglandular<br />
fibrosis of the endometrium is considered to be the<br />
major diagnosable cause of embryonic and fetal loss<br />
in older mares [15]. Recently, a comparison between<br />
mares with and without uterine cysts (a common<br />
ultrasound finding in mares with degenerative chronic<br />
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erreir<br />
eira & C. Meir<br />
eira.<br />
<strong>2011</strong>. Uterine and Luteal hemodynamic evaluation of the non pregnant mare.<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s117 - s121.<br />
endometriosis) found a reduced uterine vascular<br />
perfusion in mares with uterine cysts and a positive<br />
association between size of the cystic are and<br />
disturbed uterine hemodynamics [8].<br />
So far, only one report has evaluated the<br />
uterine hemodynamic in recipient mares and because<br />
partial results were published it is not yet concluded<br />
which parameters should be followed to the selection<br />
of mares [14]. The results from the evaluation of the<br />
beginning of pregnancy are in accordance to the ones<br />
reported before for inseminated and pregnant mares<br />
[9,14].<br />
Few are known about the hemodynamic uterine<br />
response to the infusion of inflammatory substances,<br />
such as semen. To evaluate the uterine inflammatory<br />
response and hemodynamic, Bollwein et al.<br />
(2003) [2] injected in one group of mare raw semen<br />
and in the other seminal plasma. Cytological<br />
evaluation showed that, compared to raw semen,<br />
intrauterine infusion of seminal plasma caused only<br />
mild endometritis, but both led to a remarkable<br />
increase in uterine blood flow. Nitric oxide (NO) is a<br />
potent vasodilator released during endometrial<br />
inflammation [17]. However, the result suggests that<br />
not only uterine inflammation, but also other factors<br />
are responsible for increased uterine blood flow, such<br />
as prostaglandins and estrogens present in seminal<br />
plasma [7]. Further investigations are necessary to<br />
determine whether changes in uterine and ovarian<br />
perfusion after insemination are related to fertility in<br />
mares.<br />
2.2 CORPUS LUTEUM (CL) EVALUATION<br />
Ovarian artery of the mare supplies the ovary<br />
as well as the oviduct and therefore can be used for<br />
evaluation of these areas. The CL evaluation can also<br />
be done by the percentage of luteum area with<br />
colored signals as an indicator of the extent of blood<br />
flow. The percentage of the CL area with colored<br />
signals is determined subjectively by images<br />
observations in real time [12] and/or by a freezing<br />
Power Doppler cross-section image with the<br />
maximum number of color pixels taped and the total<br />
number of color pixels is assessed by a computer<br />
analysis system [1].<br />
The first evaluation of the ovarian hemodynamic<br />
was reported in 2002 from data collected<br />
of the right and left ovarian arteries [3]. In contrast to<br />
the uterine arteries, ovarian arteries revealed significant<br />
differences between sides in blood supply. PI values<br />
were sifnificantly lower in the ovarian artery ipsilateral<br />
to the corpus luteum. In this side, the resistance to<br />
blood flow declined after a short rise until D6 and<br />
increased afterwards again until the end of diestrus<br />
[3].<br />
During the estrous cycle an extensive angiogenesis<br />
takes place in the CL [13] which is essential<br />
for the substrate supply needed for the biosynthesis<br />
and secretion of progesterone [6]. Vascular perfusion<br />
and progesterone concentrations showed high<br />
correlation, both parameters rose noticeably in the first<br />
days after ovulation [1,10]. In a first report, the CL<br />
circulation reached its maximum on D5, the<br />
progesterone concentration in peripheral blood<br />
increased until D7 [1] and in a posterior report,<br />
maximum perfusion was achieved two days after the<br />
maximum progesterone concentration (D8) [10].<br />
Blood flow reduced between D10 - D14 some days<br />
before the plasma progesterone decrease [1, 10] and,<br />
during the luteolytic period (D15 - D17), the decline<br />
in CL blood-flow area was greater than blood flow<br />
decrease [10].<br />
During the luteolytic period, blood flow was<br />
constant during the ascending portion and decreased<br />
during the descending portion of the PGFM pulses<br />
[11].<br />
III. FINAL CONSIDERATIONS<br />
Uterine alterations caused by inflammation,<br />
response from seminal plasma infusion, hormonal<br />
variations during estrous and diestrus, pregnancy and<br />
action of various vasoactive factors influence on the<br />
vascular perfusion detected by Doppler ultrasound.<br />
As well as a high correlation between plasma<br />
progesterone concentration and CL blood flow allows<br />
the evaluation of the CL in this technique.<br />
Doppler ultrassonography add knowledge to<br />
uterine viability and CL functionality and it can be<br />
easily used by field veterinarians. It is a non invasive<br />
technique and which supplies a real time result. However,<br />
because of the short time, further investigations<br />
are still necessary to the establishment of normal and<br />
pathological patterns.<br />
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F.S.<br />
Ignácio, J.C. Fer<br />
erreir<br />
eira & C. Meir<br />
eira.<br />
<strong>2011</strong>. Uterine and Luteal hemodynamic evaluation of the non pregnant mare.<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s117 - s121.<br />
REFERENCES<br />
1 Bollwein H., Mayer R., Weber F., Stolla R. & 2002. Luteal blood flow during the estrous cycle in mares. Theriogenology.<br />
65: 2043-2051.<br />
2 Bollwein H., Sowade C. & Stolla R. 2003. The effect of semen extender, seminal plasma and raw semen on uterine and<br />
ovarian blood flow in mares. Theriogenology. 60: 607-616.<br />
3 Bollwein H., Weber F., Kolberg B. & Stolla R. 2002a. Uterine and ovarian blood flow during the estrous cycle in mares.<br />
Theriogenology. 65: 2129-2138.<br />
4 Bollwein H., Maierl R. & Stolla R. 1998. Transrectal color Doppler sonography of the A. uterine in cyclic mares.<br />
Theriogenology. 49: 1483-1488.<br />
5 Cacciatore B.N., Simber B.N., Fusaro P. & Tiitinen A. 1996. Transvaginal Doppler study of uterine artery blood flow in in<br />
vitro fertilization - embryo transfer cycles. Fertility and Sterility. 66: 130-134.<br />
6 Carr B.R., Macdonald P.C. & Simpson E.R. 1982. The role of lipoproteins in the regulation of progesterone secretion by<br />
the corpus luteum. Fertility and Sterility. 38: 303-311.<br />
7 Claus R., Dimmick M.A., Gimenez T. & Hudson L.W. 1992. Estrogens and prostaglandin F2α in the semen and blood<br />
plasma of stallions. Theriogenology. 38: 687-693.<br />
8 Ferreira J.C., Gastal E.L., Ginther O.J. 2008. Uterine blood flow and perfusion in mares with uterine cysts: effect of the size<br />
of the cystic area and age. Reproduction. 135: 541-550.<br />
9 Ferreira J.C., Ignácio F.S. & Meira C. 2010. Uterine vascular perfusion and spectral-Doppler measurements during early<br />
gestation in mares: new concepts of evaluation. Animal Reproduction Science. 121(S): 281-283.<br />
10 Ginther O.J., Gastal E.L., Gastal M.O., Utt M.D. & Beg M.A. 2007. Luteal blood flow and progesterone production in<br />
mares. Animal Reproduction Science. 99: 213-220.<br />
11 Ginther O.J., Rodrigues B.L.,, Ferreira J.C., Araujo R.R. & Beg M.A. 2008. Characterisation of pulses of 13,14-dihydro-<br />
15-keto-PGF2alpha (PGFM) and relationships between PGFM pulses and luteal blood flow before, during, and after<br />
luteolysis in mares. Reproduction, Fertility and Development. 20: 684-693.<br />
12 Ginther, O.J. & Utt, M.D. 2004. Doppler ultrasound in equine reproduction: principles, techniques, and potential. Journal<br />
of Equine Veterinary Science. 24: 516-526.<br />
N<br />
13 Hansel W. & Blair R.M. 1996. Bovine corpus luteum: a historic overview and implications for future research.<br />
Theriogenology. 45: 1267-1294.<br />
14 Ignácio F.S., Camargo J.C., Melo R.P., Abreu N.C., Araújo G.H. & Meira C. <strong>2011</strong>. Uterine hemodynamic evaluations in<br />
recipient mares: preliminary results. In: II Congreso Argentino de Reproducción Equina. (Argentina). pp.533-536.<br />
15 Kenney R.M. 1978. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early<br />
embryonic death. Journal of the American Veterinary Medical Association. 172: 241-226.<br />
16 Silva L.A., Gastal E.L., Beg M.A. & Ginther O.J. 2005. Changes in vascular perfusion of the endometrium in association<br />
with changes in location of the embryonic vesicle in mares. Biology of Reproduction. 72: 755-761.<br />
17 Still J.G. & Greiss F. 1978. The effect of prostaglandins and other vasoactive substances on uterine blood flow and<br />
myometrial activity. American Journal of Obstetrics Gynecoly. 130(1): 1-8<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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L.A. Silva. <strong>2011</strong>. Local Effect of the Conceptus on Uterine Vascular Perfusion and Remodeling during Early Pregnancy<br />
in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s123 - s134.<br />
ISSN 1679-9216 (Online)<br />
Local Eff<br />
ffec<br />
ect t of the Conc<br />
onceptus on Uter<br />
erine<br />
Vascular Per<br />
erfusion and Remo<br />
emodeling<br />
during Early Pregnancy in Mares - New Findings by Doppler Ultrasonography<br />
Luciano Andrade Silva<br />
ABSTRACT<br />
Background: The mammalian reproductive tract is the only organ system in the body where entire tissue layers and structures<br />
are in physiologically dynamic and cyclic changes. Angiogenesis is well known to be critical to assure blood supply for tissue<br />
growth and remodeling. Ovarian-produced steroids control reproductive tract remodeling, and cyclic rhythmicity of the<br />
hypothalamic-ovarian axis.<br />
Review: We have proposed that uterine remodeling during pregnancy is modulated by the conceptus. Special attention was<br />
paid to conceptus modulation of the uterine vascular and architectural changes prior to implantation in equids. Our studies<br />
using Doppler ultrasonography have described vascular and morphological endometrial changes during early pregnancy in<br />
mares. The most important vascular changes observed were: 1) Transient changes in endometrial vascular perfusion accompany<br />
the embryonic vesicle as the vesicle changes location during embryo mobility. 2) The continued presence of the vesicle in the<br />
same horn for an average of 7 min stimulated an increase in vascularity of the endometrium of the middle segment of the horn<br />
during mobile phase. 3) After fixation, endometrial vascularity was progressively higher in the following sequence: horn<br />
without the vesicle, horn with the vesicle, and area of endometrium surrounding the fixed vesicle. 4) After fixation, an early<br />
vascular indicator of the future position of the embryo proper was discovered by color-Doppler imaging and consisted of a<br />
colored spot in the image of the endometrium close to the wall of the embryonic pole. In addition to the observed vascular<br />
changes, morphological changes also were observed. They are related to asymmetrical encroachment of the uterine wall, N<br />
resulting from differential thickening of the upper turgid uterine wall at the mesometrial attachment, in which is normally<br />
observed in mares after embryonic vesicle fixation. The thickening of the endometrium was studied during mobile phase and<br />
after fixation, and the thickness of the endometrium at the mesometrial aspect of the vesicle divided by the thickness at the<br />
antimesometrial aspect was termed the encroachment ratio. The most important morphological changes observed were: 1)<br />
Differential dorsal thickening of the endometrium that surrounds the embryonic vesicle began during the later days of the<br />
mobility phase. 2) After fixation, the differential dorsal thickening or endometrial encroachment upon the vesicle increased<br />
rapidly and was more than four times thicker than ventrally by 3 days after fixation. 3) The increase in vascularity began before<br />
the increase in the encroachment ratio in the endometrium at the site of future fixation. 4) The increase in the encroachment<br />
ratio between 1 and 3 days after fixation was more rapid than during -4 to 0 days before fixation. 5) Embryonic vesicle<br />
dysorientation was associated with a flaccid uterus and defective encroachment of the dorsal endometrium. 6) Asymmetric<br />
enlargement of the allantoic sac spontaneously corrected the disorientation of the embryo proper in mares with apparently<br />
normal uterine tone and endometrial encroachment, so that orientation of the umbilical cord attachment was at a normal<br />
position near 12 o’clock.<br />
Conclusion: Our data provided insight into the architectural and vascular changes in the reproductive tract of equids. These<br />
results set the stage for future experiments to understand more completely the role of the conceptus in regulating the uterine<br />
environment in favor of its development.<br />
Keywords: doppler ultrasonography, conceptus, mares, uterus, vascular perfusion, remodeling.<br />
Departamento de Zootecnia, Faculdade de Zootecnia e Engenharia de Alimentos - FZEA, Universidade de São Paulo (USP), Campus de<br />
Pirassununga, SP, Brazil. CORRESPONDÊNCIA: L.A. Silva [luciano.vetmed@usp.br - PHONE: +55 (19) 3565-4092]. USP - Campus de<br />
Pirassununga. Avenida Duque de Caxias Norte, no. 229. CEP 13635-900 Pirassununga, SP, Brazil.<br />
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in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
I. EARLY PREGNANCY IN EQUIDS<br />
II. DOPPLER ULTRASONOGRAPHY<br />
III. BLOOD FLOW ASSESSED BY DOPPLER ULTRA-<br />
SONOGRAPHY DURING PREGNANCY<br />
IV. LOCAL EFFECT OF THE CONCEPTUS ON UTE-<br />
RINE VASCULAR PERFUSION AND REMODELING<br />
DURING EARLY PREGNANCY<br />
4.1 Mobility phase and Fixation.<br />
4.2 Fixation and Orientation<br />
4.3 Embryonic Vesicle Dysorientation<br />
V. GENERAL DISCUSSION<br />
I. EARLY PREGNANCY IN EQUIDS<br />
Equids are the only common eutherian<br />
mammals in which real-time images of conceptus<br />
(embryo, extraembryonic membranes and fluid)<br />
migration, fixation, and orientation and conversion<br />
from yolk sac to allantoic sac placentation can be<br />
studied sequentially in vivo and detectable without<br />
disturbance. This research capability results from the<br />
availability of transrectal ultrasonography, the large<br />
size of the fluid-filled embryonic vesicle (conceptus),<br />
and the close proximity of the uterine wall to the rectal<br />
wall [17].<br />
The equine embryonic vesicle enters a uterine<br />
horn on Day 6 (ovulation = Day 0) [3,35]. When first<br />
detected by transrectal ultrasonography on Days 9<br />
or 10, the vesicle is frequently (60% of time) in the<br />
uterine body [15,25]. Thereafter, the frequency of<br />
entries into the uterine horns increases and a phase<br />
of maximum mobility begins, involving all parts of<br />
the uterus. Maximum mobility extends over Days 12-<br />
14 when the vesicle grows from about 9 to about 15<br />
mm in diameter. The mobility favors physiologic<br />
exchange between the relatively small conceptus and<br />
large uterus [15]. In this regard, results of confinement<br />
of the conceptus to one uterine horn indicate that the<br />
conceptus locally stimulates uterine turgidity and<br />
edema, as well as contractility [24], and that<br />
movement throughout the uterus is required to prevent<br />
the bilaterally active uterine luteolytic mechanism<br />
[24,26]. Cessation of mobility is called fixation and<br />
occurs on Days 15-17 [16]. The site of fixation is at a<br />
flexure in the caudal segment of one of the uterine<br />
horns without regard to the side of ovulation. It has<br />
been postulated that fixation occurs at the flexure<br />
because it is a physical impediment to continued<br />
mobility of the growing vesicle [14,16].<br />
Orientation of the embryonic vesicle refers<br />
to the position of the embryonic disc or embryo<br />
proper at the periphery of the vesicle (embryonic pole)<br />
relative to the position of the mesometrial attachment.<br />
The pattern of orientation (antimesometrial versus<br />
mesometrial) is fairly constant within species but<br />
differs among species [28,29]. When first detected<br />
by ultrasound (Days 19 to 22), the equine embryo<br />
proper is in the ventral hemisphere of the embryonic<br />
vesicle or opposite to the mesometrial attachment<br />
[16]. It is unlikely that orientation occurs before<br />
embryo mobility ceases. In this regard, simulated<br />
embryonic vesicles rotated or rolled during<br />
intrauterine location changes [17]. These observations<br />
indicate that orientation occurs between the day of<br />
fixation (Day 16) and the earliest reported day of<br />
ultrasonic identification of the embryo proper (Day<br />
19). It has been postulated [16,17] that equine<br />
embryo orientation results from the interaction of at<br />
least three factors: 1) differences in tensile strength<br />
between the thin (two cell layers) and thick (three<br />
layers) portions of the vesicle wall; 2) asymmetrical<br />
encroachment of the uterine wall on the vesicle,<br />
resulting from differential thickening of the upper<br />
turgid uterine wall at the mesometrial attachment; and<br />
3) the massaging action of uterine contractions. A<br />
distinct, smooth, and strong capsule encloses the<br />
embryonic vesicle until about Day 21 [4] and is an<br />
additional factor that likely favors the orientation<br />
process. The surface of the equine embryonic vesicle<br />
develops adhesive qualities [8] which may aid in<br />
anchoring the vesicle after orientation is completed.<br />
Disproportional thickening of the dorsal uterine wall<br />
occurs by Day 17 and accounts for the nonspherical<br />
shapes of the vesicle as it begins to encroach upon<br />
the two-layered membrane [16].<br />
The beginning of the implantation process in<br />
mares starts around Day 40 of pregnancy but the<br />
beginning of the functional placenta is not observed<br />
until Day 60, with complete formation of microcotyledons<br />
about Day 120 [1,32]. Based on the<br />
number of tissues separating maternal from fetal<br />
blood, the equine placenta is classified as epitheliochorial<br />
as all six tissue layers (epithelium, stroma,<br />
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and endothelium) are present in both maternal and<br />
fetal sides [2].<br />
After entering the uterus, the embryo must<br />
be detected by the mother and the luteolytic mechanism<br />
abrogated so as to maintain progesterone synthesis<br />
by the corpus luteum [31]. This is the first luteal<br />
response to pregnancy, better known as maternal<br />
recognition of pregnancy. High embryo loss rates are<br />
common at this time in all domestic animals and<br />
women, and are higher if the conception resulted from<br />
assisted reproductive techniques [9,27,37]. The<br />
mobility phase of the equine embryonic vesicle is<br />
well established as an important event to maintain<br />
luteal function [26]. The early pregnancy is a critical<br />
time for embryo survival in mares, likely to in others<br />
mammals species. Many are the factors involved in<br />
early embryo loss and, securely, the uterine vascular<br />
and architectural changes will support an adequate<br />
uterine environment for embryo survival and<br />
development. High rate of embryo are reported<br />
during the two first months of pregnancy ranging from<br />
2.6% to 24.0% [7,37,39].<br />
II. DOPPLER ULTRASONOGRAPHY<br />
Transrectal B-mode (gray scale) ultrasonography<br />
revolutionized diagnosing and monitoring<br />
of biologic and pathologic reproductive events<br />
in cattle and horses. An important advantage of this<br />
technique is that a structure can be evaluated in real<br />
time while the area is being scanned systematically.<br />
B-mode is used not only to identify and measure<br />
structures, but also to assess physiologic status.<br />
Doppler ultrasound adds blood-flow information to<br />
the B-mode image about anatomy and function [22].<br />
Doppler ultrasound involves two modalities<br />
(spectral mode and color-flow mode) with distinctly<br />
different methods for targeting an area of interest.<br />
For the spectral mode, the blood-flow characteristics<br />
in a focused area of a vessel are assessed by<br />
placement of a sample gate cursor into the B-mode<br />
or color-mode image of the lumen of a targeted<br />
vessel. Color-flow imaging estimates blood velocities<br />
and encodes and displays it as colored regions superimposed<br />
on the B-mode image. The extent of local<br />
perfusion or blood flow area within the tissues can<br />
be estimated with color flow and quantified directly<br />
at the level of the tissue [19].<br />
III. BLOOD FLOW ASSESSED BY DOPPLER<br />
ULTRASONOGRAPHY DURING PREGNANCY<br />
Uterine blood flow changes during pregnancy<br />
have been an object of interest of many studies. Using<br />
electromagnetic probes, it was shown that blood flow<br />
increased in the uterine artery ipsilateral but not<br />
contralateral to the conceptus between Days 13 and<br />
15 in sheep [30] and Days 15 and 17 in cattle [11].<br />
Swine have embryos in both horns and blood flow<br />
transiently increases in both uterine arteries 12 and<br />
13 days after insemination, but when embryos are<br />
experimentally confined to one horn, the blood flow<br />
increases only on that side [12]; furthermore, blood<br />
flow to uterine segments containing a conceptus is<br />
greater than for segments that do not contain a<br />
conceptus [13]. Blood flow to the pregnant uterus<br />
has been shown to be increased in the uterine artery<br />
ipsilateral to the embryo proper on Days 14-18 and<br />
after Day 25 in heifers [11]. A brief review of the<br />
earliest studies on uterine blood flow changes is<br />
provided by [10].<br />
Transrectal Doppler ultrasonography was used<br />
for noninvasive study of the blood flow in the uterine<br />
arteries during early pregnancy in mares [5,6]. Time<br />
averaged maximum velocity (TAMV) was higher and N<br />
resistance index (RI) was lower in the arteries of<br />
pregnant mares than in nonpregnant mares beginning<br />
on Day 11. From Days 15 to 29 of pregnancy, TAMV<br />
was higher and RI lower in the uterine artery ipsilateral<br />
to the conceptus than in the opposite artery. The<br />
authors indicated that an increase in TAMV<br />
represented greater blood flow in the arteries, and a<br />
decrease in RI represented reduced resistance to blood<br />
flow in the vasculature distal to site of assessment. It<br />
was not determined whether conceptus fixation had<br />
occurred in at least some mares by the day of detection<br />
of a difference in blood flow between the ipsilateral<br />
and contralateral arteries. Thus, a local effect of the<br />
embryonic vesicle on the uterine vasculature in<br />
association with mobility of the conceptus was not<br />
demonstrated. Data on uterine vascular changes during<br />
early pregnancy in mares assessed by color-mode<br />
Doppler ultrasonography have been published<br />
[21,33,34] and will be detailed in the next topics of<br />
this review. The effect of the conceptus mediating<br />
uterine perfusion changes during mobility phase and<br />
after embryonic vesicle fixation was studied. In<br />
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addition, the effects of uterine vascular changes on<br />
the endometrial ultrasonographic morphology were<br />
also investigated.<br />
IV. LOCAL EFFECT OF THE CONCEPTUS ON UTERINE<br />
VASCULAR PERFUSION AND REMODELING DURING<br />
EARLY PREGNANCY<br />
4.1 Mobility phase and Fixation.<br />
In our first study [33], color Doppler<br />
ultrasonography was used to study the relationships<br />
of endometrial vascular perfusion and uterine blood<br />
flow to the mobility of the embryonic vesicle during<br />
early pregnancy in mares. The equine embryonic<br />
vesicle is mobile on Days 12-14 (Day 0 = ovulation),<br />
when it is about 9-15 mm in diameter. Movement<br />
from one uterine horn to another occurs on average<br />
about 0.5 times per hour. Mobility ceases (fixation)<br />
on Days 15-17. Transrectal color Doppler ultrasonography<br />
was used to study the relationship of embryo<br />
mobility (experiment 1) and fixation (experiment<br />
2) to endometrial vascular perfusion (Figure 1). In<br />
experiment 1, mares were bred and examined daily<br />
from Days 1-16 and were assigned, retrospectively,<br />
to a group in which an embryo was detected (pregnant<br />
mares; n=16) or not detected (n=8) by Day 12.<br />
Endometrial vascularity (scored 1-4, none to maximal)<br />
did not differ on Days 1-8 between groups or between<br />
the side with and without the corpus luteum.<br />
Endometrial vascularity scores were higher (P < 0.05)<br />
on Days 12-166 in both horns of pregnant mares than<br />
in mares with no embryo. In pregnant mares, the<br />
scores increased (P < 0.05) between Days 10-12 in<br />
the horn with the embryo and were higher (P < 0.05)<br />
than in the opposite horn on Days 12–15 (Figure 2).<br />
In experiment 2, 14 pregnant mares were examined<br />
from Day 13 to 6 d after fixation (Figure 3). Endometrial<br />
vascularity scores and number of colored<br />
pixels per cross section of endometrium were greater<br />
(P < 0.05) in the endometrium surrounding the fixed<br />
vesicle than in the middle portion of the horn of<br />
fixation. Results supported the hypothesis that<br />
transient changes in endometrial vascular perfusion<br />
accompany the embryonic vesicle as the vesicle<br />
changes locations during embryo mobility.<br />
4.2 Fixation and Orientation<br />
Orientation of the embryonic vesicle refers<br />
to the position of the embryo proper at the periphery<br />
of the vesicle relative to the position of the mesometrial<br />
attachment. In mares, the embryonic pole of<br />
the vesicle is antimesometrial after completion of<br />
orientation. Day of vesicle fixation, differential<br />
thickening of the endometrium near the mesometrial<br />
attachment, and orientation of the embryonic vesicle<br />
were studied in 30 ponies in our second study<br />
presented in this review [34], using B-mode and<br />
color-Doppler transrectal ultrasonography (Figure 4).<br />
The thickness of the endometrium at the mesometrial<br />
aspect of the vesicle divided by the thickness at the<br />
antimesometrial aspect was termed the encroachment<br />
ratio (Figure 4). An early vascular indicator of the<br />
future position of the embryo proper was discovered<br />
by color-Doppler imaging and consisted of a colored<br />
spot in the image of the endometrium close to the<br />
wall of the embryonic pole (Figure 5). The early<br />
indicator was detected in each mare 0.5 ± 0.1 days<br />
after fixation and 2.5 ± 0.2 days before first detection<br />
of the embryo proper. The position of the early<br />
indicator when first detected at the periphery of the<br />
embryonic vesicle was not different significantly from<br />
the position of the embryo proper when first detected.<br />
At the future site of fixation, the first increase (P <<br />
0.05) in the encroachment ratio occurred between 4<br />
and 1 day before fixation (Figure 6). Results supported<br />
the hypothesis that differential thickening of the<br />
endometrium precedes orientation and indicated that<br />
orientation occurs immediately after fixation.<br />
4.3 Embryonic Vesicle Dysorientation<br />
The phenomenon of dysorientation of the<br />
embryonic vesicle is the subject of our third study<br />
[21]. Orientation of the embryo proper at the periphery<br />
of the equine embryonic vesicle is normally<br />
antimesometrial or on the ventral aspect of the embryonic<br />
vesicle (6 o’clock relative to 12 o’clock at<br />
the center of the mesometrial attachment). An early<br />
ultrasonographically detectable vascular endometrial<br />
indicator of the future position of the embryo proper<br />
has been reported previously and was first detected<br />
in a mean 2.5 days before detection of the embryo<br />
proper. In the present study, four occurrences of<br />
dysorientation of the embryo proper were found in a<br />
group of 30 mares (incidence, 13%; Figure 7). When<br />
first detected, the early indicator of the clock-face<br />
position of the embryonic pole for the dysorientation<br />
and normal orientation group, respectively, was 1.3<br />
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Figure 1. Two images of cross-sections of uterine horns showing minimal (left panel) and maximal (right panel) colored areas of the<br />
endometrium from the Doppler flow mode. The sample gate (sg, left panel) indicates the area sampled in the mesometrial attachment<br />
(mm=mesometrium) for generating the spectrum used by the scanner in calculating time averaged maximum velocity and pulsatility index. The<br />
arrows for each panel delineate the endometrium (reproduced with permission [33]).<br />
N<br />
Figure 2. Means ± SEM scores for endometrial vascularity and uterine contractility in bred mares in which an embryo was either detected or not<br />
detected by Days 9–12. Number of mares was 16 and 8 for the mares with and without an embryo, respectively, except that only 3, 11, and 15<br />
mares with a detected embryo were available on Days 9, 10, and 11, respectively. The day effect was significant (P < 0.0001) for contractility<br />
on Days 1–8, and the day-by-group interaction on Days 9–16 was significant for contractility (P < 0.02) and vascularity (P < 0.0001). An<br />
asterisk (*) indicates a day of a difference (P < 0.05) between horns within the pregnant group and between days within a group. The pound mark<br />
(#) indicates the days of a difference (P < 0.05) between pregnant and nonpregnant groups. CL = corpus luteum. Contra = contralateral. Ipsi =<br />
ipsilateral. Experiment 1 (reproduced with permission [33]).<br />
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in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
Figure 3. Means ± SEM for uterine color Doppler end points and contractility in the middle segment of the uterine horn<br />
of fixation and the opposite horn. The upper two panels include data for the area of endometrium at the location of the<br />
fixed vesicle. The scores for vascularity and contractility were from 1–4 for none, minimal, intermediate, and maximal.<br />
Significant main effects (G, group; D, day) and the interaction (GD) are shown for the days of and after fixation. An<br />
asterisk (*) indicates a day with a difference (P < 0.05) between the group above and the group below the asterisk<br />
within a day. Experiment 2 (reproduced with permission [33]).<br />
Figure 4. Color mode sonogram depicting the o’clock method of determining the position of a structure at the periphery of the embryonic vesicle<br />
(A) and B-mode sonogram illustrating the method for determining the endometrial encroachment ratio (EER; B). The numbers (3, 6, 9, 12) refer<br />
to o’clock positions. Alignment with the area of mesometrial attachment is at 12:00 o’clock or 12 h. Endometrial thickness (B) at 10:30 was<br />
divided by the thickness at 4:30 (green arrows) and the thickness at 1:30 was divided by the thickness at 7:30 (yellow arrows). The average<br />
endometrial encroachment ratio in this illustration is 2.7 (reproduced with permission [34]).<br />
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in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
Figure 5. Sonograms illustrating (arrows) early endometrial indicators of the future position of<br />
the embryo proper (A, B) and the embryo proper (C, D). The arrows (A, B) indicate sites where<br />
the color of the early indicator appears to permeate the vesicle wall, a small embryo proper (note<br />
the small color spot; C), and a more developed embryo proper (D). The color-Doppler assessment<br />
was confined to the area delineated by the dotted lines (A) to improve the color flow resolution<br />
(reproduced with permission [34]).<br />
N<br />
Figure 6. Means (±SEM) for endometrial encroachment ratio (dorsal thickness divided by ventral thickness) and three end<br />
points for assessing the extent of vascular perfusion of the endometrium centralized to the day of fixation (n=9 mares).<br />
Horn of fixation before Day 0 refers to the future horn of fixation as determined retroactively. The opposite horn refers to<br />
the horn in which fixation did not occur. An asterisk indicates a significant difference (P < 0.05) between days combined<br />
for the two horns. For each panel, lower-case letters above the day axis indicate days of differences between means within<br />
the horn of fixation; any two days without a common lower-case letter are different (P < 0.05; reproduced with permission<br />
[34]).<br />
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L.A. Silva. <strong>2011</strong>. Local Effect of the Conceptus on Uterine Vascular Perfusion and Remodeling during Early Pregnancy<br />
in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
Figure 7. Color-Doppler ultrasonograms of the conceptus at Day 19 for normal orientation and dysorientation. Encroachment or thickening of<br />
the endometrium between the mesometrial attachment (upper right) and the embryonic vesicle is prominent for normal orientation but is not<br />
apparent for dysorientation. Arrows indicate the periphery of the endometrium. E = embryo proper (reproduced with permission [21]).<br />
± 0.3 and 0.4 ± 0.1 hours from the 6 o’clock position<br />
(P < 0.008). The extent of dysorientation increased<br />
progressively over Days 16 to 19, so that the embryo<br />
proper was at 3, 9, 9, or 10 o’clock (Figure 8). Dysorientation<br />
was associated with a flaccid uterus and<br />
defective encroachment of the dorsal endometrium<br />
upon the vesicle in three of the four mares. In a second<br />
study, dysorientation of the embryo proper occurred<br />
in two mares with apparently normal uterine tone and<br />
endometrial encroachment. When first detected, the<br />
embryo proper was at 9 or 10 o’clock. However,<br />
asymmetric enlargement of the allantoic sac<br />
spontaneously corrected the dysorientation, so that<br />
orientation of the umbilical-cord attachment was at a<br />
normal position near 12 o’clock (Figure 9), at the<br />
area of the mesometrial attachment.<br />
per. The embryonic disc is a very active developmental<br />
area and the vascular system is the first organ system<br />
to be formed during embryogenesis. More detailed<br />
V. GENERAL DISCUSSION<br />
Our studies were the first in the literature to<br />
use the color-mode of Doppler ultrasonography to<br />
assess blood flow in the uterus of mares. In spite of<br />
the subjectivity of the color-Doppler evaluation during<br />
scored system evaluations, we have demonstrated in<br />
all of our experiments with the use of objective<br />
validation methods, that this technique is reliable and<br />
precise in detecting small variations in blood flow.<br />
The early indicator which consists of a colored<br />
spot in the endometrium close to the wall of the<br />
embryonic pole and its detection is a good example<br />
of the sensitivity of the technique. Based on the early<br />
indicator, it was possible to map the position of the<br />
embryonic disc from the day of fixation of the embryonic<br />
vesicle until visualization of the embryo pro-<br />
Figure 8. Mean (± SEM) for encroachment ratio and clock-face<br />
position of the embryonic pole (number of hours from 6 o’clock).<br />
The interaction between group (normal orientation and dysorientation)<br />
and day was significant for both end points. Difference between<br />
groups was significant (P < 0.05) for Days 18 and 19 for endometrium<br />
and for each day for embryonic pole (reproduced with permission<br />
[21]).<br />
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L.A. Silva. <strong>2011</strong>. Local Effect of the Conceptus on Uterine Vascular Perfusion and Remodeling during Early Pregnancy<br />
in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
N<br />
Figure 9. Comparison of the expansion of the allantoic sac for a conceptus with normal orientation and dysorientation<br />
of the embryo proper (E). Correction of the dysorientation of the embryo proper occurred by asymmetric expansion<br />
of the allantoic sac, so that the orientation of the umbilical-cord attachment of the fetus (F) was in a normal position<br />
in the mesometrial area. In the correction of dysorientation of the embryo proper, one end of the opposing<br />
membranes of the yolk and allantoic sacs was first to reach the mesometrial area and apparently became adhered.<br />
The other unadhered end continued to move toward 12 o’clock as the allantoic sac continued to expand (reproduced<br />
with permission [21]).<br />
studies should be done to identify the exact origin of<br />
this early indicator or Doppler signals observed in<br />
our study. It could reflect endometrial blood flow<br />
stimulated by embryonic factors which stimulate<br />
vessels in this area of intimate contact through a<br />
paracrine action. Another possibility is that the early<br />
indicator is formed by contractions of the cardiac<br />
muscle cells at the embryonic disc area even before<br />
their organization as a heart. Doppler ultrasonography<br />
detects signals produced by structures in movement.<br />
Contraction of the embryonic cardiac cells interacting<br />
with tissues in the immediate area may be sufficiently<br />
strong to create tissue movement producing the<br />
observed Doppler echoes.<br />
In mares, transient changes in endometrial<br />
vascularity accompanied conceptus location changes<br />
during the mobility phase and continued presence of<br />
the conceptus in the same horn (7-min average) stimulated<br />
an increase in vascularity. After fixation,<br />
endometrial vascularity was higher in the endome-<br />
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L.A. Silva. <strong>2011</strong>. Local Effect of the Conceptus on Uterine Vascular Perfusion and Remodeling during Early Pregnancy<br />
in Mares - New Findings by Doppler Ultrasonography. Acta Scientiae Veterinariae. 39(Suppl 1): s123 - s134.<br />
trium surrounding the fixed conceptus, than in other<br />
areas of the ipsilateral horn, or in the opposite horn.<br />
Differential dorsal thickening of the endometrium<br />
preceded embryonic orientation. Based on our studies<br />
assessing uterine blood flow during early pregnancy<br />
of mares and cows with color Doppler and in the<br />
previous studies which other authors used others<br />
techniques to assess uterine blood flow in cows, sows<br />
and ewes, Equids exhibited the most precocious<br />
increase in uterine perfusion during early pregnancy,<br />
when compared with bovids [35]. Uterine blood flow<br />
changes started to be detected on Day 12 of pregnancy.<br />
Two distinct mechanisms could be collaborating<br />
to stimulate the increase in uterine vascular perfusion<br />
during early pregnancy in mares and these two<br />
mechanisms are probably related to two distinct phases<br />
of early pregnancy in Equids; the mobility phase<br />
and post-fixation phase of the embryonic vesicle. We<br />
suggest that the embryonic vesicle could stimulate<br />
vasodilation and angiogenesis in the endometrium.<br />
These two processes are probably combined.<br />
However, during mobility phase, transient rapid<br />
changes in uterine blood flow were shown to be related<br />
to location of the embryonic vesicle in the uterus.<br />
Slight encroachment of the dorsal endometrial was<br />
detected during the final days of mobility phase. These<br />
results suggest that the rapid transient changes in<br />
uterine blood flow reflect vasodilation of blood vessels<br />
in the endometrium. More research is necessary to<br />
determine which factors provoke this kind of<br />
stimulation. The vesicle produce large amounts of<br />
estrogens and prostaglandins at this time and these<br />
hormones could be involved in vasodilatory<br />
mechanisms. In addition, physicochemical interaction<br />
of the embryonic vesicle capsule with the endometrial<br />
luminal epithelium during conceptus movement may<br />
be also considered as a possible factor of stimulation.<br />
Angiogenesis is also likely present toward the end of<br />
the mobility phase based on the initial dorsal<br />
endometrial encroachment and slight increase in<br />
endometrial perfusion observed at this time. This idea<br />
is further supported by results of the morphometric<br />
study [36] which demonstrated increased growth of<br />
blood vessels, as well as increased angiogenic factors,<br />
in the endometrium adjacent to the fixated conceptus.<br />
After fixation, the increase in blood flow and encroachment<br />
of the dorsal endometrium is dramatic.<br />
Presumably, most of the vascular stimulant factors<br />
produced by the embryonic vesicle are highly<br />
localized at the site of fixation. Our findings suggest<br />
that, while presumptive vasodilation occurs during<br />
the mobility phase, the predominant angiogenic stimulus<br />
occurs post-fixation.<br />
Orientation of the embryonic vesicle occurred<br />
immediately after fixation. Embryonic dysorientation<br />
was associated with a flaccid uterus and defective<br />
encroachment of the dorsal endometrium. Asymmetric<br />
enlargement of the allantoic sac spontaneously<br />
corrected dysorientation. Adherence points were<br />
found between the yolk sac surface and the dorsal<br />
endometrium [36]. These are new findings showing<br />
step by step the dynamic interactions between the<br />
embryonic vesicle and the uterine wall for the express<br />
purpose of aligning the future site of umbilical cord<br />
formation to the richest vascular area, the dorsal<br />
endometrium and mesometrium. Orientation of the<br />
embryonic disc in the ventral endometrial area permits<br />
direct contact of the bilaminar layer of the yolk sac at<br />
the abembryonic pole with the richest glandular and<br />
vascularized area of the endometrium. This positioning<br />
favors embryo-maternal exchange guaranteeing<br />
survival and development of the early conceptus.<br />
It is interesting to observe in Equids that the<br />
embryo proper forms ventrally but during development<br />
of the allantois is translocated to the dorsal<br />
area, the richest endometrial area. At this position,<br />
the umbilical cord develops. We have observed that<br />
abnormal orientation normally terminates the<br />
pregnancy but, in two specific cases when the uterus<br />
presented slight tone, the embryonic vesicles were<br />
able to correct their orientation by continuing to<br />
expand the yolk sac-allantois boundary until it<br />
impinged on the dorsal endometrium, permitting<br />
formation of the umbilical cord in the correct position.<br />
Adherence points on the surface of the conceptus,<br />
specifically the bilaminar layer of the yolk sac, are<br />
important to maintain conceptus orientation. Studies<br />
of the composition of the equine capsule have demonstrated<br />
carbohydrate changes during early<br />
pregnancy. However, visualization of the adherence<br />
points on the yolk sac surface offers a more specific<br />
area with which to study biochemical interactions<br />
between the surface of the capsule and the endometrial<br />
luminal epithelium.<br />
Modulation of the uterine vascular system by<br />
the conceptus includes two very distinct and balanced<br />
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events. The first event consists of stimulation of the<br />
uterine architectural and vascular system changes.<br />
From the earliest stages of pregnancy to its culmination,<br />
the conceptus presumably releases factors driving<br />
tissue changes in the uterus, including vascular<br />
remodeling, to favor survival and development. However,<br />
equally important is the need to limit these uterine<br />
changes to prevent overdevelopment. Such may be<br />
the case with large offspring syndrome, for example,<br />
in which abnormalities in placental vascularization are<br />
observed. Many questions are still unanswered, such<br />
as whether tissue and vascular remodeling during<br />
pregnancy is self-limited or whether this process is<br />
mediated by conceptus-produced factors. The processes<br />
involved in tissue remodeling of the pregnant<br />
uterus and in abnormal conditions of tissue growth,<br />
as cancer are similar in appearance. However, during<br />
pregnancy tissue growth is limited or controlled in<br />
contrast to pathological conditions such as cancer.<br />
These thoughts set the stage for a more complete future<br />
of investigation. Knowledge of the mechanisms<br />
which regulate angiogenic and tissue remodeling of<br />
pregnancy would represent an enormous advance<br />
in science. Understanding the mechanisms regulating<br />
angiogenesis and tissue remodeling in the pregnant<br />
reproductive tract will help in the development of<br />
therapies for pathological conditions which exhibit<br />
abnormal tissue growth.<br />
In summary, our results set the stage for future<br />
experiments to understand more completely: 1) the<br />
role of the conceptus in regulating the uterine<br />
environment to favor its development, including<br />
understanding the balance in stimulating and limiting<br />
uterine architectural and vascular changes, 2) the role<br />
of vascular changes in the regulation of physiological<br />
processes in the reproductive tract during cyclicity<br />
and pregnancy, and 3) the role of blood flow changes<br />
as a practical diagnostic measurer of normal organ<br />
and tissue functionality.<br />
REFERENCES<br />
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3 Battut I., Colchen S., Fieni F., Tainturier D. & Bruyas J.F. 1997. Success rates when attempting to nonsurgically collect<br />
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two days after ovulation: observations on fresh specimens. Journal of Anatomy. 135: 191-209.<br />
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mares. Theriogenology. 60(4): 597-605.<br />
6 Bollwein H., Weber F., Woschée I. & Stolla R. 2004. Transrectal Doppler sonography of uterine and umbilical blood flow<br />
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7 Carnevale E.M. & Ginther O.J. 1992. Relationships of age to uterine function and reproductive efficiency in mares.<br />
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8 Denker H.W. 2000. Structural dynamics and function of early embryonic coats. Cells Tissues Organs. 166(2): 180-207.<br />
9 Diskin M.G. & Morris D.G. 2008. Embryonic and early foetal losses in cattle and other ruminants. Reproduction of<br />
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10 Ford S.P. 1985. Maternal recognition of pregnancy in the ewe, cow and sow: Vascular and immunological aspects.<br />
Theriogenology. 23: 145-159.<br />
11 Ford S.P., Chenault J.R. & Echternkamp S.E. 1979. Uterine blood flow of cows during the oestrous cycle and early<br />
pregnancy: effect of the conceptus on the uterine blood supply. Journal of Reproduction and Fertility. 56(1): 53-62.<br />
12 Ford S.P. & Christenson R.K. 1979. Blood flow to uteri of sows during the estrous cycle and early pregnancy: local effect<br />
of the conceptus on the uterine blood supply. Biology of Reproduction. 21(3): 617-624.<br />
13 Ford S.P., Christenson R.K. & Ford J.J. 1982. Uterine blood flow and uterine arterial venous and luminal concentrations<br />
of estrogens on days 11, 13, and 15 after estrus in pregnant and nonpregnant sows. Journal of Reproduction and Fertility.<br />
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14 Gastal M.O., Gastal E.L., Kot,K. & Ginther O.J. 1996. Factors related to the time of fixation of the conceptus in mares.<br />
Theriogenology. 46(7): 1171-1180.<br />
15 Ginther O.J. 1983. Mobility of the early equine conceptus. Theriogenology. 19(4): 603-611.<br />
16 Ginther O.J. 1983. Fixation and orientation of the early equine conceptus. Theriogenology. 19(4): 613-623.<br />
17 Ginther O.J. 1985. Dynamic physical interactions between the equine embryo and uterus. Equine Veterinary Journal.<br />
3(Suppl 1): 41-47.<br />
18 Ginther O.J. 1992. Reproductive biology of the mare, basic and applied aspects. 2nd edn. Cross Plains: Equiservices<br />
Publishing, pp.1-40 and 173-290.<br />
19 Ginther O.J. 2007. Ultrasonic Imaging and Animal Reproduction: Color-Doppler Ultrasonography - Book 4. Cross<br />
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20 Ginther O.J., Gastal E.L., Gastal M.O. & Beg M.A. 2005. Regulation of circulating gonadotropins by the negative effects<br />
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21 Ginther O.J. & Silva L.A. 2006. Incidence and nature of disorientation of the embryo proper and spontaneous correction<br />
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23 Girling J.E. & Rogers P.A.W. 2005. Recent advances in endometrial angiogenesis research. Angiogenesis. 8(2): 89-99.<br />
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26 McDowell K.J., Sharp D.C., Grubaugh W., Thatcher W.W. & Wilcox C.J. 1988. Restricted conceptus mobility results in<br />
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27 Mclean J.M. 1987. Early embryo loss. The Lancet. 329: 1033-1034.<br />
28 Mossman H.W. 1971. Orientation and site of attachment of the blastocyst: A comparative study. In: The Biology of the<br />
Blastocyst. Blandau R.J. (Ed). Chicago: Chicago Press, pp.27-48.<br />
29 Mossman H.W. 1987. In: Vertebrate Fetal Membranes. New Brunswick: Rutgers University Press, pp.79-83.<br />
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the conceptus and the ovary bearing the corpus luteum. Journal of Animal Science. 58(2): 423-429.<br />
31 Roberts R.M., Xie S. & Mathialagan N. 1996. Maternal recognition of pregnancy. Biology of Reproduction. 54: 294-302.<br />
32 Sharp D.C. 2000. The early fetal life of the equine conceptus. Animal Reproduction Science. 60–61: 679-689.<br />
33 Silva L.A., Gastal E.L., Beg M.A. & Ginther O.J. 2005. Changes in vascular perfusion of the endometrium in association<br />
with changes in location of the embryonic vesicle in mares. Biology of Reproduction. 72(3): 755-761.<br />
34 Silva L.A. & Ginther O.J. 2006. An early endometrial vascular indicator of completed orientation of the embryo and the<br />
role of dorsal endometrial encroachment in mares. Biology of Reproduction. 74(2): 337-343.<br />
35 Silva L.A. & Ginther O.J. 2010. Local effect of the conceptus on uterine vascular perfusion during early pregnancy in<br />
heifers. Reproduction. 139(2): 453-463.<br />
36 Silva L.A., Klein C., Ealy A.D. & Sharp D.C. <strong>2011</strong>. Conceptus-mediated endometrial vascular changes during early<br />
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37 Vanderwall D.K. 2008. Early Embryonic Loss in the Mare. Biology of Reproduction. 28: 691-702.<br />
38 Weber J.A., Freeman D.A., Vanderwall D.K. & Woods G.L. 1991. Prostraglandin E 2<br />
secretion by oviductal transportstage<br />
equine embryos. Biology of Reproduction. 45(4): 540-543.<br />
39 Woods G.L., Baker C.B., Baldwin J.L., Ball B.A., Bilinski J., Cooper W.L., Ley W.B., Mank E.C. & Erb H.N. 1987. Early<br />
pregnancy loss in brood mares. Journal of Reproduction and Fertility. 35(Suppl 1): 455-459.<br />
www.ufrgs.br/actavet<br />
39 (Suppl 1)<br />
s134
M.M. Franc<br />
anco, A. Pellegr<br />
ellegrin,<br />
R.A. Figueir<br />
igueiredo<br />
edo, et al. <strong>2011</strong>. The Innovation Network in Animal Reproduction: EMBRAPA’s<br />
Experience in Organizing a Research Project on Reprodutive... Acta Scientiae Veterinariae. 39(Suppl 1): s135 - s138.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s135 - s138.<br />
ISSN 1679-9216 (Online)<br />
The Innovation Net<br />
etwor<br />
ork in Animal Repr<br />
eproduc<br />
duction:<br />
EMBRAP<br />
APA’s s Exper<br />
erienc<br />
ience e in<br />
Organizing a Research Project on Reproductive Biotechnology<br />
Maurício Machain Franco, Aiesca Pellegrin, Ricardo Alamino Figueiredo, Alexandre Rossetto<br />
Garcia, Luiz Sergio Almeida Camargo, Hymerson Costa Azevedo & João Henrique Moreira Viana<br />
ABSTRACT<br />
Background: In 2007 a broad Network project was proposed, aiming to organize in a more efficient way the different research<br />
and development actions related to new reproductive technologies in Embrapa. The initial proposal focused to develop new<br />
technological solutions to increase Brazilian livestock competitiveness; generate basic knowledge to support development of<br />
new technologies; to promote genetic improvement and evaluate dairy and beef animal models for the different ecosystems,<br />
to improve laboratorial infrastructure and promote capacity building; and to promote equal development of the technologies<br />
for the different livestock species. The final proposal, titled “Innovation Network in Animal Reproduction” was approved for<br />
the period 2008-2012.<br />
Review: The Network Project includes research activities in animal reproduction and in the interfaces of reproduction and<br />
animal health, nutrition, and genetics. A consortium of 12 of the Embrapa units, 14 Universities, 2 <strong>International</strong> Research<br />
Centers and 5 private companies are engaged in the project. The main structure follows the general guidelines of the Embrapa´s<br />
standard of network projects, being organized in 10 thematic Component Projects (CPs): CP1- Activities related to the<br />
organization of the Network, as the coordination of the CPs, financial management, promotion of meetings and workshops,<br />
and compilation of results and evaluations; CP2 – Development and evaluation of new technologies for the sanitary control<br />
of semen, oocytes, and embryos; CP3- Evaluation of nutritional strategies to improve reproductive efficiency; CP4- Development<br />
N<br />
of methods to improve the quality and quantity of gametes used in assisted reproductive technologies; CP5- Development of<br />
in vivo and in vitro embryo production systems; CP6- Establishment of protocols for the isolation, culture and maintenance of<br />
cell lines aiming the production of animal clones; CP7- Establishment of alternative protocols to the production of transgenic<br />
or intragenic animals; CP8- Identification, selection, use and conservation of genetic resources; CP9- Validation and monitoring<br />
of technologies; CP10- Technological innovation.<br />
Conclusions: The establishment of the network allowed the organization of Embrapa’s different ongoing research actions in<br />
animal reproduction in a large and multidisciplinary project. As consequences, there was a larger interaction among the<br />
different research groups of the company and external partners. The proposal of collaborative research improved the scientific<br />
production of the group and also the development of products, processes, and technical information to the private sector. The<br />
technology transference and capacity building activities were strategically reorganized in line with the scope of the Network<br />
Project. A last consequence of the network was the possibility of applying for consortium research funding opportunities. The<br />
group also expects to improve scientific collaboration and to increase the relevance of R&D projects in animal reproduction,<br />
to improve the interplay with the government agencies in charge of the establishment of laws and rules for reproductive<br />
biotechnology used in livestock, and to improve their capacity of identifying and measuring the impact of the new technologies<br />
in the different livestock production systems.<br />
Keywords: Network research, embryo technologies, R&D projects.<br />
Brazilian Agricultural Research Corporation. CORRESPONDENCE: J.H.M. Viana [jhmviana@cnpgl.embrapa.br - FAX: +55 (32) 3249-<br />
4701]. Rua Eugênio do Nascimento n. 610, Dom Bosco. CEP 36038-330 Juiz de Fora, MG, Brazil.<br />
s135
M.M. Franc<br />
anco, A. Pellegr<br />
ellegrin,<br />
R.A. Figueir<br />
igueiredo<br />
edo, et al. <strong>2011</strong>. The Innovation Network in Animal Reproduction: EMBRAPA’s<br />
Experience in Organizing a Research Project on Reprodutive... Acta Scientiae Veterinariae. 39(Suppl 1): s135 - s138.<br />
I. INTRODUCTION<br />
II. THE NETWORK STRUCTURE<br />
III. RESULTS OF THE NETWORK PROJECT<br />
IV. FUTURE CHALLENGES AND PERSPECTIVES<br />
I. INTRODUCTION<br />
The development and use of new reproductive<br />
biotechnologies such as in vitro embryo production,<br />
nuclear transfer and sperm sorting had a deep impact<br />
in the Brazilian livestock sector in the last decade. A<br />
remarkable example of this was the increase of the<br />
bovine embryo industry, in which the country reached<br />
a leading position, accounting for about 1/3 of the<br />
world embryo transfers [1]. The Brazilian Agricultural<br />
Research Corporation (Embrapa) took part of this<br />
revolution, providing research and development<br />
solutions by its different regional centers. Besides a<br />
significant scientific contribution, publishing many<br />
studies in national and international Journals, the<br />
Embrapa’s research team was also engaged in the<br />
introduction of emerging technologies in the country<br />
(OPU, cloning), in capacity building (ET and IVF<br />
courses), in the interplay with the productive sector<br />
(The IVEP Lab Network) and in the organization of<br />
scientific societies and promotion of workshops and<br />
congresses in this field (CBRA, <strong>SBTE</strong>). There was<br />
not, however, a formal instrument to coordinate the<br />
research actions developed by the different Centers<br />
and in cooperation with other institutions. Therefore,<br />
it was quite difficult to evaluate the impact of the<br />
development of new technologies on the productive<br />
sector. In 2007 a first broad Network project was<br />
proposed, aiming “To order in a more efficient way<br />
the different research and development actions related<br />
to new reproductive technologies and correlated<br />
areas”. The initial proposal focused in the most<br />
important challenges 1) To promote genetic<br />
improvement and evaluate dairy and beef animal<br />
models for the different ecosystems, specially the tropical<br />
ones, and with proper sanitary control; 2)<br />
Develop new technological solutions to increase<br />
Brazilian livestock competitiveness; 3) Improve<br />
laboratorial infrastructure and promote capacity<br />
building to support the adoption of new technologies;<br />
4) Promote equal development of the technologies<br />
for the different livestock species (bovine, ovine,<br />
goats, buffalos, equine and swine); 5) Generate basic<br />
knowledge in the field of the project to support<br />
development of new technologies for animal and<br />
human health. The final proposal was approved in<br />
the following year to be implemented in the period<br />
2008-2012, with the title “Innovation Network in<br />
Animal Reproduction”.<br />
II. THE NETWORK STRUCTURE<br />
The Network Project includes research<br />
activities in animal reproduction and in the interfaces<br />
of reproduction and animal health, nutrition, and<br />
genetics, and also technology validation and<br />
promotion of their use. A consortium of 12 of the<br />
Embrapa units (Embrapa Headquarters, Cenargen,<br />
CNPGL, CNPGC, CPACT, CPAP, CPATU, CPATC,<br />
CPAC, CPAFAC, CNPC, CPPSUL), 14 Universities<br />
(public, private and from abroad), 2 <strong>International</strong><br />
Research Centers and 5 private companies are<br />
engaged in the project. The main structure follows<br />
the general guidelines of the Embrapa´s standard of<br />
network projects, being organized in thematic<br />
Component Projects (CPs). Each CP includes different<br />
activities related to each research line. The Innovation<br />
Network in Animal Reproduction includes 10 Component<br />
Projects, as described below:<br />
CP1- Activities related to the organization of<br />
the Network, as the coordination of the CPs, financial<br />
management, promotion of meetings and workshops,<br />
and compilation of results and evaluations. The Project<br />
has a consulting committee composed of the leaders<br />
of each PC and members from other institutions,<br />
helping in the strategic decisions. This committee has<br />
meetings each semester to monitor and evaluate the<br />
project. The CP1 is also in charge of looking for<br />
funding opportunities, intellectual property and visual<br />
identity (Figure 1) and actualization of the project<br />
home page (http://www.macroprograma1.cnptia.embrapa.br/biotecanimal).<br />
CP2 – Development and evaluation of new<br />
technologies for the sanitary control of semen,<br />
oocytes, and embryos. The activities of this CP are in<br />
the interface reproduction/animal health, and were<br />
proposed mainly focusing in the concern about the<br />
possibility of dissemination of pathogens by the use<br />
of reproductive biotechnologies, and the need to<br />
establish diagnose and control strategies. The research<br />
actions included in the CP range from pathogen<br />
molecular characterization to the development of<br />
specific diagnosis kits.<br />
CP3- Evaluation of nutritional strategies to<br />
improve reproductive efficiency. This CP is in the<br />
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R.A. Figueir<br />
igueiredo<br />
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Experience in Organizing a Research Project on Reprodutive... Acta Scientiae Veterinariae. 39(Suppl 1): s135 - s138.<br />
Figure 1. Logo of the Innovation Network in Animal Reproduction.<br />
interface reproduction/nutrition and has a general aim<br />
of to evaluate the influence of nutrition and body<br />
score condition on reproductive efficiency and on<br />
the production and survivability of gametes in<br />
livestock animals. The research actions deal not only<br />
with the effects of diet quantity and quality, but also<br />
with the effects of specific components of the diet.<br />
Besides, these effects are measured from the<br />
interference with the intra-follicular environment to<br />
pregnancy rates.<br />
CP4- Development of methods to improve<br />
the quality and quantity of gametes used in assisted<br />
reproductive technologies. This CP aims to support<br />
the improvement of in vivo and in vitro technologies<br />
by the study of farm animal gametogenesis, of<br />
gamete collection procedures and laboratory<br />
processing, and their subsequent use in artificial<br />
conditions. The relationship of environment, genetics,<br />
and folliculogenesis; the kinetics of nuclear<br />
maturation; the association of follicular development<br />
and DNA metilation; the effects of freezing on<br />
chromosome structure and cytoskeleton; the<br />
development of new procedures for sperm recovery<br />
and molecular markers of sperm fertility; and the use<br />
of sperm sorting techniques; are under study.<br />
CP5- Development of in vivo and in vitro<br />
embryo production systems. The aim of this CP is to<br />
identify bottlenecks in embryo production systems<br />
and develop technologies to improve embryo production,<br />
quality, and cryotolerance. The research<br />
subjects are the development of new embryo cryopreservation<br />
procedures, the improvement of hormonal<br />
synchronization and superovulation protocols, the<br />
evaluation of different approaches to improve the<br />
efficiency of in vitro embryo production, the<br />
development of new techniques of gamete and<br />
embryos micromanipulation, and the evaluation of<br />
gene expression patterns related to in vitro embryo<br />
development.<br />
CP6- Establishment of protocols for the<br />
isolation, culture and maintenance of cell lines aiming<br />
the production of animal clones. This CP focus in the<br />
development of cloning techniques, by the<br />
establishment of stable somatic and embryonic cell<br />
lines; by the molecular study of imprinted genes<br />
related to cloning; and by the development of new<br />
nuclear transfer techniques.<br />
CP7- Establishment of alternative protocols<br />
to the production of transgenic or intragenic animals.<br />
This CP aims to evaluate different approaches to the<br />
production of genetically modified cells, embryos,<br />
and animals; which could be able to express<br />
recombinant factors of interest. The effect of different<br />
chemicals in the transgene expression and alternative<br />
methods to produce transgenic embryos and animals N<br />
are under investigation.<br />
CP8- Identification, selection, use and conservation<br />
of genetic resources. This CP aims to evaluate<br />
the efficiency and impact of new reproductive<br />
biotechnologies in animal breeding programs. The<br />
project also evaluates the potential use of reproductive<br />
biotechnologies in the formation of specialized beef<br />
and dairy crossbred herds.<br />
CP9- Validation and monitoring of<br />
technologies. This CP has a transversal character, and<br />
aims to monitor the development of animals<br />
generated with the different biotechnologies<br />
developed in the others CPs, mainly those using in<br />
vitro culture systems and genetic manipulation.<br />
CP10- Technological innovation. Just like the<br />
CP9, this Project has a transversal character, and aims<br />
to promote the use of technologies generated and, or<br />
tested in the other CPs. The main strategy is to promote<br />
interplay of the research and development sector with<br />
the private activity, and generate an information<br />
network which could support large scale use and<br />
evaluation of new technologies, focusing technological<br />
innovation.<br />
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anco, A. Pellegr<br />
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igueiredo<br />
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Experience in Organizing a Research Project on Reprodutive... Acta Scientiae Veterinariae. 39(Suppl 1): s135 - s138.<br />
III. RESULTS OF THE NETWORK PROJECT<br />
The establishment of the network allowed the<br />
organization of Embrapa’s different ongoing research<br />
actions in animal reproduction in a large and<br />
multidisciplinary project. As consequences, there was<br />
a larger interaction among the different research<br />
groups of the company and external partners. The<br />
proposal of collaborative research and the<br />
consolidation of results improved the scientific<br />
production of the group and also the development of<br />
products, processes, and technical information to the<br />
private sector. This fact is demonstrated by the range<br />
of publications in basic and applied scientific journals,<br />
books, technical bulletins, patents and intellectual<br />
property requests [2]. The technology transference<br />
and capacity building activities were strategically<br />
reorganized in line with the scope of the Network<br />
Project. An example was the promotion of a special<br />
course in animal biotechnologies to the staff of the<br />
Agriculture Ministry. The network also converged<br />
some ongoing <strong>International</strong> Collaboration projects,<br />
as with East Timor, South Korea, Kenya and<br />
Tanzania. A last consequence of the network was the<br />
possibility of applying for consortium research<br />
funding opportunities.<br />
IV. FUTURE CHALLENGES AND PERSPECTIVES<br />
A network project has an increased complexity<br />
and demand a larger institutional infrastructure<br />
and personal support. Consequently, the success of<br />
this kind of project may requires the use of new<br />
coordination strategies, use of web tools to organize<br />
information, multi-institutional contracts with<br />
suppliers, etc., and the support of innovation agencies.<br />
We expect with the formation of this network,<br />
however, to improve scientific collaboration and to<br />
increase the relevance of R&D projects in animal<br />
reproduction. Another future possibility is the increase<br />
of the use of our technological platform in areas as<br />
wildlife conservation, bio-pharmaceutical production,<br />
reduction of sanitary barriers, etc., as well as the<br />
integration with other network projects such as animal<br />
genomics.<br />
The network may also improve the interplay<br />
with the government agencies in charge of the<br />
establishment of laws and rules for reproductive<br />
biotechnology used in livestock, allowing a better<br />
identification of the research demands of the<br />
productive sector and providing proper technical<br />
background for official decisions.<br />
A final goal of network project is to improve<br />
our capacity in identifying and measuring the impact<br />
of the new technologies in the different livestock<br />
production systems and, consequently, to show the<br />
importance of R&D for the agribusiness and for the<br />
people.<br />
REFERENCES<br />
1 Viana J.H.M., Siqueira L.G.B., Palhão M.P. & Camargo L.S.A. 2010. Use of in vitro fertilization technique in the last<br />
decade and its effect on brazilian embryo industry and animal production. Acta Scientiae Veterinariae. 38(2): 661-674.<br />
2 Empresa Brasileira de Pesquisa Agropecuária. 2010. Relatório de Acompanhamento de projeto. 196p.<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s139 - s146.<br />
ISSN 1679-9216 (Online)<br />
The<br />
Year 2009 Wor<br />
orld<br />
ldwide Sta<br />
tatistics tistics of Embryo Transf<br />
ansfer in Domestic Far<br />
arm Animals<br />
Summary of the Inter<br />
erna<br />
national Embryo Transf<br />
ansfer (IETS) Data Retr<br />
etrie<br />
ieval Committ<br />
ommittee<br />
Report<br />
Brad Stroud 1 & Gabriel A. Bó 2,3,4<br />
ABSTRACT<br />
Background: For the nineteenth consecutive year, the <strong>International</strong> Embryo Transfer Society (IETS) Data Retrieval Committee<br />
has global embryo transfer (ET) statistics to report. The goal of the IETS Data Retrieval Committee is to collect complete in<br />
vivo and in vitro embryo collection and transfer statistics from every ET practitioner in the world. Although, not all the ET<br />
activity that is performed world-wide is included in this report, it is the best report available and gives us a good indication<br />
about the trends and directions of the activity different parts of the world.<br />
Review: The number of bovine in vivo derived (IVD) embryos collected/flushed worldwide in 2009 was 704,000 compared to<br />
746,000 embryos in 2008. That’s a 5.6% decline. However, the number of embryos transferred is down by only 0.5% (from 539,000<br />
to 536,000). North America reported the largest downward slide. A depressed US economy coupled with the discovery of some<br />
lethal recessive genes in a popular breed of beef cattle are some of the main reasons for the drop in volume of ET activity there.<br />
The number of frozen IVD embryos transferred outnumbered fresh transfers by almost 50,000 (292,000 frozen and 244,000 fresh).<br />
The total number of transferrable bovine in vitro produced (IVP) embryos worldwide was 379,000 in 2009 compared to<br />
331,000 in 2008. This represents a 12.7% increase in production. Brazil again leads the global field of in vitro embryo<br />
production and transfers. The total number of IVP embryos transferred worldwide was 307,845. The efficiency of frozen IVP<br />
embryos will likely determine the acceptance of in vitro technology by other countries. So far, the majority of the IVP embryos N<br />
transferred have been fresh, not frozen (i.e. only 7% of the IVP embryos transferred in 2009 were frozen). However, that data<br />
varies according to different regions of the world. For example, Asia and Europe both reported that 58% of the IVP embryos<br />
transferred in those continents were frozen. That’s very similar to the percent of in vivo embryos transferred that are frozen in<br />
those same regions. Consequently, North America (primarily the US) and South America (primarily Brazil) both reported that<br />
only 4% of the in vitro produced embryos that had been transferred were frozen. Including in vivo and in vitro fresh and frozen,<br />
there were 49,465 more bovine embryos transferred in 2009 (843,862) as compared to 2008 (794,397). This represents a<br />
healthy 5.9% increase. Many data collectors could not separate beef and dairy embryo production so no attempt was made to<br />
estimate that ratio on a global basis. If all species are considered including in vivo and in vitro production, there were 1,139,981<br />
viable embryos collected / produced and 872,120 embryos transferred into recipients. Equine ET activity was down slightly<br />
in 2009. The number of reported flushes (36,971) was down by 7300 compared to 44,338 in 2008. However, the number of<br />
transfers (24,491) was only down by about 2500. Small Ruminant ET activity was up by about 40% over the previous year<br />
(36,199 transferable embryos were collected in 2009). Australia was the clear leader in ovine embryo production and transfers.<br />
Swine ET activity is very low worldwide.<br />
Conclusion: The volume of ET activity reported from all the committee’s regional data collectors indicates that the embryo<br />
transfer industry is doing well. Is also important to note that this report does not include every country’s statistics, and very few,<br />
if any, country has 100% of its activity represented; however, it is the best world-wide report available about the commercial<br />
embryo transfer business.<br />
Keywords: IETS, statistics, embryo transfer, in vivo, in vitro.<br />
Descritores: IETS, estatísticas, transferência de embriões in vivo, in vitro.<br />
1<br />
Chairman of the IETS Data Retrival Committee; Stroud Veterinary Embryo Service, Inc Weatherford TX, USA. 2 President of the IETS;<br />
Instituto de Reproduccion Animal de Cordoba (IRAC), Cordoba, Argentina, and 3 Instituto de Ciencias Básicas, Carrera de Medicina Veterinaria,<br />
Universidad Nacional de Villa Maria, Villa del Rosario, Cordoba, Argentina. CORRESPONDENCE: G.A. Bo [gabrielbo@iracbiogen.com.ar].<br />
Instituto de Reproduccion Animal de Cordoba (IRAC), Paraje Pozo del Tigre, Zona Rural General Paz, 5145, Cordoba, Argentina.<br />
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I. INTRODUCTION<br />
II. REPORT OF BOVINE IN VIVO DERIVED EMBRYOS<br />
IN 2009<br />
III. REPORT OF BOVINE IN VITRO PRODUCED<br />
EMBRYOS IN 2009<br />
IV. THE OVERALL ACTIVITY OF ET IN OTHER<br />
SPECIES IN 2009<br />
V. CONCLUSIONS<br />
I. INTRODUCTION<br />
For the nineteenth consecutive year, the<br />
<strong>International</strong> Embryo Transfer Society (IETS) Data<br />
Retrieval Committee has global embryo transfer (ET)<br />
statistics to report [1]. The goal of the IETS Data<br />
Retrieval Committee is to collect complete in vivo<br />
and in vitro embryo collection and transfer statistics<br />
from every ET practitioner in the world. Although it<br />
is very difficult to collect the data from so many<br />
different regions in the world, previous chairperson<br />
of the committee, Dr. Michel Thibier of France, did a<br />
superb job creating data templates and selecting regional<br />
collectors who were willing to sacrifice their<br />
personal time calling on practitioners in their<br />
geographical areas to submit their previous year’s<br />
information. Countries that are members of regional<br />
ET societies or associations i.e., the American Embryo<br />
Transfer Association (AETA), the European Embryo<br />
Transfer Association (AETE), the Canadian Embryo<br />
Transfer Association (CETA), the Brazilian Embryo<br />
Technology Society (<strong>SBTE</strong>) to name a few, are well<br />
organized and have established collection protocols<br />
that make reporting consistent from year-to-year.<br />
Additionally, many countries do not have associations<br />
and data collectors in those countries must call on<br />
the ET practitioners that they know personally to<br />
provide them with data. Certainly, not all the ET<br />
activity that is performed worldwide is included in<br />
this report. However, it is the best report available of<br />
the activity world-wide and efforts are on-going to<br />
improve the system.<br />
II. REPORT OF BOVINE IN VIVO DERIVED EMBRYOS IN<br />
2009<br />
For the third consecutive year the reported<br />
number of flushes and the number of IVD embryos<br />
transferred into recipients has declined (Table 1).<br />
Although Asia, Europe and Oceania have reported healthy<br />
increases in the number of flushes (18%, 13%, and<br />
400% respectively), North America, South America,<br />
and Africa, all reported decreases. Overall, there was<br />
a 7% reduction in the number of flushes reported<br />
worldwide from 111,806 in 2008 to 104,282 in 2009.<br />
The most noteworthy decline in the number of flushes<br />
came from North America where there was a reduction<br />
of 16,277 from 2008 to 2009.<br />
The biggest decline in that region came from<br />
the US where there was a reduction of 15,147 flushes<br />
reported, which represents a 29% drop in one year.<br />
Mexico, under the direction of regional collector S Romo,<br />
has collected data from 14 ET teams which is the most<br />
from this country in the short history of this committee.<br />
Collectively, they reported 1875 flushes and 10,270<br />
transferrable embryos collected. The ratio of beef to dairy<br />
donors flushed was about 4:1 in favor of beef. Close to<br />
9000 bovine embryos were reported transferred in Mexico<br />
in 2009. That number however includes both IVD and<br />
IVP embryos. Even with a sharp decline in the volume<br />
of IVD embryos transferred in 2009 North America still<br />
accounts for 46% of the reported in vivo derived embryo<br />
transfers worldwide. In the US, for at least a decade the<br />
ratio of beef to dairy collections has been 2:1. In 2009<br />
that changed to 1.5:1. There was a drop of 7,000 dairy<br />
flushes in 2009 and a drop of 9,000 beef flushes in the<br />
US. Canada reported a high preponderance of dairy to<br />
beef flushes (4.8:1 ratio) in 2009. The ratio in 2000 was<br />
about 3:1, so dairy is playing a more significant role in<br />
Canada in recent years.<br />
Africa’s flush numbers were down from 2389 in<br />
2008 to 1446 in 2009. However, there was an increase in<br />
the number of frozen embryos transferred in 2009, which<br />
overall brought them back equal to last year’s overall<br />
number of transfers. The majority of Africa’s ET data is<br />
generated from the Republic of South Africa.<br />
Asian data generated in 2009 came exclusively<br />
from Japan. One hundred percent of the flushes and fresh<br />
transfers that were reported by Asia were done in Japan.<br />
Based on reported data Asia is responsible for transferring<br />
about 15% of the world’s in vivo derived embryos.<br />
Japan showed an increase in flushes, fresh transfers,<br />
and frozen transfers. A serious effort to recruit data<br />
collectors from Asia is necessary. No doubt ET is being<br />
performed in at least some of the 44 countries in that<br />
continent that are currently not reporting. As of now<br />
there are only five countries with designated data<br />
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collectors; Dochi from Japan, Nguyen from Vietnam,<br />
R Parnpai from Thailand, and Seok, Lee, and Son<br />
from S Korea, plus SN Lee from Taiwan. However,<br />
as previously mentioned, Dochi from Japan is the<br />
only one reporting data for the 2009 calendar year.<br />
There are reports from Parnpai that Thailand is trying<br />
to organize an ET society which will help them gather<br />
data from member practitioners in the future.<br />
Europe, a continent of 47 countries, has 25<br />
countries with data collectors for AETE. As always,<br />
they are very thorough and prompt with their data<br />
reporting. The number of flushes is up from 2008 by 13%<br />
(from 14,894 to 16,856). <strong>Annual</strong>ly, Europe is responsible<br />
for transferring about 18% of the world’s IVD embryos.<br />
Globally, more frozen in vivo derived<br />
embryos were transferred in 2009 than fresh embryos<br />
(291,000 and 244,000 respectively). That statistic held<br />
true for every continent except South America where<br />
four times as many fresh embryos were transferred<br />
than frozen. One of the reasons is due to the large<br />
number of available recipients in Brazil and Argentina.<br />
Also, many more in vitro produced embryos<br />
(Table 2) are transferred in Brazil where fresh transfers<br />
are preferred to frozen.<br />
Brazil reported a significant drop of 25,000<br />
in the number of in vivo derived embryos transferred<br />
in 2009. However, the difference was made up with<br />
256,000 in vitro produced embryos being transferred,<br />
which was an increase of 34,000 from 2008. Uruguay<br />
now has two regional data collectors, S Kmaid and P<br />
Bañalas, who reported 779 flushes of which 21% was<br />
dairy and 79% beef. Argentina reported close to 450<br />
dairy collections along with 3400 beef flushes. They<br />
also reported 14,385 fresh and frozen transfers<br />
combined.<br />
Oceania, especially Australia, has a history<br />
of reporting only a small percentage of the ET that is<br />
actually done there. Although Australia has a<br />
Table 1. Bovine In Vivo Derived Embryo Activity in 2009.<br />
CONTINENT<br />
Flushes<br />
Transferrable<br />
Embryos<br />
Number of Transferred Embryos<br />
Fresh Frozen Total %<br />
Asia 10,924 112,783 22,958 53,172 76,130 14.23%<br />
N<br />
Europe 16,856 106,495 43,999 51,074 95,073 17.77%<br />
N. America 52,921 347,531 111,106 137,599 248,705 46.47%<br />
S. America 12,065 67,093 42,876 17,220 60,096 11.23%<br />
Oceania 10,070 60,200 18,522 27,573 46,095 8.54%<br />
Total 104,282 704,230 243,885 291,279 535,164<br />
2008 Totals 111,806 746,250 242,006 297,677 539,683<br />
Per Cent Decline -6.73% -5.63% 0.78% -2.15% -0.84%<br />
Table 2. The Top Five Countries Outside Europe and North America<br />
in 2009 (based on number of bovine in vivo embryos transferred).<br />
Number Bovine Embryos<br />
Country Number of Flushes<br />
Transferred<br />
Japan 10,924 75,706<br />
Australia 10,020 46,095<br />
Brazil NA 42,383<br />
Argentina 3846 14,385<br />
S Africa 1,421 8,753<br />
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veterinary society, the Australian Reproduction<br />
Veterinarians (ARV)) that encompasses the science<br />
of ET, a veterinarian must first be a member of the<br />
Australian Veterinary Association (AVA) before<br />
joining the ARV. There is also a contingency of nonveterinary<br />
ET practitioners that are not members of<br />
the veterinary association and are not organized. It’s<br />
been very difficult for the regional collector, R Pashen,<br />
to communicate with both groups. As a result of the<br />
confusion the bovine data from Australia is not complete.<br />
There was no separation between dairy and beef ET<br />
activity reported in Australia. According to the regional<br />
data collector (L Frers) New Zealand does not flush many<br />
donors (35 dairy and 15 beef) in 2009, but leaned<br />
more towards in vitro embryo production using sex<br />
sorted semen.<br />
III. REPORT OF BOVINE IN VITRO PRODUCED<br />
EMBRYOS IN 2009<br />
Globally, the number of in vitro produced<br />
(IVP) embryos was up by 47,000 (12%) from a year<br />
ago (Table 3). Brazil was responsible for 68% of the<br />
IVP production, while Japan produced 20% of the<br />
world’s IVP embryos (Table 3). The number of in<br />
vitro produced embryos transferred also showed a<br />
healthy 17% increase from 254,000 in 2008 to<br />
307,000 in 2009. One of the factors that could be<br />
attributing to a decrease in the in vivo activity of cattle<br />
worldwide is the increase of in vitro embryo production,<br />
especially in South America. Again, the one<br />
valuable resource that Brazil has that some countries<br />
don’t is a large number of recipients. With a national<br />
herd estimated at almost 200 million head, Brazil<br />
alone is amply supplied with potential recipients.<br />
If one compares the number of IVD to the<br />
number of IVP produced embryos transferred annually<br />
over the past decade (see Figure 1) the lines are beginning<br />
to converge meaning that the number of in vivo and<br />
in vitro embryos transferred could be on a collision<br />
course for similarity. However, Figure 2 illustrates that<br />
much of the in vitro production is coming from South<br />
America, mainly Brazil. Asia comes in second place,<br />
but their numbers of transferred IVP embryos are<br />
declining in recent years. China did not report any<br />
data in 2009 after having reported over 50,000 in<br />
vitro produced embryos transferred in 2005. Currently<br />
there is no regional data collector for the country of<br />
China, so Figure 2 could be misleading as to the rise<br />
and fall of the transfer of in vitro produced embryos<br />
from Asia. The rest of Figure 2 clearly shows that<br />
other continents are stable with the activity of IVP<br />
embryos, and the number of transfers is much lower<br />
in those regions of the world.<br />
It will be interesting to see in the coming years if<br />
this shift in technology will continue to trend in countries<br />
other than those in South America and Asia towards the<br />
favor of in vitro embryo production.<br />
The efficiency of frozen IVP embryos will likely<br />
determine the acceptance of in vitro technology by<br />
other countries. So far, the majority of the IVP<br />
embryos transferred have been fresh, not frozen. Data<br />
from Table 3 indicates that overall only 7% of the<br />
IVP embryos transferred in 2009 was frozen.<br />
However, that data varies according to different regions<br />
Table 3. Bovine In Vitro Produced Embryos in 2009.<br />
Continent<br />
Transferrable<br />
Embryos<br />
Number of Transferred Embryos<br />
Fresh Frozen Total %<br />
Afria 400 100 0 100 0.03%<br />
Asia (Japan) 80775 5923 7764 13687 4.46%<br />
Europe<br />
(8 countries)<br />
7653 2389 3419 5808 1.89%<br />
N. America 20390 17850 807 18657 6.07%<br />
S. America 257701 247518 10093 257611 83.85%<br />
Oceania 11325 10666 683 11349 3.69%<br />
Total 378244 284446 22766 307212<br />
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Figure 1. Comparison of the number of in vivo and in vitro embryos transferred annually for the past decade.<br />
N<br />
Figure 2. The trends of in vitro produced embryos transferred by continents since 2000.<br />
of the world. For example, Asia and Europe both<br />
reported that 58% of the IVP embryos transferred in<br />
those continents were frozen. That’s very similar to<br />
the percent of in vivo embryos transferred that are<br />
frozen in those same regions. Consequently, North<br />
America (primarily the US) and South America<br />
(primarily Brazil) both reported that only 4% of the<br />
in vitro produced embryos that had been transferred<br />
were frozen.<br />
In Europe eight of the twenty-five countries<br />
reporting ET statistics either produced or transferred<br />
in vitro produced embryos. Germany led with 3271<br />
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in vitro embryos produced, whereas Italy transferred<br />
the most IVP embryos - 2006. The Netherlands were<br />
second in both embryos produced and transferred<br />
(2377 and 1930 respectively).<br />
Japan’s in vitro production came mainly from<br />
abattoir derived oocytes, but hey reported 1225 OPU<br />
sessions which produced 12,919 oocytes and 2256<br />
transferrable embryos (17.5% efficiency). Additionally,<br />
they produced 78,519 viable embryos from 674,584<br />
abattoir oocytes (11.6% efficiency). South Korea, who<br />
has reported in vitro production in past years, failed<br />
to report any ET data in 2009.<br />
Of the five countries reporting from Africa only<br />
Kenya reported in vitro activity. They produced 400<br />
IVP embryos of which one hundred were transferred<br />
fresh and the others were frozen.<br />
Canada, USA, and Mexico, for the first time, all<br />
reported in vitro produced embryos from North America.<br />
Canada produced 564 dairy and 20 beef IVP embryos.<br />
All of the US in vitro produced embryos were a<br />
product of OPU. 4885 OPU sessions yielded 80,505<br />
oocytes and 19,806 transferrable embryos for an<br />
efficiency rate of almost 25%, which could be<br />
misleading if some of the ET teams failed to report<br />
their oocytes and only reported transferrable embryos.<br />
Two ET groups in Mexico reported a combined 238<br />
OPU procedures resulting in 617 transferrable<br />
embryos. Including transfer data from a third<br />
practitioner, Mexican teams reported 504 fresh IVP<br />
embryo transfers of which 86 were frozen abattoir<br />
derived IVP embryos.<br />
As previously stated Brazil is the major IVP<br />
player in South America, but it should be noted that<br />
Uruguay reported 350 OPU sessions producing 1398<br />
embryos. Uruguay also produced 270 abattoir derived<br />
IVP embryos. In total, Uruguay reported transferring<br />
1613 In Vitro Produced embryos.<br />
Oceania produced a fair number of in vitro<br />
produced embryos. New Zealand reported 896 OPU<br />
sessions and 1725 transferrable embryos. Australia did<br />
not report the number of OPUs, but did produce 9600<br />
transferrable in vitro produced embryos. The reported<br />
total number of bovine In Vivo Derived and In Vitro<br />
Produced embryos transferred in 2009 worldwide was<br />
843,862 which is an increase of 49,462 transfers from<br />
the 2008 total of 794,397.<br />
IV. THE OVERALL ACTIVITY OF ET IN OTHER SPECIES IN<br />
2009<br />
In this report, statistics are recorded for three<br />
species of small ruminants; sheep, goats, and deer (Table<br />
4). The reported number of viable sheep embryos flushed<br />
in 2009 was 13,000 more than in 2008 (32,768 and 18,828<br />
respectively). That’s close to a 40% increase. The<br />
number of transfers in sheep was probably up too, but<br />
Australia did not report how many of their world leading<br />
25,000 embryos collected were transferred. South Africa<br />
followed Australia in sheep embryo production. They<br />
flushed 925 sheep and collected 5426 viable embryos,<br />
but only transferred 35 fresh. Mexico was third with 170<br />
flushes and 1056 transfers. In Europe, Turkey and the<br />
Czech Republic combined to collect 197 viable embryos,<br />
and transferred 143. R Mapletoft of Canada reported<br />
137 flushes and 565 transferrable embryos recovered in<br />
his country. He also reported work done in Chile by a<br />
Canadian ET practitioner where 105 frozen embryos were<br />
transferred. Mapletoft also reported sheep ET performed<br />
in Mexico by a Canadian team. Forty donors were flushed<br />
resulting in 241 transferrable embryos of which all were<br />
transferred fresh. Mapletoft again reported a Canadian<br />
team transferring 104 ovine embryos in the Bahamas.<br />
Table 4. Small ruminant ET activity in 2009.<br />
Species<br />
Transferrable<br />
Embryos<br />
Number of Transferred Embryos<br />
Fresh Frozen Totals<br />
Sheep Total 32768 1326 408 1734<br />
Goat Total 2478 206 146 352<br />
Cervids Total 953 941 0 941<br />
Total 36199 2473 554 3027<br />
*The number of sheep embryos transferred is misleading. Australia reported collecting<br />
25,000 transferrable embryos from 4100 flushes in 2009, but no transfers.<br />
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From South America, Uruguay reported 96 flushes<br />
and 502 fresh ovine transfers.<br />
Goat collections and transfers were also down<br />
significantly in 2009. Only 352 embryos were<br />
reported being transferred worldwide compared to<br />
an all time high of 20,000 in 2006. Although Australia<br />
reported collecting 450 caprine embryos they did not<br />
report any transfers. Goat embryo activity in the US<br />
is vastly underreported based on verbal<br />
communication of the committee chairperson with<br />
goat breeders across the country. Only a few bovine<br />
ET practitioners in the US are performing caprine ET.<br />
The small ruminant ET practitioners seem disinterested<br />
in joining the AETA so they don’t get ET data surveys<br />
sent to them by the association. This is potentially a<br />
problem in other countries as well. However, the<br />
combined CETA/AETA 2010 annual conference had<br />
a small ruminant wet lab that could open the door to<br />
this group in the future, at least in North America.<br />
Mexico reported 32 goat flushes resulting in 170<br />
transferable embryos all of which were transferred<br />
fresh. Mapletoft again gets credit for reporting goat<br />
transfers in the United Arab Emirates by a Canadian<br />
team. They reported 19 flushes, 173 viable embryos<br />
recovered, and 36 fresh transfers.<br />
The volume of cervid ET has remained constant<br />
from 2008 to 2009. New Zealand is the only country<br />
reporting cervid activity, but the regional collector in New<br />
Zealand was not the reporter. Instead, Mapletoft reported<br />
that one of his teams from Canada collected 953 in<br />
vivo produced embryos, and transferred 941 fresh<br />
embryos in that country.<br />
The total number of equine flushes was down<br />
from 44,000 in 2008 to 37,000 in 2009 (Table 5). Based<br />
on the number of transfers the top three countries<br />
performing equine ET are Brazil (41% of world’s activity),<br />
Argentina (31% of activity), and the United States (20%<br />
of activity). North America reported the largest drop from<br />
13,500 to 10,000. P McCue, the new regional data collector<br />
from the USA did a thorough job of soliciting the equine<br />
breed associations in the US for accurate data. The<br />
American Quarter Horse Association (AQHA), which<br />
is the largest breed association that allows registration of<br />
ET foals, reported an all time high of 4069 ET foals in<br />
2007. The last two years showed consecutive declines<br />
of ET foals of 3288 and 2458 respectively. Brazil remained<br />
stable with 14,100 flushes which was only 100 fewer<br />
than 2008. Argentina was down in flushes but stable with<br />
transfers. Canada was down from 42 mares flushed in<br />
2008 to 26 in 2009. Collectively, Europe reported 1024<br />
flushes in 2009, which was down from 1216 the previous<br />
year. Although the specific country is unclear, S Merton<br />
from Europe reported that 60 in vitro produced equine<br />
embryos were transferred in 2009 (Italy) which is up<br />
from 48 in 2008. There was no equine activity reported N<br />
from Asia in 2009 or 2008. The Republic of South Africa<br />
was the only country reporting equine flushes in 2009<br />
from the African continent. They more than doubled their<br />
Table 5. Equine ET Activity in 2009.<br />
Country<br />
Flushes<br />
Transferrable<br />
Embryos<br />
Number of Transferred Embryos<br />
Fresh Frozen Total %<br />
Argentina 10800 7560 7500 7500 30.65%<br />
Brazil 14100 10100 10100 15 10115 41.34%<br />
Canada 26 22 22 0 22 0.09%<br />
Europe 1024 1037 1037 0 1037 4.24%<br />
South Africa 162 120 120 0 120 0.49%<br />
Australia 910 710 710 710 2.90%<br />
USA 9933 4966 4966 0 4966 20.29%<br />
Total 36955 24515 24455 15 24470<br />
2008 Totals 44338 27082 26606 379 26985<br />
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2008 count of 56 flushes to 120 in 2009. South<br />
America dominated the equine ET world by reporting<br />
72% of all that species activity. Oceania, by way of<br />
Australia, reported 910 flushes (up from 24 in 2008)<br />
and 710 transfers.<br />
The reported number of swine flushes is down<br />
by 80% in 2009 (Table 6). The USA was largely<br />
responsible for the sharp decline. In 2008 the US<br />
reported 134 flushes, but only 9 flushes for 2009.<br />
Those 9 collections were reported by Mapletoft from<br />
Table 6. Swine ET activity in 2009.<br />
Country<br />
Flushes<br />
Transferrable<br />
Embryos<br />
Number of Transferred Embryos<br />
Fresh Frozen<br />
Total &<br />
Percentage<br />
Canada 10 325 380 380 48.72%<br />
Czech Republic NR 716 20 0 20 2.56%<br />
USA* 9 132 0 0 0 0.00%<br />
France** 10 325 380 0 380 48.72%<br />
Total 29 1498 780 0 780<br />
2008 Totals 149 3800 3092<br />
*All embryos exported **325 embryos exported.<br />
Canada. It’s unclear if swine ET activity is actually<br />
down or the data is not being reported. Again, the<br />
swine ET industry is not associated with the AETA in<br />
the USA, so the regional collector has no connections<br />
to anyone performing swine embryo transfers.<br />
V. CONCLUSIONS<br />
The volume of ET activity reported from all<br />
the committee’s regional data collectors indicates that<br />
the embryo transfer industry is doing well. As always<br />
some country’s data is up and others are down.<br />
Anyone reading this report should take into<br />
consideration that it does not include every country’s<br />
statistics, and very few, if any, country has 100% of<br />
its activity represented. To guess what percent of the<br />
world’s actual ET is represented in this document<br />
would be unprofessional at best, so no attempt will<br />
be made to do so; however, it is the best world-wide<br />
report available about the commercial embryo<br />
transfer business.<br />
Acknowledgments. The chairman would like to thank all<br />
the regional and country collectors that spend a<br />
considerable portion of their personal time each summer or<br />
winter (depending on the hemisphere) calling and emailing<br />
their fellow practitioners for all their data. It is a thankless<br />
job, but the Statistics and Data Retrieval Committee is<br />
perhaps one of the most important the IETS has. For 19<br />
consecutive years the data has been gathered, assimilated,<br />
and published for the world to see. For as many man hours<br />
that it takes to prepare the report a great deal of gratitude<br />
should be given to those involved. Lastly, we would like to<br />
thank Dr. Michel Thibier, the former committee chair, for his<br />
sincere dedication and time.<br />
REFERENCE<br />
1 Stroud B. 2010. IETS Statistics and Data Retrieval Committee Report. The year 2009 worldwide statistics of embryo transfer in<br />
domestic farm animals, IETS Newsletter December 2010, <strong>International</strong> Embryo Transfer Society, 2441 Village Green Place,<br />
Champaign, IL 61822 USA. 28(4): 11-21. Available at:<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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.W. Tha<br />
hatcher<br />
cher. <strong>2011</strong>. Temporal Historical Observations, Rapidly Expanding Technological Tools, and Integration of<br />
Scientific Disciplines to Enhance Reproductive Performance... Acta Scientiae Veterinariae. 39(Suppl 1): s147 - s169.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s147 - s169.<br />
ISSN 1679-9216 (Online)<br />
Temp<br />
empor<br />
oral Histor<br />
oric<br />
ical Obser<br />
bservations<br />
tions, Rapidly Expanding Technolo<br />
echnologic<br />
gical<br />
Tools<br />
ols, and<br />
Integr<br />
egration of Scientific Disciplines to Enhance Repr<br />
eproduc<br />
ductiv<br />
tive Per<br />
erfor<br />
ormanc<br />
mance of<br />
Lacta<br />
tating ting Dair<br />
airy Cows:<br />
The Fost<br />
oster Mothers of The Human Rac<br />
ace<br />
William<br />
W. Tha<br />
hatcher<br />
ABSTRACT<br />
Background: Lactating dairy cow of the 21st century is considered to be sub-fertile after intensive selection for milk yield.<br />
production of the dairy cows exemplifies the progress that can be made in the application of technology and holistic dairy cow<br />
management to optimize production. The hormonal and metabolic responses, associated with homeorrhetic and homeostatic<br />
regulatory responses to partition nutrients for lactation, coupled with intensive management contribute to the reduction in<br />
fertility. The numerous postpartum reproductive and metabolic disorders are associated with sub-optimal fertility in the<br />
breeding period. Continual advancements have been made in reproductive/lactation physiology, endocrinology, nutrition,<br />
herd health and management to improve reproductive herd fertility on commercial dairies. Objective of this presentation is to<br />
focus on current technologies and experimental approaches relative to their application in unraveling certain biological<br />
windows, which will further impact our ability to enhance reproductive efficiency coupled with increased food production<br />
and well-being of both animals and humans.<br />
Review: Feeding of omega 6- and omega 3- polyunsaturated fatty acids exert pro- and anti- inflammatory effects on the innate<br />
immune system that increases subsequent reproductive performance. Chronic exposure to a GnRH-agonist induced a marked<br />
atrophy of the postpartum uterus. Colostrum contains a pleothera of growth factors (lactocrine secretions) that influence<br />
uterine developmental programming in the pig, and neonatal exposure to estrogens/progesterone in pigs or cattle alters early<br />
programming of the uterus leading to dysfunctional reproductive tract consequences in the adult. Physiological systems to<br />
optimize ovarian and uterine function have led to timed insemination fertility programs that have enhanced herd pregnancy<br />
N<br />
rates. Sequencing of the bovine genome has provided an array of new technological approaches to unravel the multi-factorial<br />
control systems to support conceptus-placental development throughout gestation and avoid pregnancy failure. A bovine<br />
microarray identified genes that were differentially expressed in conceptus and endometrial tissues at day 17 post-LH surge in<br />
cyclic and pregnant cows that were lactating or nonlactating. Expression of PAG genes within the conceptus and endometrium<br />
of pregnant cows and their association with other genes determined by standard partial correlation analyses infer a possible<br />
role of PAG in pregnancy maintenance and implantation by regulation of embryo development, trophoblast cell invasion,<br />
immune regulation, and prostaglandin metabolism. The associations detected are suggestive of potential pathways for<br />
investigation in early pregnancy at day 17 involving potential direct and indirect effects of PAG 11 produced by the conceptus.<br />
Development of microarrays of single nucleotide polymorphisms (SNP) across the bovine genome has led to Genomic<br />
Predicted Transmitting Abilities (GPTA) for various production traits including daughter pregnancy rates. More specific<br />
physiological reproductive traits have sufficient heritabilities that warrant consideration for selection. Furthermore, current<br />
technological advances are identifying candidate “fertility” genes for potential genetic selection. selection for production,<br />
health and reproductive traits will be the wave of the future as genomic and bioinformatic tools continue to be expanded and<br />
refined.<br />
Conclusion:This manuscript targets biological windows and technological advancements to improve reproductive performance<br />
of lactating dairy cows. Epidemiological analyses reveal that healthy postpartum lactating dairy cows are indeed fertile.<br />
Chronic exposure to a GnRH agonist induced postpartum uterine atrophy warranting additional research as to potential<br />
strategies to improve uterine health. Feeding of nutraceuticals such as polyunsaturated omega-6 and omega-3 fatty acids<br />
improves postpartum innate immune function and subsequent reproductive performance. Lactocrine secretions in colostrum<br />
and neonatal exposure to estrogens and progesterone influence uterine developmental programming related to subsequent<br />
reproductive competence. Reproductive management programs that optimize ovarian and uterine function permit a fertile<br />
single timed insemination to first and second inseminations. The sequencing of the bovine genome has led to thorough<br />
University of Florida, Department of Animal Sciences, Institute of Food and Agricultural Sciences. CORRESPONDENCE: W.W. Thatcher<br />
[thatcher@ufl.edu – PHONE: +1 (352) 392-5590]. Department of Animal Sciences, Institute of Food and Agricultural Sciences, PO Box<br />
110910, Gainesville, FL 32611-0910, USA.<br />
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.W. Tha<br />
hatcher<br />
cher. <strong>2011</strong>. Temporal Historical Observations, Rapidly Expanding Technological Tools, and Integration of<br />
Scientific Disciplines to Enhance Reproductive Performance... Acta Scientiae Veterinariae. 39(Suppl 1): s147 - s169.<br />
characterizations of the endometrium and conceptus transcriptones in response to key physiological periods such as pregnancy<br />
and lactation. Early expression of Pregnancy Associated Glycoprotein (PAG) genes within the conceptus and endometrium of<br />
pregnant cows and their association with other genes infer a possible role of PAG in pregnancy maintenance and implantation.<br />
The array of SNPs across the bovine genome and specific SNPs within candidate genes related to reproductive processes and<br />
fertility will enhance genetic selection for fertility along with production and health associated traits.<br />
Keywords: genomics, timed insemination, immunosuppression, transcriptome analysis, neonate, colostrum, uterus.<br />
I. INTRODUCTION<br />
Modern dairy practices require considerably<br />
fewer resources than dairying in 1944 with 21% of<br />
animals, 23% of feedstuffs, 35% of the water, and<br />
only 10% of the land required to produce the same 1<br />
billion kg of milk in the USA. Increased production<br />
of the dairy cows exemplifies the progress that can<br />
be made in the application of technology. Continual<br />
and expanding advances in technology across<br />
scientific disciplines hopefully will meet the majority<br />
of the global needs for food production to feed a<br />
continual expanding world population, since land use<br />
and water availability for agriculture is limited. Such<br />
efforts will require multi-interdisciplinary scientific<br />
efforts, political re-programming to internationalize<br />
agricultural production and distribution, and<br />
education of both producers and consumers as to the<br />
application of technology to produce safe foods.<br />
The lactating dairy cow of the 21st century is<br />
considered to be sub-fertile after intensive selection<br />
for milk yield. The hormonal and metabolic responses,<br />
associated with homeorrhetic and homeostatic<br />
regulatory responses to partition nutrients for lactation,<br />
coupled with management contribute to the reduction<br />
in fertility. Continual advancements have been made<br />
in reproductive/lactation physiology, endocrinology,<br />
nutrition, herd health and management to improve<br />
reproductive herd fertility on commercial dairies.<br />
Current scientists and those in training have<br />
exciting repertoires of experimental models and<br />
technological tools to bring to bear in improving animal<br />
productivity with the use of the whole animal<br />
and various aspects of molecular and cell biology.<br />
However, an understanding of the scientific past is<br />
very insightful and can improve the efficiency of one’s<br />
career and that of their cooperative multi-interdisciplinary<br />
efforts. To make this point, in particular<br />
to trainee’s, a vivid example of the past that was<br />
extremely insightful to the author was the publication<br />
of Dr. Joseph Halbane’s (Figure 1;[29]). “EmbryonoalImpulse”<br />
represents rapid growth of the fetal<br />
mammary gland in the 8 th and 9 th months of<br />
s148<br />
pregnancy. Neonatal swelling and regression of the<br />
mammary gland and uterus were due to active<br />
substances from the placenta and their withdrawal.<br />
There is a “Puberty Impulse” on the mammary gland<br />
and uterus due to activity of the ovaries, and this was<br />
further characterized by ovariectomy and retransplantation<br />
experiments. After puberty, periodic<br />
swelling of the mammary gland and uterus occurs<br />
with re-occurring estrous cycles (i.e., in humans the<br />
“MenstrualImpulse”). There is the “Pregnancy<br />
Impulse” of rapid proliferation with hyperplasia of<br />
the glandular tissue but at a much greater rate than<br />
post-pubertal changes leading to the inference that<br />
placenta produces more regulatory substances than<br />
does the ovary. Pregnancy changes were not due to<br />
fetus but due to the placenta since growth proceeds<br />
after death of the fetus and subsides with loss of placenta.<br />
Secretions produced by epithelium of the placenta<br />
(i.e., tropholast and chorionic epithelium) and<br />
not stromal tissue were inferred. The CL persists<br />
during pregnancy under influence of the placenta.<br />
Changes in the maternal and fetal uterus were<br />
correlated due to the fact that both regress in the<br />
postpartum or puerperium period. Puerperal<br />
involution of the maternal uterus occurs only after<br />
delivery of the placenta and was considered a true<br />
atrophy. Emptying of the uterus was considered<br />
critical to onset of milk secretion (i.e., 3-4 days after<br />
birth). Secretion before placental delivery was characterized<br />
as colostrum not milk. Suckling did not induce<br />
milk secretion and only maintained secretion after<br />
the uterus is emptied and suckling was associated<br />
with quiescence of the ovary.<br />
Insightful components in Figure 1 relate to<br />
several physiological periods such as the periparturient<br />
period in which distinct changes are being<br />
programmed on both the newborn relative to potential<br />
function of the uterus, mammary gland and well-being<br />
of the newborn, and the maternal processes of<br />
mammary gland lactogenesis, regression of the uterus,<br />
homeorethetic/homeostatsis responses to support<br />
lactation, and immune status. All of these processes<br />
influence efficiency of production and reproduction.
W.W<br />
.W. Tha<br />
hatcher<br />
cher. <strong>2011</strong>. Temporal Historical Observations, Rapidly Expanding Technological Tools, and Integration of<br />
Scientific Disciplines to Enhance Reproductive Performance... Acta Scientiae Veterinariae. 39(Suppl 1): s147 - s169.<br />
Also note that the distinct differences Halbane [29]<br />
deduced following pregnancies in the non-suckled<br />
and suckled states. Non-suckled or non-lactation<br />
resulted in a coupled decrease in both the uterus and<br />
mammary gland and an earlier recrudescence of<br />
ovarian cycles. In contrast, the suckled or lactating<br />
state resulted in a greater rate of uterine regression<br />
and a marked delay in recrudescence of ovarian<br />
cycles. Presently, following 105 years after these initial<br />
observations in a human clinical practice and a<br />
pleothera of animal husbandry publications to<br />
improve animal management and production, major<br />
new findings and experimental approaches are being<br />
applied to further optimize the productivity and wellbeing<br />
of animals and humans.<br />
Objective of this presentation is to focus on<br />
current technologies and experimental approaches<br />
relative to their application in unraveling certain<br />
biological windows described by Halbane [29], which<br />
will further impact our ability to increase food<br />
production and well-being of both animals and<br />
humans.<br />
II. PERIPARTUM AND POSTPARTUM EFFECTS ON<br />
MATERNAL AND NEONATAL REPRODUCTIVE FUNCTION<br />
Genetic and environmental differences<br />
among cows in the peripartum period are associated<br />
with various endocrine and biochemical systems<br />
regulating postpartum milk secretion, ovarian<br />
recrudescence, uterine regression, and health of the<br />
maternal unit as well as the newborn. Furthermore,<br />
the influence of the peripartum period on subsequent<br />
reproductive processes has been somewhat of a<br />
myoptic view that has not integrated the collated<br />
physiological endocrine, immune and nutritional<br />
physiology on reproductive processes. The<br />
conceptus (i.e., fetus and placenta) may be central to<br />
regulation of these various processes [59]. A series<br />
N<br />
Figure 1. Original figure translated from German entitled : “The Inner secretion of the Ovary and Placenta and its Importance for Function<br />
of the Mammary Gland” (J. Halban 1905 Archiv fur Gynakologie 75:353; in: Obstet Gyn 1955 6(5):559-565); Translations:Mamma<br />
(Mammary Gland), Lunarmonat (lunar month), Geburt ( birth), Wochen postpartum (weeks postpartum), Kindheit (childhood), Pubertat<br />
(puberty), Menstruelle Schwellungen (menstrual swellings), Puerperale Invol. Ohne Stillen (postpartum involution without breast<br />
feeding), Klimakterium (menopause), Senium (senescence).<br />
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.W. Tha<br />
hatcher<br />
cher. <strong>2011</strong>. Temporal Historical Observations, Rapidly Expanding Technological Tools, and Integration of<br />
Scientific Disciplines to Enhance Reproductive Performance... Acta Scientiae Veterinariae. 39(Suppl 1): s147 - s169.<br />
of experiments demonstrated quantitative hormonal<br />
and physiological differences among cows: in the<br />
peripartum period attributable to cows selected for<br />
milk yield versus a non-selected control line [17,18];<br />
first calf Holstein cows bearing either Holstein,<br />
Holstein*Angus or Holstein*Brahman conceptuses<br />
[27, 28]; and cows managed during the prepartum<br />
period under a shade heat abatement system versus<br />
a control no-shade environment [13,38]. Distinct<br />
differences in free and sulfated estrogens,<br />
progesterone, prolactin, 13-14 dihydroxy- 15 keto<br />
PGF 2α<br />
, thyroxine and triiodothyronine, as well as<br />
physiological differences in conceptus birth weights,<br />
postpartum milk yield, and uterine regression were<br />
detected in response to these distinct experimental<br />
differences.<br />
The transition period, typically considered<br />
from the time 3 weeks prepartum until 3 weeks postpartum,<br />
is marked by declining dry matter intake,<br />
negative energy status once lactation is initiated, and<br />
inadequate innate immunity that increases the risk<br />
of uterine diseases. A major issue facing dairy cows<br />
under intensive management systems is the high<br />
incidence of health problems, particularly those that<br />
affect the reproductive tract and those of metabolic<br />
origin that affect subsequent reproductive<br />
performance. Data from 5,719 postpartum dairy<br />
cows evaluated daily for health disorders from seven<br />
dairy farms were compiled [50]. Cows were subjected<br />
to presynchronized timed AI programs. Only 55.8%<br />
of them were considered healthy and did not develop<br />
any disease event in the first 60 days postpartum.<br />
Incidence of clinical diseases (calving related<br />
problems, 14.6%; metritis, 16.1%; clinical<br />
endometritis, 20.8%; fever, 21.0%; mastitis, 12.2%;<br />
ketosis, 10.4%; lameness, 6.8%; digestive problems,<br />
2.8%; pneumonia, 2.0%) was high and 27.0% of the<br />
cows were diagnosed with a single disease event,<br />
whereas 17.2% had at least 2 disease events in the<br />
first 2 months of lactation. In spite of similar milk<br />
yield, cows diagnosed with health problems were<br />
less likely to be cyclic at 65 days postpartum. Calving<br />
related disorders and those that affect the<br />
reproductive tract were the major contributors for<br />
the depression in cyclicity (cyclic: healthy 84.1 %<br />
versus 70.7% > 1 disease). Diagnosis of health<br />
disorders in early lactation markedly depressed the<br />
risk of cows to become pregnant at the first<br />
postpartum AI (pregnancy per TAI: healthy 51.4%<br />
versus 34.7% > 1 disease), and increased the risk of<br />
pregnancy loss in the first 60 days of gestation<br />
(pregnancy loss %: healthy 8.9% versus 15.8% > 1<br />
disease). These responses indicate that reduction in<br />
morbidity by prevention of periparturient diseases has<br />
the potential to enhance fertility of dairy cows by<br />
improving resumption of postpartum ovulation,<br />
increasing pregnancy per AI, and minimizing the risk<br />
of pregnancy loss. A major focus point to be gleaned<br />
is that restoration of uterine/ovarian function and<br />
optimization of immune function are considered<br />
important researchable areas to sustain reproduction<br />
in lactating dairy cows. Furthermore, epidemiological<br />
data analyses are a powerful tool to identify<br />
reproductive inefficiencies and potential causative<br />
associations but do not prove cause and effect.<br />
2.1 Restoration of postpartum uterine function<br />
Silvestre et al. [52] documented clearly that<br />
at 2 + 1 days postpartum chronic treatment subcutaneously<br />
with a non-degradable implant containing 5<br />
mg of the GnRH-Agonist Deslorelin had a profound<br />
effect on uterine involution. The Deslorelin implant<br />
suppressed follicular development, enhanced physical<br />
involution of the uterus and cervix, increased tonicity<br />
of the uterine wall, reduced frequency of abnormal<br />
cervical discharges, and decreased inflammatory processes<br />
of the reproductive tract. Likewise, a degradable<br />
Deslorelin implant enhanced uterine involutionary<br />
processes but also delayed onset of ovulations, which<br />
offset the potential benefit of improved uterine<br />
regression and health [55]. Future research should<br />
focus on GnRH agonist delivery systems in which<br />
initiation and termination of treatments could be<br />
manipulated practically into a restricted time frame<br />
through 22 days postpartum to optimize uterine<br />
involution. Also the mechanism of Deslorelin action<br />
needs further investigation as to either direct or indirect<br />
effects on the uterus. This may avoid a prolonged<br />
period of anovulation. If the agonist is directly<br />
effective, can a molecule be developed that exerts a<br />
uterine effect without desensitizing gonadotrophs that<br />
secrete LH and FSH Perhaps the decreased secretion<br />
of LH and/ or FSH has direct effects on the uterus<br />
since clearly follicle development and turnover is<br />
reduced. This is the same endocrine status of suckled<br />
beef cows that have a greater rate of uterine regression<br />
and less uterine health problems than lactating dairy<br />
cows. Induced atrophy of the uterus via suppression<br />
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of gonadotrophin secretion, possibly independent of<br />
oxytocin secretion, is an intriguing hypothesis.<br />
2.2 Nutritional modulation of postpartum innate<br />
immunity and subsequent fertility<br />
Nutritional management to alter innate<br />
immunity in the transition period followed by a dietary<br />
change that enhances subsequent pregnancy rates in<br />
high producing dairy cows are strategies that are<br />
complementary to reproductive management<br />
programs and potentially more acceptable to both<br />
producers and consumers. Silvestre et al. [53,54]<br />
designed an experiment to evaluate the effects of<br />
differential temporal supplementation of various<br />
calcium (Ca) salts of fatty acids on reproduction,<br />
production and innate immune responses. Holstein<br />
cows (n = 1,380) were assigned randomly to be fed<br />
either transition diets supplemented with 1.5% of the<br />
DM as Ca salts of either palm oil (PO; mostly saturated<br />
and monounsaturated fatty acids) or safflower oil (SO;<br />
mostly linoleic acid). At 31days postpartum, cows<br />
within each transition diet were randomized to receive<br />
either PO or Ca salts containing fish oil (FO; enriched<br />
in :5 n-3<br />
that leads to suppression in the biosynthesis of<br />
inflammatory molecules) until 160 days postpartum.<br />
Feeding SO during the transition period<br />
improved early postpartum neutrophil bactericidal<br />
function, abundance of the L-selectin adhesion<br />
molecule, and neutrophil production of proinflammatory<br />
cytokines [53]. The SO dietary supplement<br />
elevated plasma concentrations of haptoglobin<br />
and fibrinogen. Conversely, feeding FO during the<br />
breeding period attenuated cytokine secretion from<br />
neutrophils.<br />
Transition and breeding diets did not affect<br />
the proportion of cows cycling at 74 days postpartum<br />
(80.0%; [54]). Overall first service pregnancy per AI<br />
at 30 and 60 days after insemination were 39.3% and<br />
33.3%, respectively, and there were no effects of diets.<br />
However, fewer cows fed FO diets lost their pregnancy<br />
(6.3 versus 13.6%). Perhaps the anti-inflammatory<br />
effects of the omega-3 FA in FO [53] fed during the<br />
breeding period reduced pregnancy losses.<br />
Furthermore, cows fed FO had an increased<br />
pregnancy per AI to the second service at both 30<br />
days (36.2 vs 27.2%) and 60 days (34.5 vs 23.7%)<br />
after insemination. At second service, cows fed SO<br />
in the transition period followed by FO during the<br />
breeding period had the highest proportion pregnant<br />
(i.e., 43.3% at 30 days and 43.1% at 60days). The<br />
combination of reduced pregnancy loss at first AI<br />
and increased pregnancy at the second AI resulted in<br />
a greater proportion of cows fed FO that were pregnant<br />
after two postpartum inseminations. The mechanisms<br />
regarding the positive effects of FO supplementation<br />
are speculative, but could be associated with an<br />
improved ratio of IGF-2 to IGF-1 gene expression,<br />
and/or anti-inflammatory effects within the<br />
endometrium that are complementary to the<br />
immunosuppressive and anti-luteolytic effects of the<br />
conceptus that maintain pregnancy.<br />
Collectively, strategic supplementation of fatty<br />
acids benefitted immune function early postpartum<br />
and exerted immunosuppressive effects during the<br />
breeding period, which could explain some of the<br />
improvement observed in fertility [53,54]. Clearly<br />
feeding nutriceutical components in the diet that<br />
improve reproductive performance will be perceived<br />
by the consumer as a more acceptable management<br />
strategy compared to treatment with safe and<br />
improved hormones.<br />
2.3 Maternal regulation of neonatal passive immunity<br />
and lactocrine programming of uterine development<br />
Parturition marks the cessation of intrauterine N<br />
life but does not end maternal effects in the sense<br />
that the newborn calf suckles colostrum (e.g., beef<br />
calf) or receives via supplementation fresh or stored<br />
frozen/thawed colostrum, during the first 24 h after<br />
birth (e.g., intensive management of a commercial<br />
dairy) to acquire passive immunity. Whether the mean<br />
transfer of 248 g of IgG or 7 kg of colostrum to the<br />
newborn [41] contributes to the period of postpartum<br />
immunosuppression of the mother and is related to<br />
peripartum reproductive disorders (e.g.<br />
puberalmetritis, endometritis, subclinical endometritis)<br />
warrants investigation [30]. Perhaps periparturient<br />
cows need to be acutely supplemented with<br />
immunoglobulins to compensate for major losses in<br />
the colostrum during the first 24 h of lactation.<br />
Colostrum contains a plethora of growth<br />
factors (e.g., EGF, IGF-1, IGF-2, and other unidentified<br />
factors) that target the neonate to affect<br />
differentiation of anterior pituitary mammotropes,<br />
gastrointestinal tract development, and maturation of<br />
the immune system [11, 44]. The term lactocrine was<br />
coined to describe a mechanism through which a<br />
bioactive factor or hormone reaches the neonatal<br />
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circulation shortly after birth, as a specific consequence<br />
of nursing [68]. Lactocrine-acting factors,<br />
whether natural or man-made, may affect developmental<br />
events associated with programming of female<br />
reproductive tract tissues. This has been demonstrated<br />
vividly in the pig based upon the following lines of<br />
evidence[4]: (1) colostrum is a source of bioactive<br />
relaxin; (2) relaxin can be detected in the circulation<br />
of newborn pigs only if they are allowed to nurse;<br />
(3) the neonatal uterus is RLX receptor (RXFP1)<br />
positive at birth and prior to onset of endometrial<br />
estrogen receptor (ESR1) expression; (4) administration<br />
of exogenous relaxin to newborn gilts stimulates<br />
both uterine ESR1 and vascular endothelial<br />
growth factor (VEGFA) expression by postnatal day<br />
2; (5) such effects of relaxin administered from birth<br />
can be attenuated by pretreatment of gilts with the<br />
ESR1 antagonist ICI 182,780 indicative that relaxin<br />
may act, in part, via cross talk with the estrogen signaling<br />
system.<br />
Other mammary born bioactive factors or<br />
cooperative lactocrine mechanisms affect the neonatal<br />
uterine developmental program based upon<br />
comparisons between newborn pigs that nursed ad<br />
libitum or received a milk replacer with or without<br />
exogenous relaxin for 48 h after birth [11]. Colostrum<br />
consumption was required for normal uterine protein<br />
and/or transcript expression on postnatal day 2 of<br />
RXFP1, ESR1, VEGFA, and matrix metalloproteinase<br />
9 (MMP9). In contrast, uterine ESR1, VEGFA, and<br />
MMP9 protein levels were below detection limits in<br />
replacer-fed gilts. Supplemental relaxin increased<br />
uterine ESR1 protein and mRNA in nursed gilts, as<br />
well as VEGFA protein in nursed and VEGFA mRNA<br />
in both nursed and replacer-fed gilts. Relaxin<br />
treatment did not affect uterine MMP9 mRNA levels.<br />
When compared with replacer-fed gilts on postnatal<br />
day 2, uterine RXFP1 mRNA was reduced in nursed<br />
gilts and in relaxin-supplemented replacer-fed gilts.<br />
The authors concluded that establishment of the<br />
neonatal porcine uterine developmental program<br />
requires maternal lactocrine support beyond just the<br />
presence of relaxin.<br />
In porcine neonates, estradiol valerate<br />
exposure from birth to postnatal day 13 increased<br />
uterine RXFP1 gene expression, and both ESR1 and<br />
VEGFA proteins at postnatal day 14 [12]. When<br />
uterine responses were examined in mature gilts at<br />
day 12 of pregnancy, endometrial RXFP1 mRNA<br />
remained elevated, while ESR1 protein was reduced<br />
in gilts that were postnatally treated with estradiol<br />
valerate. Early estradiol valerate treatment decreased<br />
neonatal uterine WNT7A but increased HOXA10<br />
expression. The expression of WNT7A was reduced<br />
in estradiol valerate-treated adults. Transient EV<br />
exposure increased MMP9 transcripts at postnatal day<br />
14, whereas both latent and active MMP9 activity<br />
was increased in adults at day 12 of pregnancy due<br />
to early estradiol valerate treatment. These findings<br />
support the hypothesis that transient estrogeninduced<br />
disruption of porcine uterine development<br />
from birth alters early programming events that lead<br />
to functional consequences in the adult.<br />
The lactocrine regulation of uterine development<br />
in the bovine neonate is essentially<br />
unknown. As described above there are various<br />
growth factors in colostrum of cattle and in particular<br />
very high concentrations of IGF-1 [43]. Relaxin<br />
concentrations compared to the pig are low or perhaps<br />
non-existent in cattle; although a relaxin-like factor<br />
(RLF), a new member of the insulin-relaxin gene<br />
family, is expressed in bovine ovarian follicular thecal<br />
cells (33). Bartol et al. [3] demonstrated that exposure<br />
of neonatal heifer calves to progesterone and estradiol<br />
benzoate (PE) delivered from a commercial growthpromoting<br />
implant either at birth (day 0), 21 or 45<br />
days of age altered adult (i.e., 15 mo of age in the<br />
luteal phase of an induced estrous cycle) uterine<br />
morphology and uterine luminal protein content<br />
compared to non-implanted control heifers. Treatment<br />
did not affect plasma progesterone concentrations<br />
(3.2ng/ml). Regardless of age at treatment, neonatal<br />
PE exposure reduced uterocervical wet weight by<br />
35% (112.8 < 173.9 g), myometrial area by 23%<br />
(126.3 < 162.8 +8.5 mm 2 ), and endometrial area by<br />
27% (33.3 < 45.4 +2.7 mm 2 ) compared with the<br />
untreated controls. Endometrial gland density was<br />
reduced by 40% in treated heifers. This effect was<br />
related to age at implant placement. Uterine gland<br />
density was reduced by 66% in heifers treated at birth,<br />
while reductions of 22 and 33% were observed for<br />
heifers treated on neonatal days 21 or 45, respectively.<br />
Consistently, ULF protein content was lower in the<br />
treated heifers (2.67
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occurs. Identification of the complete array and role<br />
of factors that define the uterine developmental<br />
program and the subsequent impact of reproductive<br />
tract development on fertility will provide critical<br />
management insight into factors regulating herd<br />
fertility. This is an area warranting further investigation<br />
in cattle.<br />
III. OPTIMIZATION OF REPRODUCTIVE PROGRAMS<br />
FOR TIMED ARTIFICIAL INSEMINATION (TAI)<br />
It is essential that dairy producers, farm staff,<br />
nutritionists, and veterinarians understand the<br />
physiological underlying reasons why certain<br />
components of the reproductive management<br />
program are able to improve reproductive performance<br />
or conversely why a misunderstanding of the<br />
program can lead to catastrophic pregnancy results.<br />
No one reproductive breeding program is practical<br />
and economically optimal for all dairy production<br />
units due to differences in available facilities, size of<br />
the unit, labor that places reproduction as a high<br />
priority, and a functionally dynamic record system.<br />
What is essential is the implementation of optimal<br />
programs that increase pregnancy rates and these<br />
programs become fertility programs that achieve more<br />
than just inseminating all cows.<br />
Optimization of stage of the estrous cycle (i.e.,<br />
days 5 to 9) at the onset of the Ovsynch protocol<br />
(i.e.GnRH_5-7days_PGF 2α−<br />
2-3days-GnRH-16h-AI)<br />
is important to achieve a subsequent synchronized<br />
ovulation at the second GnRH preceding the TAI.<br />
Programming the stage of the estrous cycle at the<br />
time the Ovsynch protocol is implemented (e.g., Days<br />
5-9 of estrous cycle) has multiple effects: increases<br />
the probability that the first injection of GnRH will<br />
induce ovulation of the first wave follicle and<br />
subsequent recruitment of a new follicle wave; that<br />
there is progesterone availability throughout the<br />
period between the first injection of GnRH and<br />
injection of PGF 2α<br />
, that there is a CL to respond to<br />
the luteolytic injection of PGF 2α<br />
, and producing a<br />
viable oocyte for fertilization and development of a<br />
robust CL upon induction of ovulation to the second<br />
GnRH. Indeed ovulation of the first follicle wave<br />
results in presence of both the original CL and an<br />
accessory CL, induced by the GnRH injection, which<br />
are responsive to the injection of PGF 2α<br />
.<br />
The Ovsynch protocol preceded by a PGF 2α<br />
program (Presync-Ovsynch) has become the nucleus<br />
program for reproductive management in the industry.<br />
Successful use of such a program is dependent highly<br />
upon obtaining good compliance in implementing<br />
all component parts of the protocol. The original<br />
Presynch-Ovsynch program entailed two injections<br />
of PGF 2?<br />
given 14 days apart with the Ovsynch<br />
protocol initiated 12 days after the second injection<br />
of PGF 2?<br />
for presynchronization [41]. This system<br />
increased pregnancy rates compared to Ovsynch<br />
alone, and it is essential to start the Ovsynch program<br />
between 10 to 12 days after presynchronization (i.e.,<br />
the second injection of PGF 2α<br />
) to obtain good<br />
pregnancy rates to the TAI. A 14-day interval may<br />
be convenient for producers but is not optimal to<br />
obtain maximal fertility. An 11–day interval after presynchronization<br />
(i.e., cows would be predominately<br />
on days 5-8 of the estrous cycle) was better than a<br />
14-day interval to begin the timed AI protocol [23].<br />
High-producing lactating dairy cows have a<br />
greater incidence of two waves of follicle growth<br />
during the estrous cycle compared with growing<br />
heifers that are more likely to have three follicular<br />
waves. The interval from follicle emergence to estrus<br />
is 3.5 days greater for cows with two follicular waves<br />
than for those with three follicular waves [7];<br />
consequently the period of follicular dominance Nis<br />
greater and fertility to TAI in cows with greater periods<br />
of follicle dominance is reduced. One means of<br />
reducing the period of ovulatory follicle dominance<br />
is to shorten the interval from follicle recruitment to<br />
luteal regression (i.e., implement a 5-day interval<br />
between GnRH and PGF 2α<br />
injection) to increase<br />
pregnancy per TAI in lactating dairy cows. Following<br />
two presynchronization injections of PGF 2α<br />
at 36 and<br />
50 days in milk, Santos et al. [50]randomly assigned<br />
933 cows to a Cosynch 72 h protocol (CoS72: day<br />
61 GnRH, day 68 PGF 2α<br />
, day 71 GnRH) or to a 5<br />
day-Cosynch 72 h with two injections of PGF 2α<br />
(5<br />
day-CoS2: day 61 GnRH, day 66 and day 67 PGF 2α<br />
,<br />
day 69 GnRH). Regression of CL was lesser (91.5<br />
vs. 96.3%) and pregnancy/TAI greater (39.3 vs.<br />
33.9%) for 5 day-CoS2 than CoS72, respectively. It<br />
was essential to inject two doses of PGF 2α<br />
given 24 h<br />
apart (i.e., day 66 and day 67) to insure complete<br />
regression of the CL.<br />
An additional study tested timing of the GnRH<br />
associated with timed insemination at first service and<br />
development of a re-synchronization TAI program<br />
for second service of non-pregnant cows [6].<br />
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Following two injections of PGF 2?<br />
at 46 and 60 days<br />
in milk, 1227 cows were randomly assigned to a 5-<br />
day OVS56 h (day 72 GnRH, days 77 and 78 PGF 2α<br />
,<br />
day 79 [56 h] GnRH TAI at 72 h or to a 5-<br />
dayCosynch72h (i.e., GnRH and TAI occurred<br />
concurrently). Pregnancy/TAI did not differ between<br />
groups when evaluated at either day 32 (45.9%) or<br />
day 60 (39.7%) after TAI. Thus, the 5-dayCosynch<br />
72 h program with two injections of PGF 2α<br />
is very<br />
efficient in getting cows pregnant. Cows diagnosed<br />
as nonpregnant by transrectal ultrasonography on day<br />
32 after the first TAI postpartum (112 ± 3 days in<br />
milk) were blocked by parity and method of<br />
synchronization at first AI [6]. Within each block,<br />
cows were assigned randomly to one of two treatments.<br />
All cows received an injection of GnRH 2<br />
days after the pregnancy diagnosis at day 34 after<br />
the previous AI. They were treated with an injection<br />
of PGF 2α<br />
5 and 6 days after the first GnRH. A second<br />
injection of GnRH was administered 56 h after the<br />
first PGF 2α<br />
, and insemination was performed 16 h<br />
later. Cows assigned to the control group (n = 334)<br />
did not receive any further treatment, whereas cows<br />
assigned to supplemental progesterone (n = 341)<br />
received an intravaginal insert containing 1.38 g of<br />
progesterone (Eazi-Breed CIDR Cattle Insert, Pfizer<br />
Animal Health, New York, NY) from the first GnRH<br />
to the first PGF 2α<br />
of the resynchronization protocol.<br />
Treatment cows supplemented with progesterone had<br />
greater pregnancy/AI compared with<br />
unsupplemented cows (51.3 vs. 43.1%). Premature<br />
ovulation tended to be greater for control than<br />
progesterone treated cows (7.5 vs. 3.6%).<br />
Presently in <strong>2011</strong>, these systems have been<br />
developed to optimize the beginning of a TAI<br />
program, controlling the period of follicle dominance<br />
to improve fertility, recognition of the need to sustain<br />
progesterone exposure throughout the period of<br />
follicular synchronization, the essentiality of<br />
obtaining complete regression of the CL in lactating<br />
dairy cows, and finally the need to optimize timing<br />
of AI relative to induction of ovulation with GnRH.<br />
These advancements allow on farm pregnancy rates<br />
of 40 to 50% for first and second service. However,<br />
they require maximal compliance in protocol implementation,<br />
integration with on farm computer<br />
monitoring for lists of cows to be handled, treated,<br />
and monitoring the efficiency of the system. It is<br />
essential that studies designed to examine specific<br />
effects, in all areas of dairy management, on pregnancy<br />
per AI have sufficient number of animals per<br />
treatment group for example to have at least a 95%<br />
chance (P 0.05) of detecting a 7-percentage-unit<br />
increase in pregnancy per AI (e.g., 34 vs. 41%) with<br />
90% protection against a type II error.<br />
Implementation of such programs are<br />
successful as depicted by 21-day pregnancy rates for<br />
consecutive 21 day intervals from July, 2009 through<br />
the following March, 2010 in a Florida commercial<br />
dairy herd (Figure 2). The dairy was comprised of<br />
4,000 cows managed in a free-stall facility with an<br />
average milk production of 10,000 Kg/cow. The<br />
bench mark 21-day pregnancy rate that was the goal<br />
to be achieved on the farm was 20%, whereas the<br />
average pregnancy rate obtained was 23% (Figure<br />
2). Pregnancy rate during the hot periods of July<br />
through September was a mean of 18.5%, with a carryover<br />
period of reduced pregnancy rates until early<br />
November. Pregnancy rates increased to 29% in<br />
January. The overall 23% pregnancy rate would be<br />
equivalent to achieving an overall 70% estrous<br />
detection rate and a 32.8% conception rate to<br />
inseminations in cows detected in estrus. Usual estrous<br />
detection rates are 50% with conception rates of 30%,<br />
which would approximate a 21 day conception rate<br />
of 15%. Heat detection rates and conception rates<br />
can drop to 15% and 12% in the summer periods of<br />
Florida, which is equivalent to a 2% pregnancy rate.<br />
Thus the chronic use of this fertility management<br />
program was quite effective.<br />
Future advancements to further improve<br />
efficiency of the programs will entail early diagnosis<br />
of pregnancy and online monitoring of ovarian cycles<br />
and health status in the milking parlor with the use of<br />
nano-technology. Such technology combined with<br />
optimized housing to maximize animal comfort,<br />
health and well-being will further allow high<br />
producing lactating dairy cows to successfully<br />
reproduce and yet sustain high levels of milk<br />
production. Assisted reproductive technology such<br />
as Timed Embryo Transfer with in vitroproduced<br />
embryos is further effective in improving fertility with<br />
implementation of improved culture media [8].<br />
However, advancements in embryo freezing of in<br />
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vitroproduced embryos to achieve normal pregnancy<br />
rates are still needed for more worldwide implementation<br />
of this technology.<br />
IV. GENOMICS AND ENDOMETRIAL TRANSCRIPTOME<br />
ANALYSES OF THE ENDOMETRIUM<br />
4.1 Evolutionary Genomics<br />
Since completion of the human genome<br />
sequencing project in 2001, the genome for the<br />
bovine has been sequenced in 2004. It was further<br />
refined in 2009 to allow for evolutionary comparisons<br />
with other species and more detailed identification<br />
of expressed genes and their proteins. Sequencing of<br />
the bovine genome was a world-wide endeavor (for<br />
more information on the project and selected<br />
references see www.hgsc.bcm.tmc.edu/projects/<br />
bovine). It has been estimated that the cattle genome<br />
contains approximately 3 billion nucleotides with<br />
roughly 1% coding for functional genes. The high<br />
N<br />
Figure 2. Twenty-one day pregnancy rate and number of pregnant cows per 21-day interval for twelve<br />
21- intervals beginning 7/21/09 and ending 3/16-10 at Alliance Dairy, Florida. Mean 21-Day pregnancy<br />
rate of 23% compared to a pre-established management goal of 20%.<br />
degree of conservation of genetic sequences across<br />
different species is providing valuable comparisons<br />
of genomic sequences to help in the discovery of<br />
genes and to map their location to bovine<br />
chromosomes. Latest estimates have identified at least<br />
22,000 protein coding genes and 496 miRNA genes<br />
that are capable of differentially regulating gene<br />
expression.<br />
What applications and inferences do<br />
technology associated with elucidation of the bovine<br />
genome offer to dairy producers First there are the<br />
evolutionary implications associated with the<br />
ruminant and importance of lactation [19]. Seventysix<br />
percent (778 out of 1020) of sequential duplications<br />
corresponded to complete or partial gene<br />
duplications with high sequence identity (median<br />
98.7%). This suggests that many of these gene<br />
duplications are specific to either the Bos lineage (i.e.,<br />
s155<br />
wild and domestic cattle) and tend to encode proteins<br />
that often interface with the external environment,<br />
particularly immune proteins and sensory and/or<br />
olfactory receptors and include defensins, and<br />
pregnancy-associated glycoproteins. Duplications that<br />
are present exclusively in cattle may have functional<br />
implications for their unique physiology, environment<br />
that they subside in, and diet of cattle. An<br />
overrepresentation of genes involved in reproduction<br />
in cattle is associated with several gene families<br />
expressed in the ruminant placenta. These gene<br />
families encode the intercellular signaling proteins<br />
pregnancy-associated glycoproteins, interferon tau<br />
(IFN-α) and prolactin-related proteins These genes<br />
regulate ruminant-specific aspects of early pregnancy<br />
recognition, fetal growth, maternal adaptations to<br />
pregnancy, and the coordination of parturition. Examples<br />
of genes varying in cattle relative to mouse
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include a cluster of b-defensin genes, which encode<br />
antimicrobial peptides. Compared to the human and<br />
mouse genome, the cattle genome has increased<br />
changes in the numbers of interferon genes and the<br />
number and organization of genes involved in<br />
adaptive immune responses. This extensive<br />
duplication and divergence of genes involved in<br />
innate immunity may be because of the substantial<br />
load of microorganisms present in the rumen of cattle,<br />
which increases the risk of opportunistic infections at<br />
mucosal surfaces and positive selection for the traits<br />
that enabled stronger and more diversified innate<br />
immune responses at these locations. Another<br />
possibility is that immunity may have been under<br />
selection due to the herd structure, which can promote<br />
rapid disease transmission. Also, immune function–<br />
related duplicated genes have gained nonimmune<br />
functions, e.g., IFN-α that in addition to regulating<br />
antiviral activity is involved in maintenance of the<br />
corpus luteum in early pregnancy by its actions on<br />
the uterus to ultimately suppress secretion of<br />
prostaglandin F2α; the C-class lysozyme genes,<br />
which are involved in microbial degradation in the<br />
abomasum.<br />
A summary of these evolutionary comparisons<br />
among species indicate that the biological<br />
systems most affected by changes in the number and<br />
organization of genes in the cattle lineage include<br />
reproduction, immunity, lactation, and digestion.<br />
These changes in the cattle lineage probably reflect<br />
metabolic, physiologic, and immune adaptations due<br />
to microbial fermentation in the rumen, the herd<br />
environment and its influence on disease transmission,<br />
and the reproductive strategy of cattle. Mapping of<br />
the cattle genome and associated resources will<br />
facilitate the identification of novel functions and<br />
regulatory systems as well as the tools for genetic<br />
improvement within the dairy industry.<br />
4.2 Functional genomics<br />
Gene discovery says nothing of gene function.<br />
However, searching databases from other species<br />
and now the bovine genome is helping to predict<br />
gene function, particularly for single gene traits. Other<br />
methods are also helping us to identify functional<br />
roles of genes and include gene chips or microarrays,<br />
gene knockouts and gene knockdowns. For example<br />
Affymetrix Inc. now markets a bovine genotyping<br />
chip, allowing broader translation of the genome<br />
project into applications. Microarrays are nylon or<br />
glass slides or “chips”, as they are commonly called,<br />
that are spotted with partial gene coding sequences.<br />
Chips are incubated with fluorescently tagged<br />
complementary DNA (cDNA) from tissues of interest<br />
to determine what genes are being expressed.<br />
As a very specific integrated example for the<br />
use of this technology, we have taken a conceptus/<br />
endometrial analytical transcriptome approach to<br />
elucidate potential biological effects of cycle versus<br />
pregnancy of nonlactating (NL) and lactating (L)<br />
dairy cows at day 17 after a programmed LH surge<br />
[10,61]. We first characterized an experimental<br />
platform to evaluate lactation and pregnancy effects<br />
for subsequent global transcriptome analyses.<br />
Pregnant heifers (n=33) were assigned randomly after<br />
calving to L (n=17) and NL ( n=16) groups. The L<br />
group was fed a total mixed ration (1.65 Mcal NEL/<br />
kg, 16.5% CP) and the NL group fed a maintenance<br />
ration (1.45 Mcal NEL/kg, 12.2% CP). Blood was<br />
collected thrice weekly for 8 weeks and analyzed for<br />
insulin, IGF-1, NEFA, BHBA, glucose, and BUN.<br />
Rectal temperatures, ovarian ultrasonography, body<br />
weight (BW) and body condition score (BCS) were<br />
measured during the study. All cows were presynchronized<br />
and enrolled in a timed artificial<br />
insemination (TAI) protocol; 10 cows in the L and<br />
12 in the NL were TAI. On d 17 after GnRH/TAI, all<br />
cows were slaughtered and endometrial and<br />
conceptus tissues collected. The Bovine was used to<br />
assess conceptus and endometrial gene expression.<br />
Temporal changes in BCS and BW did not<br />
differ between L and NL cows. L cows had higher<br />
body temperature than NL cows (38.4 vs 38.2ºC),<br />
and NL cows cycled earlier than L (26.3 vs 34.7<br />
days postpartum). Concentrations of NEFA did not<br />
differ between NL and L cows; however, cows in L<br />
group had greater concentrations of BHBA (4.90 vs<br />
2.97 mg/dL) and BUN (11.6 vs 6.5 mg/dL) and lower<br />
concentrations of glucose (74.0 vs 79.9 mg/dL) than<br />
NL cows. Mean plasma concentrations of insulin<br />
postpartum did not differ between NL and L (1.28 vs<br />
1.24 ng/mL). Concentration of IGF-1 was lower (P <<br />
0.01) for L compared with NL (140.5 vs 198.2 ng/<br />
mL), and also different (P = 0.01) between cyclic<br />
and pregnant (147.6 vs 191.0 ng/mL). Insulin was<br />
not correlated (P > 0.10) with any of the metabolites<br />
measured in both simple and partial correlations.<br />
Concentrations of IGF-1 had a +0.25 correlation (P<br />
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< 0.01) with glucose, but this correlation was not<br />
significant when adjusted for lactation. Negative<br />
correlations (P < 0.01) between IGF-1 and NEFA (r =<br />
-0.33), and BUN (r = -0.25) were detected. Among<br />
metabolites, the highest correlation was between<br />
BHBA and BUN (P < 0.01; r = +0.59). Concentration<br />
of progesterone from GnRH or TAI (d 0) until d 17<br />
was lower for L cows than NL cows.<br />
Metabolomic responses such as concentrations<br />
of NEFA, BHBA, BUN, glucose, insulin and<br />
IGF-1 in plasma are indicative that L cows underwent<br />
metabolic changes associated with homeorhetic processes<br />
in response to lactogenesis and galactopoiesis.<br />
The alterations in metabolites reflect mobilization of<br />
lipids and proteins during a period of negative energy<br />
balance postpartum. In the present study, metabolic<br />
changes related to lactational status were observed<br />
even though there were no differences in BW and<br />
BCS between LC and NL cows.<br />
RNA from conceptus and intercaruncular<br />
endometrial tissues was extracted using<br />
TRIzol ® reagent (Invitrogen Corporation, Carlsbad,<br />
CA, USA) according to instructions provided by the<br />
manufacturer. Samples were purified<br />
(PureLink ® Micro-to-Midi kit; Invitrogen Corporation,<br />
Carlsbad, CA) and RNA concentrations and purity<br />
were determined (Agilent 2100 Bioanalyzer, Agilent<br />
Technologies, Inc., Santa Clara, CA, USA). All<br />
samples were further processed for amplification and<br />
labeling and had a RNA integrity number > 7.5, which<br />
is related to the ratio of 18S and 28S ribosomal<br />
subunits. Samples were placed in aliquots and stored<br />
at -80 o . Amplification and biotin labeling were<br />
performed with an initial 200 ng of RNA by using<br />
the MessageAmp III (Applied Biosystems, Inc.,<br />
Foster City, CA, USA) according to manufacturer’s<br />
guidelines. Samples were then tested in the<br />
bioanalyzer for quality determination and<br />
subsequently submitted for fragmentation and<br />
hybridization in the bovine microarray (Affymetrix ®<br />
Bovine Genome Array, Affymetrix ® , Inc., Santa Clara,<br />
CA, USA).<br />
Only pregnant (L, n=8; NL, n=6) and noninseminated<br />
cyclic (L, n=7; NL, n=4) cows were<br />
analyzed. Differentially expressed genes were<br />
selected with P-value < 0.01 and absolute expression<br />
> 40. In addition, a fold effect > 1.5 was used as a<br />
criterion for genes affected by pregnancy. Analyses<br />
of the endometrium detected 210 genes differentially<br />
regulated by lactation (136 down-regulated and 74<br />
up-regulated), 702 genes differentially regulated by<br />
pregnancy (407 down-regulated and 295 upregulated)<br />
and 61 genes were responsive in an<br />
interactive manner between pregnancy and lactation.<br />
Genes up- and down-regulated in pregnant cows were<br />
associated with several gene ontology (GO) terms,<br />
such as defense response (GO:0006952), interferon<br />
regulatory factor (IPR001346), cell adhesion<br />
(GO:0007155) and extracellular matrix<br />
(GO:0031012). Gene ontology (GO) analyses of upand<br />
down-regulated genes of lactating cows revealed<br />
terms related to immunoglobulin-like fold (<br />
(IPR013783), immune response (GO:0006985),<br />
COMM domain (IPR017920) and non-membrane<br />
bounded organelle (GO:0043228).<br />
For purposes of this presentation, we have<br />
chosen to focus on expression of Early Pregnancy<br />
Associated Glycoproteins (PAGs), also known as<br />
Pregnancy Specific Protein B (PSPB; [9]) and<br />
Pregnancy Serum Protein of Mr 60 kDa (PSP60; [39])<br />
in early pregnancy at day 17 in both conceptus and<br />
endometrial tissues. Adequate synchronization of<br />
embryonic development and remodeling of the<br />
endometrium are crucial to support conceptusplacental<br />
development throughout gestation and<br />
N<br />
avoid pregnancy failure. The conceptus-derived<br />
placental cells (trophoblasts) fuse to the endometrium<br />
and deliver secretory products into the maternal<br />
system. Numerous molecules, including proteins,<br />
cytokines, hormones, and growth factors coordinate<br />
the conceptus-maternal interface and systemically<br />
moderate maternal anatomy, endocrinology,<br />
immunology and physiology to create an appropriate<br />
environment for conceptus development and survival<br />
[5,22]. In cattle, 18 distinct PAG genes and 14<br />
pseudogenes have been identified [56]. The<br />
measurement of PAG in maternal blood is an<br />
alternative reproductive management tool that can<br />
be used for early pregnancy diagnosis in cattle (i.e.,<br />
day 27) and may be an indicator of conceptus/fetal<br />
wellbeing and pregnancy loss [26,51,62]. However,<br />
the functional role (s) of these molecules is still<br />
unclear. The function (s) of PAG family genes may<br />
be combined with their spatial and temporal<br />
expression throughout pregnancy and may be<br />
involved in adhesion, implantation and remodeling<br />
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of the fetal-maternal unit [67], immune<br />
regulation[1,16,31] and prostaglandin synthesis and<br />
regulation [14, 65].<br />
Around 40% of total embryonic losses are<br />
estimated to occur between d 8 and 17 of pregnancy<br />
[58]. An important event on d 16 to 17 after estrus is<br />
the maintenance of the CL. This process is established<br />
by the ability of the conceptus to secrete sufficient<br />
amounts of IFN-?, which regulates secretion of<br />
PGF 2?<br />
in the uterine endometrium [57]. Changes<br />
involved in the process from a cyclic to a pregnant<br />
state not only depend on adequate production of<br />
antiluteolytic signals from the conceptus, but also the<br />
response of the endometrium to those signals. A precise<br />
understanding of the dialogue between<br />
conceptus-maternal-placental units is needed to<br />
reduce these early losses and improve reproductive<br />
efficiency of cattle. Thus, elucidating the mechanisms<br />
that control embryo and endometrial development<br />
in early gestation is important for identification of<br />
genes regulating implantation, placentogenesis, and<br />
maintenance of pregnancy in lactating dairy cows.<br />
In the present study all conceptuses (n = 13)<br />
expressed PAG 2, 8, 11 and 12, which are all members<br />
of the ancient PAG group indicating that these genes<br />
are expressed by both trophectoder mmononucleated<br />
and binucleated cells (Table 1). The PAG11 was the<br />
PAG family gene member most abundantly expressed<br />
by d 17 conceptuses (i.e., expression level = 53,831<br />
± 2,620). Additional PAG family genes were<br />
expressed by some of the conceptuses: PAG 1 (n=1),<br />
PAG 7 (n=3), PAG 9 (n=1), PAG 10 (n=1), PAG 17<br />
Figure 3. Conceptus gene expression; simple (r) and standard partial (pr) correlations of<br />
PAG11 with prostaglandin regulatory genes: PGH2 synthase (prostaglandin H2 synthase);<br />
PTGES (prostaglandin E synthase; PTGES3 (prostaglandin E synthase 3 [cytosol]); and<br />
PGF2αR (prostaglandin F2α receptor). Grey shadow represents genes that were correlated<br />
with Table PAG 1. 11 Conceptus in both simple and partial endometrial correlation expression analyses. of PAG genes at d 17 of<br />
Table 1. Conceptus and endometrial expression of PAG genes at d 17 of pregnancy. pregnancy.<br />
Gene Expression Level<br />
PAG Genes<br />
Conceptus (13) a Endometrium Endometrium Pregnant<br />
Cyclic Cows<br />
Cows<br />
2 28,818.95 ± 1,435 4.22 (11) 52.78 ± 36 (4/14) a<br />
8 10,352.84 ± 1,892 4.22 (11) 8.87 (1/14)<br />
11 53,831.91 ± 2,620 4.23 (11) 84.15 ± 39 (7/14)<br />
12 9,187.08 ± 1,170 4.22 (11) 11.73 ± 5.9 (3/14)<br />
a<br />
Number of animals in parenthesis.<br />
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(n=2), PAG 18 (n=1), PAG 20 (n=1) and PAG 21 (n=5).<br />
These reflected other ancient and modern (i.e.,<br />
expressed in binucleate cells) PAG family genes<br />
expressed by some of the conceptuses.<br />
Simple correlation (r) analyses were analyzed<br />
with PAG 11 that was present and most abundant in<br />
all conceptuses. The following correlations of PAG<br />
11 with genes expressed in conceptuses were detected:<br />
PGH2 synthase (prostaglandin H 2 synthase [Cox-<br />
2]; r = 0.87; P < 0.01), PTGES (prostaglandin E<br />
synthase; r = 0.76; P < 0.01), PTGES3 (prostaglandin<br />
E synthase 3 [cytosol]; r = 0.69; P < 0.02) and<br />
PGF 2α<br />
(prostaglandin F 2α<br />
receptor; r = -0.73; P < 0.01).<br />
Standard partial correlation (pr) analyses (Figure 3)<br />
holding PGH2 synthase as a constant showed a<br />
decrease in the correlations of PAG 11 with PTGES<br />
(pr = 0.50; P < 0.09), PTGES3 (pr = 0.17; P < 0.59)<br />
and PGF 2α<br />
(pr = -0.47; P < 0.11). Moreover, PAG11<br />
and PGH2 synthase were still highly correlated when<br />
standard partial correlation analyses were run holding<br />
PTGES (pr = 0.76; P < 0.01), PTGES3 (pr = 0.74; P <<br />
0.01) or PGF 2α<br />
(pr = 0.78; P < 0.01) constant (Figure<br />
3). Prostaglandin related expression of genes in the<br />
conceptus was not affected by lactation. These series<br />
of analyses suggest that PAG 11 is highly associated<br />
with gene expression of PGH2 Synthase in the<br />
conceptus and that downstream influences of PAG<br />
11 on expression of enzymes in the PGE cascade as<br />
well as the PGF 2α<br />
were possibly manifested by the<br />
upstream regulation of PGH2 Synthase expression.<br />
The PAG genes, which are expressed<br />
exclusively by trophoblast cells (i.e., ancient PAG)<br />
were also observed in the endometrium of pregnant<br />
cows at d 17 of pregnancy. Among the PAG genes<br />
expressed in the endometrial tissue were PAG 2 (n=4),<br />
PAG 8 (n=1), PAG 11 (n=7) and PAG 12 (n=3) (Table<br />
1). PAG 11 was expressed in a greater number of<br />
cows and at a higher level than the other PAG genes<br />
observed in the endometrial tissue at day 17 of<br />
pregnancy (i.e., expression level = 84.15 ± 39), which<br />
is well before detection in the blood at day 27 [62].<br />
Analyses of simple correlations in endometriual<br />
tissue detected associations of PAG 11 with<br />
expressed genes of trophoblast cells such as TKDP1<br />
N<br />
Figure 4. Endometrium gene expression; standard partial correlations (pr) associations of PAG 11 with other<br />
functional genes. TKDP1 (trophoblast Kunitz domain protein 1); TP1 (trophoblast protein 1); PTX3 (Pentraxin-<br />
Related Gene); DKK1 (Dickkopf Homolog 1); HPGD (Hydroxyprostaglandin dehydrogenanse 15); PGRMC2<br />
(progesterone receptor membrane component 2); PTGDS (prostaglandin D2 synthase) and PIBF1 (progesterone<br />
induced blocking factor 1). Grey shadow represents the genes that were correlated with PAG11 in simple<br />
correlation analyses. The standard partial correlation, pt is the correlation between the expression of two genes<br />
with expression of a third gene held constant and also adjusted for treatments (N/S = not significant).<br />
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(trophoblastKunitz domain protein 1; r = 0.86; P <<br />
0.01) and TP1 (trophoblast protein 1; r = 0.67; P <<br />
0.01). PAG 11 also was correlated with genes<br />
expressed in the endometrium associated with<br />
conceptus invasion and implantation, PTX3<br />
(pentraxin-related gene; r = 0.50; P < 0.01) and DKK1<br />
(dickkopf homolog 1; r = 0.43; P < 0.02); prostaglandin<br />
regulatory genes such as HPGD (hydroxyprostaglandin<br />
dehydrogenanse 15; r = 0.56; P <<br />
0.01), and a gene related to progesterone regulation,<br />
PGRMC2 (progesterone receptor membrane<br />
component 2; r = 0.35; P < 0.07).<br />
A series of standard partial (pr) correlation<br />
analyses (Figure 4) revealed a direct correlation of<br />
PAG11 with TKDP1 (pr =0.72; P < 0.01) when<br />
holding TP1 constant (Figure 4). Moreover, when pr<br />
were run holding PAG 11 as a constant, TKDP1 was<br />
correlated with TP1 (pr= 0.52; P < 0.01). However,<br />
no correlation was observed between PAG11 and TP1<br />
when TKDP1 was hold as a constant (Figure 4). In<br />
addition, associations between TP1 and PTX3 were<br />
observed when TKDP1 was held constant (pr = 0.52;<br />
P < 0.01). Moreover, having TP1 as a constant,<br />
showed that PTX3 was highly correlated with DKK1<br />
(pr = 0.61; P < 0.01). A negative association between<br />
PTX3 and PTGDS (prostaglandin D2 synthase; pr =<br />
-0.60; P < 0.01) was observed when PIBF1 (progesterone-induced<br />
blocking factor 1) was held constant.<br />
Furthermore, PIBF1 was negatively correlated with<br />
PTGDS holding PGRMC2 constant (pr = -0.70; P <<br />
0.01). PIBF1 also had a positive correlation with<br />
PGRMC2 (pr = 0.74; P < 0.01) when PTGDS was<br />
held constant. In addition, results of standard partial<br />
correlations showed that HPGD was correlated<br />
negatively with PTGDS (pr = -0.42; P < 0.04) when<br />
holding PGRMC2 constant and positively correlated<br />
with PGRMC2 (pr = 0.50; P < 0.01) when holding<br />
PTGDS constant (Figure 2). In general the absence<br />
of a pr+trt (i.e., partial correlation adjusted for both a<br />
third expression gene and treatment; not significant<br />
[N/S]) reflected that the pr correlation was mainly<br />
due to the treatment effects (e.g., pregnancy).<br />
Endometrial expression of HPGD and<br />
PGRMC2 was not affected by pregnancy or lactation.<br />
Pregnant cows had lower expression of PTGDS<br />
compared with cyclic cows. Moreover, pregnancy<br />
enhanced the expression of PIBF1, DKK1 and PTX3.<br />
In addition, DKK1 expression was inhibited by<br />
lactation. Presence of genes expressed exclusively<br />
in trophoblast cells of the endometrium, such as TP1,<br />
TKDP1 and PAG11, were observed only in pregnant<br />
cows and their expression did not differ between LC<br />
and NL cows.<br />
All conceptuses expressed PAG 2, 8, 11 and<br />
12, which are all members of the ancient PAG group<br />
indicating that these genes are expressed by both<br />
trophectoderm mononucleated and binucleated cells.<br />
Correlation analyses were conducted with conceptus<br />
and endometrial expression levels of genes with the<br />
intent of identifying associations that might be<br />
important functionally for pregnancy and to provide<br />
a basis for future studies to identify the possible<br />
function(s) of PAG genes. Different possible functions<br />
of PAG genes may be related to the diverse<br />
localization of the ancient and modern PAG groups.<br />
Ancient PAG genes, mainly expressed at the<br />
microvillar junctions of the fetal-maternal interface,<br />
may be involved in binding together the fetal maternal<br />
surfaces or establishing an immunological barrier.<br />
In contrast, the expression of modern PAG genes (i.e.,<br />
exclusively expressed in trophoblast binucleated cells)<br />
occurs mainly in the developing maternal villi of the<br />
placentomes which ideally positions them to<br />
manipulate the maternal immune system [67].<br />
In the present study, no correlations between<br />
conceptus expressed PAG11 and genes involved in<br />
immune control were observed at d 17 of pregnancy.<br />
However, simple correlation analyses showed that<br />
endometrial PAG11 was correlated with PTX3, a gene<br />
that is produced by various tissues in response to<br />
proinflammatory signals. In humans, both trophoblast<br />
conditioned medium and trophoblast explants<br />
increased PTX3 mRNA expression in endometrial<br />
stromal cells [46]. In addition, Tranguch et al. [63]<br />
documented that PTX3 null mice had compromised<br />
implantation and decidualization processes.<br />
Endometrial PAG11 of the present study was<br />
correlated with DKK1, a gene that antagonizes Wnt/<br />
-catenin signaling. DKK1 is involved in early<br />
development of head structures anterior to the<br />
midbrain and promotes trophoblast cell invasion<br />
[42,44]. PTX3 was correlated positively with DKK1,<br />
suggesting that PTX3 might be acting in combination<br />
with DKK1 in the establishment of implantation. In<br />
addition, we observed a negative correlation between<br />
PTX3 and PTGDS, which might suggest an alternative<br />
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way to attenuate PGF 2α<br />
like effects at this stage of<br />
pregnancy. It has been documented that prostaglandin<br />
D2 is converted to a biologically active PGF 2α<br />
stereoisomer<br />
(9, 11 PGF 2α<br />
; [60]), which has vasoconstrictive<br />
and smooth muscle contractile properties, therefore,<br />
possibly deleterious for the establishment of<br />
pregnancy. Moreover, results of standard partial<br />
correlation analyses showed a negative association<br />
between PTGDS and HPGD. HPGD is known to be<br />
involved in the degradation of prostaglandins such<br />
that a sequential reduction in PTGDS would enhance<br />
HPGD to increase PGF 2α<br />
metabolism in early<br />
pregnancy. Endometrial expression of HPGD is<br />
possibly associated with the tight regulation of prostaglandin<br />
activity (i.e., production and degradation)<br />
in the endometrial tissue.<br />
HPGD also was correlated positively with the<br />
expression of the PGRMC2 gene, which encodes a<br />
protein that binds progesterone. Thus, this correlation<br />
indicates that endometrial progesterone binding might<br />
be associated with prostaglandins degradation in early<br />
pregnancy. Interestingly, simple correlation analyses<br />
showed that endometrial PAG 11 was positively<br />
correlated with HPGD and PGRMC2. These<br />
associations may be important in the elucidation of<br />
functions for PAG genes. PAG genes might be<br />
involved in the prostaglandin and progesterone<br />
regulation of early pregnancy.<br />
Early expression of PAG genes within the<br />
conceptus and endometrium of pregnant cows and<br />
the association with other genes infer a possible role<br />
of PAG in pregnancy maintenance and implantation<br />
by regulation of embryo development, trophoblast<br />
cell invasion, immune regulation, and prostaglandin<br />
metabolism. The associations detected are suggestive<br />
of potential pathways for investigation in early<br />
pregnancy at day 17 involving potential direct and<br />
indirect effects of PAG 11 produced by the conceptus,<br />
but in no means do they prove cause and effect. Future<br />
investigations involving use of proteomics,<br />
metabolomics, lipidomics, lazer dissection, and in<br />
vitrocell culture experiments will help to elucidate<br />
these suggested control systems inferred from a<br />
transcriptone analytical approach [20]. Nevertheless,<br />
use of a classical statistical approach of standard partial<br />
correlation analyses is insightful in sorting out<br />
potential interrelationships of conceptus and<br />
endometrial tissues.<br />
4.3 Genomic selection<br />
Mapping of the bovine genome has facilitated<br />
the ability to complement direct genetics with<br />
traditional quantitative genetics that will benefit the<br />
dairy industry. The genetic contribution of many<br />
multi-gene traits in cattle (e.g., milk production) is<br />
well documented, and this knowledge has provided<br />
the basis for the identification and mapping of a<br />
growing number of quantitative trait loci (QTL). The<br />
only limitation to performing direct genetic<br />
experiments and identifying genes underlying these<br />
traits is the lack of a complete genome sequence,<br />
which is now available for the bovine. Sequencing<br />
the bovine genome and identifying “Single<br />
Nucleotide Polymorphisms” (SNPs) will provide<br />
additional polymorphic markers and positional<br />
candidate genes derived from the human and bovine<br />
genomic maps. Indeed due to the higher homology<br />
between the bovine with the human genome compared<br />
to the genome of the mouse, the functional genomics<br />
of the bovine is probably more applicable than<br />
using the mouse as an experimental model. The<br />
populations with designed mating generated by natural<br />
reproduction, artificial insemination or assisted<br />
reproductive technologies provides unique<br />
opportunities for selection and propagation of efficient N<br />
dairy cattle in the future that can perhaps both produce<br />
milk and reproduce efficiently. Clones can also be<br />
generated from fibroblasts or stem cells and<br />
cryopreserved.<br />
A deep draft sequence assembly of shotgun<br />
reads from a single Hereford female and comparative<br />
sequences sampled from six additional breeds were<br />
used to develop probes to interrogate 37,470 singlenucleotide<br />
polymorphisms (SNPs) in 497 cattle from<br />
19 geographically and biologically diverse breeds<br />
[25]. These data show that cattle have undergone a<br />
rapid recent decrease in effective population size from<br />
a very large ancestral population, possibly due to<br />
domestication, selection, and breed formation.<br />
Domestication and artificial selection appear to have<br />
left detectable signatures of selection within the cattle<br />
genome, yet the current levels of diversity within<br />
breeds are at least as great as exists within humans.<br />
The availability of high-throughput assays for<br />
genotyping single nucleotide polymorphisms (SNP)<br />
has led to the genotyping of thousands of dairy cattle<br />
using the BovineSNP50 BeadChip (Illumina, Inc.,<br />
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San Diego, CA, USA) or similar platforms. The SNP<br />
markers represent single base changes (A, T, C, or<br />
G) within the DNA sequence of a bull or cow. This<br />
technology provides the ability to carry out 54,000<br />
DNA SNP marker tests simultaneously; SNPs are<br />
throughout the bovine genome of approximately 3<br />
billion base pairs. Consequently, the SNPs become<br />
genetic markers for individual animals such as<br />
progeny tested bulls in artificial insemination (AI)<br />
programs or young bulls that are candidates for such<br />
programs. A study at the USDA-ARS Beltsville<br />
Agricultural Research Center established the SNP<br />
genotypes for 5,369 Holstein bulls and cows [64,66].<br />
The genotype data of the bulls were used to estimate<br />
the effects of 38,416 SNP markers on production,<br />
type, longevity, udder health and calving ability.<br />
Based on the estimated SNP associations on these<br />
phenotypic traits from this parent population, a<br />
genomic predicted transmitting ability (PTA) was<br />
determined for each of 2,035 young Holstein bulls<br />
born from 2000 to 2003 that had no progeny. In<br />
2009 the PTA of each young bull was determined<br />
from its progeny and compared with the traditional<br />
PA (Parental Average) and the genomic PTA<br />
computed from the 2004 data. The same process<br />
was performed in the Jersey breed (1361 older animals<br />
and 388 young bulls) and the Brown Swiss breed<br />
(512 older animals and 150 young bulls). Results in<br />
Table 2 show the increase in reliability (REL) due to<br />
genomic information, as compared with the REL<br />
from parent average information only. Gains in REL<br />
from genomic information were positive for almost<br />
all responses. Gains in REL for Jerseys and Brown<br />
Swiss were not as large as for Holsteins and this is<br />
largely due to a fewer number of progeny tested bulls<br />
that were genotyped. For each trait, a young animal’s<br />
PA can be combined with information from the<br />
BovinSNP50 bead Chip to obtain a genomic PTA of<br />
much greater accuracy. For a bull calf, REL of the<br />
genomic PTA is equivalent to what could be obtained<br />
by measuring performance on 25 or 30 test daughters.<br />
Weigel et al. [66] compared how well<br />
genomic evaluations were performing for the young<br />
bulls of 2000-2003 that in 2009 had both genomic<br />
data and at least 50 milking daughters. Parent averages<br />
(PA), Genomic Predicted Transmitting Abilities<br />
(GPTA); and Daughter Yield Deviations (DYD;<br />
contains no genomic information) of the bulls. A<br />
total of 238 Holstein bulls had official genomic PTAs<br />
for milk, fat, protein, somatic cell score (SCS) in<br />
January 2009 that were based solely on genomic<br />
information and the bulls had at least 50 milking<br />
daughters in August 2009. Only 60 bulls had at least<br />
50 daughters in their genetic evaluations for daughter<br />
pregnancy rate (DPR). Comparisons of reliability<br />
(REL) for PA (Jan. 2009), GPTA (Jan. 2009) and<br />
DYD (Aug. 2009) are very insightful and are<br />
presented in Table 3. The average January 2009<br />
RELs for PA was 42% for yield traits, 39% for SCC,<br />
and 26% for DPR; whereas RELs of the genomic PTA,<br />
Table 2. Changes in reliability due to the inclusion of genomic data in national<br />
genetic evaluations in the United States.<br />
Trait Holstein Jersey Brown Swiss<br />
Net Merit +24% +8% +9%<br />
Milk Yield +26% +6% +17%<br />
Fat Yield +32% +11% +10%<br />
Protein Yield +24% +2% +14%<br />
Fat Percentage +50% +36% +8%<br />
Protein Percentage +38% +29% +10%<br />
Productive Life +32% +7% +12%<br />
Somatic Cell Score +23% +3% +17%<br />
Daughter Pregnancy Rate +28% +7% +18%<br />
Table from VanRaden et al. [64].<br />
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Table 3. Comparison of January 2009 parent averages (PA) and genome-enhanced predicted transmitting<br />
abilities (GPTA) for milk, fat, protein, somatic cell score (SCS), and daughter pregnancy rate (DPR) with<br />
August 2009 daughter yield deviations (DYD) for US Holstein bulls whose first-crop daughters calved<br />
between January and August.<br />
Milk Fat Protein SCS DPR<br />
No. Bulls 238 238 238 237 60<br />
Reliability (Jan ‘09 PA) 42% 42% 42% 39% 36%<br />
Reliability (Jan ‘09 GPTA) 72% 72% 72% 67% 62%<br />
No. Daughters (Aug ’09 DYD) 71 71 71 71 62<br />
Reliability (Aug ‘09 DYD) 84% 84% 84% 67% 52%<br />
Correlation (Jan ‘09 PA, Aug ‘09 DYD) 0.444 0.540 0.476 0.376 0.213<br />
Correlation (Jan ‘09 GPTA, Aug ‘09 DYD) 0.624 0.695 0.632 0.531 0.341<br />
Table from Weigel K.A. et.al. 2010Available at: http://www.aipl.arsusda.gov/publish/other/2010/<br />
submit_9wcgalp_vanraden_kw.pdf [66].<br />
which include both pedigree and genomic<br />
information, averaged a higher 72%, 67% and 62%,<br />
respectively. When examining actual production<br />
responses of the daughters (DVD in August, 2009),<br />
the average REL(s) were 84% for yield traits, 67%<br />
for SCS and 62% for DPR. The correlations between<br />
August 2009 DYD from progeny testing and January<br />
2009 PA and GPTA for each trait were much higher<br />
with the inclusion of genomic information (Table 3).<br />
The lower REL for DPR and SCS illustrates the greater<br />
difficulty in improving lower heritable fertility and<br />
health traits through genetic selection although<br />
progress can be made. However, with good<br />
reproductive management, as described earlier, the<br />
opportunities to improve reproductive performance<br />
through selection will be enhanced. The ability to<br />
estimate genomic PTA of young bulls via genotyping<br />
of SNPs without progeny test estimates allows for<br />
the use of young bulls with some degree of<br />
confidence. This would allow dairy producers to use<br />
a larger number of young bulls that would lower the<br />
risk associated with the use of lower REL bulls.<br />
Producers who supplement their traditional sire<br />
selections with a group of superior genome-tested<br />
bulls (i.e., each used in moderation) will achieve the<br />
greatest genetic progress.<br />
Traditional reproductive fertility traits have<br />
been considered to be lowly heritable (e.g., < 0.05).<br />
Undoubtedly this has been due to fertility<br />
measurements (e.g., days open) that do not<br />
completely reflect the physiological characteristics of<br />
the cow and are confounded with environmental<br />
effects such as farm management expertise and<br />
decisions made by management (e.g., voluntary<br />
waiting period, culling, nutritional programs etc.).<br />
Royal et al. [47] pointed out that although traditional<br />
measures of fertility have low heritabilities they have N<br />
relatively high coefficients of genetic variation<br />
indicative of a potential for genetic improvement.<br />
A more specific physiological response study<br />
reported heritabilities for anovulation and pregnancy<br />
loss of 0.171 and 0.489, respectively when estimated<br />
in commercial herds of California and a research herd<br />
in Wisconsin [2]. A series of studies from the<br />
University of Nottingham documented that more<br />
specific measurements of reproductive responses<br />
measured as progesterone profiles in milk are<br />
moderately to highly heritable [47,48]. Estimates of<br />
heritability for interval to commencement of luteal<br />
activity postpartum, length of the first luteal phase<br />
postpartum, and occurrence of a certain type of<br />
persistent CL were 0.16, 0.17, and 0.13, respectively<br />
[48]. Furthermore, significant and strong genetic<br />
correlations existed between endocrine fertility traits<br />
and production traits. Heritability for the percentage<br />
of milk samples with luteal activity during the first<br />
60 d postpartum was 0.30 (3x/wk) and decreased<br />
with more infrequent sampling to 0.25, 0.20, and 0.14<br />
for weekly, twice monthly, and monthly sampling,<br />
respectively [45]. Incorporation of endocrine para-<br />
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meters of fertility, such as commencement of luteal<br />
activity measured in repeated samples at test days,<br />
into a fertility selection index may offer the potential<br />
to improve the accuracy of estimating predicted<br />
transmitting ability for fertility.<br />
Ever evolving and exciting technological<br />
strategies are now in place for development of gene<br />
markers offered by continual advances in DNA<br />
arraying technologies, the bovine genome mapping<br />
program, and deep RNA sequencing to more<br />
extensively explore the transcriptomes of conceptus<br />
and reproductive tissues. Use of genomic technology<br />
has identified a potential gene or associated locus<br />
that is related to bull fertility [21]. A Phase I<br />
comprehensive genome wide analysis of SNPs for<br />
bull fertility identified a total of 97 SNPs that were<br />
significantly associated with fertility (P < 0.01). In<br />
Phase II, the four most significant SNPs of Phase I<br />
were tested in 101 low fertility and 100 high-fertility<br />
bulls. One of the SNPs, rs41257187 (C-T) is in the<br />
coding region of the integrin beta 5 gene on<br />
chromosome 1. The SNP rs41257187 induces a<br />
synonymous (Proline - Proline) suggesting<br />
disequilibrium with the true causative locus. However,<br />
incubation of bull spermatozoa with integrin beta 5<br />
antibodies significantly decreased the ability to fertilize<br />
oocytes. These insightful findings indicate that<br />
the bovine sperm integrin beta 5 protein plays a role<br />
during fertilization and could serve as a positional or<br />
functional marker of bull fertility. This genomic<br />
approach enters into the tool box for strategies to<br />
improve dairy cattle fertility.<br />
Seven sequence variants (SVs) have been<br />
identified in exon 1 and in the promoter region upstream<br />
of the bovine gonadotrophin releasing hormone<br />
(GnRH) receptor gene [15]. The g.-108T > C<br />
allelic variants were associated with an approximately<br />
0.4 day reduction in predicted transmitting ability for<br />
days to first service. This relationship infers that<br />
selection for animals carrying the g.-108T>C group<br />
of alterations may improve fertility in the dairy cow.<br />
Furthermore, cattle with the homozygous (CC)<br />
genotype for the calpastatin gene had an additional<br />
0.82 and 0.57 PTA greater units of Daughter<br />
Pregnancy Rate (i.e., equivalent to 3.28 and 2.28 days<br />
open) compared with the TT homozygous and CT<br />
heterozygous animals [24].<br />
The laboratory of H. Khatib at the Univerisity<br />
of Wisconsin constructed an in vitrofertilization (IVF)<br />
system that has the advantages of a unified<br />
environment and well-isolated components of the<br />
embryonic development process. Utilizing this<br />
system, SNP in several genes and interactions<br />
between them have been found to be associated with<br />
fertilization and early embryonic survival rates until<br />
day 7 [34,35,37]. In order to determine SNP genotpyes,<br />
the ovaries of origin for the oocytes and sperm<br />
from bulls were genotyped for genes of interest. The<br />
candidate genes with their SNP alleles were associated<br />
differentially with in vitrofertility of the embryos. This<br />
in vitroassessment of potential fertility genes were<br />
then examined in vivoin which bulls were genotyped<br />
for SNP alleles and fertility was assessed [36].<br />
Estimated relative conception rate (ERCR) data from<br />
222 young and mature Holstein bulls were obtained<br />
from April through June 2001. Estimated relative<br />
conception rate was the difference in conception rate<br />
(nonreturn rate at 70 d) of a sire compared with other<br />
AI sires used in the same herd for first insemination<br />
of lactating cows. The ERCR values for the 222 bulls<br />
ranged from 4.99 to +5.23. Semen for each of the<br />
bulls was SNP genotyped for the following genes:<br />
FGF2 = fibroblast growth factor 2; POU1F1 =<br />
pituitary-specific positive transcription factor 1; GH<br />
= growth hormone; PRL = prolactin; GHR= growth<br />
hormone receptor; PRLR = prolactin receptor;<br />
STAT5A = signal transducer and activator of<br />
transcription 5A; OPN = osteopontin; UTMP = uterine<br />
milk protein, and the data were analyzed for<br />
association with ERCR. The ERCR was associated<br />
with FGF2 and STAT5A polymorphisms. This in vivo<br />
validation of previous in vitro assessments of fertility<br />
suggests that these genes can be used in gene-assisted<br />
selection programs for reproductive performance in<br />
dairy cattle.<br />
Collectively the various studies cited identify<br />
a number of candidate genes for inclusion in a fertility<br />
array. Such strategies involving various technological<br />
approaches and cell-animal models will likely lead<br />
to development of fertility arrays that will allow for<br />
the identification of animals at a young age with<br />
potentially high fertility (i.e., male and female) and<br />
high production. The challenge will be to manage<br />
the lactating cows to achieve their reproduction and<br />
production potentials.<br />
Markers for several recessive diseases have<br />
been developed through the use of Marker Assisted<br />
Selection. Examples of diseases that severely impact<br />
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reproductive performance, but that have been<br />
reduced to minor concerns because of the use of<br />
genetic markers, are BLAD (Bovine Leukocyte<br />
Adhesion Deficiency), DUMPS (Deficiency of<br />
Uridine - 5-Monophosphate Synthase) and CVM<br />
(Complex Vertebral Malformation).<br />
Sequencing the bovine genome and further<br />
advances in functional genomics promises great<br />
benefits to the dairy industry. As genes for production<br />
traits are identified, genetic selection strategies can<br />
be improved. One can envision making<br />
improvements in milk yields and milk fat and protein<br />
composition, as well as herd health and reproductive<br />
performance. As genes for production traits are<br />
identified, gene selection will be reduced to simply<br />
running a genetic test for the complement of gene<br />
alleles associated with the characteristics of interest.<br />
V. CONCLUSION<br />
Epidemiological data analyses are a powerful<br />
tool to identify reproductive inefficiencies and<br />
potential causative associations, but do not prove<br />
cause and effect. Healthy postpartum lactating dairy<br />
cows are indeed fertile.<br />
Induction of ovarian quiescence in response<br />
to chronic exposure of a GnRH agonist induced<br />
postpartum uterine atrophy and warrants additional<br />
investigation relative to potential impacts on improved<br />
uterine health.<br />
Dietary supplementation with polyunsaturated<br />
omega-6 and omega-3 fatty acids improves<br />
postpartum innate immune function and subsequent<br />
reproductive performance.<br />
Colostrum feeding contains lactocrine<br />
secretions that influence uterine developmental<br />
programming in the immediate postpartum period and<br />
neonatal exposure to estrogens/progesterone alters<br />
early programming of the uterus leading to<br />
dysfunctional reproductive tract consequences in the<br />
adult.<br />
Reproductive management programs that<br />
optimize ovarian and uterine function permit a single<br />
timed insemination to an induced ovulation that<br />
increases pregnancy per insemination to both first<br />
insemination and resynchronized inseminations of<br />
cows diagnosed non-pregnant.<br />
The sequencing of the bovine genome has<br />
led to thorough characterizations of the endometrium<br />
and conceptus transcriptomes in response to key<br />
physiological periods such as pregnancy and lactation.<br />
Early expression of PAG genes within the conceptus<br />
and endometrium of pregnant cows and their<br />
association with other genes determined by standard<br />
partial correlation analyses infer a possible role of<br />
PAG in pregnancy maintenance and implantation by<br />
regulation of embryo development, trophoblast cell<br />
invasion, immune regulation, and prostaglandin<br />
metabolism.<br />
Candidate genes have been identified that are<br />
related to fertility based upon in vitro and in vivo<br />
approaches. The array of SNPs across the bovine<br />
genome and specific SNPs within candidate genes<br />
related to reproductive processes and fertility will<br />
enhance genetic selection for fertility.<br />
Genomic selection for production, health and<br />
reproductive traits will be the wave of the future as<br />
genomic and bioinformatic tools continue to be<br />
expanded and refined.<br />
Acknowledgments. author is indebted to the following<br />
students and colleagues who have contributed too many<br />
of the research projects described in this manuscript over N<br />
the last 40 years and who are my mentors. Their intellectual<br />
contributions and dedicated efforts are the foundation of<br />
an interdisciplinary program that has sustained my<br />
commitment to science:<br />
Students and Trainees. Francis C. Gwasdauskas, John R.<br />
Chenault, Heriberto Roman Ponce, Luis C. Fernandes,<br />
Robert M. Eley, Frank F. Bartol, Louis A. Guilbault, Jeffrey<br />
F. Knickerbocker, LokengaBadinga, Joan S. Curl, Stephen<br />
D. Helmer, Matthew Lucy, GuenahelDanet-Desnoyers,<br />
Rodolfo de la Sota, Eric J.-P. Schmitt, Thais Diaz Zambrano,<br />
Mario Binelli, Frederico Moreira, Ricardo Mattos,<br />
SukruMetinPancarci, AydinGuzeloglu, Julian Bartolome,<br />
Todd Bilby, Flavio T. Silvestre, Leonidas A. Chow, Juan F.<br />
Troconiz, Elveria O. Valdivia, Diann S. Eley, John M.<br />
McDermott, Judy Van Cleeff, Monte Meyer, Daniel Arnold,<br />
Flavia Lopes, Isabella Thompson, Gregory S. Lewis, David<br />
Wolfenson, Timothy S. Gross, Jorge Savio, Joan Burke,<br />
Divakar Ambrose and Ronaldo Luís Aoki Cerri.<br />
Colleagues. Fuller W. Bazer, R. Michael Roberts, Charles<br />
J. Wilcox, Peter J. Hansen, Charles R.Staples, Robert J.<br />
Collier, Jose E.P. Santos, T.R. Hansen.<br />
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www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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R.C. Uliani, L.A. Silv<br />
ilva,<br />
M.A. Alv<br />
lvar<br />
arenga.<br />
<strong>2011</strong>. Mare’s Folliculogenesis: Assessment of ovarian and perifollicular<br />
vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s171.<br />
ISSN 1679-9216 (Online)<br />
Mechanisms involved in selection of a single dominant follicle and regression<br />
of the corpus luteum: Does a common differentiating cell type, the granulosa/<br />
large luteal cell, underlie these two disparate physiological events?<br />
Milo C. Wiltbank<br />
1 , Brian<br />
W. Kir<br />
irkpa<br />
patr<br />
trick<br />
1 , Michele R. Bast<br />
astos<br />
3 , Paulo D. Car<br />
arvalho<br />
alho 1 , Gulnaz<br />
Yilmazbas-<br />
Mecitaglu 1 , Chris A. Burke 1 , Alexandre H. Souza 1 & Roberto Sartori 2<br />
ABSTRACT<br />
Background: Selection of a single dominant follicle in cattle involves a sequence of endocrine, intercellular, and intracellular<br />
changes that produces a single follicle that continues to grow, whereas the non-selected follicles cease growth and eventually<br />
undergo atresia. Technical breakthroughs have helped to unravel the pattern of changes during selection but have not yet<br />
definitely established the role of each specific change in the follicle selection process. Following ovulation and luteinization,<br />
the granulosa cells become the large luteal cells. More than 80% of P4 production by the ruminant CL is due to the functional<br />
properties of the large luteal cell. Factors related to CL development and regression are described below.<br />
Review: In Holstein heifers morphologically distinguishable selection of a single dominant follicle occurs when the follicle<br />
reaches ~8.5 mm in diameter. The circulating FSH concentrations, on average, reach a nadir near this time, although individual<br />
cows show surprising variation in the magnitude and pattern of the changes in FSH that accompany follicle selection.<br />
Blockade of LH pulses by treatment with the GnRH receptor antagonist, Acyline, does not inhibit follicle growth before 8 mm<br />
but follicles do not proceed past the point of follicle selection in Acyline-treated animals. This effect is consistent with the<br />
dramatic increase in expression of LH receptors in granulosa cells near the time of follicle selection and suggests that a shift<br />
from FSH-dependence to LH-dependence occurs in the dominant follicle. Surprisingly, Acyline-treated animals do not have N<br />
the characteristic increase in LH receptor expression in granulosa cells indicating a potential role for LH pulse in induction of<br />
LH receptors in the granulosa cells of the dominant follicle. A myriad of other gene expression changes have been reported in<br />
granulosa cells near the time of follicle selection including changes that would: increase estradiol production, inhibit apoptosis,<br />
change intrafollicular paracrine regulators, change the extracellular matrix, and alter metabolism and cell proliferation. The<br />
granulosa cell is only 10 µm in diameter in the preovulatory follicle, however the LH surge and subsequent luteinization<br />
produce a cell of about 38 µm in diameter, a growth of over 50-fold (500 µm 3 to 30,000 µm 3 ) in cell volume. Although, the large<br />
luteal cells represent less than 4% of the luteal cells, their large volume represents about 40% of the total volume of the CL.<br />
This cell type is differentiated to be an amazing P4 production factory that appears to be constitutively “on” but can be turned<br />
off by the actions of prostaglandin F2α (PGF) during luteolysis. The corpus luteum acquires the ability to undergo luteolysis<br />
following PGF treatment at about 7 days after the LH surge. This has been termed “luteolytic capacity”. We have been able to<br />
demonstrate acquisition of many of the same molecular features of luteolytic capacity during luteinization of granulosa cells in<br />
vitro.<br />
Conclusions: 1) Selection of a single dominant follicle seems to involve and may depend upon molecular changes in the<br />
granulosa cell; 2) It seems likely that changes in the large luteal cells are the physiologic key to acquisition of luteolytic<br />
capacity.<br />
Keywords: follicle, corpus luteum, cattle.<br />
1<br />
Department of Dairy Science, University of Wisconsin-Madison, USA. 2 Department of Animal Science, Superior School of Agriculture “Luiz<br />
de Queiroz” (ESALQ), University of São Paulo, Piracicaba, SP, Brazil. 3 Department of Animal Reproduction and Radiology, School of<br />
Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, SP, Brazil. CORRESPONDENCE: M.C. Wiltbank<br />
[wiltbank@wisc.edu]. 1675 Observatory Drive, Madison, WI 53706-1284, USA.<br />
s171
O. Sandra. <strong>2011</strong>. Deciphering early sensor and driver properties of the endometrium: contribution of the uterus to<br />
pregnancy outcome. ssssssssssssssss ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s173 - s182.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s173 - s182.<br />
ISSN 1679-9216 (Online)<br />
Deciphering early sensor and driver properties of the endometrium:<br />
contribution of the uterus to pregnancy outcome<br />
Olivier Sandra<br />
ABSTRACT<br />
Background: Pregnancy involves a continuum of complex biological processes. Each of them can be affected by the<br />
environment surrounding the pregnant female (e. g. chemicals, nutrition, stress, infection) and data have focused on gametes<br />
quality, early blastocyst development and placental function and their perturbations by environmental insults or embryo<br />
biotechnologies. During the pregnancy period spanning the entry of the blastocyst into the uterine cavity to implantation of<br />
the embryo, biological functions of the endometrium have also been extensively studied, namely uterine receptivity (controlled<br />
by maternal factors) and maternal pregnancy recognition (that requires conceptus-produced signals). Nevertheless, recent data<br />
based on experimental perturbations have unveiled unexpected biological properties of the endometrium whose structural<br />
organization and functionality during pre-placentation period impact embryo trajectory through epigenetic alterations with<br />
subsequent consequences on pregnancy progression and final outcome.<br />
Review: A renewed vision of the endometrium is presented in this review. Several features including uterine structure, protracted<br />
pre-implantation period, epitheliochorial implantation, a clearly identified signal for pregnancy recognition (interferon-tau),<br />
high-throughput analyses tools and original experimental models make ruminants valuable models for detailing the molecular<br />
and cellular events taking place in the endometrium before placentation occurs. In ruminants as well as other species, endometrial<br />
receptivity is required for embryo implantation and is achieved through biological actions of maternal hormones including<br />
ovarian steroids. Among them, progesterone appears as a major factor whose experimental alterations of circulating levels can<br />
N<br />
significantly stimulate or inhibit conceptus elongation. Conceptus growth and survival have also been shown to fail in<br />
pregnant ewes lacking endometrial glands (UGKO). In these bovine and ovine models, endometrium exhibits altered gene<br />
expression patterns and drives embryo development independently from the quality of donor oocytes. In the context of<br />
pregnancy maternal recognition, the presence of the conceptus deeply modifies endometrial transcriptome across various<br />
stages of late pre-implantation phase. Acting as a paracrine or an endocrine factor, interferon-tau has been recognized as an<br />
indispensable factor for successful implantation through its biological actions on endometrial cells, immune cells and luteal<br />
cells. Nevertheless, beyond the global maternal reaction to conceptus secretions, distinct endometrial responses can be<br />
elicited by embryos produced by in vivo fertilization, in vitro maturation and in vitro fertilization or somatic cell nuclear<br />
transfer. These findings have been confirmed in human when endometrial stromal cells are incubated with normal or compromised<br />
embryos but only upon differentiation into decidual cells. Then mammalian endometrium can be considered as an early<br />
biosensor of embryos presenting different potentials of post-implantation development.<br />
Conclusion: Endometrium appears as a dynamic and reactive tissue. Its persistent or transient epigenetic modifications can<br />
dramatically affect pre-implantation embryo development with lasting consequences on later stages of pregnancy, including<br />
placentation, foetal development, pregnancy outcome and post-natal health. Developing diagnosis and prognosis tools based<br />
on endometrial factors will be valuable with the aims to estimate the reproductive capacity of the mother or to assess the<br />
developmental potential of the embryo, particularly when assisted reproductive technologies are applied.<br />
Keywords: endometrium; biosensor; epigenetics, pregnancy, ruminants, genomics.<br />
CORRESPONDENCE: D.P.A.F. Braga [dani@webbraga.com/dbraga@fertility.com.br - FAX: +55 (11) 3885-9858]. Fertility - Assisted<br />
Fertilization Center. Av. Brigadeiro Luis Antonio, n. 4545, CEP:01401-002, São Paulo, SP, Brazil.<br />
s173
O. Sandra. <strong>2011</strong>. Deciphering early sensor and driver properties of the endometrium: contribution of the uterus to<br />
pregnancy outcome. ssssssssssssssss ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s173 - s182.<br />
I. INTRODUCTION<br />
Pregnancy involves a continuum of complex<br />
biological processes and several checkpoints (or<br />
hurdles) that have to be passed successfully. These<br />
hurdles include production and quality of the gametes<br />
(oocytes, sperm), fertilization, luteal function rescue,<br />
early development of the embryo, implantation,<br />
development of the foeto-placental unit until term<br />
and parturition. Each of these steps is crucial for the<br />
successful delivery of a healthy offspring and their<br />
onset or progression can be affected by events taking<br />
place in the environment surrounding the parental<br />
organism. The major contributor to pregnancy is the<br />
mother who produces one of the two gametes and<br />
will host the whole gestation until term. Acute<br />
challenges, short or long periods of perturbations<br />
related to nutrition, metabolism, stress, infections or<br />
endocrine disruptors have been identified as factors<br />
that affect gametes quality and fertilization, journey<br />
of the early embryo through the oviduct, cellular<br />
interactions between endometrium and hatched<br />
blastocyst or conceptus, foeto-placental development<br />
or parturition [22,40]. In addition, biotechnologies<br />
of reproduction (or assisted reproductive technologies)<br />
associated to embryo transfer have been<br />
shown to alter biological properties of the embryo<br />
with a subsequent impact on later stages of pregnancy<br />
[64]. Therefore inadequate maternal compartment<br />
and/or suboptimal quality of the embryo may impact<br />
the two ways communication between mother and<br />
embryo, precluding completion of successful<br />
pregnancy and affecting long term health status of<br />
the offspring [19].<br />
Although the oviduct from unstimulated<br />
animals appears to be the optimal environment for<br />
early embryonic nursing [11,26], in vitro embryo<br />
production and embryo transfer have demonstrated<br />
the oviduct to be a dispensable organ for supporting<br />
progression of pregnancy to term. Until now, despite<br />
several attempts in maintaining early and late fetal<br />
life outside the uterus [12], no surrogate biological<br />
or artificial system has been derived for the uterus.<br />
In normal physiological conditions, the uterus and<br />
its internal part referred as the endometrium constitute<br />
the maternal site for embryo implantation. External<br />
events (as described in the former paragraph) or<br />
intrinsic maternal features may affect biological<br />
functions of maternal organs or tissues that will in<br />
turn impact endometrial physiology. Figure 1 depicts<br />
a personal view of early pregnancy, where the maternal<br />
organism can be represented as an organscontaining<br />
funnel channelling every event affecting<br />
maternal tissues to the narrow hole represented by<br />
the endometrium. As the ultimate and unique<br />
biological layer facing the implanting embryo,<br />
endometrium drives the development of embryonic<br />
disk and extra-embryonic tissues during the<br />
establishment of pregnancy [33]. On the other hand,<br />
recent data based on in vitro embryo manipulations<br />
have unveiled an unexpected biological property of<br />
the endometrium since this tissue is able to<br />
differentially react to embryos displaying distinct<br />
potencies to term development [43]. Based on animal<br />
data - mainly ruminants - with references to recent<br />
results published in human, this review presents a<br />
renewed vision of the endometrium whose driver and<br />
sensor properties make this maternal tissue a major<br />
epigenetic contributor for embryo development with<br />
subsequent impact on issue of pregnancy.<br />
II. BROAD LINES OF EARLY PREGNANCY IN<br />
RUMINANTS<br />
In ruminants, the bicornuate uterus is covered<br />
with the endometrium displaying two specific areas,<br />
namely the caruncles and the intercaruncular areas<br />
[15]. The caruncles represent aglandular structures<br />
of limited size and distributed over the endometrial<br />
surface. The intercaruncular areas are large and contain<br />
the endometrial glands that produce histotroph,<br />
a collection of numerous and diverse factors including<br />
cytokines and growth factors [65]. Upon oocyte<br />
fertilization and after hatching, the extra-embryonic<br />
tissue of the ruminant conceptus undergoes a<br />
progressive and critical elongation phase [18] before<br />
implanting at day 15-16 post-oestrus in the sheep and<br />
the goat gestation (gestation period: 5 months) or day<br />
19-20 post-oestrus in the cow (gestation period: 9<br />
months). Although a decidual-like process (related<br />
to hemochorial implantation) has been reported in<br />
sheep [35], the ruminant synepitheliochorial implantation<br />
is characterized by the apposition then the<br />
adhesion between the trophectoderm and the uterine<br />
luminal epithelium [7].<br />
The protracted pre-implantation phase (10 to<br />
15 days) is associated to the secretion of interferontau<br />
(IFNT), the major signal of pregnancy recognition<br />
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O. Sandra. <strong>2011</strong>. Deciphering early sensor and driver properties of the endometrium: contribution of the uterus to<br />
pregnancy outcome. ssssssssssssssss ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s173 - s182.<br />
N<br />
Figure 1. A personal view of the early pregnant cow. The funnel represents the maternal organism<br />
crossed by environmental insults that will target endometrium in a direct way or indirectly –by affecting<br />
biological functions of other maternal tissues-. Endometrial reaction can be modulated by the quality of<br />
the embryo as reported when assisted reproductive technologies with embryo transfer are used.<br />
CL: corpus luteum; CNS: central nervous system; embryo; Ut: uterus.<br />
in ruminants. IFNT is a type I interferon whose spatiotemporal<br />
production is very specific and restricted to<br />
ruminant species. During the peri-implantation period,<br />
IFNT has been found to be secreted by trophectoderm<br />
cells uniquely and this factor has been shown to be<br />
indispensable for pregnancy recognition through its<br />
antiluteolytic actions [55]. IFNT biological functions<br />
were first considered to be paracrine by inhibiting<br />
the secretion of endometrial prostaglandin-F2α whose<br />
production is associated with luteolysis in the absence<br />
of conceptus [66]. Nevertheless, in addition to the<br />
impact of IFNT on the endometrial physiology, recent<br />
publications have also demonstrated endocrine and<br />
direct IFNT biological actions on extra-uterine tissues<br />
including circulating blood cells as well as corpus<br />
luteum whose progesterone secretion is indispensable<br />
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for the establishment and maintenance of pregnancy<br />
in large animal species [50,51].<br />
The long pre-implantation phase allowing the<br />
dissection of the first cellular contacts and molecular<br />
events occurring between the conceptus and the<br />
endometrium, the synepithelial implantation making<br />
it possible the easy separation of maternal side from<br />
extra-embryonic tissue, the abundance of<br />
endometrial tissues and accessible uterine fluids as<br />
well as availability of high-throughput genomic tools<br />
dedicated to ruminants constitute solid bases for<br />
investigating driver and sensor properties of the<br />
endometrium before placentation occurs.<br />
III. UTERINE RECEPTIVITY AND DRIVER PROPERTIES<br />
OF ENDOMETRIUM<br />
One of the most striking evidences of embryo<br />
control by endometrium has been provided by the<br />
gland knock-out (UGKO) ovine model. The neonatal<br />
exposure of ewes to norgestomet -a P4 analogueleads<br />
to the UGKO phenotype. In the adults, the<br />
uterus lacks middle to deep endometrial glands and<br />
exhibits a markedly reduced surface of luminal<br />
epithelium. UGKO ewes present recurrent early<br />
pregnancy loss due to failure in conceptus elongation<br />
and survival between day 12 and day 14 post-oestrus,<br />
a phenotype also present when control embryos were<br />
transferred into UGKO recipients [28]. A microarray<br />
analysis comparing cyclic and UGKO ewes has<br />
revealed 23 differentially expressed genes in the<br />
endometrium whose majority were immunoglobulin<br />
genes, likely because of the large numbers of immune<br />
cells present in the tissue [29]. The authors suggested<br />
that different populations or altered numbers of<br />
immune cells in UGKO ewes could be involved in<br />
recurrent early pregnancy loss observed in this experimental<br />
model as reported in humans. More<br />
generally, the UGKO model illustrates that alterations<br />
of the uterine tissue architecture and functionality<br />
during early post-natal life may have long-term and<br />
detrimental consequences on fertility.<br />
Uterine receptivity can be defined as a restricted<br />
time-related period when the uterus is receptive<br />
to blastocyst attachment and implantation. Impaired<br />
uterine receptivity leads to embryo implantation<br />
failure and defining the implantation window and<br />
endometrium receptivity has represented one of the<br />
biggest challenges to increase the success of assisted<br />
reproductive technologies in both human and animals<br />
[2,20]. In sheep, embryo transfer experiments have<br />
shown that normal embryo development depends<br />
upon a sequence of changes in uterine secretions [73].<br />
Many experiments have aimed to decipher the<br />
consequences of embryo development and uterine<br />
environment asynchrony. They have shown that<br />
embryo exposure to inappropriate uterine factors (“out<br />
of phase”) may lead to irreversible induction of<br />
abnormal development [3]. In order to define<br />
endometrial receptivity, cellular and molecular events<br />
leading to morphological and functional modifications<br />
of endometrium have been extensively investigated<br />
based on candidate gene approaches or highthroughput<br />
analyses in mammals [34,49,57]. In cattle,<br />
the highest number of differentially genes across<br />
oestrous cycle has been detected between day 7 and<br />
day 13 post-oestrus [23] and factors related to<br />
endometrial remodelling, regulation of angiogenesis,<br />
cell adhesion and embryo feeding have been<br />
identified [4,47,58]. Very interestingly, a set of<br />
endometrial genes expressed at day 7 or day 14 of<br />
the bovine oestrous cycle have been suggested to be<br />
predictors of successful or failed term pregnancy after<br />
embryo transfer in the following oestrous cycle [58].<br />
In the same order of ideas, the quantitative<br />
homogeneous expression of six endometrial genes<br />
has been reported during the implantation window in<br />
women who became pregnant in the subsequent ICSI<br />
cycle profiles [1]. In fact, the picture is much more<br />
complex since a recent human microarray study has<br />
shown common and specific pathways of gene<br />
deregulation in endometrial biopsies collected during<br />
the mid-luteal phase from women undergoing<br />
recurrent pregnancy loss, recurrent miscarriage and<br />
implantation failure after IVF [39]. Collectively, these<br />
first published results support the notion that<br />
endometrial biomarkers during the non pregnant<br />
phase can predict the outcome of pregnancy when<br />
assisted reproductive technologies are used.<br />
In mammalian species including ruminants,<br />
endometrial receptivity necessary for embryo<br />
implantation is driven by ovarian steroid hormones,<br />
namely estrogens (E) and progesterone (P4).<br />
Progesterone has been shown to be the major factor<br />
controlling uterine function in mammalian species [67]<br />
and bovine or ovine animal models have been derived<br />
to investigate the biological functions of this steroid<br />
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hormone. Ovariectomized cows administered with<br />
either estradiol or P4 or in combination have been<br />
recently used to identify endometrial genes regulated<br />
by P4 or estrogen using a microarray approach [63].<br />
Experimental models displaying artificially altered<br />
levels of P4 have also been reported in cattle and<br />
sheep. Elevated concentrations of circulating P4 -<br />
within physiological ranges- during the early postconception<br />
period have been associated with the<br />
advancement of conceptus elongation in the bovine<br />
[14,27] and ovine species [61]. On the other hand, a<br />
low P4 circulating level -obtained by repeated<br />
injections of PGF2a between day 3 and 7 of the<br />
oestrous cycle- has led to a striking reduction in<br />
conceptus size after recovery at day 13 [10]. This<br />
experimental model is consistent with the impaired<br />
ability of the maternal genital tract (oviduct and/or<br />
uterus) to support embryo development reflects in<br />
postpartum dairy cows displaying reflects low P4<br />
blood levels [54]. In these bovine and ovine experimental<br />
models, altered P4 blood levels have been<br />
associated with modified patterns of endometrial gene<br />
expression [23,24,62] including numerous nutrients,<br />
nutrient sensing pathways, growth factors and extracellular<br />
molecules [8] as well as immunomodulator<br />
factors [31,38]. Very interestingly, consequences of<br />
lower or higher P4 levels on bovine embryo<br />
elongation are visible when embryos are transferred<br />
to recipient females after the treatment period and<br />
collected 6 or 7 days later [16,24]. Collectively these<br />
findings strengthen the notion that epigenetic status<br />
of the pre-implantation endometrium in adult females<br />
can be altered to an extent that totally prevents or<br />
dramatically affects embryo development at<br />
implantation, independently from the quality of the<br />
donor oocytes.<br />
IV. MATERNAL PREGNANCY RECOGNITION AND<br />
SENSOR PROPERTIES OF THE ENDOMETRIUM<br />
Whereas first steps of uterus remodelling and<br />
receptive window are programmed by maternal<br />
hormones independently from the presence of the<br />
embryo, successful pregnancy will require embryo<br />
recognition by the maternal organism with a major<br />
and crucial contribution of the uterine reaction. This<br />
reaction is achieved through embryonically derived<br />
factors that are indispensable for promoting<br />
implantation through the establishment of permanent<br />
cellular interactions between the trophectoderm and<br />
the endometrium [30]. Embryo derived signals vary<br />
according to mammalian species and they have been<br />
abundantly reviewed [8,36,72]. In ruminants, IFNT<br />
has been identified as the signal of trophectoderm<br />
origin crucial for maternal recognition signal and for<br />
rescuing the corpus lutem, an indispensable step for<br />
maintaining P4 luteal secretion [45,55]. Identifying<br />
IFNT-endometrial target genes has generated an<br />
exponential number of publications during the last<br />
decade based on in vitro endometrial cell cultures<br />
and in vivo experimental models [8]. Nevertheless<br />
IFNT is not the only factor of embryo origin affecting<br />
uterine physiology and identifying embryo-related<br />
factors triggering local endometrial reaction may<br />
benefit from in vivo experimental models such as the<br />
ovine model of unilateral pregnancy. By placing a<br />
ligature proximal to the uterine body, the conceptus<br />
is confined in the uterine horn ipsilateral to the corpus<br />
luteum [9]. By comparing the gravid and the nongravid<br />
horns, the paracrine impact of conceptussecreted<br />
factors on the endometrium can be<br />
dissociated from their paracrine actions. In this model,<br />
dynamic changes of endometrial T lymphocyte<br />
populations were reported to be independent from<br />
embryo secretions [41] whereas the localization of<br />
macrophages was affected by the presence of the N<br />
conceptus [69]. At the molecular level, we also<br />
reported the paracrine influence of the conceptus on<br />
the endometrial expression of SOCS genes, a family<br />
of intracellular factors displaying major functions in<br />
the negative control of cytokine signalling pathways<br />
[60].<br />
In ruminants, global endometrial reaction to<br />
the presence of the conceptus has been recently<br />
investigated using large-scale analyses [68]. In cattle<br />
and to a lesser extend in sheep, analyses of molecular<br />
and cellular events at the endometrial level have<br />
benefited from the development of microarray<br />
platforms [21]. Still remains some limitation in data<br />
mining since the current free or priced softwares are<br />
built and dedicated to human and rodent data and<br />
they lack records of factors interactions specifically<br />
associated to pregnancy. The expression of<br />
endometrial related factors -including innate immune<br />
response and associate factors- has been shown to<br />
be significantly altered during early and late preimplantation<br />
period compared to equivalent day of<br />
oestrous cyle in cattle (day 5 to day 16 post-oestrus<br />
[25]; day 17 [71]; day 18 [5,37]; day 20 [42]). Some<br />
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of the identified genes appeared to be preferentially<br />
regulated in the caruncles or in the intercaruncular<br />
areas, suggesting specific functions related to the<br />
development of the placentomes [42]. Collectively,<br />
these data have demonstrated an important global<br />
reaction of the endometrium –quantitatively and<br />
qualitatively- while facing the conceptus.<br />
Nevertheless recent studies first carried out in cattle<br />
then supported by human data have demonstrated a<br />
subtle property of the mammalian endometrium.<br />
In ruminants, in vitro maturation, in vitro<br />
fertilization (IVF) and subsequent in vitro embryo<br />
culture have been reported to significantly alter gene<br />
expression patterns in blastocysts and elongating<br />
embryos when compared to their in vivo derived<br />
counterparts [17,44,46]. Although the rate of success<br />
is still very low, somatic cell nuclear transfer (SCNT)<br />
can lead to term-development of cloned embryos<br />
when correct nuclear reprogramming takes place<br />
[32,48,56]. Nevertheless, severe or fatal<br />
consequences on embryo and foetal-placental development<br />
have also been reported and shown to considerably<br />
vary according to the origin of somatic cell<br />
lines and across laboratories [74]. These embryos with<br />
distinct potentials of term development were<br />
postulated to elicit different endometrial gene patterns<br />
that could account for the final outcome of a<br />
pregnancy. Our research group [43] demonstrated<br />
that late pre-implantation endometrium shows<br />
dramatic changes in gene expression related to the<br />
way the embryo was produced, namely by in vivo<br />
fertilization, in vitro maturation and IVF or SCNT.<br />
These data have led to the original concept defining<br />
the endometrium as an early biosensor of embryo<br />
quality [43]. Data mining showed immune response<br />
and metabolism as the two most affected biological<br />
functions and differential expression of candidate<br />
genes was confirmed. By comparing endometrial<br />
transcriptomes of cows recipient for IVF-derived<br />
embryos or cloned embryos generated with various<br />
somatic cells lines, several endometrial genes have<br />
also been shown to be associated with nuclear transfer<br />
procedure at day 18 post-oestrus [6]. Very<br />
interestingly, recent human data proposed that<br />
decidualizing endometrial stromal cells can sense<br />
embryo quality and would be able to eliminate compromised<br />
embryos [59,70]. Therefore the endometrium<br />
as a biosensor of embryo displaying divergent<br />
developmental potencies appears to be a feature<br />
valid across mammals, despite structural differences<br />
in implantation and placentation.<br />
V. CONCLUSION<br />
In mammalian species, placentation and foetal<br />
development represent the major part of pregnancy<br />
and last much longer than the pre- and periimplantation<br />
periods. Detrimental events taking place<br />
in the uterine environment beyond implantation have<br />
been shown to dramatically affect the outcome of<br />
pregnancy by altering placenta functions and foetus<br />
development [13]. Nevertheless, current data<br />
demonstrate that congenital anomalies, acquired<br />
diseases or perturbations of adult maternal physiology<br />
during reproductive life (e. g. stress, nutrition;<br />
endocrine disruptors, infection; figure 1) can affect<br />
endometrial function in a permanent or transient<br />
manner. Distinct endometrial responses can also be<br />
elicited by embryos presenting different postimplantation<br />
fates, making endometrium an early<br />
biosensor of embryo developmental potential. Hence<br />
mammalian endometrium appears as a dynamic and<br />
reactive tissue whose compromised or suboptimal<br />
physiology can deeply or subtly affect embryo<br />
development before implantation with visible and<br />
sometimes severe consequences on placentation<br />
process, fetal development and pregnancy outcome.<br />
Consequently, although term pregnancy issue<br />
incontestably relies on the quality of the embryo (the<br />
seed) [52], it is obvious that endometrium (the soil)<br />
has to be considered as a critical contributor for<br />
embryo trajectory as early as pregnancy initiates. In<br />
the context of assisted reproductive technologies,<br />
developing diagnosis and prognosis tools based on<br />
endometrial factors will be valuable with the<br />
objectives to estimate the reproductive capacity of<br />
the mother, to correct suboptimal endometrial<br />
functionality or to assess the developmental potential<br />
of the embryo. On a more fundamental aspect, an<br />
essential issue will be to carefully analyse situations<br />
in which embryos can overcome the control exerted<br />
by the endometrium, as illustrated by the defect in<br />
nature’s quality control proposed by Aplin and<br />
collaborators [53]. In other words, embryos that fail<br />
to develop to term would be able to implant. In this<br />
situation, the unusual properties of these embryos (e.<br />
g. producing an excess of embryo-derived signals)<br />
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O. Sandra. <strong>2011</strong>. Deciphering early sensor and driver properties of the endometrium: contribution of the uterus to<br />
pregnancy outcome. ssssssssssssssss ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s173 - s182.<br />
as well as the quality of the endometrial milieu will<br />
have to be clearly defined. Eventuallly, determining<br />
the limits of endometrial plasticity at the onset of<br />
pregnancy represents difficult tasks but essential<br />
challenges for providing new insights on the contribution<br />
of maternal environment to embryo epigenetic<br />
shaping in link with success, alterations or failure of<br />
pregnancy.<br />
Acknowledgements. O. S. was a member of the European<br />
Network of Excellence on “Embryo Implantation Control”<br />
(EMBIC) partially funded by the European Union Grant<br />
LSHN-CT-2004-512040 (FP6). O. S. is supported by grants<br />
from INRA, EMBIC and Agence National pour la Recherche<br />
(ANR-08-GENM-037).<br />
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www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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hebel.<br />
el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s183 - s202.<br />
ISSN 1679-9216 (Online)<br />
Use of Applied Repr<br />
eproduc<br />
ductiv<br />
tive Technolo<br />
echnologies (FTAI,<br />
FTET) ) to Improve the<br />
Reproductive Efficiency in Dairy Cattle<br />
Ricardo C. Chebel<br />
ABSTRACT<br />
Background: Reproductive inefficiency of dairy cattle, characterized by reduced estrous expression and detection rates,<br />
reduced pregnancy per artificial insemination (number of cows pregnant divided by number of cows inseminated), reduced<br />
pregnancy rates (number of cows pregnant divided by the number of cows eligible to become pregnant during a time interval),<br />
and increased pregnancy losses, has a large financial impact on dairy operations across the world. Although the most important<br />
component of reduced reproductive efficiency in dairy cattle is unquestionably poor management and diseases that result<br />
from it, the genetic selection and the resulting increased milk yield have caused physiological changes in lactating dairy cows<br />
that also affect fertility. The most important of these changes is the increased feed intake and the consequent increased<br />
mesenteric and liver blood flow to supply the nutrients necessary for milk yield. This causes significant decreases in<br />
concentrations of progesterone and estradiol that affect estrous expression, follicular growth, oocyte quality, and embryo<br />
development and survival. This review will discuss reproductive technologies used in large dairy herds to mitigate the effects<br />
of these physiological changes on reproductive performance.<br />
Review: The use of ovulation/estrous synchronization protocols (OSP), pre and post-ovulation hormonal treatments, and<br />
embryo transfer (ET) in the reproductive management of lactating dairy cows was reviewed. Several OSP have been developed<br />
in the past 20 years. To achieve acceptable pregnancy per artificial insemination (P/AI) OSP should result in synchronized<br />
recruitment of a new follicular wave, growth of follicles under P4 concentration > 2 ng/mL, synchronized luteolysis, and<br />
N<br />
synchronized ovulation at the end of the protocol. When embryo recipient cows are submitted to OSP, these protocols must<br />
aim to tightly synchronize luteolysis and ovulation at the end of the protocol. The use of ET in lactating dairy cows in the U.S.<br />
has been limited to herds of registered animals, to mitigate the negative effects of exposure to heat stress, to improve genetics<br />
of expanding herds, and in a few herds to salvage repeat-breeders. Lactating dairy cows are sensitive to heat stress because of<br />
the high metabolic rate resulting from the increased feed intake necessary to supply nutrients for milk production. Several<br />
studies have demonstrated that lactating dairy cows exposed to heat stress that receive ET have improved reproductive<br />
performance compared with cows receiving AI. Finally, the use of hormonal treatments to increase P4 concentration during<br />
early diestrus was reviewed because several studies have demonstrated a strong association among P4 concentration and<br />
embryo development and pregnancy establishment. The effects of hormonal treatments during ovulation synchronization<br />
protocols, after AI or at ET on P4 concentration and reproductive outcomes are controversial and likely dependent on<br />
management, milk yield, and diet of the lactating dairy cows.<br />
Conclusion: The use of reproductive technologies in lactating dairy cows, particularly AI, is extremely well disseminated and<br />
has resulted in significant improvements in milk yield in the past 50 years. Recent developments in the understanding of<br />
reproductive physiology of lactating dairy cows have resulted in ovulation synchronization protocols that optimize fertility<br />
after AI or ET.<br />
Keywords: Lactating dairy cow, synchronization, artificial insemination, embryo transfer.<br />
CORRESPONDENCE: R.C. Chebel [chebe002@umn.edu – PHONE: +1 ( 612) 625-3130]. Department of Veterinary Population Medicine,<br />
University of Minnesota, 1365 Gortner Ave, ZIP CODE: 551098, Saint Paul, MN, USA.<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
I. INTRODUCTION<br />
II. DISCUSSION<br />
2.1 Physiological Changes Associated with Reduced<br />
Fertility<br />
2.2 Ovulation synchronization protocols<br />
2.2.1 Presynchronization.<br />
2.2.2 Resynchronization.<br />
2.2.3 Reducing the period of dominance of the ovulatory<br />
follicle (5d-Cosynch).<br />
2.2.4 Low P4 concentration and fertility.<br />
2.3 Embryo transfer<br />
2.3.1 Mitigation of effects of heat stress on reproductive<br />
efficiency.<br />
2.3.2 Reproductive performance of repeat-breeder cows.<br />
2.3.3 Effects of P4 concentration on embryo survival.<br />
2.4 Hormonal treatments to increase P4 concentration<br />
during diestrus and pregnancy after AI or ET.<br />
III. CONCLUSIONS<br />
I. INTRODUCTION<br />
Fertility in lactating dairy cows has been decreasing<br />
in the past 50 years. This decrease in fertility<br />
has been associated with a steady increase in milk<br />
yield, which is a consequence of genetic selection<br />
for milk yield and improvements in nutritional<br />
management. According to the National Animal<br />
Health Monitoring System [49], reproductive failure<br />
is the most important cause of involuntary culling.<br />
The costs of reproductive inefficiency to the cattle<br />
industry are extremely important and have been<br />
recognized as such for decades. Senger et al. [71]<br />
suggested that the dairy industry loses approximately<br />
$ 300 million per year because of poor estrous<br />
detection rate and accuracy. The estimated average<br />
value of a pregnancy is $ 275 [20] and that of an<br />
abortion is between $ 555 and $ 640 [20,80]. These<br />
values are dependent on lactation number, milk yield,<br />
days in milk (DIM), price of milk, and cost of replacement<br />
animals.<br />
In technical terms, compromised reproductive<br />
inefficiency involves fertilization failure -<br />
observed from the day of AI to 5-6 d after AI, early<br />
embryonic loss - observed from 5-6 d after AI to 17-<br />
24 d after AI, late embryonic loss - observed from<br />
17-24 d after AI to 42 d after AI, and fetal loss -<br />
observed from 42 d after AI to term [64]. Reduced<br />
fertilization and increased early embryonic loss are<br />
usually observed as increased return to estrus after<br />
AI. Increased late embryonic loss is observed as altered<br />
inter-estrus interval or increased abortions if the first<br />
pregnancy diagnosis takes place before 42 d after<br />
AI. Increased fetal losses are observed as increased<br />
number of abortions after 42 d after AI (Figure 1).<br />
Ultimately, in large dairy herds, reproductive failure<br />
is observed as reduced pregnancy per AI (P/AI;<br />
number of pregnancies divided by the number of<br />
cows inseminated), increased number of abortions,<br />
and decreased pregnancy rates (number of cows<br />
pregnant within a time period divided by the number<br />
of cows eligible to become pregnant during the same<br />
period).<br />
Figure 1. Characterization of reproductive failure in cattle.<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
Different studies have evaluated the fertilization<br />
of oocytes and quality of embryos from<br />
lactating dairy cows, heifers, and non-lactating dairy<br />
cows [10,12,17,21,67]. Although not all studies<br />
compared production and quality of embryos by<br />
lactating dairy cows directly with that of heifers and<br />
non-lactating dairy cows, it is estimated that nonsuperovulated<br />
lactating dairy cows have approximately<br />
76% of recovered structures fertilized, whereas<br />
non-lactating cows and heifers have 78% and 100%,<br />
respectively, of recovered structures fertilized.<br />
Furthermore, approximately 66% of fertilized<br />
structures recovered from lactating dairy cows are<br />
classified as excellent/good quality embryos but<br />
approximately 74% and 72% of fertilized structures<br />
recovered from non-lactating dairy cows and heifers,<br />
respectively, are classified as excellent/good quality<br />
embryos. Consequently, among all oocyte-embryo<br />
recovered from lactating dairy cows, 50% are<br />
classified as excellent/good embryos, whereas 58%<br />
and 72% of all oocyte-embryo recovered from nonlactating<br />
cows and heifers, respectively, are classified<br />
as excellent/good embryos. In recent studies<br />
conducted by our group in several herds across the<br />
U.S.A. we observed P/AI of lactating dairy cows to<br />
be between 35 and 40% at 30 to 38 d after first<br />
postpartum AI [13,14,62]. Therefore, it is expected<br />
that 25% of all excellent/good quality embryos will<br />
be lost between 6 and 35 d after first postpartum AI<br />
of lactating dairy cows, representing 1.78% of<br />
embryonic losses per day.<br />
Summarization of data from 15 different<br />
studies conducted in the U.S.A. that reported late<br />
embryonic loss demonstrates that pregnancy losses<br />
from 27-31 to 38-50 d after AI is approximately 13%<br />
with a range of 3 to 43%. This represents pregnancy<br />
losses of approximately 0.85% per day during this<br />
period. Furthermore, incidence of late embryonic/fetal<br />
losses from approximately 40 to 120 d after AI has<br />
been reported to range from 8.3 to 10.7%, which<br />
represents daily losses of approximately 0.11% of<br />
pregnancies diagnosed at 40 d after AI. On the other<br />
hand, according to data from six published<br />
manuscripts (total of 7,426 AI) P/AI at 38 d after first<br />
AI in virgin heifers ranges from 55 to 70% and only<br />
approximately 3% of heifers lose pregnancy from<br />
38 to 120 d of gestation, resulting in daily pregnancy<br />
loss of approximately 0.05%.<br />
From these data it is obvious that the stages<br />
of greatest risk for reproductive failure are fertilization,<br />
embryo development, maternal recognition of<br />
pregnancy, and placentation. Furthermore, it is clear<br />
that lactating dairy cows are less likely to conceive<br />
and to carry out the pregnancy to term than virgin<br />
heifers. Although the factors associated with reduced<br />
fertility in lactating dairy cows are multiple and multifaceted,<br />
they all originate from the ability or lack<br />
thereof of lactating dairy cows to cope with the<br />
nutritional demands associated with the extremely<br />
elevated milk yield. With the onset of colostrum/milk<br />
production lactating dairy cows face severe nutritional<br />
demands that are usually not fully met by feed intake<br />
and result in negative energy balance, metabolic<br />
diseases, immune suppression, and increase incidence<br />
of diseases. In this manuscript we will discuss the<br />
effects of increased milk yield on physiological<br />
alterations that affect reproductive efficiency, and we<br />
will discuss reproductive technologies used to mitigate<br />
the effects of these physiological alterations on<br />
reproductive performance.<br />
II. DISCUSSION<br />
2.1 Physiological Changes Associated with Reduced<br />
N<br />
Fertility<br />
There are several hormones that are extremely<br />
important to the reproductive function of ruminants<br />
[e.g. progesterone (P4), estradiol, GnRH, LH, FSH,<br />
and prostaglandin (PG) F 2α<br />
]. In this section, we will<br />
briefly discuss the importance of P4 and estradiol,<br />
their concentrations, and metabolism in lactating dairy<br />
cows.<br />
Estradiol is produced by antral ovarian<br />
follicles. Under reduced concentrations of P4 (< 1<br />
ng/mL) estradiol is responsible for signs of estrus and<br />
a positive feed-back on the hypothalamus, which<br />
stimulates secretion of GnRH that causes the pituitary<br />
gland to produce an ovulatory LH-peak. Furthermore,<br />
priming of the uterus with estradiol during the<br />
proestrus is expected to reduce the binding capacity<br />
of oxytocin to its endometrial receptors [43], reducing<br />
the positive feed-back of oxytocin on endometrial<br />
production of PGF 2α<br />
. For example, ovariectomized<br />
cows treated with exogenous estradiol, mimicking<br />
concentrations of estradiol during the proestrus, had<br />
smaller concentrations of PGF 2α<br />
metabolite after<br />
oxytocin challenge compared with oxytocin-challen-<br />
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R.C. Cheb<br />
hebel.<br />
el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
ged ovariectomized cows not treated with estradiol<br />
[43].<br />
Several studies have found a link between<br />
metestrus and early diestrus P4 concentrations and<br />
embryo development and elongation and the<br />
subsequent establishment of pregnancy. Mann and<br />
Lamming [42] demonstrated that cows that had the<br />
largest embryos at 16 d after AI were also the cows<br />
that had the greatest P4 concentrations starting at<br />
approximately d 5 after AI and that larger embryos<br />
produced greater quantities of interferon-τ. This<br />
indicates that increased P4 concentrations during early<br />
diestrus should result in hastened development of<br />
embryos and improved signaling from the embryo<br />
for maternal recognition of pregnancy [42]. Despite<br />
the fact that mRNA for P4 receptors can be identified<br />
in nuclei of cells of early bovine embryos, in vitro<br />
exposure of cleaved embryos to elevated P4<br />
concentrations did not affect subsequent development<br />
to the blastocyst stage, nor recovery rates of 14 d old<br />
in vitro produced (IVP) embryos 7 d after transfer<br />
[16]. On the other hand, supplementation with P4<br />
between d 3 and 7 of pregnancy did not alter the<br />
morphology of embryos recovered in the morula to<br />
blastocyst stage, but conceptus from heifers<br />
supplemented with P4 were significantly larger at d<br />
13 and 16 after AI [9]. Similarly, when multiple<br />
embryos were transferred into superstimulated<br />
recipient heifers it was observed that embryos<br />
transferred into these heifers were significantly larger<br />
at d 13 than embryos transferred into nonsuperstimulated<br />
heifers, indicating a strong<br />
association between P4 concentration and embryo<br />
development after the blastocyst stage [40]. The<br />
establishment of a uterine environment conducive to<br />
embryo growth and elongation appears to be P4<br />
dependent, because alterations in uterine gene<br />
expression are induced by increased P4 concentrations.<br />
For example, messenger RNA expression<br />
for transport and secretory proteins (e.g. lipoprotein<br />
lipase and connective tissue growth factor) present<br />
in the bovine endometrium, thought to contribute to<br />
uterine histotroph and thus conceptus elongation,<br />
were expressed earlier during diestrus and at higher<br />
levels in cows with elevated P4 concentrations [26].<br />
Studies have compared the concentrations of<br />
estradiol and P4, the diameter of ovulatory follicles,<br />
and corpora lutea (CL) volume between lactating<br />
dairy cows and non-lactating cows or heifers. Lopez<br />
et al. [41] demonstrated that high producing lactating<br />
dairy cows (47 kg/day) had smaller peak concentration<br />
of estradiol during estrus (6.8 pg/mL) compared<br />
with low producers (32 kg/day - 8.6 pg/ml) and<br />
heifers (11.3 pg/mL), despite having larger ovulatory<br />
follicles (high producers = 18.6 mm, low producers<br />
= 17.4 mm, and heifers = 15 mm). Consequently,<br />
high producing dairy cows had shorter duration of<br />
estrus (high producers = 7 h, low producers = 11.9 h,<br />
and heifers = 11.3 h) and had fewer standing events<br />
during estrus (high producers = 6.5, low producers =<br />
9.8, and heifers = 16.8 mounts). Furthermore, Sartori<br />
et al. [66] demonstrated that lactating dairy cows had<br />
smaller concentration of estradiol during estrus than<br />
non-lactating dairy cows (7.9 vs. 11.3 pg/mL). Besides<br />
affecting estrus detection rates, this reduced estradiol<br />
concentration is expected to result in prolonged<br />
interval from luteolysis to ovulation (lactating cows<br />
= 5.2 ± 0.2 d, non-lactating cows = 4.6 ± 0.1 d) [66]<br />
because the estradiol threshold necessary to stimulate<br />
an LH surge would take longer to be reached in<br />
lactating dairy cows. Extended interval from luteolysis<br />
to ovulation compromises oocyte quality because<br />
pre-ovulatory follicles are exposed to reducing P4<br />
concentrations and increasing LH pulsatility for longer<br />
periods resulting in premature oocyte maturation.<br />
Lactating dairy cows also have reduced P4<br />
concentrations compared with heifers starting as early<br />
as d 5 of the estrous cycle [66]. The reduced P4 concentrations<br />
to which cows are exposed during metestrus<br />
and diestrus may result in exposure of follicles<br />
to increasing pulsatile release of LH, which causes<br />
premature oocyte maturation and reduced embryo<br />
quality [8,38]. Oocytes collected on d 8 of the estrous<br />
cycle from cows with P4 concentration declining from<br />
1.7 to 0.6 ng/mL from estrous cycle d 6 to 9 were<br />
more likely to be in stage II of meiosis compared<br />
with oocytes from cows that had P4 concentration<br />
increasing from 1.4 to 3.1 ng/mL during the same<br />
period [38]. Furthermore, cows exposed to P4 < 1<br />
ng/mL before ovulation are at higher risk for short<br />
luteal phase, because the lack of P4 priming results<br />
in premature increase in estradiol receptors in the<br />
endometrium following ovulation and consequently<br />
premature expression of oxytocin receptors in the<br />
endometrium, which leads to premature secretion of<br />
PGF 2α<br />
and luteolysis [31,88]. Exposure of cows to<br />
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reduced P4 concentration after AI may affect embryo<br />
growth and consequently production of IFN-τ,<br />
compromising maternal recognition of pregnancy and<br />
pregnancy establishment as described previously.<br />
It is not clear whether reduced production of<br />
estradiol and P4 or increased metabolism of estradiol<br />
and P4 or both are the cause for reduced estradiol<br />
and P4 concentrations in lactating dairy cows, but<br />
the latter is more likely. Studies conducted in<br />
Wisconsin have demonstrated that the rate of metabolism<br />
of steroidal hormones in lactating dairy cows<br />
is greater than that of non-lactating dairy cows [61].<br />
This seems to be directly correlated with the increased<br />
feed intake of lactating dairy cows and the consequent<br />
hyperthrophy and hyperplasia of the liver and organs<br />
of the gastrointestinal tract. This results in increased<br />
blood flow through the liver and greater metabolism<br />
of steroidal hormones. Sangsritavong et al. [61]<br />
demonstrated that unfed cows have reduced blood<br />
flow through the liver compared with cows fed 7.8<br />
lb/d, 23.4 lb/d, and 33.4 lb/d (Figure 2), and that faster<br />
decreases in P4 and estradiol concentrations are<br />
observed after feeding [61]. Similarly, cows receiving<br />
100 and 50% of NRC (2001) recommendations had<br />
significantly reduced P4 concentrations compared<br />
with cows receiving 25% of NRC recommendations<br />
or unfed cows (Figure 3) [85].<br />
This is clear evidence that high yield lactating<br />
dairy cows have reduced estradiol and P4 concentrations<br />
as a consequence of increased feed intake,<br />
N<br />
Figure 2. Effect of feed intake on liver blood flow. Adapted from Sangsritavong et al. [27].<br />
Figure 3. Effect of feed intake on progesterone concentration. Adapted from Vasconcelos et al. [28].<br />
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which is a consequence of increased milk yield. This<br />
poses significant challenges to the reproductive<br />
efficiency of these animals because of the importance<br />
of estradiol and P4 for reproductive efficiency,<br />
explaining in part the significant decreases in<br />
reproductive efficiency observed in the past decades.<br />
2.2 Ovulation synchronization protocols<br />
Fixed time AI (TAI) protocols were developed<br />
in 1995 with the goal of synchronizing follicular<br />
growth, luteolysis, and ovulation. The first protocol<br />
developed was the Ovsynch, which consists of one<br />
injection of GnRH on d 0, one injection of PGF 2α<br />
on<br />
d 7, a second GnRH injection approximately 56 h<br />
after the PGF 2α<br />
injection and TAI at 12-16 h later<br />
[54]. The first GnRH injection synchronizes a new<br />
follicular wave, whereas the PGF 2α<br />
injection<br />
synchronizes luteolysis, and the last GnRH injection<br />
synchronizes ovulation. Subsequent studies<br />
demonstrated that the ideal time to initiate the<br />
Ovsynch protocol is between d 5 and 9 of the estrous<br />
cycle, because at this stage of the estrous cycle more<br />
lactating dairy cows ovulate to the first GnRH<br />
injection of the protocol [86]. Later, it was<br />
demonstrated that the ovulation to the first GnRH<br />
injection of the Ovsynch protocol is critical for<br />
embryo quality [9] and P/AI [15] of lactating dairy<br />
cows, because cows that do not ovulate to the first<br />
GnRH injection have prolonged dominance period<br />
of the ovulatory follicle [9] and ovulate aged oocytes<br />
[45]. Thus, presynchronization protocols were<br />
developed in an attempt to maximize the number of<br />
cows that start the timed AI protocol between d 5<br />
and 9 of the estrous cycle.<br />
2.2.1 Presynchronization.<br />
The first presynchronization protocol<br />
developed at the University of Florida was based on<br />
two injections of PGF 2α<br />
given 14 d apart (Presynch)<br />
[47]. In this study, cows submitted to the Ovsynch<br />
protocol 12 d after receiving the Presynch had P/AI<br />
approximately 12 percentage points greater than<br />
those not presynchronized [47]. By giving 2 injections<br />
of PGF 2α<br />
14 d apart the percentage of cows that<br />
display estrus from 2 to 6 d after the second injection<br />
is expected to be 65% [15], depending on compliance<br />
to the protocol and the percentage of anovular<br />
cows in the herd. Therefore, it is expected that by<br />
starting the Ovsynch protocol 10 to 12 d after the last<br />
PGF 2α<br />
injection the majority of cows would be<br />
between d 4 and 10 of the estrous cycle.<br />
In an attempt to simplify the Presynch-<br />
Ovsynch protocol by giving most injections on the<br />
same of the week, Navanukraw et al. [50] compared<br />
the fertility of cows submitted to the Ovsynch protocol<br />
alone with the fertility of cows submitted to a<br />
Presynch-Ovsynch with the last PGF 2á<br />
injection<br />
given14 d before the start of the Ovsynch (14-14<br />
Presynch-Ovsynch). In this study, cows receiving the<br />
Presynch-Ovsynch (14-14) had greater P/AI than<br />
cows receiving the Ovsynch alone.<br />
Galvão et al. [29] compared the fertility of<br />
cows submitted to the 14-14 Presynch-Ovsynch to<br />
that of cows submitted to a 14-11 Presynch-Ovsynch<br />
(interval between the last PGF 2α<br />
injection of the<br />
Presynch and the start of the Ovsynch = 11 d). In this<br />
study, cows receiving the 14-11 Presynch-Ovsynch<br />
had P/AI 6 percentage points higher than cows<br />
receiving the 14-14 Presynch-Ovsynch [29]. This<br />
improvement in fertility seems to result from the<br />
increased percentage of cows that ovulated to the first<br />
GnRH injection of the Ovsynch protocol when<br />
submitted to the 14-11 Presynch-Ovsynch compared<br />
with the 14-14 Presynch-Ovsynch [29].<br />
More recently, presynchronization protocols<br />
based on GnRH and PGF 2α<br />
injections have been<br />
developed. Double-Ovsynch is the most know of<br />
these protocols as more peer-reviewed data exists<br />
[74]. As the name suggests, cows are submitted to a<br />
‘presynchronizing-Ovsynch’ and 7 d after its end<br />
cows are submitted to a ‘breeding-Ovsynch’. This<br />
protocol has the following potential benefits:<br />
improved synchrony of the estrous cycle, anovular<br />
cows are more responsive to it than to the Presynch-<br />
Ovsynch, and more cows are likely to have growth<br />
of the ovulatory follicle under P4 concentrations > 2<br />
ng/ml. The studies published recently comparing the<br />
Double-Ovsynch and the Presynch-Ovsynch,<br />
however, reported improvements in P/AI only in<br />
primiparous cows submitted to Double-Ovsynch, but<br />
not in multiparous cows [74]. It is unclear why only<br />
primiparous cows benefited from the Double-<br />
Ovsynch, but one could speculate that because<br />
greater percentage of primiparous cows are expected<br />
to be anovular early in lactation compared with multiparous<br />
cows, the former would benefit the most from<br />
the additional GnRH injections given during the<br />
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Double-Ovsynch. It is important to point out,<br />
however, that cows submitted to the Double-Ovsynch<br />
are less likely to be observed in estrus because of the<br />
multiple GnRH injections that they receive and this<br />
would affect estrous detection rates.<br />
Therefore, the recommended protocol for first<br />
postpartum AI for herds with good estrous detection<br />
rate is the Presynch-Ovsynch, with the interval<br />
between the last PGF 2α<br />
injection of the Presynch and<br />
the start of the Ovsynch of 10 to 12 d.<br />
2.2.2 Resynchronization.<br />
Most researchers agree that resynchronization<br />
protocols to which cows diagnosed non-pregnant are<br />
submitted to have to be optimized. Resynchronization<br />
protocols used are dependent on herd size, estrous<br />
detection rate, and diagnosis of pregnancy by<br />
ultrasonography or manual palpation per rectum.<br />
Most published research on resynchronization<br />
protocols evaluated the effect of timing of initiation<br />
of the resynchronization protocol (usually the<br />
Ovsynch protocol or an adaptation thereof) on P/AI.<br />
It is clear from these published manuscripts that<br />
starting the resynchronization protocols before d 28<br />
post-AI will result in reduced P/AI (Figure 4). This is<br />
likely because a large percentage of cows that start<br />
the resynchronization protocol before d 28 post-AI<br />
would be in proestrus, estrus, or metestrus at the start<br />
of the resynchronization protocol, affecting ovulation<br />
to the first GnRH injection, P4 concentration during<br />
ovulatory follicle growth, and synchrony of luteolysis.<br />
Theoretically, if the length of the estrous cycle<br />
of lactating dairy cows is 23 d [66], the ideal interval<br />
from AI to start the resynchronization protocol would<br />
be between 28 and 32 d post-AI (d 5 to 9 of the new<br />
estrous cycle). It is interesting to note, however, that<br />
recent studies from our laboratory in collaboration<br />
with other researchers demonstrated that starting the<br />
resynchronization protocol at different intervals after<br />
d 27 post-AI does not affect P/AI [6]. In light of the<br />
fact that only 52% of cows non-pregnant to a previous<br />
AI are observed in estrus between 20 and 24 d post-<br />
AI, this finding is not surprising (Chebel personal<br />
communication, 2010). Several factors are likely to<br />
affect the pattern of return to estrus: 1. approximately<br />
15% of cows submitted to ovulation synchronization<br />
protocols do not have the estrous cycle properly<br />
synchronized and 10 to 15% of cows inseminated<br />
based on signs of estrus are not truly in estrus; 15%<br />
of postpartum cows are anovular cows that have<br />
shorter luteal phase after first postpartum AI; and, 3.<br />
approximately 18% of inseminated cows are<br />
expected to have early/late embryonic death. Thus,<br />
it is not surprising that we can poorly predict the stage<br />
of the estrous cycle that cows are in at the start of<br />
resynchronization protocols, making necessary<br />
strategies to presynchronize the estrous cycle of nonpregnant<br />
cows before the start of resynchronization.<br />
N<br />
A limiting factor to controlling the estrous cycle of<br />
inseminated cows before the start of resynchronization<br />
protocols is the fact that PGF 2á<br />
cannot be<br />
used until cows are diagnosed non-pregnant.<br />
Figure 4. Effects of interval between AI and initiation of the resynchronization protocol (Ovsynch or a variation<br />
thereof) on pregnancy per AI.<br />
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Although the indicated label use of CIDR<br />
inserts is to improve return to estrus when used from<br />
14 to 21 d after initial AI, the use of CIDR inserts<br />
according to label recommendations has proven to<br />
be inefficient, as the interval to re-insemination and<br />
proportion of cows re-inseminated prior to pregnancy<br />
diagnosis is not improved [15,29].<br />
Recently, presynchronization protocols for<br />
non-pregnant cows before the start of the<br />
resynchronization have been explored. In one study,<br />
non-pregnant cows were either resynchronized with<br />
the Ovsynch protocol starting at 33 d post-AI or with<br />
a presynchronizing injection of PGF 2α<br />
at 34 d post-<br />
AI and the Ovsynch protocol at 46 d post-AI [72].<br />
Cows presynchronized with PGF 2α<br />
had greater P/AI<br />
than cows resynchronized with the Ovsynch alone<br />
(35.2 vs. 25.6%) [72]. Similarly, cows resynchronized<br />
with the Double-Ovsynch (start of the<br />
‘presynchronization-Ovsynch’ 22 d post-AI, nonpregnancy<br />
diagnosis 29 d post-AI by ultrasonography,<br />
and start of the ‘breeding-Ovsynch’ 39 d post-<br />
AI) had greater P/AI than cows resynchronized with<br />
the Ovsynch alone starting at 32 d post-AI (38.5 vs.<br />
30%) [32]. Although these experiments clearly<br />
demonstrate that improvements in P/AI to<br />
resynchronized AI could be obtained from presynchronizing<br />
the resynchronization protocol, these<br />
protocols resulted in inter-AI interval 7 to 13 d longer<br />
compared with resynchronizing with the Ovsynch<br />
alone, which could offset the improvements in P/AI.<br />
Our laboratory has recently conducted several<br />
experiments evaluating different resynchronization<br />
protocols. In the most recent study [24], cows at 31<br />
± 3 d post-AI were selected to receive one of three<br />
resynchronization protocols: Cosynch72 starting at<br />
non-pregnancy diagnosis; CIDRsynch =<br />
Cosynch72+CIDR starting at non-pregnancy<br />
diagnosis; or G7G = GnRH injection at enrollment<br />
and start the Ovsynch at non-pregnancy diagnosis.<br />
All cows were examined for pregnancy 7 d after<br />
enrollment, at 38 ± 3 d post-AI. Throughout this study,<br />
cows observed in estrus were re-inseminated on the<br />
same day. Among cows re-inseminated at fixed (after<br />
the completion of the resynchronization protocol),<br />
cows in the G7G and CIDRsynch treatments had the<br />
greatest P/AI (Cosynch72 = 22.1%, G7G = 31.2%,<br />
CIDRsynch = 29.5%) [24]. When we evaluated the<br />
data from all cows, including those re-inseminated<br />
in estrus, the differences in overall P/AI after reinsemination<br />
were smaller (Cosynch72 = 28%, G7G<br />
= 32%, CIDRsynch = 31%) [46]. That was mainly<br />
because the presynchronizing GnRH injection given<br />
to G7G cows and the treatment with CIDR during<br />
the resynchronization protocol reduced the percentage<br />
of G7G cows and CIDR cows that were reinseminated<br />
in estrus (Figure 5) [46]. Among cows<br />
submitted to the Cosynch72 treatment the P/AI of<br />
those re-inseminated in estrus was significantly better<br />
than that of cows re-inseminated at fixed time, which<br />
increased their overall P/AI (Figure 6) [46].<br />
In a subsequent study, we evaluated the<br />
effects of a presynchronizing GnRH injection given<br />
at different intervals post-AI. In this study, cows<br />
received a presynchronizing GnRH injection at 17<br />
or 24 d post-AI and started the Ovsynch 7 d later [7,<br />
46]. Thus, there were 4 treatments: EGGPG – d 17<br />
Figure 5. Percentage of cows re-inseminated in estrus at different intervals in relation to the start of the<br />
resynchronization protocol (Day 0) and at fixed time. Adapted from Mendonça et al. [42].<br />
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Figure 6. Pregnancy per AI according to resynchronization protocol and re-insemination procedure<br />
(AI in estrus or at fixed time (TAI)). Adapted from Mendonça et al. [42].<br />
GnRH, d 24 GnRH, d 31 PGF 2α<br />
(if diagnosed nonpregnant),<br />
d 33 GnRH, and d 34 TAI; EOVS – same<br />
as EGGPG without the presynchronizing GnRH on<br />
d 17 post-AI; LGGPG – d 24 GnRH, d 31 GnRH (if<br />
diagnosed non-pregnant), d 38 PGF 2α<br />
, d 40 GnRH,<br />
and d 41 TAI; and, LOVS – same as LGGPG without<br />
the presynchronizing GnRH on d 24 post-AI [7,23].<br />
Cows were re-inseminated at any time if observed in<br />
estrus. Percentage of cows re-inseminated in estrus<br />
was smallest for EGGPG treatment and greatest for<br />
LOVS treatment (EGGPG=23.7, EOVS = 41.6,<br />
LGGPG = 49.0, LOVS = 57.6%) and the interval to<br />
re-insemination was slightly shorter for EGGPG and<br />
EOVS cows (EGGPG = 13.7 ± 0.2, EOVS = 11.6 ±<br />
0.2, LGGPG = 15.4 ± 0.3, LOVS = 14.6 ± 0.3 d)<br />
[7,23]. Overall P/AI [including cows re-inseminated<br />
in estrus or at fixed time (TAI) upon completion of<br />
the resynchronization protocol] was not different<br />
among treatments (EGGPG = 26.2, EOV = 29.1,<br />
LGGPG = 30.5, LOV = 30.5%) [7,23]. Regardless<br />
of treatment or farm, cows re-inseminated in estrus<br />
had greater P/AI at 66 d post-AI than cows that<br />
received TAI (36.0 vs. 23.9%). Among cows receiving<br />
TAI upon completion of the resynchronization<br />
protocol treatment did not affect P/AI (EGGPG = 26.1,<br />
EOV = 19.4, LGGPG = 25.3, LOV = 23.8%) [7,23].<br />
The use of CIDR within the resynchronization<br />
protocol has also been evaluated by our group in<br />
collaboration with other researchers [6]. In this study<br />
non-pregnant cows were initiated in the<br />
resynchronization protocol at 32 or 39 d post-AI and<br />
receive or did not receive a CIDR insert during the<br />
resynchronization protocol [6]. In this study, we<br />
observed that the interaction between time of initiation<br />
of the resynchronization protocol and CIDR treatment<br />
affected P/AI, because CIDR treatment tended to<br />
increase P/AI of cows starting the resynchronization<br />
protocol at 39 d post-AI (28 vs. 23.7%) but had no<br />
effect on P/AI of cows that started the<br />
resynchronization protocol at 32 d post-AI (no CIDR<br />
N<br />
= 26.9 and CIDR = 24.2%) [6].<br />
In summary, resynchronization protocols<br />
should start after 28 d post-AI. Up to date, no presynchronization<br />
protocols or additional hormonal treatments<br />
(e.g. CIDR during the resynchronization) have<br />
been able to increase P/AI of cows that start the<br />
resynchronization protocol between 28-34 d post-AI.<br />
On the other hand, cows that start the resynchronization<br />
protocol at 35-41 d post-AI should be presynchronized<br />
(e.g. PGF 2α<br />
or GnRH or Ovsynch) or<br />
treated with CIDR during the resynchronization<br />
protocol to increase P/AI to resynchronized TAI.<br />
Regardless of the resynchronization protocol chosen,<br />
herds that have good estrous detection accuracy and<br />
rate should not use 100% timed AI for re-insemination<br />
of non-pregnant cows because more than likely P/AI<br />
of cows re-inseminated in estrus will be higher than<br />
that of cows re-inseminated upon completion of<br />
resynchronization protocols.<br />
2.2.3 Reducing the period of dominance of the ovulatory<br />
follicle (5d-Cosynch).<br />
Cows submitted to the Ovsynch protocol are<br />
expected to have an interval from ovulatory follicle<br />
emergence to ovulation of 8.5 d approximately.<br />
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According to Cerri et al. [10] reducing the interval<br />
from emergence to ovulation in 2.3 d (from 8.1 to<br />
5.8 d) results in significant improvements in embryo<br />
quality. Thus, we have tested the hypothesis that<br />
reducing the interval from the first GnRH injection<br />
of the timed AI protocol to insemination would<br />
improve fertility.<br />
Because to achieve this reduction in interval<br />
from the first GnRH injection to AI we would have<br />
to treat cows with PGF 2α<br />
5 d after the GnRH injection,<br />
which could result in suboptimal luteolysis, in a pilot<br />
study, we compared the percentage of cows that had<br />
luteolysis when PGF 2α<br />
was given on d 7 (COS72;<br />
GnRH on d 0, PGF2α on d 7, and GnRH+TAI on d<br />
10), on d 5 (COS5d1; GnRH on d 0, PGF2α on d 5,<br />
and GnRH+TAI on d 8), or on d 5 and 6 (COS5d2;<br />
GnRH on d 0, PGF2a, on d 5 and 6, and GnRH+TAI<br />
on d 8) after the first GnRH injection [62]. As<br />
expected the percentage of cows that had luteolysis<br />
was smallest for those receiving one injection of<br />
PGF 2α<br />
on d 5 after the GnRH (COS72 = 79.0,<br />
COS5d1 = 59.1, COS5d2 = 95.7%) [62].<br />
In a subsequent study, 933 cows were<br />
submitted to the Presynch and 12 d later to either the<br />
COS72 or the COS5d2 described previously [62].<br />
Cows receiving the COS5d2 had smaller ovulatory<br />
follicles (18.4 ± 0.3 and 16.8 ± 0.3 mm), reflecting<br />
the shorter interval from follicle recruitment to<br />
ovulation, and were more likely to have luteolysis<br />
(96.3%) compared with COS72 cows (91.5%) [62].<br />
Because greater luteolysis in COS5d2 cows could<br />
confound the effect of treatment on P/AI, we<br />
analyzed P/AI including only cows that had luteolysis<br />
and observed that COS5d2 cows had higher P/AI<br />
than COS72 cows at 38 (39.3 and 33.9%) and 66<br />
(36.7 and 32.5%) d after AI [62]. Thus, COS5d2<br />
treatment increased P/AI by reducing the dominance<br />
period of the ovulatory follicle [62].<br />
2.2.4 Low P4 concentration and fertility.<br />
The start of the timed AI protocols at 5 to 9 d<br />
of the estrous cycle is not only important to maximize<br />
the percentage of cows that ovulate to the first GnRH<br />
injection of the protocol and to assure that synchronized<br />
luteolysis will occur at the end of the protocol,<br />
but also to assure that ovulatory follicles grow under<br />
elevated P4 concentrations. As mentioned above,<br />
reduced concentrations of P4 before ovulation may<br />
result in increased exposure of follicles to pulsatile<br />
release of LH, which causes premature oocyte<br />
s192<br />
maturation and reduced embryo quality [8,38]. In<br />
recent studies conducted by my laboratory [23,56]<br />
we evaluated whether the reduced P/AI observed in<br />
anovular cows and cows induced to ovulate follicles<br />
of the first follicular wave was caused by the exposure<br />
of ovulatory follicles to reduced concentrations of P4.<br />
In these two studies we demonstrated that reduced P/<br />
AI of anovular cows and cows induced to ovulate the<br />
dominant follicle of the first follicular wave is a<br />
consequence of compromised embryo quality because<br />
of exposure to P4 concentration < 2 ng/mL during<br />
follicle growth (Figure 7 and 8). Interestingly, there<br />
was no effect of P4 concentration during growth of<br />
the ovulatory follicle on percentage of cows with short<br />
luteal phase after AI [46], indicating that the benefits<br />
of P4 concentration were likely associated with health<br />
of oocyte.<br />
2.3 Embryo transfer<br />
Embryo transfer (ET) is not a new technology<br />
as in 1891 Heape [36] reported the transfer of two 4-<br />
cell Angora embryos into inseminated Belgium rabbits<br />
and the production of four Belgium and two Angora<br />
young from the same dam. Only six decades later<br />
successful ET pregnancy [80] and birth of an ET calf<br />
[90] were reported. Since then, the growth of<br />
commercial application of ET in the cattle industry<br />
has been significant and, in 2007, 823,160 embryos<br />
were transferred [78].<br />
According to the National Animal Health<br />
Monitoring System [49], approximately 11.5% of<br />
U.S.A. dairy herds have transferred at least one embryo<br />
into a lactating dairy cow or heifer in 2006.<br />
Interestingly, a similar percentage of operations<br />
transferred embryos into only heifers or cows (8.9<br />
and 8.6, respectively) and slightly more operations<br />
transferred fresh than frozen embryos (8.2 vs. 7.7%).<br />
Although ET is primarily seem as a technique to<br />
improve the genetic composition of the herd, in recent<br />
years, ET has been used in dairy operations in an<br />
attempt to improve reproductive performance of<br />
lactating dairy cows.<br />
2.3.1 Mitigation of effects of heat stress on reproductive<br />
efficiency.<br />
High production dairy cows (daily milk yield<br />
> 35 kg) often consume 20 to 30 kg of dry matter per<br />
day. The increased dry matter intake (DMI) results in<br />
significant increases in metabolic rates and heat<br />
production, such that the daily heat production by
R.C. Cheb<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
Figure 7. Correlation among P4 concentration during the superstimulation protocol (P4) and<br />
percentage of cows producing at least one transferable (solid line; percentage of cows producing<br />
at least one transferable embryo = 33.7 + (35.2 x P4) – (5.4 x P4 2 ); r 2 (Adj.) = 0.96) or one<br />
freezable (dashed line; percentage of cows producing at least one freezable embryo = 33.6 +<br />
(34.6 x P4) – (5.5 x P4 2 ); r 2 (Adj.) = 1.0) embryo. Adapted from Rivera et al. [47].<br />
N<br />
Figure 8. Percentage of cows conceiving after first postpartum AI according to progesterone<br />
concentration during ovulatory follicle growth. Progesterone category: 0 = 0 to 0.99 ng/ml;<br />
1 = 1 to 1.99 ng/ml; 2 = 2 to 2.99 ng/ml; 3 = 3 to 3.99 ng/ml; 4 = 4 to 4.99 ng/ml; and, 5 ><br />
5 ng/ml. Pregnancy per AI (P/AI) = 24.2 + (6.9 x P4) – (0.76 x P4 2 ); r 2 (Adj.) = 75.8%.<br />
Adapted from Denicol et al. [46].<br />
lactating dairy cows increased 30 MJ since the 50s<br />
[4]. This has prompted researchers to re-evaluate<br />
characterization of heat stress in lactating dairy cows<br />
and to determine that increases in milk yield from 34<br />
kg/d to 46 kg/d result in decreased threshold temperature<br />
of 9°F [5]. Consequently, heat stress affects<br />
high producing dairy cows more dramatically than<br />
lower producing dairy cows and non-lactating<br />
animals [1,66].<br />
Oocytes and embryos of lactating dairy cows<br />
are damaged by heat stress. Several reports have<br />
demonstrated that exposure to heat stress degenerates<br />
thecal and granulosa cells, and reduces percentage<br />
of fertilized oocytes and percentage of recovered<br />
structures classified as excellent-good quality embryos<br />
in lactating dairy cows, and in vitro and in vivo<br />
embryonic development [28,34,44,47,50]. Embryos<br />
older than 3 to 4 d of age produced in vivo from<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
donors not exposed to heat stress or produced in vitro,<br />
however, are more resistant to heat stress because<br />
they are capable to produce heat-shock protein 70<br />
and because they have greater number of cells [69].<br />
Several studies have demonstrated that,<br />
during heat stress, reproductive performance of<br />
lactating dairy cows receiving embryos is improved<br />
compared with lactating dairy cows receiving AI<br />
[28,30,51,72]. In a recent study conducted in Brazil<br />
[82] lactating dairy cows were assigned to receive<br />
fixed time AI or ET after having their ovulation<br />
synchronized with one of two protocols. Although<br />
no differences in percentage of cows pregnant were<br />
observed between cows receiving AI or ET during<br />
the mild weather months, during the summer months<br />
a significant decrease in percentage of cows pregnant<br />
after AI was observed, whereas season did not affect<br />
percentage of cows pregnant at ET (Table 1).<br />
In a study conducted in two commercial dairy<br />
farms in TX, cows were randomly selected to receive<br />
AI or IVP embryos, fresh or vitrified, during heat<br />
stress season [77]. In vitro produced embryos were<br />
inseminated with sex-sorted semen. Because embryo<br />
recipient cows only received embryos if they had a<br />
CL on the day of ET, whereas all cows assigned to<br />
the AI treatment received AI, the researchers calculated<br />
two reproductive outcomes: pregnancy rates<br />
(including all cows enrolled in each treatment,<br />
regardless if recipient cows received an embryo or<br />
not), and P/AI or pregnancy per ET (P/ET; including<br />
only cows that received AI or ET) [77]. Cows<br />
receiving fresh IVP embryos had the best<br />
reproductive performance (P/ET, pregnancy rates, and<br />
percentage of cows producing a live calf) followed<br />
by cows receiving vitrified IVP embryos and cows<br />
receiving AI, respectively (Table 2). Furthermore, 80<br />
to 85% of offspring of cows receiving IVP embryos<br />
were female calves (Table 2). Even though the cost<br />
of a dose of semen was $ 20 and the cost of IVP<br />
embryos was $ 60, the significant improvements in<br />
reproductive performance during heat stress and the<br />
increased number of live heifers produced from<br />
transfer of IVP embryos compared with cows<br />
receiving AI, transfer of IVP embryos resulted in an<br />
estimated return over investment of $ 22-42/lactating<br />
cow/year [19].<br />
Table 1. Reproductive performance of lactating dairy cows from two TX dairy herds exposed to heat<br />
stress and submitted to fixed time AI (TAI), in vitro produced (IVP) fresh and vitrified embryos.<br />
Adapted from Vasconcelos et al. [63].<br />
Items Winter Spring Summer Fall P - value<br />
Pregnancy rate 28 d, %<br />
TAI 37.1 a 32.9 a 21.6 b 27.0 ab 0.08<br />
ET 45.1 44.7 41.2 45.1 0.48<br />
Pregnancy rate 60 d, %<br />
TAI 33.7 a 28.4 ab 18.2 b 25.8 ab 0.02<br />
ET 38.3 37.1 35.9 39.1 0.50<br />
a,b<br />
Within a row, means without a common superscript differed (P < 0.05).<br />
Table 2. Reproductive performance of lactating dairy cows from two TX dairy herds exposed to heat stress and submitted to fixed time AI<br />
(TAI), in vitro produced (IVP) fresh and vitrified embryos. Adapted from Bilby et al. [64].<br />
Items AI IVP fresh embryo IVP vitrified embryo P - value<br />
Number of cows receiving AI or ET 219 134 188<br />
Pregnancy per AI or ET 1 ,% 22.9 a,A 45.5 b 30.9 B < 0.01<br />
Pregnancy rates, % 18.3 a 42.1 b 29.3 c < 0.01<br />
Cows delivering a life calf, % 14.6 a 27.5 b 17.1 a < 0.01<br />
Percentage of live calves of female sex 2 , % 50 a,A 79.1 b 72.5 B < 0.01<br />
1<br />
Data regarding cows with properly synchronized estrous cycle. 2 Deliveries referent to the services performed during the study.<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
Unquestionably, improved in vivo and in vitro<br />
production of embryos has favored the use ET-based<br />
reproductive strategies to improve reproductive<br />
performance of lactating dairy cows, particularly<br />
during seasons of heat stress. The more disseminated<br />
use of IVP embryos for the reproductive management<br />
of lactating dairy cows, however, depends on the<br />
creation of cryopreservation procedures that result<br />
in higher and more consistent P/ET.<br />
2.3.2 Reproductive performance of repeat-breeder cows.<br />
Dairy cows are classified as repeat breeders<br />
once they have received more than 3 AI and do not<br />
conceive. Considering that the average pregnancy<br />
rate of dairy herds in the U.S.A. is approximately<br />
16% and P/AI approximately 30%, it is not surprising<br />
that nearly 10 to 20% of lactating dairy cows in the<br />
U.S.A. may be considered as repeat-breeders. The<br />
causes of the reproductive failure of repeat-breeder<br />
cows are not completely elucidated. Even though the<br />
definition of repeat-breeder cows is sub-fertility of<br />
animals that do not present anatomical or infectious<br />
abnormalities at the time of diagnosis, it is likely that<br />
dystocia, occurrence of postpartum diseases (e.g.<br />
mastitis, metritis, endometritis, displacement of<br />
abomasum), and exposure to heat stress, among other<br />
things, are predisposing factors to this condition.<br />
Therefore, it is obvious that correction of predisposing<br />
factors that cause repeat-breeders is the best solution<br />
to this problem. Nonetheless, in situations in which<br />
oocyte quality and/or uterine environment are<br />
compromised, the use of ET to improve reproductive<br />
performance seems to be a good alternative. Recently,<br />
researchers have demonstrated in a large retrospective<br />
study (n = 9,551) that pregnancy outcomes of repeatbreeder<br />
cows was significantly improved when they<br />
received ET (41.7%) compared with repeat-breeder<br />
cows that received AI (17.9%) [58].<br />
2.3.3 Effects of P4 concentration on embryo survival.<br />
Although the role of P4 on the growth of<br />
embryos is well known, conflicting results have been<br />
reported regarding the effects of P4 concentration on<br />
establishment of pregnancy after ET. Although a few<br />
studies with Bos indicus influenced beef heifers and<br />
lactating dairy cows in tropical environment indicated<br />
that P4 concentration at the time of transfer affected<br />
P/ET [17,28,39] studies with Bos taurus influenced<br />
beef and lactating dairy cows demonstrated no<br />
correlation between P4 concentration at the time of<br />
s195<br />
transfer and P/ET [40,44,60,64,84]. Similarly, P4<br />
concentrations 5 to 7 d after transfer did not affect P/<br />
ET in lactating dairy cows according to a few studies<br />
[30,81] but increased P/ET in beef and lactating dairy<br />
cows according to others [39,51].<br />
An important consideration that must be made<br />
when evaluating published data regarding the effect<br />
of P4 concentration at the time of ET or 7 d later on<br />
P/ET is whether the synchrony of the estrous cycle<br />
of the recipient cows was determined and controlled<br />
for. In a recent study conducted by our group [39],<br />
we determined synchrony of the estrous cycle of<br />
recipient lactating Holstein cows based on P4<br />
concentrations at each injection of the OVP and at<br />
the time of ET. Cows that did not have a properly<br />
synchronized estrous cycle had greater P4<br />
concentration on the day of ET (3.9 ± 0.2 vs. 2.2 ±<br />
0.2 ng/ml), but had lower P4 concentration 7 d after<br />
ET (4.5 ± 0.3 vs. 5.6 ± 0.2 ng/ml). That was mainly<br />
because among cows with properly synchronized<br />
estrous cycle only 3.6% had luteolysis from the day<br />
of ET to 7 d later, whereas among cows that did not<br />
have a properly synchronized estrous cycle 20% had<br />
luteolysis from the day of ET to 7 d later [39].<br />
Consequently, P/ET 60 d after ET of cows that did<br />
not have a properly synchronized estrous cycle was N<br />
lower than P/ET of cows that had a properly<br />
synchronized estrous cycle (17.1 vs. 27.2%). In this<br />
study, when we excluded from the analysis cows that<br />
did not have the estrous cycle properly synchronized<br />
and evaluated the effects of P4 concentration 7 d after<br />
ET on P/ET, we observed a significant correlation<br />
between them (Figure 9) [39].<br />
2.4 Hormonal treatments to increase P4 concentration<br />
during diestrus and pregnancy after AI or ET.<br />
Because of the importance of P4 concentration<br />
on embryo development, attempts have been<br />
made to increase P4 concentrations during metestrus<br />
and diestrus of cows receiving AI or ET through<br />
different hormonal treatments during the OVP or after<br />
presumptive ovulation.<br />
Several research groups have attempted to<br />
increase P4 concentration during metestrus and<br />
diestrus by treating cows with human chorionic<br />
gonadotropin (hCG) or GnRH at different intervals<br />
after presumptive ovulation. This is expected to cause<br />
ovulation of large/dominant follicles present in the<br />
ovary at the time of treatment, formation of an<br />
accessory CL, and increase P4 concentrations.
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Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
Treatment with 1,500 to 3,300 IU of hCG at 5 to 7 d<br />
after presumptive ovulation results in greater percentage<br />
of cows forming accessory CL and, in most<br />
studies, significant increases in P4 concentrations<br />
approximately 7 d later [12,24,50,52]. The effects of<br />
hCG given 5 to 7 d after AI or presumptive ovulation<br />
on pregnancy outcomes, however, are a little more<br />
conflicting with some studies reporting higher<br />
pregnancy for hCG treated cows (51,53,60,65] and<br />
other studies reporting no effect of hCG treatment<br />
on pregnancy outcomes [41,51,78] (Figure 10).<br />
The effects of GnRH treatment after presumptive<br />
ovulation on P4 concentration and pregnancy<br />
outcomes also are conflicting. Lactating dairy cows<br />
[14,17,35] receiving GnRH 5 to 7 d after presumptive<br />
ovulation and lactating dairy cows receiving GnRH<br />
11 d after presumptive ovulation [89] had slightly<br />
higher P4 concentrations 5 to 7 d after treatment than<br />
Figure 9. Correlation between P4 concentration 7 d after ET and pregnancy per ET (P/ET) 53 d after ET.<br />
Average (± SEM) P4 concentrations on 7 d after ET according to quartiles were: quartile 1 = 2.7 ± 0.2, quartile<br />
2 = 4.7 ± 0.1, quartile 3 = 5.9 ± 0.1, and quartile 4 = 8.4 ± 0.3 ng/ml. P/ET = 12.7 + (6.0 x P4); r 2 (Adj.) =<br />
98.5%. Adapted from Kenyon et al. [74].<br />
Figure 10. Effect of hCG treatment 5 to 7 d after presumptive ovulation on pregnancy outcomes after AI or ET<br />
in dairy heifers [Schmitt et al. (69)], beef cows [Nishigai et al. (51);Wallace et al. (89)], and dairy cows [Santos<br />
et al. (64); Hanlon et al. (69); Galvão et al. (30); Vasconcelos et al. (81)].<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.<br />
non-treated cows. The increases in P4 concentration<br />
after GnRH treatment are smaller and less consistent<br />
compared with those observed after hCG treatment<br />
because hCG has a longer half-life than GnRH (30<br />
vs. 5 h) and, consequently, a more potent LH-like<br />
activity that not only causes new ovulations but<br />
extends the functional life of CL already present in<br />
the ovaries [18]. Lactating dairy cows treated with<br />
GnRH had higher pregnancy outcomes according to<br />
some [30,81] but not all studies [24,46]. In a metaanalysis<br />
conducted by Peters et al. [53], the effects of<br />
GnRH treatment 11 to 14 d after presumptive<br />
ovulation on pregnancy outcomes was evaluated<br />
based on 16 studies and 8,535 inseminations.<br />
According to this meta-analysis, the interaction<br />
between study and treatment affected the pregnancy<br />
outcomes, which deems this meta-analysis quite<br />
inconclusive [86]. When the authors included in the<br />
logistic regression important independent variables<br />
that could affect the pregnancy outcomes (e.g. OVP<br />
to which cows were submitted, cattle breed), however,<br />
GnRH treatment did not affect pregnancy outcomes<br />
[86]. In recent studies we conducted in dairies in CA,<br />
MN, and TX, we demonstrated that GnRH treatment<br />
at 17 or 31 d after presumptive ovulation did not<br />
reduce pregnancy losses from 31 to 66 d after AI<br />
[87], but cows treated with GnRH at 17 and 24 d<br />
after AI had greater P4 concentration at 31 d after AI<br />
and reduced pregnancy losses from 31 to 66 d after<br />
AI (7.5 vs. 11.9%) [88]. It is interesting to point out<br />
that according to this later study [88] there was a<br />
strong correlation between P4 concentration at 31 d<br />
after AI and incidence of pregnancy loss from 31 to<br />
66 d after AI (Figure 11).<br />
Treatment of cows and heifers with equine<br />
chorionic gonadotropin (eCG) during OVP has<br />
garnered interest for its potential to increase P4<br />
concentration after ovulation and increase pregnancy<br />
per AI or ET. In general, the studies that evaluated<br />
the effects of eCG treatment on P4 concentration and<br />
pregnancy outcomes attempted to promote hastened<br />
growth and ovulation of dominant follicles and,<br />
N<br />
Figure 11. Correlation between progesterone concentration 31 d after AI (P4) and pregnancy loss from 31<br />
to 66 d after AI. Pregnancy loss = 87.41 – (26.5 x P4) + (2.1 x P4 2 ); r 2 (Adj.) = 99.7%. Adapted from<br />
Scanavez et al. [88].<br />
consequently, increased P4 concentration after AI or<br />
ET by treating animals with eCG close to the expected<br />
time of ovulation [32,36,47,56,90]. Although these<br />
studies do not promote consensus regarding the<br />
effects of eCG treatment on pregnancy outcomes, it<br />
appears that anovular cows and cows with low body<br />
condition score would benefit the most from eCG<br />
treatment. On the other hand, treatment of embryo<br />
recipient lactating dairy cows with eCG around the<br />
time of follicular wave emergence to promote<br />
superstimulation and higher P4 concentration at the<br />
time of ET does not seem to improve P/ET (control =<br />
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el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
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32.8 vs. eCG = 37.3%) [39]. According to this study,<br />
treatment of cows with 800 IU of eCG around the<br />
time of follicular wave emergence resulted in reduced<br />
percentage of cows with synchronous estrous cycle<br />
(61.0 vs. 71.7%) and reduced the percentage of cows<br />
selected to receive ET (79.1 vs. 87.5%) [39]. It is not<br />
clear how eCG affects synchrony of the estrous cycle<br />
in lactating dairy cows, but the eCG’s long half-life<br />
is likely to be involved.<br />
Important factors that may affect the effects<br />
of hormonal treatments during the OVP and after<br />
presumptive ovulation on pregnancy outcomes are<br />
body condition score, lactation status, environmental<br />
conditions, and cattle breed.<br />
III. CONCLUSIONS<br />
The constant pressure for more efficient milk<br />
production demands that high producing dairy cows<br />
be used by dairy operations. Because of the<br />
dependency of milk yield on dry matter intake, it is<br />
unavoidable that lactating dairy cows will continue<br />
to demonstrate physiological changes (e.g. reduced<br />
steroidal hormone concentration and increase<br />
sensitivity to heat stress) that predispose them to<br />
compromised reproductive performance. The use of<br />
fixed time AI protocols and ET in the reproductive<br />
management of lactating dairy cows will continue to<br />
be important, particularly in situations of heat stress.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s203 - s221.<br />
ISSN 1679-9216 (Online)<br />
From Hatching into Fetal Life in the Pig<br />
Poul Hyttel, Kristian M. Kamstrup & Sara Hyldig<br />
ABSTRACT<br />
Background: Potential adverse effects of assisted reproductive technologies may have long term consequences on embryonic<br />
and fetal development. However, the complex developmental phases occurring after hatching from the zona pellucida are less<br />
studied than those occurring before hatching. The aim of the present review is to introduce the major post-hatching<br />
developmental features bringing the embryo form the blastocyst into fetal life in the pig.<br />
Review: In the pre-hatching mouse blastocyst, the pluripotency of the inner cell mass (ICM) is sustained through expression<br />
of OCT4 and NANOG. In the pre-hatching porcine blastocyst, a different and yet unresolved mechanism is operating as OCT4<br />
is expressed in both the ICM and trophectoderm, and NANOG is not expressed at all. Around the time of hatching, OCT4<br />
becomes confined to the ICM. In parallel, the ICM is divided into a ventral cell layer, destined to form the hypoblast, and a<br />
dorsal cell mass, destined to form the epiblast. The hypoblast gradually develops into a complete inner lining along the<br />
epiblast and the trophectoderm. Upon hatching (around Day 7-8 of gestation), the trophectoderm covering the developing<br />
epiblast (Rauber´s layer) is lost and the embryonic disc is formed by development of a cavity in the epiblast, which subsequently<br />
“unfolds” resulting the establishment of the disc. In parallel, the epiblast initiates expression of NANOG in addition to OCT4.<br />
The blastocyst enlarges to a sphere of almost 1 cm around Day 10 of gestation. Subsequently, a dramatic elongation of the<br />
embryo occurs, and by Day 13 it has formed a thin approximately one meter long filamentous structure. This elongation is<br />
paralleled with the initiation of placentation along with which, the embryonic disc undergoes gastrulation. The latter process<br />
is preceded by a thickening of the posterior region of the epiblast, putatively developing as a consequence of an absence of<br />
inhibitory signals from a condensed portion of the hypoblast underlying the anterior epiblast. The thickened posterior epiblast N<br />
expresses the primitive streak marker BRACHYURY. Subsequently, the epiblast thickening extends in an anterior direction<br />
forming the primitive streak; also expressing BRACHYURY. Gastrulation is hereby initiated, and epiblast cells ingress through<br />
the primitive streak to form mesoderm and endoderm; the latter is inserted into the dorsal hypoblast whereas the mesoderm<br />
forms a more loosely woven mesenchyme between the epiblast and the endoderm. The anterior mesoderm, ingressing through<br />
the anterior end of the primitive streak, referred to as the node, forms the rod-like notochord interposed between the epiblast<br />
and the endoderm. During the subsequent neurulation, which is a process overlapping with gastrulation in time, the notochord<br />
induces the overlying epiblast to form neural ectoderm, which sequentially develops into the neural plate, neural groove, and<br />
neural tube, whereas the lateral epiblast develops into the surface ectoderm. In parallel with the development of the somatic<br />
germ layers, ectoderm, mesoderm, and endoderm, the primordial germ cells, the predecessors of the germ line, develop in the<br />
posterior epiblast and initiates a migration finally bringing them to the genital ridges of the developing embryo. In parallel, the<br />
ectoderm gives rise to the epidermis and neural tissue, the mesoderm develops into the cardiovascular system as well as the<br />
urogenital and musculoskeletal systems, whereas the endoderm forms the gastrointestinal system and related organs as the<br />
liver and pancreas.<br />
Conclusions: Porcine embryonic and fetal development is controlled by molecular mechanisms that to some degree differ<br />
from those operating in the mouse. It is of importance to uncover the molecular control of development in ungulates as it has<br />
great implications for assisted reproductive technologies as well as for biomedical model research.<br />
Keywords: Biomedical models, embryology, blastulation, gastrulation, neurulation, embryonic staging.<br />
CORRESPONDENCE: P. Hyttel [poh@life.ku.dk – FAX: +45 353 32547]. Department of Basic Animal and Veterinary Sciences, Faculty of<br />
Life Sciences, University of Copenhagen, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark.<br />
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hebel.<br />
el. <strong>2011</strong>. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency in<br />
Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.<br />
I. INTRODUCTION<br />
II.BLASTULATION: DEVELOPMENT OF TRO-<br />
PHECTODERM, ICM, EPIBLAST, HYPOBLAST,<br />
AND EMBRYONIC DISC<br />
III. GASTRULATION: DEVELOPMENT OF MESO-<br />
DERM, ENDODERM, AND ECTODERM<br />
3.1 The primitive streak<br />
3.2 Ingression of cells forming mesoderm and endoderm<br />
IV. NEURULATION: DEVELOPMENT OF THE NEU-<br />
RAL ECTODERM AND NEURAL CREST<br />
4.1 Neural ectoderm<br />
4.2 Neural crest<br />
V. DEVELOPMENT OF THE PRIMORDIAL GERM<br />
CELLS (PGCS)<br />
VI. FURTHER DEVELOPMENT OF THE EMBRYO<br />
6.1 The ectoderm and its early derivatives<br />
6.2 The mesoderm and its early derivatives<br />
6.3 Paraxial mesoderm<br />
6.4 Paraxial mesoderm<br />
6.5 Lateral plate mesoderm and body folding<br />
6.6 Blood and blood vessel formation<br />
6.7 The endoderm and its early derivatives<br />
VII. PLACENTATION AND FORMATION OF EXTRA-<br />
EMBRYONIC MEMBRANES AND CAVITIES<br />
7.1 Development of extra-embryonic membranes and<br />
cavities<br />
7.2 Placentation<br />
VIII. STAGING OF EMBRYONIC DEVELOPMENT<br />
IX. CONCLUSIONS<br />
I. INTRODUCTION<br />
The wide-spread use of in vitro production<br />
of, in particular, bovine embryos in animal husbandry<br />
has paved the way for a detailed morphological and<br />
molecular understanding of oocyte maturation,<br />
fertilization and initial embryonic development until<br />
the time of hatching. Hence, studies on these life processes<br />
have become facilitated by the easy<br />
accessibility of oocytes, zygotes, and embryos.<br />
Cloning by somatic cell nuclear transfer (SCNT) is<br />
another technology, which over the past decade has<br />
resulted in alternative in vitro production of<br />
considerable numbers of both bovine and porcine<br />
embryos adding to the accessibility of embryos for<br />
research. Development of the embryo to the blastocyst<br />
stage includes several complex processes as e.g. the<br />
activation of the embryonic genome (for review, see<br />
Oestrup et al. [31]). It is also clear, however, that<br />
success in developing into a blastocyst is not necessarily<br />
a guarantee for further development into a fetus<br />
and newborn. Hence, it is well-documented that in<br />
vitro embryo culture may impose long-term effects,<br />
which are revealed later during embryonic and fetal<br />
development [40]. This phenomenon becomes even<br />
more exaggerated when embryos are produced by<br />
SCNT, which imposes an even higher risk of<br />
embryonic and fetal aberrations as well as neonatal<br />
loss [9,35].<br />
In order to evaluate embryonic and fetal<br />
development resulting from assisted reproductive<br />
technologies more properly, increasing focus should<br />
be put on the normality of some of the complex posthatching<br />
processes, as e.g. gastrulation, neurulation,<br />
placentation and initial organogenesis, which are<br />
prerequisites for full term development. These processes<br />
are the focus of the present review. Over the<br />
past decade the pig has attracted increasing attention<br />
as a useful biomedical model, due to which the<br />
presented data will mainly be derived in this species.<br />
Comparative notes will be made to cattle, whenever<br />
the variation between these two species are pronounced<br />
as e.g. at placentation. First, important developmental<br />
processes of the general embryology including<br />
blastulation, gastrulation, neurulation, and development<br />
of the germ line will be presented, and,<br />
second, a short summary of the special embryology,<br />
i.e. the development of the organ systems, will be<br />
given.<br />
II. BLASTULATION: DEVELOPMENT OF<br />
TROPHECTODERM, ICM, EPIBLAST, HYPOBLAST, AND<br />
EMBRYONIC DISC<br />
Blastulation (from the Greek term blastos<br />
meaning sprout) is the process by which the embryo<br />
develops into a fluid-filled structure in which the cells<br />
have segregated into lines destined to produce the<br />
embryo proper (the ICM and epiblast) and such<br />
developing into the extra-embryonic membranes (the<br />
trophectoderm and hypoblast).<br />
In the pig, the blastocyst forms at around Day<br />
5 of gestation. The porcine embryo initiates compaction<br />
as early as the 8-16-cell stage, when the<br />
embryo assumes a spherical appearance with a<br />
smoother surface where the protrusions of the individual<br />
blastomeres are no longer seen. The outer cells,<br />
allocated to the trophectoderm, become connected<br />
by tight junctions and desmosomes sealing the<br />
developing blastocyst cavity where the ICM forms<br />
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Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.<br />
as a cluster of lucent cells. Adjacent ICM cells communicate<br />
through scarce gap junctions [12]. The<br />
trophectoderm is divided into a polar portion,<br />
covering the ICM, and a mural portion sealing the<br />
blastocyst cavity. In advance of hatching, the ICM<br />
develops into a distinct ventral cell layer, destined to<br />
form the hypoblast, and a dorsal mass of cells destined<br />
to form the epiblast (Figure 1).<br />
A dynamic change in gene expression is the<br />
driving force for the first cell differentiation: i.e. the<br />
segregation of the compacting blastomeres into the<br />
ICM and trophectoderm. In the mouse, the ICM develops<br />
a stable regulatory circuit, in which the transcription<br />
factors NANOG [5,25], OCT4 [27,36],<br />
SOX2 [1], and the more recently identified SAL4 [7],<br />
2006; [49]Zhang, et al., 2006) promote pluripotency<br />
and suppress differentiation. In contrast, in the trophectoderm-destined<br />
cells, the transcription factors<br />
CDX2 and EOMES are upregulated together with<br />
ELF5 and TEAD4, which are transcription factors<br />
that act upstream of CDX2 to mediate trophectoderm<br />
differentiation [26,28,47]. On the other hand,<br />
expression of the trophectoderm-associated transcription<br />
factors, CDX2, TEAD4, and ELF5, are<br />
repressed in the ICM by the regulatory circuit of<br />
NANOG, SOX2, and OCT4[34]. In the pig, the<br />
expression of CDX2 during preimplantation development<br />
appears conserved as compared with the<br />
mouse [19]. OCT4 is, on the other hand, expressed<br />
in both the ICM and trophectoderm as opposed to<br />
the mouse [17,18], and NANOG expression has not<br />
been observed in the porcine ICM [11]. Hence, there<br />
are marked species differences with respect to the<br />
molecular background for ICM and trophectoderm<br />
specification.<br />
The embryo expands in size and hatches from<br />
the zona pellucida by Days 7 to 8, and in parallel the<br />
OCT4 expression becomes confined to the ICM [43],<br />
whereas expression of NANOG is still lacking (Figure<br />
2; [46]). At the time of hatching, the ICM is in the<br />
N<br />
Figure 1. Transmission electron micrograph of porcine Day 6 blastocyst<br />
showing the zona pellucida (ZP), polar trophectoderm (Te) and the inner<br />
cell mass, which has already divided into ventral cells (VC), developing<br />
into the hypoblast, and dorsal cells (DC), developing into the epiblast. BC:<br />
Blastocyst cavity. Insert: Light micrograph of the same blastocyst showing<br />
the inner cell mass (ICM).<br />
Figure 2. Confocal laser scanning micrographs of Day 8-9 hatched porcine blastocyst displaying OCT4 expression in the inner<br />
cell mass, whereas NANOG expression is lacking. E-CADHERIN is used as an epithelial marker.<br />
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Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.<br />
process of separating into two distinct cell<br />
populations. Hence, the most “ventral” cell layer<br />
towards the blastocyst cavity flattens and, finally,<br />
delaminates forming the hypoblast. The “dorsal” cell<br />
population establishes the epiblast. The hypoblast<br />
subsequently extends along the inside of the<br />
trophectoderm forming a complete inner epithelial<br />
lining of the embryo. The polar trophectoderm<br />
covering the epiblast (known as the Rauber´s layer)<br />
becomes very thin around Day 9 of gestation and<br />
gradually disintegrates exposing the epiblast to the<br />
uterine environment. Before the shedding of Rauber’s<br />
layer, tight junctions are formed between the<br />
outermost epiblast cells to maintain the epithelial<br />
sealing the embryo despite the loss of the polar<br />
trophectoderm. Apparently, the porcine epiblast forms<br />
a small cavity, which finally opens dorsally followed<br />
by an “unfolding” of the complete epiblast upon the<br />
disintegration of Rauber’s layer (Figure 3; [12]). After<br />
the loss of this component about Day 10 of gestation,<br />
the epiblast is discernable in the stereo microscope<br />
as a circular lucent structure known as the embryonic<br />
disc (Figure 4; [43]). Along with the formation of the<br />
embryonic disc, the blastocyst enlarges, and by Day<br />
10 it reaches a diameter of more than half a<br />
centimetre. In parallel, with the formation of the<br />
embryonic disc, the porcine epiblast starts to express<br />
not only OCT4, but also NANOG (Figure 5; [46]).<br />
At this stage of development, the first sign of<br />
anterior-posterior polarization develops in the<br />
embryonic disc: As mentioned earlier, the epiblast is<br />
underlaid by the hypoblast, and an area of increased<br />
density of closely apposed hypoblast cells develops.<br />
This area is approximately the same size as the em-<br />
Figure 3. Light micrograph of sections of the same porcine Day 9 blastocyst. (A) Rauber’ layer (RL), continuous with the remaining portion of<br />
the trophectoderm (Te), covers the epiblast (Ep), in which a cavity (C) has developed. The epiblast is underlaid by the epiblast-related taller<br />
hypoblast (Ep-Hy) and the trophectoderm by the trophectoderm-related lower hypoblast (Te-Hy). (B) Another section from the same epiblast<br />
showing the opening of the cavity towards the external environment and the “unfolding” (arrows) of the epiblast to form the embryonic disc.<br />
Figure 4. Porcine Day 10 blastocyst. (A) Stereo micrograph showing the blastocyst presenting the embryonic disc (arrow). (B) Light<br />
micrograph of section of the embryonic disc showing the dome-shaped epiblast (Ep) underlaid by the hypoblast (Hy). The epiblast is continuous<br />
with the trophectoderm (Te) indicated by the arrows.<br />
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Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.<br />
bryonic disc, but it is dislocated about one third of its<br />
diameter anteriorly as compared with the epiblast of<br />
the embryonic disc (Figure 6; [13,46]). It is likely<br />
that this dense hypoblast region emits signals to the<br />
epiblast which suppress mesoderm-formation in the<br />
anterior epiblast regions [13]. In this sense, the hypoblast<br />
may carry the blue-print for the specification of<br />
the epiblast.<br />
During Days 11 to 12, the embryonic disc<br />
develops into an oval shape, and a crescent-shaped<br />
accumulation of cells are found in the posterior region<br />
of the disc [43]. This crescent includes mesodermal<br />
progenitors which express the mesodermal markers,<br />
T (BRACHYURY) and GOOSECOID [3,45], and<br />
apparently ingression of BRACHYURY-expressing<br />
extra-embryonic mesoderm is initiated from this<br />
crescent even before the “true” gastrulation starts with<br />
the appearance of the primitive streak (see later; [45]).<br />
A porcine embryo displaying BRACHYURY expression<br />
in the posterior epiblast is displayed in Figure 6.<br />
With the development of the embryonic disc,<br />
a very peculiar pattern of OCT4 and NANOG expression<br />
develops in the porcine epiblast: The majority of<br />
epiblast cells express OCT4, but small groups or<br />
islands of cells are OCT4 negative [46]. The latter<br />
cells, on the other hand, express NANOG resulting in<br />
a mutually exclusive expression pattern (Figure 7).<br />
Subsequently, NANOG expression is lost in almost<br />
the entire epiblast, except for a few cell in the most<br />
posterior region of the embryonic disc, in which OCT4<br />
is also expressed [46]. The latter cells are believed to<br />
be the primordial germ cells (PGCs).<br />
N<br />
Figure 5. Confocal laser scanning micrographs of Day 9-10 hatched porcine blastocyst displaying OCT4 and NANOG expression in the<br />
epiblast.<br />
Figure 6. Confocal laser scanning micrographs of Day 10-11 porcine blastocyst displaying expression of T (BRACHYURY) in the posterior<br />
portion of the epiblast and of FOXA2 in the hypoblast. The open arrowheads in mark the periphery of the elongated embryonic disc. The<br />
asterisks mark the hypoblast area with higher cell density, which is about one third dislocated anteriorly as compared with the embryonic disc.<br />
A: Anterior; P: Posterior. Modified from Wolf et al. (<strong>2011</strong>b)[45].<br />
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Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.<br />
III. GASTRULATION: DEVELOPMENT OF MESODERM,<br />
ENDODERM, AND ECTODERM<br />
Gastrulation (from the Greek term gastrula<br />
meaning small stomach) is the process by which the<br />
three somatic germ layers, ectoderm, mesoderm, and<br />
endoderm, as well as the PGCs (see later) are formed.<br />
3.1 The primitive streak<br />
During Days 11-12 of gestation, the porcine<br />
embryo initiates a dramatic elongation that over a<br />
couple of days results in the transformation of the<br />
spherical blastocyst to an approximately 1 m long,<br />
extremely thin filamentous structure. Gastrulation in<br />
the porcine embryo is initiated around the time, when<br />
elongation is in its initial progress [45]. Porcine<br />
gastrulation is not dependent on implantation, as it is<br />
in man and mouse [3,10]. “True” gastrulation is defined<br />
by the presence of the primitive streak (Figure<br />
8). The porcine primitive streak apparently develops<br />
as an anterior extension of the BRACHYURY and<br />
GOOSECOID expressing crescent of epiblast cells<br />
[3,45]. The streak elongates in an anterior direction<br />
and forms a depression, termed the primitive groove,<br />
at the midline. The porcine primitive streak expresses<br />
BRACHYURY throughout its extension and<br />
GOOSECOID at least in the anterior portion [23,45].<br />
An example of a BRACHYURY expressing porcine<br />
primitive streak is visualized in Figure 9. At approximately<br />
Days 13-14 of gestation, the primitive<br />
streak extends from the posterior pole of the epiblast<br />
and approximately two thirds of the length of the<br />
embryonic disc [42]. A key embryonic signalling cen-<br />
Figure 7. Confocal laser scanning micrographs of Day 10 porcine blastocyst displaying mutually excluding epiblast cell populations expressing<br />
OCT4 and NANOG. Modified from Wolf et al. (<strong>2011</strong>a)[46].<br />
Figure 8. Median section though embryonic disc from Day 12-13 porcine embryo. The epiblast (Ep) is continuous with the trophectoderm (Te),<br />
indicated by the arrows. In the posterior two third of the epiblast, more loosely arranged cells ingress through the primitive streak (PS) to the<br />
space between the epiblast and the hypoblast (Hy/En), which is gradually exchanged by final endoderm. Loose mesoderm (Me) is also located<br />
in this area. A: Anterior; P: Posterior; D: Dorsal; V: Ventral.<br />
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tre during gastrulation, found at the anterior end of<br />
the primitive streak, is the organizer region, termed<br />
the node [37]. In the porcine embryo, the node is<br />
morphologically evident as a thickening of cells in<br />
the anterior part of the early primitive streak (Figure<br />
9); [42].<br />
3.2 Ingression of cells forming mesoderm and<br />
endoderm<br />
Formation of the primitive streak involves<br />
extensive movement of cells, where the epiblast cells<br />
first gather at the posterior end of the embryonic disc,<br />
then rearrange to extend anteriorly in the streak itself,<br />
and, finally, undergo an epithelial-mesenchymal<br />
transition through the primitive streak to become<br />
either mesoderm or definitive endoderm. When the<br />
primitive streak has formed, epiblast cells continue<br />
to enter this structure, which, thus, contains a dynamic<br />
ever changing cell population. The cells, which ingress<br />
to the space between the epiblast and hypoblast<br />
form mesodermal and endodermal precursors. Until<br />
recently it was generally believed that the definitive<br />
endoderm derived from the primitive streak replaced<br />
the hypoblast cell layer. Recently however, it was<br />
shown that in the gastrulating murine embryo the newly<br />
formed definitive endoderm cells insert themselves<br />
into the hypoblast epithelium in a dispersed manner<br />
N<br />
Figure 9. Confocal laser scanning micrographs of Day 12-13 porcine embryo. (A–C) Side-view of the embryonic disc. Note the expression of<br />
T (BRACHYURY) in the primitive streak and the posterior epiblast, the intensive FOXA2 expression in the hypoblast, and the co-expression<br />
of the two markers in the node (arrow and arrowhead in C). The periphery of embryonic disc marked with open arrowheads in A. Note the<br />
expression of T in the primitive streak as well as in intra- and extra embryonic mesoderm (EEM) underlying the epiblast and trophectoderm,<br />
respectively. (D-F) Optical transversal sections of the embryonic disc corresponding to the broken line in A. Note the expression of T in the<br />
primitive streak as well as in intra- and extra embryonic mesoderm underlying the epiblast and trophectoderm, respectively. A: Anterior; P:<br />
Posterior.<br />
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Dairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s203 - s221.<br />
[20]. Whether this is the case in the porcine embryo<br />
is not known. The mesodermal cells arrange<br />
themselves as an intermediate cell layer between the<br />
two developing epithelial layers, i.e. the epiblast and<br />
endoderm.<br />
The cells entering the primitive streak are<br />
exposed to distinct signaling factors at different concentrations<br />
dependent on where in the primitive streak<br />
the cells ingress through. Cell tracing studies has<br />
shown that the fate of a given cell is related to the site<br />
of ingression through the primitive streak: Cells ingressing<br />
through anterior streak and node become<br />
prechordal plate mesoderm, notochord, and endoderm,<br />
cells ingressing through “mid” streak become<br />
paraxial mesoderm, and cells migrating through the<br />
posterior streak become extra-embryonic and lateral<br />
plate mesoderm. These cell movements are, beside<br />
geometrical differences, being conserved from<br />
reptiles to mouse [24].<br />
When the primitive streak has reached its<br />
maximum extension of about two thirds of the length<br />
of the embryonic disc, a subsequent posterior<br />
retraction the streak occurs. Until recently, it was<br />
thought that the primitive streak actually shortened<br />
during this retraction. However, new investigations<br />
in the mouse have shown that the node does not<br />
regress posteriorly, but that the streak becomes<br />
relatively shorter due to the longitudinal growth of<br />
the embryonic disc (Yamanaka et al., 2007). Along<br />
with this process, cells ingressing through the node<br />
forms the notochord; a rod-shaped structure<br />
interposed between the epiblast and the endoderm<br />
extending from the rostral end of the embryonic disc<br />
posteriorly to the node, from which it grows in a posterior<br />
direction (Figure 10). The notochord posterior<br />
to the node is apparently formed by particular cells<br />
migrating posteriorly from the node [48]. The<br />
notochord expresses BRACHYURY [45].<br />
With the formation of the three somatic germ<br />
layers; ectoderm, mesoderm, and endoderm and the<br />
PGCs (see later), the progenitors of all fetal tissue<br />
lineages are formed.<br />
IV. NEURULATION: DEVELOPMENT OF THE NEURAL<br />
ECTODERM AND NEURAL CREST<br />
Neurulation is the process leading to the<br />
formation of the neural tube, the precursor of the central<br />
nervous system including the brain and spinal<br />
Figure 10. Confocal laser scanning micrographs of Day 14 porcine embryo. (A) Dorsal view of the embryonic disc showing the epiblast and<br />
developing ectoderm, identified from persisting OCT4 expression, and T (BRACHYURY) expression in the primitive streak (PS) posteriorly,<br />
in the node (N), and in the notochord (No) anteriorly. Note the cluster of OCT4 expressing primordial germ cells posteriorly (arrow). (B) Optical<br />
side view of the embryonic disc displaying the same features. A: Anterior; P: Posterior; D:Dorsal; V: Ventral. Modified from Wolf et al.<br />
(<strong>2011</strong>b)[45].<br />
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cord. This organ system is the first to initiate its<br />
development; functionally, however, it is overtaken<br />
by the later developing vascular system. Timewise,<br />
the process of neurulation overlaps with that of<br />
gastrulation: Along with the posterior retraction of<br />
the primitive streak, neurulation progresses in an anterior<br />
to posterior direction. Hence, over a certain<br />
period of time, the embryonic disc presents both the<br />
primitive streak posteriorly and the developing neural<br />
system anteriorly.<br />
4.1 Neural ectoderm<br />
The epiblast cells anterior to the primitive node<br />
are induced to differentiate at the second gestational<br />
week [44]. The notochord’s signalling molecules,<br />
including Sonic hedgehog, induce the overlying<br />
epiblast to differentiate into neuroectoderm, whereas<br />
the remaining more lateral portion of the epiblast<br />
differentiates into surface ectoderm. This notochordinduced<br />
neurulation is referred to as primary neurulation.<br />
The first morphological sign of primary<br />
neurulation is a dorsal thickening in the anterior ectoderm<br />
forming an elliptical region referred to as the<br />
neural plate. Subsequently, the neural plate undergoes<br />
a shaping which converts it into a more elongated<br />
key-hole shaped structure with broad anterior and<br />
narrow posterior regions. Neural plate shaping is<br />
followed by the development of two lateral elevations,<br />
the neural folds, on either side of a depressed<br />
midregion referred to as the neural groove. In pigs<br />
and cattle the neural folds become clear during the<br />
third week of development (Figure 11).<br />
N<br />
Figure 11: Porcine Day 14-15 embryos. (A) Stereo micrograph of the embryonic disc showing the primitive streak (PS) posteriorly, delineated<br />
by arrowheads, and the neural groove (NG) anteriorly, delineated by arrows. CAF: Chorioamniotic folding. (B) Section of embryonic disc along<br />
the broken line in A. Note the thick neural ectoderm (NE) continuous with the trophectoderm at the arrows. The mesoderm (Me) is seen between<br />
the neural ectoderm and the endoderm (En). The mesoderm also forms extra-embryonic portions lining both the trophectoderm and the yolk sac<br />
(YS) with the extra-embryonic coelom (EC) between the layers. The latter opens into the primitive gut forming the hindgut (Hg) and the foregut<br />
(Fg). CAF: Chorioamniotic folding. (C) Section through the dorsal portion of the neural tube (NT) showing the neural ectoderm (NE)<br />
overlaid by the surface ectoderm (SE) characterized by expression of Pankeratin. Note the PAX7 expressing neural crest cells.<br />
A: Anterior; P: Posterior; D:Dorsal; V: Ventral.<br />
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The neural folds continue to elevate, appose<br />
in the midline, and, eventually, fuse to create the neural<br />
tube which becomes covered by the surface<br />
ectoderm. Primary neurulation creates the brain and<br />
most of the spinal cord, whereas in the tail bud, the<br />
posterior neural tube is formed by secondary neurulation,<br />
where the spinal cord initially forms as a<br />
solid mass of epithelial cells, and a central lumen<br />
develops secondarily by cavitation.<br />
The primary neurulation is accompanied by<br />
a bending of the neural plate, which occurs at three<br />
principal sites: the median hinge point (MHP), overlying<br />
the notochord, and the paired dorsolateral hinge<br />
points (DHLP) at the points of attachment of the surface<br />
ectoderm. The MHP is induced by signals from<br />
the notochord.<br />
Gradually, the neural folds approach each<br />
other in the midline, where they eventually fuse.<br />
Cellular protrusions extend from apical cells of the<br />
neural folds as they approach one another in the<br />
dorsal midline and interdigitate as the folds come<br />
into contact. This allows a first cell-cell recognition<br />
and provides an initial adhesion pending later<br />
establishment of permanent cell contacts.<br />
The subsequent fusion of the neural folds<br />
begins in the cervical region and proceeds in a zipperlike<br />
fashion anteriorly and posteriorly from there. As<br />
a result of these processes, the neural tube is formed<br />
and separated from the overlying surface ectoderm.<br />
Until fusion is complete, the anterior and posterior<br />
ends of the neural tube communicate with the<br />
amniotic cavity via two openings, the anterior and<br />
posterior neuropores. Closure of the neuropores<br />
occurs at approximately Days 24 to 26 and Days 15<br />
to 16 in cattle and pig, respectively; the anterior<br />
neuropore one to 2 days prior to the posterior.<br />
Neurulation is then complete. The central nervous<br />
system is represented at this time by a closed tubular<br />
structure with a narrow posterior portion, the anlage<br />
of the spinal cord, and a much broader cephalic<br />
portion, the primordium of the encephalon. During<br />
neurulation, the neuroepithelium is entirely proliferative;<br />
cells do not begin to exit the cell cycle and<br />
start neuronal differentiation until after the neural tube<br />
closure is complete.<br />
During neurulation, cell proliferation is accompanied<br />
by some degree of apoptosis in the neuroepithelium.<br />
The rate of apoptosis appears to be<br />
finely tuned and it seems to be equally detrimental if<br />
the intensity of apoptosis is increased or decreased.<br />
Apoptosis at the tips of the neural folds may serve a<br />
special function. After opposing neural folds have<br />
made contact and adhered to each other, midline<br />
epithelial remodelling by apoptosis breaks the<br />
continuity between the neuroepithelium and surface<br />
ectoderm.<br />
4.2 Neural crest<br />
Along with the elevation and fusion of the<br />
neural folds, certain cells at the lateral border or crest<br />
of the neural folds become detached. This cell<br />
population, known as the neural crest cells, will not<br />
participate in formation of the neural tube; instead<br />
they migrate widely and participate in the formation<br />
of many other tissues, such as the integument<br />
(melanocytes), other parts of the nervous system<br />
(including neurons for the central, sympathetic and<br />
enteric nervous system as well as glial and Swann<br />
cells), and large parts of the craniofacial mesenchymal<br />
derivatives [16].<br />
The mechanism whereby the neural crest cells<br />
detach from the neural folds is comparable with that<br />
occurring during ingression of epiblast cells in the<br />
primitive streak and node - a second example of<br />
epithelio-mesenchymal transition. The term mesenchyme<br />
refers to loosely organized embryonic tissue<br />
regardless of germ layer origin. Thus, both neuroectoderm<br />
(through the neural crest cells) and<br />
mesoderm (at gastrulation) may give rise to mesenchyme.<br />
The induction of neural crest cells is possibly<br />
mediated by a gradient of BMP4, BMP7, and WNT<br />
secreted by the surface ectoderm. In the chick and<br />
pig, the neural crest cells express the transcription<br />
factor PAX7 [2].<br />
V. DEVELOPMENT OF THE PRIMORDIAL GERM CELLS<br />
(PGCS)<br />
The development of the germ line involves<br />
specification of the cell linage and subsequent<br />
migration of the individual cells through various embryonic<br />
tissues to the final destination in the genital<br />
ridges. After reaching the genital ridges, the cells of<br />
the germ line integrate and initiate the final steps of<br />
differentiation towards mature germ cells; a process<br />
not completed until adulthood.<br />
At the stage where the embryo presents a clear<br />
primitive streak the OCT4 expression gradually<br />
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decreases in the epiblast. When that happens, the<br />
putative PGC precursors can be identified by their<br />
continuous expression of this marker (Figure 12;<br />
Hyldig, unpublished data). In addition, they also<br />
express NANOG another well known germ line<br />
marker. These cells are seen dispersed within the caudal<br />
third of the embryonic disc [33]. From their position<br />
within the porcine embryonic disc, the putative<br />
PGC precursors move in caudal direction to the extra-embryonic<br />
yolk sac wall. Initially, OCT4 positive<br />
cells are dispersed in both the embryonic and the extra-embryonic<br />
part of the yolk sac wall. A small cluster<br />
can be identified in the junction between embryonic<br />
and extra-embryonic tissue (Figure 12). As the yolk<br />
N<br />
Figure 12. Schematic presentation of the position of the porcine germ line during early development. Sections of the porcine<br />
embryo are depicted below drawings of embryo proper. Broken lines across embryo proper indicate section sites. Red dots<br />
represent PGCs. At embryonic Day 12, the putative germ line precursors are positioned in the caudal third of the embryo proper,<br />
scattered around the primitive streak. At Day 13 the distribution is similar, but with some PGCs in the extra-embryonic yolk sac wall<br />
where a specific cluster of PGCs is formed. At Day 15 the PGCs are seen in the ventral wall of the hind gut in all its length.<br />
Subsequently, the population moves in dorsal direction towards the genital ridges so that by Day 20, most PGCs reside in this tissue.<br />
The Day 28 gonads are beginning to form and PGCs are restricted to these organs. PS: Primitive streak; NG: Neural groove; Mn:<br />
Mesonephros; Me: Mesenterium; Li: Liver.<br />
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sac by Day 14-15 folds under the caudal area of the<br />
embryo to form the ventral wall of the hind gut, PGCs<br />
becomes restricted to this. Subsequently, the PGC<br />
follow the migration path from the ventral to dorsal<br />
side of the hind gut and further dorsolateral into the<br />
genital ridges. Although a few PGCs are seen in the<br />
genital ridge at Day 17, the vast majority is resided<br />
in the hindgut area at least until Day 18. By Day 20<br />
most PGCs are positioned in and around the<br />
attachment site of the elongated mesentery, however<br />
still with a substantial part of the population<br />
positioned in the lower mesentery and hindgut area.<br />
The tubular mesonephric tissue forms voluminous<br />
bulges, forcing the genital ridges in towards the<br />
midline and effectively discontinuing the linear contact<br />
between them and the PGCs in the dorsal<br />
mesentery[14]. The final colonisation of the genital<br />
ridges occurs around E23-24 [15]. The integration<br />
of the PGCs into the genital ridge tissue and the<br />
subsequent differentiation of the germ line is to our<br />
knowledge largely unexplored in the porcine species.<br />
The gonadal tissue begins organising by Day 42.<br />
Germ cell cords are present in both male and female<br />
gonads, though larger and more regular in males.<br />
Male gonads are rounded with only a slim cellular<br />
connection to the mesonephros [46].<br />
In the newly formed PGCs, DNA is highly<br />
methylated, as it is in their epiblast progenitors. However,<br />
by the time the PGC have entered the genital<br />
ridge, DNA has become largely hypomethylated. The<br />
demethylation is well studied in the murine species<br />
and studies of various repeats and differentially<br />
methylated domain (DMD) sequences of imprinted<br />
genes in porcine embryos show a comparable<br />
demethylation. The process is completed by Day 28-<br />
31, where after remethylation is started [4,38].<br />
Immunostainings of genomewide CpG methylation<br />
have indicated that the demethylation is initiated<br />
around Day 15 during PGC migration toward the<br />
genital ridges [14]. During the subsequent<br />
gametogenesis, when oocytes and spermatozoa are<br />
formed from the PGC derivatives, de novo<br />
methylation of DNA occurs. Importantly, this<br />
genome-wide demethylation and remethylation also<br />
includes the sex-specific DNA methylation of particular<br />
loci, forming the basis of genomic imprinting.<br />
VI. FURTHER DEVELOPMENT OF THE EMBRYO<br />
The three germ layers, ectoderm, mesoderm,<br />
and endoderm, form the basis for the further<br />
development of organ systems collectively referred<br />
to as the area of special embryology (for review, see<br />
Hyttel et al. [16]).<br />
6.1 The ectoderm and its early derivatives<br />
The development of the neuroectoderm has<br />
already been described in a former paragraph. After<br />
having allocated cells for endoderm, mesoderm, the<br />
germ line, and neuroectoderm, most of the remaining<br />
more laterally located epiblast will differentiate into<br />
surface ectoderm. In parallel with the closure of the<br />
neuropores, two bilateral thickenings of the surface<br />
ectoderm, the otic placode and the lens placode, are<br />
established in the embryonic cephalic ectoderm (Figure<br />
13A). The otic placode invaginates to form the<br />
otic vesicle, which will develop into the inner ear for<br />
hearing and balance, while the lens placode<br />
invaginates and forms the lens of the eye. The<br />
remaining surface ectoderm gives rise to the<br />
epidermis and associated glands of the skin, as well<br />
as the epithelium covering the oral and nasal cavities<br />
and the caudal portion of the anal canal. The<br />
epithelium covering the oral cavity gives rise to the<br />
enamel of the teeth and also part of the pituitary gland,<br />
the adenohypophysis.<br />
6.2 The mesoderm and its early derivatives<br />
Formation of the notochord provides an<br />
embryonic midline axis as a template for the axial<br />
skeleton. Initially, cells of the mesoderm form a thin<br />
sheet of loosely woven mesenchyme on either side<br />
of the notochord. Soon, however, the mesoderm closest<br />
to the notochord (the paraxial mesoderm) proliferates<br />
and forms pairs of segmental thickened structures<br />
known as somites (Figure 13). This process<br />
starts in the occipital region of the embryo, and in<br />
large animal species, somites are formed at a rate of,<br />
on average, about six pairs a day. The number of somites<br />
formed during this phase of development<br />
therefore forms a basis for estimating embryonic age.<br />
More laterally, the mesoderm remains thin and<br />
is therefore referred to as the lateral plate mesoderm.<br />
The lateral plate mesoderm is continuous with the<br />
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Figure 13. Stereo micrographs of porcine embryos. (A) Day 15-16 embryo showing lens placode (LP), otic placode (OP), somites (S),<br />
developing heart (H), yolk sac (YS), and allantois (Al). (B) Day 18-19 embryo showing pharyngeal arches (PA), developing heart with atrial<br />
(At) and ventricular (Ve) compartments, forelimb bud (FB), hind limb bud (HB), and prominent mesonephros (Mn).<br />
extra-embryonic mesoderm. The extra-embryonic<br />
mesoderm is split into an outer component lining<br />
trophectoderm and an inner component lining the<br />
hypoblast, and the cavity between these two<br />
components is referred to as the extra-embryonic<br />
coelom (Figure 11). With the continued development<br />
of the coelom, an intra-embryonic coelom similarly<br />
divides the lateral plate mesoderm in such a way that<br />
the so-called somatic mesoderm associates with the<br />
surface ectoderm to constitute the somatopleura while<br />
the so-called visceral mesoderm associates with the<br />
endoderm to form the splanchnopleura. Between the<br />
paraxial and lateral plate mesoderm, the intermediate<br />
mesoderm is established.<br />
6.3 Paraxial mesoderm<br />
As a general rule, development proceeds in<br />
an anterior to posterior direction (one exception to<br />
this was the development of the primitive streak). Accordingly,<br />
formation of somites progresses from the<br />
occipital region posteriorly. Each somite subsequently<br />
differentiates into three components: The ventromedial<br />
part of the somite associates with the notochord<br />
establishing the sclerotome which patterns formation<br />
of the vertebral column. The dorso-lateral part of each<br />
somite forms regionalized precursors of both dermal<br />
and muscle tissue, the dermamyotome. From this<br />
structure, a dorso-medially located cell population<br />
forms the myotome and a dorso-laterally located<br />
group becomes the dermatome. The myotome of each<br />
somite contributes to muscles of the back and limbs,<br />
while the dermatome disperses and forms the dermis<br />
and subcutis of the skin. Later, each myotome and<br />
dermatome will receive its own segmental nerve<br />
component.<br />
6.4 Intermediate mesoderm<br />
The intermediate mesoderm, which connects<br />
paraxial and lateral plate mesoderm, differentiates into<br />
structures of both the urinary system and the gonads,<br />
together referred to as the urogenital system. The N<br />
urinary system is first developed as an abortive<br />
anteriorly located paired pronephros, succeeded by<br />
a very prominently developing paired mesonephros<br />
(Figure 13B). The mesonephros develops excretory<br />
ducts; the mesonephric ducts (Wollfian ducts). Finally,<br />
the even further posteriorly located paired metanephos<br />
develops and the persisting kidneys. The gonads<br />
develop on the medial aspect of the mesonephros,<br />
initially as the genital ridges which receive the primordial<br />
germ cells. In the male, the mesonephric<br />
ducts develops into the epididymal ducts and the<br />
ductus deferens. In the female, however, another duct,<br />
the paramesonephric duct (the Müllerian duct), forms<br />
parallel to the mesonephric duct and develops into<br />
the oviduct, the uterus, and the cranial portion of the<br />
vagina.<br />
6.5 Lateral plate mesoderm and body folding<br />
Through anterior-posterior and lateral foldings,<br />
the subdivision of the coelom into intra- and<br />
extra-embryonic cavities becomes progressively<br />
better defined and the embryonic body gradually assumes<br />
the shape of a closed tube enclosing another<br />
tube, the primitive gut. The somatopleura will form<br />
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the lateral and ventral body wall enclosing the intraembryonic<br />
coelom of which the somatic mesoderm<br />
will provide the inner lining (the mesothelium of the<br />
peritoneum and pleura) and the ectoderm the outer<br />
lining (the epidermis). The splanchnopleura will form<br />
the wall of the primitive gut and its derivatives in<br />
which the endoderm and the visceral mesoderm will<br />
provide the inner lining (the lamina epithelialis of<br />
the tunica mucosa) and outer lining (the lamina<br />
epithelialis of the tunica serosa) respectively. The<br />
visceral mesoderm will also form the connective<br />
tissue and muscular components of the gut and its<br />
derivatives. Soon, the intra-embryonic coelom will<br />
be divided into the peritoneal, pleural and pericardial<br />
cavities.<br />
6.6 Blood and blood vessel formation<br />
Both blood and blood vessels appear to arise<br />
from common mesoderm precursor cells, the<br />
hemangioblasts. These differentiate into hematopoietic<br />
stem cells (forming blood cells) and angioblasts<br />
that form endothelial cells which coalesce to<br />
form blood vessels. The first sign of blood and blood<br />
vessel formation is seen in the visceral mesoderm of<br />
the splanchnopleura covering the yolk sac (see later).<br />
However, this appears to be only a transient<br />
phenomenon; later, hematopoiesis moves first to the<br />
liver and spleen and then to the bone marrow. The<br />
heart is also of mesodermal origin; though with some<br />
contribution of neural crest cells (Figure 13).<br />
6.7 The endoderm and its early derivatives<br />
The inner epithelial lining of the<br />
gastrointestinal tract and its derivatives is the main<br />
component derived from the endoderm. With the<br />
anterior-posterior and lateral foldings of the embryo,<br />
the endoderm-enclosed primitive gut becomes<br />
enclosed within the embryo, whereas the hypoblastenclosed<br />
yolk sac becomes localized outside the<br />
embryo.<br />
The primitive gut comprises cranial (foregut),<br />
middle (midgut) and caudal (hindgut) parts. The<br />
midgut communicates with the yolk sac through the<br />
vitelline duct (Figure 14). This duct is wide initially<br />
Figure 14. Schematic drawing of the extra-embryonic membranes and cavities in the pig. The outer membrane, chorion,<br />
is formed by trophectoderm underlaid by extra-embryonic mesoderm. The allantois (green), which is a diverticulum<br />
from the hindgut, is lined on the inside by endoderm covered by extra-embryonic mesoderm. The fusion between the two<br />
membranes, the chorion and allantois, results in the chorioallantoic membrane, which forms foldings engaged in<br />
placentation. The yolk sac (red), which is a diverticulum connected with the midgut through the vitelline duct, is<br />
rudimentary. The amnion (blue) surrounds the embryo and is fused with the chorion in the mesamnion (MA).<br />
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but, as development proceeds, becomes long and narrow<br />
and is eventually incorporated into the umbilical<br />
cord. The endoderm forms the epithelium of the<br />
gastro-pulmonary system and the parenchyma of its<br />
derivatives. Endoderm of the foregut gives rise to<br />
the pharynx and its derivatives, including the middle<br />
ear, the parenchyma of the thyroid gland, the<br />
parathyroid glands, the liver and the pancreas, and<br />
the reticulated stroma of the tonsils and thymus, as<br />
well as the oesophagus, stomach, liver, and pancreas.<br />
At its anterior end, the foregut is temporarily closed<br />
by an ectodermal-endodermal membrane, the<br />
buccopharyngeal membrane. At a certain stage of<br />
development, this membrane ruptures and open<br />
communication between the amniotic cavity and the<br />
primitive gut is established. The midgut gives rise to<br />
most of the small and the large intestine down to the<br />
transverse colon whereas the hindgut gives rise to<br />
the transverse and descending colon as well as the<br />
rectum and part of the anal canal. At its caudal end,<br />
the hindgut temporarily dilates to form the cloaca, a<br />
cavity transiently common to both the developing<br />
gastrointestinal and urogenital systems. The cloaca<br />
is separated from the amniotic cavity by the cloacal<br />
membrane, composed of closely apposed ectoderm<br />
and endoderm, like the buccopharyngeal membrane.<br />
After separation of the gastrointestinal and urinary<br />
systems, the cloacal membrane breaks down, opening<br />
the two systems via the anus and urogenital sinus,<br />
respectively.<br />
VII. PLACENTATION AND FORMATION OF EXTRA-<br />
EMBRYONIC MEMBRANES AND CAVITIES<br />
7.1 Development of extra-embryonic membranes and<br />
cavities<br />
During the early phases of gastrulation, the<br />
trophectoderm becomes lined by a thin layer of extra-embryonic<br />
mesoderm, the two layers together<br />
constituting the outer extra-embryonic membrane, the<br />
chorion (Figure 14). During gastrulation, the chorion<br />
forms folds, the chorioamniotic folds, which surround<br />
the embryonic disc. Gradually, the folds extend<br />
upwards to meet and fuse above the embryonic disc<br />
thereby enclosing the disc in a sealed amniotic cavity.<br />
The term amnion is generally used collectively for<br />
the cavity and its wall. The inner epithelium of the<br />
amnion originates from the trophectoderm and so, at<br />
the embryonic disc, it is continuous with the epiblast<br />
and later the embryonic surface ectoderm. The outside<br />
covering of the amnion is composed of extraembryonic<br />
mesoderm.<br />
The site where the chorioamniotic folds meet<br />
and fuse is known as the mesamnion. In cattle and<br />
pig, the mesamnion persists; as a result, the amnion<br />
gets torn during parturition and offspring are generally<br />
born without covering membranes.<br />
With the body foldings and the formation of<br />
the endoderm-lined primitive gut, the hypoblast-lined<br />
yolk sac is transformed into an extra-embryonic cavity<br />
communicating with the primitive gut through the<br />
vitelline tube. The outside of the yolk sac is lined by<br />
visceral mesoderm. In cattle and pig, the yolk sac<br />
serves a hematopoietic function for a short period of<br />
time, but subsequently it regresses within one to two<br />
weeks after its formation and never attains other<br />
important functions.<br />
During the second or third week of<br />
development, depending on the species, the allantois<br />
is formed as an outgrowth from the hindgut into the<br />
extra-embryonic coelom. In ruminants and the pig,<br />
the allantois assumes a T-shaped appearance with the<br />
top bar of the T being located as a transverse cavity<br />
just caudal to the embryo proper and the stem of the<br />
T connected with the hindgut. Like the vitelline duct, N<br />
the allantoic duct, connecting the allantoic cavity and<br />
the hindgut, becomes incorporated into the umbilical<br />
cord as a consequence of embryonic foldings.<br />
Since the allantois is a diverticulum of the hindgut,<br />
its wall is composed of an inner epithelial lining of<br />
endodermal origin and an outer layer derived from<br />
the visceral mesoderm. As the allantois enlarges, the<br />
visceral mesodermal part of its wall fuses with the<br />
somatic mesoderm of the chorion and, finally, more<br />
or less covers the amnion. The fusion of the allantoic<br />
and chorionic walls forms the embryonic part of the<br />
chorioallantoic placenta found in the domestic<br />
animals. The intra-embryonic proximal portion of<br />
the allantoic duct, extending from the hindgut to the<br />
umbilicus, is referred to as the urachus and gives rise<br />
to the urinary bladder. Throughout gestation, the allantoic<br />
cavity serves as a repository of the wastes<br />
excreted through the embryo’s developing urinary<br />
system.<br />
Prior to attachment the conceptus is solely<br />
nourished by uterine glandular secretions<br />
(histiotrophe), but with attachment of the chorio-<br />
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allantoic placenta to the endometrial wall an exchange<br />
of fetal/maternal blood-borne nutrients (hemotrophe)<br />
also contributes. Areolae, chorionic indentations<br />
opposite the endometrial glands, are scattered in the<br />
diffuse porcine placenta and remain present during<br />
gestation [21].<br />
7.2 Placentation<br />
The placenta can be classified according to<br />
the structure of the chorioallantoic surface and its<br />
interaction with the endometrium. Areas where the<br />
chorioallantois interacts with the endometrium and<br />
engages in placental formation are referred to as<br />
chorion frondosum, in contrast to the smooth chorion<br />
leave not included in the placenta. In the pig, chorion<br />
frondosum is diffusely distributed over the entire<br />
chorioallantoic surface and so the placenta is<br />
categorized as being diffuse. The porcine chorioallantoic<br />
surface area is increased by foldings,<br />
revealed as primary plicae and secondary rugae, and<br />
is thus referred to as being folded. In cattle, the chorion<br />
frondosum is organized as arborizing chorionic<br />
villi assembled into larger macroscopically visible<br />
tufts called cotyledons. Hence, the bovine placenta<br />
is known as cotyledonary or multiplex and villous.<br />
The cotyledons combine with endometrial<br />
prominences known as caruncles, forming<br />
placentomes in which the chorioallantoic villi of the<br />
cotyledon extend into crypts of the caruncle.<br />
The placenta can also be classified based on<br />
the number of tissue layers separating the fetal and<br />
maternal circulations, thereby forming the placental<br />
barrier. There are always three fetal extra-embryonic<br />
layers in the chorioallantoic placenta: the endothelium<br />
lining the allantoic blood vessels; chorioallantoic<br />
mesenchyme, originating from the fused somatic<br />
(chorionic) and visceral (allantoic) mesoderm; and<br />
the chorionic epithelium developed from the<br />
trophectoderm and in the placenta referred to as the<br />
trophoblast. However, the numbers of layers retained<br />
in the maternal portion of the placenta varies with<br />
species. Before placentation, the endometrium presents<br />
three layers that could contribute to the placental<br />
barrier: the endometrial epithelium, connective tissue,<br />
and vascular endothelium.<br />
In cattle and pig, the placenta is epitheliochorial<br />
and the chorionic and endometrial epithelia<br />
are apposed, and there is no loss of maternal tissue.<br />
The epitheliochorial placenta in ruminants is modified<br />
as particular trophoblast cells cross into, and fuse with,<br />
some of the endometrial epithelial cells. Hence, the<br />
placenta is referred to as synepitheliochorial.<br />
VIII. STAGING OF EMBRYONIC DEVELOPMENT<br />
Simple measures have over the time been used<br />
as a reference for embryonic development including<br />
length in mm [32], days of gestation [22], numbers<br />
of somites [15, 41], or external features [8]. Within<br />
human embryology, a painstaking work has been put<br />
into developing the Carnegie system; staging system<br />
providing a precise frame of reference of embryonic<br />
development [29,30]. This staging system utilizes<br />
macroscopic as well as microscopic features in a<br />
developing embryo and fetus. The Carnegie system<br />
has been implemented in bats [6] and mouse [39].<br />
We are currently working on development of a<br />
Carnegie-based porcine staging system for the<br />
domestic pig based on the examination of approximately<br />
600 specimens.<br />
IX. CONCLUSIONS<br />
An improved understanding of post-hatching<br />
embryonic development holds an important key to<br />
not only a more proper evaluation of the success or<br />
failure of assisted reproductive technologies; it also<br />
forms an important basis for the understanding of<br />
stem cell differentiation and cell replacement therapy.<br />
A wealth of contemporary data are published on the<br />
molecular regulation of the initial lineage segregation<br />
and cell differentiation taking place in the embryo,<br />
and it is a great challenge to align all the complex<br />
sets of information into integrated networks gradually<br />
guiding the well-orchestrated embryonic and fetal<br />
development.<br />
REFERENCES<br />
1 Avilion A.A., Nicolis S.K., Pevny L.H., Perez L., Vivian N. & Lovell-Badge R. 2003. Multipotent cell lineages in early<br />
mouse development depend on SOX2 function. Genes & Development. 17: 126-140.<br />
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ançanares<br />
es, M.L.V. Alb<br />
lber<br />
ert om, C.E. Ambrósio<br />
mbrósio, et al,.<br />
<strong>2011</strong>. Abnormalities in<br />
bovine conceptus development during the embryonic phase... Acta Scientiae Veterinariae. 39(Suppl 1): s223 - s226.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s223 - s226.<br />
ISSN 1679-9216 (Online)<br />
Abnormalities in bovine conceptus development during the embryonic phase after In<br />
Vitr<br />
itro Fer<br />
ertiliza<br />
tilization tion (IVF) and cloning by nuclear transf<br />
ansfer (NT)<br />
Antônio Chaves de Assis Neto 1 , Álvaro Riveiros Galdos 1 , Ana Carolina Furlanetto Mançanares 1 ,<br />
Míryan Lança Vilia Alb<br />
lber<br />
erto 1 , Car<br />
arlos Eduar<br />
duardo do Ambrósio<br />
2 , Phelip<br />
helipe e Oliv<br />
liveir<br />
eira Favar<br />
aron<br />
1 , Fla<br />
lavio<br />
Vieir<br />
ieira<br />
Meirelles 2 & Maria Angelica Miglino 1<br />
ABSTRACT<br />
Background: Embryonic mortality is a major cause of reproductive failure in cattle, resulting in a lot of problems to the<br />
industry. Recently, many techniques have been used in the production of genetically modified animals mainly related to<br />
improve the animal production. The possibility to genetically manipulate living organisms through the addition or inactivation<br />
of genes has revolutionized the understanding of biological and molecular mechanisms. Herein, we showed data about what<br />
is known about the abnormalities in bovine conceptus using IVF and NT techniques.<br />
Review: The establishment of pregnancy results from the interaction between the trophoblast and maternal tissues. Embryonic/<br />
fetal loss occurs throughout pregnancy in cattle; however, it is concentrated mainly in the first 42 days after breeding. Recently<br />
data have showed that approximately 50% of cloned bovine embryos do not establish the gestation after the transference of the<br />
blastocyst. In addition, placental abnormalities occur in high levels in cloned animals during the initial and in the end of<br />
gestation. Low viability of cloned embryos is mainly expressed by the reduction in the rate of deployment, the increased rate<br />
of perinatal mortality and fetal, and the various anomalies observed in newborn animals. Among the pregnancy complications<br />
from placental transfer of manipulated embryos (IVF and NT) there are changes in the morphology of the placentome, in the<br />
region of the fetal-maternal contact, increase of allantoic or amniotic fluid, vascularity, lower number of cotyledons, and increased<br />
N<br />
of the interplacentomal area “Offspring Syndrome”. In addition, the placenta, fetal membranes and umbilical cord from cloned<br />
animals have rudimentary development. According to Wells et al., the overall efficiency of cloning in bovine is limited to 5-<br />
6%. In IVF pregnancies the placentome percentage, fetal villi, density and volume of binucleated cells is reduced. However,<br />
the volume of blood vessels in increased maternal wattles, believing there is a compensatory mechanism in the vascular<br />
network of the placentae.<br />
Conclusion: Many technological innovations could help to obtain a better quality production and significant improvements in<br />
animal breeding such as those brought about by nuclear transfer and in vitro fertilization. New studies on the dynamics of<br />
development in cattle embryology derived from nuclear transfer techniques and in vitro fertilization are necessary, focusing on<br />
different systems in order to find greater success in the artificial producing and selection of interest characteristics.<br />
Keywords: Bovine, In Vitro Fertilization, Nuclear transfer, Cotyledonary Placenta.<br />
CORRESPONDENCE: M.A. Miglino [miglino@usp.br - FAX: +55 (11) 3091-7805]. 1 Department of Surgery, Faculty of Veterinary Medicine,<br />
University of São Paulo (USP),São Paulo, SP, Brazil. 2 Faculty of Animal Sciences and Food Engineering, USP, Campus de Pirassununga, SP,<br />
Brazil. University of São Paulo, Av. Prof Orlando Marques de Paiva n. 87, CEP 05508-270 SP, São Paulo, Brazil.<br />
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ançanares<br />
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lber<br />
ertom,<br />
C.E. Ambrósio<br />
mbrósio, et al,.<br />
<strong>2011</strong>. Abnormalities in<br />
bovine conceptus development during the embryonic phase... Acta Scientiae Veterinariae. 39(Suppl 1): s223 - s226.<br />
I. INTRODUCTION<br />
II. PREGNANCY PROGRESS AND RELATED<br />
PROBLEMS<br />
III. PERSPECTIVES<br />
IV. CONCLUSIONS<br />
I. INTRODUCTION<br />
Embryonic mortality is a major cause of<br />
reproductive failure in cattle, resulting in delayed<br />
pregnancy, fewer calves born, reduced in milk production,<br />
and slower genetic progress, with a concomitant financial<br />
loss to the industry. Recently, many techniques have been<br />
used in the production of genetically modified animals<br />
mainly related to improve the animal production.<br />
The possibility to genetically manipulate living<br />
organisms through the addition or inactivation of genes<br />
has revolutionized the understanding of biological and<br />
molecular mechanisms [3].<br />
A number of groups of scientists have been<br />
worked with this theme in order to establish new protocols<br />
and techniques which result in an artificial embryo<br />
production and to understanding the normal development<br />
of the bovine conceptus.<br />
II. PREGNANCY PROGRESS AND RELATED PROBLEMS<br />
The establishment of pregnancy results from the<br />
interaction between the trophoblast and maternal tissues.<br />
Furthermore, numerous internal and external factors can<br />
affect follicular development: oocyte quality, the endocrine<br />
factors, the receptivity of the uterus, the capacity of the<br />
embryo to signal its presence and determine whether or<br />
not pregnancy will be successfully established and<br />
maintained.<br />
Embryonic/fetal loss occurs throughout<br />
pregnancy in cattle; however, it is concentrated mainly in<br />
the first 42 days after breeding. Recently data have<br />
showed that approximately 50% of cloned bovine<br />
embryos not establish the gestation after the transference<br />
of the blastocyst inside the receptor mother [4]. In<br />
addition, placental abnormalities occur in high levels in<br />
cloned animals during the initial and in the end of gestation<br />
[9,18].<br />
The pregnancy loss in cloned cattle occur in the<br />
first six months affecting approximately 30% of the clones<br />
that grow until delivery, and low viability of cloned embryos<br />
is mainly expressed by the reduction in the rate of<br />
deployment, the increased rate of perinatal mortality and<br />
fetal, and the various anomalies observed in newborn<br />
animals [2].<br />
Among the pregnancy complications from<br />
placental transfer of manipulated embryos (IVF and NT)<br />
there are changes in the morphology of the placentome,<br />
in the region of the fetal-maternal contact, hydrallantois<br />
(increase of allantoic fluid) hidroâmnio (increased<br />
amniotic fluid), vascularity, lower number of cotyledons,<br />
and increased of the interplacentomal area “macrosomic<br />
Calf Syndrome” (offspring syndrome), which treats the<br />
more recent phenomenon associated with embryos<br />
produced by IVF and somatic cell nuclear transfer<br />
(SCNT).<br />
Hill et al. [7], Hill et al. [8] and Batchelder et al.<br />
[1] report that placentae from cloned animals have<br />
rudimentary development containing cuboidal trophoblastic<br />
epithelium, reduced vascularity and small area with some<br />
parts cotyledonal bleeding. Other results showed that the<br />
third trimester of gestation of cloned fetuses are<br />
characterized by a high incidence of delayed development<br />
and placental insufficiency, which are not associated with<br />
chromosomal abnormalities [14].<br />
Hashizume et al. [6], Numabe et al. [13], Miglino<br />
et al. [11] and Hoffert-Goeres et al. [10] argue that the<br />
high incidence of placental edema and hydrallantois<br />
hampering development of lymphatic vessels, extraembryonic<br />
circulation, or that the permeability of blood<br />
vessels are altered in many pregnancies resulting from<br />
the cloning process.<br />
As for the clones, the mortality rates of pre-and postnatal<br />
period are significantly higher compared with controls.<br />
Wakayama et al. [15] and Yanagimachi [17] found high<br />
rates of embryo implantation (57 - 71%) but low fetal<br />
rates (5-16%) and very low in development to term (2 -<br />
3% or less) after the transfer using adult somatic cell<br />
nuclear. By using embryonic cells to high incidence of<br />
abortion is the 40th day of gestation, accompanied by<br />
poor development of the specimens [9]. Edwards et al.<br />
[4] confirms the findings of Hill et al. [9] reporting that<br />
during the 30 to 60 days of pregnant the embryo mortality<br />
can occur in 50 to 100% of pregnancies. Wells et al.<br />
[16] that complements the overall efficiency of cloning<br />
in bovine is limited to 5-6% at most.<br />
Miglino et al. [11] describe the umbilical cord<br />
and edematous fetal membranes with placentome fusion,<br />
which result in an increased size and decreased number<br />
of placentomes. Increased number of functional small<br />
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cotyledons or accessories (
A.C.A. Net<br />
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aldos, A.C.F. Mançanar<br />
ançanares<br />
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lber<br />
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C.E. Ambrósio<br />
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12 Miles, J. R. Farin, C. E. Rodrigues, K. F. Alexander, J. E. & Farin P. W. 2004. Angiogenesis and morphometry of bovine<br />
placentas in late gestation from embryos produced in vivo or in vitro. Biology of Reproduction. 71(6): 1919-1926.<br />
13 Numabe, T. Oikawa, T. Kikuchi, T. & Horiuchi T. 2000. Birth weight and birth rate of heavy calves conceived by transfer<br />
of in vitro or in vivo produced bovine embryos. Animal Reproduction Science. 64(1): 13-20.<br />
14 Sousa, P. A. de King, T. Harkness, L. Young, L. E. Walker, S. K. & Wilmut I. 2001. Evaluation of gestational deficiencies<br />
in cloned sheep fetuses and placentae. Biology of Reproduction. 65(1): 23-30.<br />
15 Wakayama, T. Perry, A. C. Zuccotti, M. Johnson, K. R. & Yanagimachi R. 1998. Full-term development of mice from<br />
enucleated oocytes injected with cumulus cell nuclei. Nature. 349(6691): 369-374.<br />
16 Wells, D. Misica, P. & Tervit H. 1999. Production of cloned calves following nuclear transfer with cultured adult mural<br />
granulose cells. Biology of Reproduction. 60: 735-745.<br />
17 Yanagimachi R. 2002. Cloning: experience from the mouse and other animals. Molecular and Cell Endocrinology. 187(1-<br />
2): 241-248.<br />
18 Young, L. E. Sinclair, K. D. & Wilmut I. 1998. Large offspring syndrome in cattle and sheep. Journal of Reproduction and<br />
Fertility. 3(3): 155-163.<br />
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.Cha<br />
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ette-P<br />
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ee, S. Camous<br />
amous, et al,.<br />
<strong>2011</strong>. The Placenta of Bovine Clones. fffffffffffffffffffffffffffffffffffffffffffffff<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s227 - s242.<br />
ISSN 1679-9216 (Online)<br />
The Placenta of Bovine Clones<br />
Pascale Chavatte-Palmer 1,2,3 , Rita Sau Fong Lee 4 , Sylvaine Camous 1,2 , Nicolas Le Cleac’h 1,2<br />
Hélène Jammes 1,2 & Michel Guillomot 1,2<br />
1,2 ,<br />
ABSTRACT<br />
Background: Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer<br />
(SCNT) in various mammals has increased significantly. The incidence of pregnancy failure and fetal death, however, is still<br />
very high, whatever the species, and impairs the commercial development of this technology, even in the bovine species where<br />
the success rates are highest compared to other species.<br />
Review: In cattle, most gestation losses are initially due to abnormal implantation and poor placental development leading to<br />
fetal demise during the early post-implantation period (30 to 70 days of pregnancy). Thereafter, in continuing pregnancies, losses<br />
usually occur in the last third of gestation and affect about 25% of the on-going pregnancies, with very large differences<br />
according to phenotype. These are currently referred to as the Large Offspring Syndrome (LOS), Large Placenta Syndrome or<br />
Abnormal Offspring Syndrome. In all cases, the placenta appears to be central to the onset of the pathology, with placentomegaly<br />
and hydrallantois being the most common features. Clinically, transabdominal ultrasound monitoring of fetal and placental<br />
development as well as the masurement of maternal plasma concentrations of pregnancy associated glycoproteins (PAG) are<br />
recommended in order to monitor the pregnancies. Humane termination of the pregnancies by Caesarian section or slaughtering<br />
of the affected animals is recommended when the pathology onset is diagnosed more than 2 weeks prior to term. Underlying<br />
mechanisms include abnormal placental vascularization, which is present early in SCNT placental development. Enzymatic<br />
response to oxidative stress is also modified. In the first trimester, several genes expressed in the trophoblast have been found<br />
to be differentially expressed between SCNT and control conceptuses, including placental lactogen (PL), the PAG, prolactin N<br />
related protein-1 (PRP-1) and Dickkopf-1(DKK-1), to name a few. All these proteins are expressed in the Binucleate cells (BNC)<br />
of the trophoblast and thus, indicate that BNC function may be affected in SCNT from very early in gestation, thereby compromising<br />
placental development. Later in pregnancy, it has been shown that transplacental exchanges are disturbed, in particular those<br />
related to glucose metabolism. Moreover, endocrine function is altered compared to controls, with decreased estrogen secretion<br />
and modifications in PAG secretion, resulting in largely elevated maternal plasma concentrations. Gene expression patterns are<br />
affected, with most prominent functional effects involving cell cycle, cell signaling pathways, molecular transport, DNA replication,<br />
recombination and repair. Most of the affected genes are downregulated. Finally, many of the pathologies reported with SCNT<br />
pregnancies resemble abnormalities reported with either mutations or deletions of imprinted genes or dysregulation of imprinted<br />
gene expression, and the expression of several imprinted genes have been shown to be abnormal in SCNT placenta.<br />
Conclusions: In conclusion, pregnancy failure after SCNT is due to multiple factors affecting, implantation, placental development,<br />
morphology, vascularization, responses to oxidative stress and the epigenetic control of gene expression. If abnormal nuclear<br />
reprogramming may induce long term effects in bovine SCNT, these effects may also be due to fetal programming due to<br />
abnormal placental function and perturbed fetal development.<br />
Keywords: Cloning, bovine, placenta, SCNT.<br />
CORRESPONDENCE: P. Chavatte-Palmer [pascale.chavatte@jouy.inra.fr - TEL: +33 (1) 34652558]. INRA, UMR 1198 Biologie du<br />
Développement et Reproduction, F-78350 Jouy en Josas, France. 1 INRA, UMR 1198 Biologie du Développement et Reproduction, Jouy en<br />
Josas, France. Maisons-Alfort, France. 3 PremUp Foundation, Paris, France; 4 Reproductive Technologies, AgResearch, Hamilton, New Zealand.<br />
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I. INTRODUCTION<br />
II. Clinical and pathological observations<br />
2.1 Early pregnancy losses<br />
2.2 Late pregnancy losses<br />
III. VETERINARY CARE OF THE BOVINE SCNT PREG-<br />
NANCY<br />
3.1 Detection of the early losses<br />
3.2 Prenatal monitoring - Identification of the high risk<br />
pregnancy<br />
IV. Underlying mechanisms<br />
4.1. Placental vascularization<br />
4.2 Placental function and exchanges<br />
4.3 Oxidative stress<br />
4.4 Gene expression<br />
4.5 Epigenetics and the role of imprinted genes<br />
V. CONCLUSIONS<br />
I. INTRODUCTION<br />
Since the first success in sheep cloning with<br />
the production of Dolly [93], many other mammals<br />
have been cloned by somatic cell nuclear transfer<br />
(SCNT). The incidence of pregnancy failure and fetal<br />
death, however, is still very high, whatever the species.<br />
In cattle, most gestational losses are initially due to<br />
early embryonic losses during the preimplantation<br />
period [49] and thereafter to abnormal placentation<br />
associated with an overgrowth of the fetus known as<br />
the Large Offspring Syndrome (LOS) [45]. In all cases,<br />
the placenta appears to be central to the onset of<br />
clone pregnancy losses [23,50]. This article reviews<br />
what is known about the events leading to the early<br />
and late fetal losses in SCNT pregnancies and the<br />
underlying mechanisms.<br />
II. CLINICAL AND PATHOLOGICAL OBSERVATIONS<br />
Pregnancy losses after transfer of SCNT<br />
embryos mostly occur in the first and the third trimester<br />
of pregnancy (Figure 1) [45].<br />
2.1 Early pregnancy losses<br />
Following transfer of cloned bovine embryos, Day<br />
30 pregnancy rates per recipient can approach 50%,<br />
whether or not one or several embryos have been<br />
transferred into each recipient [43,44]. After this initial<br />
pregnancy diagnosis, embryonic losses greater than 50%<br />
are common for nuclear transfer pregnancies in sheep,<br />
cattle and goats and especially for clones produced from<br />
somatic cells [21,56,89,90,93]. In contrast, only 2-4% of<br />
naturally conceived pregnancies are lost early during the<br />
first trimester (Day 30) and about 10% of in vitro<br />
produced (IVP) embryos have failed by Day 60. Survival<br />
of SCNT embryos to term is approximately half to 1/4 of<br />
that of in vitro fertilized embryos with most of these<br />
losses occurring in the first trimester [45].<br />
Figure 1. Schematic comparison of fetal losses throughout gestation after transfer of bovine embryos produced by<br />
IVF, NT of embryonic cells, fetal or adult fibroblasts, showing the important fetal losses in SCNT in the first and<br />
last trimester of pregnancy [45].<br />
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In naturally conceived cattle, early fetal losses<br />
may be due to abnormalities of the embryo or its placenta,<br />
alterations in maternal uterine environment or fetomaternal<br />
interactions. Possible mechanisms include<br />
chromosomal abnormalities in the embryo, hormonal<br />
changes, environmental influences, asynchronous embryo<br />
transfer and immunological rejection [92]. In SCNT<br />
pregnancies, these same factors generally still apply. The<br />
lack of, or abnormal placentome development, initially<br />
observed in bovine fetuses NT from embryonic cells [83],<br />
is the most common observation at Day 35-50. First<br />
trimester SCNT fetuses display a wide variety of<br />
placental morphologies with poor allantoic vascularization,<br />
retarded or even advanced cotyledon development [58]<br />
and formation of smaller numbers of placentomes<br />
[6,28,41,50,90]. SCNT fetuses have very variable<br />
numbers of placentomes, which suggests that the<br />
completeness of placental development varies widely in<br />
cloned animals. When placentome numbers are decreased<br />
by as much as 80%, the is a very high risk for the<br />
pregnancy to be lost before Day 90 of gestation [58].<br />
Similar placental abnormalities were also shown to occur<br />
in first trimester bovine IVF fetuses, in relation to the use<br />
of sub-optimal embryo culture media prior to embryo<br />
transfer [32,33].<br />
Fetuses recovered shortly after death from such<br />
pregnancies are grossly normal most of the time, and<br />
thus it appears that lack of normal placentation may be a<br />
major contributor to embryonic death, rather than fetal<br />
abnormalities per se. Abnormal placental development<br />
and impaired function, such as inadequate maternal-fetal<br />
contact and poor transfer of nutrients likely contribute to<br />
starvation of the developing fetus [11,17].<br />
2.2. Late pregnancy Losses<br />
In bovine SCNT pregnancies, fetal losses occur<br />
sporadically throughout the second and third trimesters.<br />
In many of these cases, decreased numbers of<br />
placentomes are over-compensated with increased total<br />
placental mass [16,23,66,72]. Enlarged placentomes are<br />
not only larger in area but coronal sections are much<br />
thicker. A similar reduction in placentome numbers<br />
together with an increase in placentome size has been<br />
achieved in sheep following surgical carunculectomies<br />
of the uterus prior to getting the females pregnant;<br />
however, growth compensation of the placentomes<br />
was not sufficient to allow the fetuses to grow at a<br />
normal rate [77], in contrast to what happens in<br />
clones, where fetal oversize is a common feature<br />
despite reduced placentome numbers [9,19,23].<br />
Occasionally, microplacentomes are observed,<br />
where large numbers of non-discrete placentomes<br />
form “mats” in the uterus. This and the observation<br />
of reduced numbers of placentomes suggest<br />
that placentomes, which should only form in relation<br />
to discrete areas, the endometrial caruncles, have<br />
failed to form on many of the caruncles or have fused<br />
together or are forming indiscriminately on the endometrium<br />
(mats of microplacentomes). This may also<br />
be the result of physiological adaptations to try to<br />
increase placental transfer capacity [66].<br />
Eventually, a high proportion of the fetal losses<br />
may be ascribed to placental-associated abnormalities,<br />
such as hydrallantois and edema of the fetal membranes<br />
and placental tissues. The enlarged placentomes and<br />
edematous membranes can be visualized by trans-abdominal<br />
or trans-rectal ultrasonography (Figure 2) or<br />
diagnosed by trans-rectal palpation.<br />
Euthanasia of pathological cases showed that<br />
placentome enlargement sometimes seems to affect only<br />
part of the placenta with areas with normal and areas<br />
with edematous placentomes. The lesions probably spread<br />
as gestation continues. During perfusion fixation of some<br />
clone placentomes, what appears to be microthrombi have N<br />
sometimes been observed. Histological examination of<br />
edematous placentomes from clones showed that although<br />
edema was present, there was little or no inflammatory<br />
process and the fetal and maternal tissue in the placentome<br />
both appeared grossly normal [23]. However, together<br />
with hydrallantois, fetal lesions, including omphalocele,<br />
ascites, cardiac enlargement, liver steatosis [18,19] are<br />
often present, together with frequent renal lesions such<br />
as hydronephrosis, some of which may be related to the<br />
observed placental abnormalities [11,18].<br />
III. VETERINARY CARE OF THE BOVINE SCNT<br />
PREGNANCY<br />
3.1. Detection of the early losses<br />
Ultrasonography in farm animals has been<br />
useful to demonstrate that a high rate of fetal death in<br />
SCNT pregnancies occurs from the first to third month<br />
of gestation [17,28,48,72]. In the early stages, the<br />
fetuses that are destined to die become progressively<br />
more growth retarded for their gestational age but it<br />
is not easy to precociously predict, using ultrasound<br />
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Figure 2. Transabdominal ultrasound view of an abnormal placentome in a SCNT pregnancy showing the hyperechogenic texture and<br />
the edema surrounding the placentome and cloudy fluids A) image obtained with a Vetscan®, 3.5 MHz probe, B) image obtained with<br />
a Voluson i, (GE Medical Systems) equipped with a transabdominal multifrequency probe (2.2-6.5 MHz). C) Photograph of an<br />
enlarged placentome with edema.<br />
A<br />
examination, which fetuses are ultimately non-viable<br />
[17]. A reasonable rate for monitoring SCNT<br />
pregnancies by trans-rectal scanning is every 15 days,<br />
beginning at Day 35. At 70-80 days of age, after the<br />
completion of implantation, the amniotic sacs of some<br />
cloned fetuses already appear to be enlarged and, at<br />
post-mortem examination, these fetuses had enlarged<br />
livers [47].<br />
In the first trimester, placental development can<br />
also be evaluated by maternal levels of serum proteins<br />
such as pregnancy specific protein B (PSPB) [82],<br />
pregnancy associated glycoprotein (bPAG) [98] and<br />
pregnancy serum protein 60, a protein of 60 kDa (PSP60)<br />
[65]. Except for some biochemical differences, these<br />
proteins all belong to the large family of Pregnancy-<br />
Associated Glycoproteins or the PAGs. PSP60, secreted<br />
by the binucleate cells of the placenta, as most PAGs, is<br />
a specific marker of pregnancy in cattle and easily<br />
assayed from maternal blood samples. Decreasing<br />
concentrations over a period of 15 days (two samples<br />
at 2 weekly interval) is indicative of fetal death while<br />
the presence of significantly higher concentrations<br />
than controls at Day 50 appeared to be correlated<br />
with impending fetal death [45]. It has not been<br />
possible, however, to determine threshold<br />
concentrations that could be predictive of fetal demise<br />
in the clones, since the concentration of PAGs is<br />
consistently higher in SCNT pregnancies by Days 60-64<br />
of gestation [17,24].<br />
3.2 Prenatal monitoring - Identification of the high risk<br />
pregnancy<br />
Late fetal loss occurs in 100% to 0% of SCNT<br />
pregnancies, depending on the breed, nuclear transfer<br />
technique and donor cell genotype and lineage, with an<br />
average figure of about 25-50% of pregnancies<br />
[46,64,70,72,91]. Fetal loss is associated with placental<br />
abnormalities leading to severe fetal hydrops,<br />
therefore necessitating rigorous monitoring of<br />
pregnancies in order to electively terminate pregnancies<br />
before maternal and fetal suffering become<br />
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an issue [16]. This is particularly important as ethical<br />
concerns regarding the welfare of fetal and neonatal<br />
clones as well as clone recipients have been the basis<br />
of the current debate leading to the postponement of<br />
the adoption of the European directive for Novel<br />
Foods [25], based on reports from the European Food<br />
Safety Agency and the European Group on Ethics in<br />
Science and New Technologies [30].<br />
High risk pregnancies can be identified by<br />
external examination and clinical observations in<br />
conjunction with ultrasonographic imaging to detect<br />
abnormal edematous placentation and increased allantoic<br />
fluid, and clinical tests to detect urinary ketones and<br />
elevated maternal plasma PSP60 concentrations<br />
[16,48,64]. Regular trans-rectal palpation of the uterus<br />
can also be used to detect abnormally rapid fluid<br />
accumulation and edematous fetal membranes. When<br />
the uterus becomes atonal, the situation is critical [16].<br />
Trans-abdominal ultrasonography is recommended for<br />
monitoring fetal viability and as an aid in detecting increased<br />
allantoic fluid volumes. Fetal heart rate has not proven to<br />
be useful for diagnosing fetal distress, as heart rates could<br />
not be measured in very severe cases where there was<br />
so much fetal fluid that it became impossible to locate the<br />
fetus [17,20,23]. Moreover, long periods of observation<br />
were not possible and continuous monitoring may be<br />
needed [13,55,68]. More research is needed as<br />
abnormalities in the fetal heart rate have been reported<br />
in IVP bovine fetuses as well [11]. In addition, although<br />
aortic diameter has been reported to be related to fetal<br />
size in the horse [1,75], it was not very useful for<br />
diagnosing very large SCNT bovine fetuses because even<br />
normal-sized SCNT fetuses may have enlarged hearts,<br />
thus making interpretation of the measurements difficult.<br />
The presence of placental edema or excessive allantoic<br />
fluid often indicates a poor prognosis for the fetus. The<br />
monitoring of the ultrasound appearance of placentomes<br />
is one of the most helpful tools for establishing a diagnosis<br />
of LOS. The edematous appearance, rather than the size<br />
of the placentomes (Figure 2), is indicative of the placental<br />
pathology in SCNT. Fetal ascites, which is often associated<br />
with abnormal placentation in SCNT, can also be<br />
monitored. In sheep, the fetal abdomen was punctured<br />
under trans-abdominal ultrasonographic guidance to<br />
retrieve the abdominal fluids from an SCNT fetus (Figure<br />
3), but the abdomen filled up rapidly again and<br />
this procedure did not prevent fetal demise (P.<br />
Chavatte-Palmer and O. Picone, personal<br />
observations).<br />
Monitoring of maternal body weight, feed<br />
intake, abdominal circumference, heart/respiratory<br />
rate and body temperature also provides valuable data<br />
to assess the health of the recipient animal. It has also<br />
been observed that some cows carrying SCNT fetuses<br />
have a fever of unexplained origin during the last<br />
few weeks of gestation. The presence of ketonuria is<br />
not necessarily a bad prognostic sign, but it does warn<br />
of potential for trouble. Significant reduction in feed<br />
intake or rapid gain in body weight or abdominal<br />
circumference indicate the development of<br />
hydrallantois and are prognostic of a poor outcome<br />
for the fetus.<br />
Although at present there is no useful therapy<br />
for improving the prognosis for severe cases of<br />
hydrallantois, less severe cases may be nursed to term<br />
by paying careful attention to the feed intake and metabolic<br />
status of the dam. Ruminants in particular are very<br />
prone to metabolic disorders (ketosis and fatty liver)<br />
caused by the increased uterine volume and reduced<br />
rumen capacity that accompany hydrallantois. In any<br />
case, a differential diagnosis for enlarged abdomen is<br />
always multiple fetuses when multiple embryos have been<br />
transferred. Rapidly growing SCNT fetuses may easily<br />
cause metabolic problems in ruminants and regular<br />
monitoring of urinary ketones will allow early intervention. N<br />
The gestational age at the onset of the pathology is an<br />
important prognostic indicator of outcome: in our<br />
laboratory, we consider that the fetus is likely to survive<br />
to term if the pathology manifests itself in the last two<br />
weeks prior to term. In severe cases, however, although<br />
the cow may be delivered with a live fetus close to term,<br />
the survival of the offspring is often compromised.<br />
Poorly viable third trimester fetuses may have<br />
significant circulatory abnormalities (hypoxia, liver<br />
congestion, placental edema). This is likely to be<br />
secondary to inadequate placental development and thus<br />
the placental circulatory system requires more careful<br />
evaluation. As abnormal placental circulation would place<br />
undue circulatory and metabolic stresses on the fetus,<br />
prospective studies on SCNT pregnancies are currently<br />
being performed to evaluate placental blood flow with<br />
3D power Doppler ultrasound after validating the method<br />
in the sheep (Figure 4) [67].<br />
IV. UNDELYING MECHANISMS<br />
Although sometimes, gross morphology<br />
appears normal in the placentomes of SCNT fetuses,<br />
many observations indicate that placental function<br />
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Figure 3. Coronal view of a cloned goat fetus with abdominal ascites (Voluson V8, GE Medical Systems, 3.5-5 MHz probe). A) before transuterine<br />
abdominal puncture, B) after trans-uterine abdominal puncture.<br />
may be impaired or modified. The overall mean<br />
surface area of placentomes has been shown to be<br />
increased in abnormal SCNT pregnancies and<br />
differences in placentome types have been observed<br />
[9,12]. Microscopically, stereological analyses of<br />
histological sections of placentomes have shown that<br />
the proportion of fetal connective tissue was increased<br />
and that of maternal epithelium decreased in SCNT.<br />
It seems as if these structural modifications were<br />
occurring to increase the efficiency of maternal-fetal<br />
exchanges [23].<br />
4.1 Placental vascularization<br />
Enlarged and prominent placental and umbilical<br />
blood vessels have often been observed in SCNT<br />
fetuses. The study of the expression of genes involved<br />
in vasculogenesis has shown that the two major<br />
factors controlling neovascularization in the placenta<br />
were affected in bovine SCNT, in a sexually<br />
dimorphic way. VEGFR-2 was overexpressed in the<br />
placenta of SCNT males and bFGF expression was<br />
decreased in the placenta of SCNT females when<br />
compared to controls [14].<br />
Figure 4. Transabdominal 3D power Doppler view (Voluson I, GE Medical Systems) of a placentome (ABC: power doppler view from 3<br />
different planes, 3D: 3D reconstruction of the total volume of the placentome) in a cloned pregnancy. Evaluations are currently being<br />
undertaken to evaluate the interest of such a technique for the diagnosis of abnormal placental function in clone cattle.<br />
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4.2 Placental function and exchanges<br />
The placenta is an organ that exists for the<br />
duration of the gestation solely to provide for the needs<br />
of the developing and growing fetus. Key functions would<br />
be the supply of adequate nutrients to the fetus, removal<br />
of fetal waste products, gaseous exchange, production<br />
of pregnancy-specific hormones and protection of the<br />
fetus against pathogens.<br />
SCNT placental function is compromised as late<br />
gestation cloned fetuses and newborn calves have been<br />
found to be hypoxic [36], hypoglycemic and<br />
hyperfructosemic [8,9] and with a certain degree of anemia<br />
[8,19], the latter being possibly related to the placental<br />
edema observed in pathological pregnancies, as observed<br />
in humans [26,78]. Moreover, the analysis of the fetal/<br />
placental weight ratio also indicates that placental<br />
efficiency is reduced in late pregnancy (Figure 5) [23].<br />
As a result of dysregulated placental development, a<br />
delicate balance exists between the capacity of the placenta<br />
to supply nutrients and the demands of a rapidly<br />
growing fetus.<br />
All placental functions themselves require large<br />
amounts of energy. An important energy source for<br />
the growing fetus is glucose, which is transported<br />
from the maternal circulation through the placenta to<br />
the fetus. Biochemical analyses of fetal fluids in one<br />
study at Days 50, 100 and 150 of gestation showed<br />
that in general, the mean glucose values were not<br />
different between SCNT and normal pregnancies,<br />
except at Day 50 of gestation, when the mean amniotic<br />
fluid glucose concetrations were lower in SCNT [60].<br />
Fetal membrane weights of these SCNT pregnancies<br />
were significantly higher at day 50 when compared<br />
with the controls and this may account for the greater<br />
utilization of the glucose, making less available to<br />
the fetus. However, once the placenta was formed, it<br />
appeared that glucose supply to the SCNT fetus was<br />
not significantly different compared with pregnancies<br />
generated by artificial insemination (AI) or with IVP<br />
embryos, as determined by mean amniotic fluid<br />
glucose levels. However, fetal fluid glucose levels in<br />
apparently failing SCNT pregnancies at Day 100 were<br />
unusually low, particularly in pregnancies with<br />
exceptionally large placentas or, in one case, a<br />
developmentally-retarded fetus with only 36 placentomes.<br />
Placental tissues themselves use 60-75% of the glucose<br />
leaving the uterine circulation and, with the excessive<br />
overgrowth of the SCNT placenta in the second half of<br />
gestation, the large placental mass could out-compete<br />
the fetus for available glucose. Glucose metabolized by<br />
the placenta and fetus contributes to a large part to<br />
the lactate present in fetal fluids and plasma. Highly<br />
elevated lactate levels, accompanied with reduced N<br />
glucose and fructose concentrations, were seen in<br />
Figure 5. Mean ± SD feto/placental weight ration in control and clone<br />
pregnancies between 180 and 220 days (N=5 controls and N=9 clones) and<br />
after 220 days of gestation (N=10 controls and N=9 clones) (after [24]).<br />
*indicates a significant difference (P < 0.05).<br />
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allantoic fluid from SCNT fetuses with enlarged placentas<br />
and in some hydrops cases.<br />
At term, the expression of the glucose<br />
transporter GLUT3 is increased in the SCNT placenta,<br />
and there is a negative correlation between maternal<br />
blood glucose prior to parturition and birth weight,<br />
in the absence of neonatal hypoglycemia, indicating<br />
that glucose uptake from the maternal circulation and<br />
transfer to the fetus is increased in late gestation in<br />
SCNT, probably to meet increased fetal requirements<br />
[51].<br />
Another function of the ruminant placenta is to<br />
synthesize fructose from glucose. Generally, the ability<br />
of the SCNT placenta to synthesize fructose did not<br />
appear to be impaired. However, some allantoic fluid<br />
samples from SCNT pregnancies did have lower glucose<br />
and fructose and these pregnancies were associated with<br />
increased placental and fetal weights, which imply that<br />
placental fructose synthesis in these cases was being<br />
limited by glucose availability at this stage [60]. In contrast,<br />
at term, hyperfructosemia in SCNT neonatal calves at<br />
birth seems to indicate either increased production or<br />
decreased utilization near term [9].<br />
The bovine placenta is very important source of<br />
steroid hormones during pregnancy [22,86]. Although it<br />
has the potential to produce both progesterone and<br />
estrogens, it does not have to capacity to produce enough<br />
progesterone in the absence of a corpus luteum to support<br />
the pregnancy until after 200 days of gestation. The placenta,<br />
however, is the main source of estrone and there<br />
are two stages in gestation when estrone production is<br />
up-regulated. The first rise in estrone levels takes place<br />
at the beginning of the second trimester. It is crucial for<br />
pregnancy progression. Retrospective analysis of maternal<br />
plasmas showed that the majority of SCNT<br />
pregnancies that fail around mid-gestation did not have<br />
this rise in estrone levels (Morrow and Lee, unpublished<br />
observations). In addition to possible effects on fetal<br />
development, estrogens also likely affect placental<br />
development and function, as estrogen receptors were<br />
found to be present in placental tissues [53].<br />
The second rise in estrogen levels take place<br />
near the end of the gestation and one function for these<br />
estrogens is the preparation of the mammary gland for<br />
lactation. We have observed that those recipient cows<br />
with SCNT fetuses that showed no signs of preparation<br />
for parturition also show poor mammary development<br />
near term. This could be due to inadequate estrogen<br />
production or excessive metabolism of estrogens, and<br />
it is likely to be associated with the absence of a prepartum<br />
decline in maternal progesterone<br />
concentrations, as observed in a few cases [48]. The<br />
placenta regulates the amount of estrogens that both<br />
itself and the fetus are exposed to, by expressing<br />
estrogen sulfotransferase, which catalyzes the<br />
sulfoconjugation of estrogens for excretion. Excess<br />
sulfoconjugation was found to be a possible cause<br />
for the poor parturition signal frequently encountered<br />
in bovine SCNT pregnancies [52].<br />
An abnormal increase in the concentration of<br />
PAGs has been reported in maternal circulation of SCNT<br />
recipients that subsequently developed placental anomalies<br />
[45]. PAGs, secreted in maternal blood, are synthesized<br />
by trophoblastic binucleate cells (BCN) that migrate and<br />
fuse with uterine epithelial cells, forming short lived<br />
trinucleate cells [94]. In cattle, other proteins such as<br />
placental lactogens (PL) are also produced by the BNC<br />
[95]. In a recent study, we reported that maternal plasma<br />
PAG concentrations were not different at Day 32,<br />
but significantly higher in SCNT than in AI and IVP<br />
control pregnancies at Day 62 and during the third<br />
trimester. Circulating bPL concentrations were<br />
undetectable at early stages and were not different in<br />
the third trimester between clone and control pregnancies.<br />
Placental tissular ratios of PAG or bPL to total proteins<br />
were not different between the two groups at all stages<br />
studied. Moreover, there was no difference in the<br />
percentage of PSP60-positive BNC in placental tissues<br />
between SCNT and control pregnancies. These results<br />
demonstrate that highly elevated maternal levels of PAG<br />
during abnormal SCNT pregnancies do not result from<br />
the placental hypertrophy, nor from an increased<br />
expression of the proteins by the placenta or a higher<br />
proportion of BNC, but could be due to changes in the<br />
composition of terminal glycosylation which result in a<br />
decrease of the clearance of PAG from the circulation<br />
[24].<br />
In the bovine placenta, approximately 15-20%<br />
of the trophoblast is composed of giant trophoblastic cells<br />
that can have two or more nuclei (BNC, trinucleate<br />
cells..). It was shown that there is a lesser proportion of<br />
diploid to tetraploid cells in the central region of<br />
placentomes and in microplacentomes (
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compared to maternal tissue in late gestation,<br />
abnormal, SCNT placentas [23].<br />
4.3 Oxidative stress<br />
In pigs, available evidence shows that aberrant<br />
expression of antioxidant enzyme proteins and associated<br />
oxidative stress in the extra-embryonic tissue derived from<br />
of Day 26 SCNT conceptuses is a major factor<br />
contributing to low birth rate [15]. Oxidative stress during<br />
early placental development is associated in other species<br />
with pregnancy-related disorders and pathologies in<br />
humans, such as embryonic resorption, spontaneous<br />
abortion, intra-uterine growth restriction, fetal death,<br />
preeclampsia, and preterm labor and delivery [2,54,87].<br />
Adequate placental antioxidant status during early<br />
pregnancy could prevent those disorders induced by<br />
oxidative stress that lead to impairment of placental<br />
function and development, fetal growth and poor<br />
pregnancy outcomes. Indeed, the content of<br />
malondialdehyde (MDA), as an index of Reactive<br />
Oxygen Species (ROS) oxidative stress, is significantly<br />
lower in the chorionic tissue of bovine SCNT compared<br />
to AI controls at Day 62 of pregnancy [3]. Similarly,<br />
although the activities of the key anti-oxidant enzymes<br />
Super Oxide Dismutase 1 and 2 (SOD1 and 2) and<br />
Glutathione Peroxidase (GPX), were not different<br />
between the chorion of AI and SCNT conceptuses, at<br />
Days 32 and 62 of gestation, catalase (CAT) activity was<br />
significantly decreased at Day 32 and significantly<br />
increased at Day 62 in SCNT chorion, demonstrating<br />
that the activity of some of the anti-oxidant enzymes is<br />
up regulated in the chorion of post-implantation SCNT<br />
fetuses [3].<br />
4.4 Gene expression<br />
To understand how placental development in<br />
SCNT pregnancies is different from AI or IVP<br />
pregnancies, several gene expression profiling studies<br />
have been carried out. Different microarray/macroarrays<br />
with different sets of genes have been used in these<br />
studies, making it difficult to compare the findings between<br />
studies. In addition, the stochastic nature of the<br />
abnormalities, the different SCNT procedures used, and<br />
the stage of gestation at which the placental samples<br />
were collected and how the tissue was sampled makes it<br />
difficult to find sets of genes or pathways that are<br />
commonly affected. This is reflected in the microarray<br />
analysis of placenta collected from AI, IVP and SCNT<br />
pregnancies [31], where cluster analysis revealed that<br />
most of the variation in gene expression can be<br />
accounted for by the stage of gestation (pre-term<br />
versus term), the source of the placental samples (AI,<br />
IVP and SCNT) and the fetal pathology. However,<br />
the compound groupings in each cluster indicate that<br />
many factors contribute to the gene expression<br />
patterns. Interestingly, the majority of the AI term<br />
samples fell into a unique cluster that excluded IVP<br />
and SCNT placenta. IVP samples clustered more with<br />
the SCNT than with the AI samples, consistent with<br />
earlier observations that the placentas from IVP<br />
pregnancies, like those from SCNT, show greater<br />
gross morphometric variability than AI placentas [58].<br />
This indicates that the environment of the preimplantation<br />
embryo can have long-term effects on<br />
placental development and gene expression profiles.<br />
Many of the differentially expressed genes and<br />
overlapping functional networks among the IVP and<br />
SCNT gene lists support the hypothesis that early<br />
embryo culture conditions likely affect the same<br />
pathways in both SCNT and IVP. However, SCNT<br />
itself affects the expression of an additional > 200<br />
genes that ultimately may contribute to the abnormal<br />
placental phenotype seen only in SCNT. The most<br />
prominent functional effects involve cell cycle, cell<br />
signaling pathways, molecular transport, DNA N<br />
replication, recombination and repair; most of these<br />
molecular pathways and functions appear to be down<br />
regulated in SCNT.<br />
Other studies have looked for differences in gene<br />
expression at earlier stages of gestation (peri-implantation<br />
and in the early stages of placentome formation), prior to<br />
the development of severe pathologies but before the<br />
early losses have occurred. However, these studies<br />
focused only on the extra-embryonic membranes or<br />
cotyledonary tissues and therefore cannot be compared<br />
directly with studies using tissues from entire placentomes.<br />
Several genes expressed in the trophoblast have been<br />
found to be differentially expressed between SCNT and<br />
control conceptuses, including placental lactogen (PL),<br />
the PAG, prolactin related protein-1 (PRP-1) [41] and<br />
Dickkopf-1(DKK-1)[57], to name a few. All these<br />
proteins are expressed in the BNCs of the trophoblast<br />
and thus, indicate that BNC function may be affected in<br />
SCNT from very early in gestation, thereby compromising<br />
placental development. At around Day 60 of gestation,<br />
the expression of two of the hemoglobin genes (HBA1<br />
and 2) and the erythrocyte spectrin beta gene (SPTB)<br />
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was found to be down-regulated [71]. Reduced<br />
expression of these three genes could mean that<br />
erythropoiesis could be impaired in the SCNT placenta<br />
and thus, compromise fetal survival.<br />
4.5 Epigenetics and the role of imprinted genes<br />
Cloning by nuclear transfer returns a<br />
differentiated cell to a totipotent status, a process termed<br />
nuclear reprogramming, and was often associated with<br />
epigenetic alterations. Several studies have shown that<br />
insufficient epigenetic reprogramming of the differentiated<br />
nuclear donor cell towards a pluripotent (embryonic) status<br />
plays a major role in the pathogenesis of LOS [69]. Both<br />
global- and gene-specific DNA methylation has been<br />
studied in SCNT embryos and offspring of several species<br />
including mouse, pig, buffalo and cattle. In the early stages<br />
of development, a hypermethylation of the entire bovine<br />
embryo has been reported using immunostaining<br />
techniques, mainly revealing the methylation patterns of<br />
repetitive regions and heterochromatic regions of the<br />
genome [81].<br />
Many of the pathologies reported with bovine<br />
clone pregnancies resemble abnormalities reported with<br />
either mutations or deletions of imprinted genes or<br />
dysregulation of imprinted gene expression. Embryo<br />
manipulations, such as assisted reproductive technology<br />
(ART), induce phenotypic changes in the embryo and<br />
the placenta by influencing imprinting genes, most of which<br />
being expressed in the placenta, in mouse [63], sheep<br />
[97] and human [4,34]. Studies in mouse mutants have<br />
shown that many of the genes involved in differentiation<br />
and growth of the placenta are imprinted [42,79].<br />
Examples of imprinted genes which control placental<br />
growth include Igf2r [88], Igf2, Mash2 [39], Esx1 [61],<br />
Cdkn1c/p57 Kip2 [85] and Phlda2/Ipl/Tssc3 [35].<br />
At the blastocyst stage, alteration of the<br />
epigenetic regulation of some imprinted genes is often<br />
associated with the embryo death. A loss of DMR<br />
methylation (differentially methylated regions implicated<br />
in the imprinting regulation) was found for IGF2R [62]<br />
and SNRPN [84] genes. In contrast, an overall lesser<br />
methylation in the DMR of the IGF2/H19 alleles was<br />
found in cattle when compared to the same tissues in<br />
control animals [37]. In the mouse, overexpression of<br />
Igf2 results in fetal and placental overgrowth and<br />
overgrowth of certain fetal organs [29]. The same organs<br />
are often the ones that are disproportionately large in<br />
SCNT fetuses. Although overexpression of IGF2 (with<br />
reference to house-keeping genes) was not observed in<br />
placental tissues from SCNT pregnancies, the increased<br />
placental mass means that overall, there must be more<br />
IGF2 produced and available to promote growth, assuming<br />
there is no difference in translational efficiency.<br />
The bioavailability of IGF2 is regulated through<br />
binding to the type 2 receptor (IGF2R) or to the IGF<br />
binding proteins (IGFBPs). The expression of IGF2R was<br />
shown to be altered in ovine large offspring syndrome<br />
[97]; however, this was not the case in bovine clones.<br />
Aberrant expression of the IGFBPs in bovine SCNT was<br />
reported [74], and even though the genes for these proteins<br />
are not imprinted, they regulate IGF2 bioavailability at a<br />
tissue-specific manner. The expression IGFBP2 was<br />
lower in the placental tissues from SCNT pregnancies<br />
[59]; thus, even if IGF2 production was not increased in<br />
clones, there was greater availability, as there was less<br />
IGFBPs to bind to it.<br />
The maternally expressed Phlda2 gene is included<br />
in a cluster of imprinted genes located on mouse<br />
chromosome 7, synthenic to a cluster on human<br />
chromosome 11p15.5 [73]. Phlda2 invalidated mice, with<br />
a lack of the maternal expression of Phlda2, are fertile<br />
but present enlarged placentae during pregnancy [35].<br />
Conversely, the loss of imprint of Phlda2 leading to a bi<br />
allelic expression, causes fetal and placental growth<br />
retardation [80]. Similarly, an increased expression of<br />
PHLDA2 in human placenta is associated with low birth<br />
weights through putative reduction in size and function<br />
of the placenta [5]. In bovine, a reduced expression of<br />
Phlda2 is associated with pathological overgrowth of the<br />
placentomes from SCNT pregnancies and the level of<br />
expression of PHLDA2 is correlated negatively with total<br />
placentome weight in cloned placenta [40]. Altogether<br />
these data strongly support the idea that PHLDA2 acts<br />
as regulator of placental growth and may be involved in<br />
overgrowth observed after SCNT.<br />
Imprinted genes may regulate fetal growth by<br />
affecting the overall development and growth of the placenta<br />
or its functions, such as the supply of nutrients to<br />
the fetus via specific transporters and ion channels. Some<br />
of these transporter genes, such as the SLC22A genes<br />
are linked to IGF2R. It is not known if the expression of<br />
the SLC22A genes is affected in the SCNT placentas.<br />
Although a larger placenta may have a greater surface<br />
area over which to transfer nutrients, aberrations in<br />
vascular development may alter permeability and the<br />
increased mass, in addition, requires a greater share of<br />
the nutrients to maintain function. Placental mass is not<br />
always positively correlated with fetal weight in bovine<br />
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SCNT pregnancies, so a fine balance between supply<br />
and demand is required to maintain an appropriate rate<br />
of fetal growth.<br />
In female mammals, one of the two X-<br />
chromosomes is randomly inactivated in somatic cells in<br />
the body. Some SCNT embryos, aborted fetuses or dead<br />
newborn calves or viable offspring, however, showed<br />
aberrations in X-chromosome inactivation [96]. This has<br />
been attributed to faulty reprogramming of the donor<br />
nucleus. In the placenta, which normally show preferential<br />
paternal X-inactivation due to imprinting, random X-<br />
inactivation has been reported in SCNT placenta. It has<br />
been postulated that this aberrant X-inactivation may be<br />
deleterious to the development of SCNT fetuses or<br />
contribute to abnormal placental development. However,<br />
we did not see any gross differences in the severity of<br />
gestational pathologies, either in the placenta or fetus,<br />
whether male or female donor cells are used (Lee, et al.,<br />
unpublished observations). There should not be any issues<br />
with X-inactivation when male donor cell lines are used,<br />
yet the outcome is no better compared with using female<br />
cell lines. Thus, aberrant X-inactivation is unlikely to be a<br />
major contributor to poor cloning outcomes.<br />
Finally, it must be noted that a high variability<br />
of global methylation has been reported in healthy<br />
adult clone cows [27]. Altogether, these studies<br />
highlight the importance to analyze the methylation<br />
status of specific loci in cloned embryos and offspring<br />
to understand the epigenetic disruption associated<br />
with the SCNT procedures.<br />
V. CONCLUSIONS<br />
In conclusion, SCNT affects many aspects of<br />
placental development in the bovine species, from the<br />
morphological structure to physiological function, gene<br />
expression and epigenetic marks. It is now well<br />
recognized that events occurring in the embryonic and<br />
fetal period may induce long term health effects in the<br />
offspring as developed through the study of the<br />
Developmental Origins of Health and Disease (DOHaD)<br />
[7,10,38]. Studying long term effects of cloning therefore<br />
must encompass the study of the effects of nuclear<br />
transfer per se as well as the secondary long term effects<br />
due to a perturbed placental function.<br />
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www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
s242
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s243 - s252.<br />
ISSN 1679-9216 (Online)<br />
Clinical disorders observed during the first 30 days of life of cloned Zebu calf<br />
Eduar<br />
duardo do Harr y Bir<br />
irgel Junior<br />
1 , Flávio<br />
Vieir<br />
ieira Meir<br />
eirelles<br />
elles 1 , Eliza Rossi Komninou<br />
2 , Mar<br />
ariana<br />
Tik<br />
ikuma<br />
Nunes<br />
2 , Fábio Celidonio Pogliani<br />
2 , Paulo Fan<br />
antina<br />
tinato Net<br />
eto 2 , Melina Mar<br />
arie<br />
Yasuok<br />
asuoka 2 , José Rodr<br />
drigo<br />
V.<br />
Pimentel1, Flávia Saldanha Kubrusly 3 & Maria Angélica Miglino 2<br />
ABSTRACT<br />
Background:The advent of animal cloning from embryos reconstructed with nuclei from cells at different stages of differentiation<br />
has been responsible for the appearance of several anomalies in calves, with some alterations diagnosed during pregnancy and<br />
others after the birth of cloned animals. Despite efforts over the past five years, the rate of mortality after the birth of cloned<br />
calves is still high, at about 50%. Below is reported the research experience gained at the University of São Paulo in the period<br />
from 2005 to 2010, related to the birth of cloned calves from Nellore.<br />
Review: Research showed that cloned calves present cardiopulmonary disorders characterized by tachycardia with episodes<br />
of arrhythmia with bradycardia, increase in the 1 st heart sound, presence or absence of heart murmurs in the 1 st and 2 nd heart<br />
sound associated with dyspnea, breathing harsh and crackling dry, moist and crackling. During the first days of life of cloned<br />
calves that died, ultrasound examination demonstrated the presence of concentric hypertrophy congenital cardiac and turbulent<br />
flow at the foramen of botal towards the right heart to left heart, indicating reversal of blood flow to the pattern observed in fetal<br />
life. As a result, mixture of arterial and venous blood occurs, compromising the ability of oxygen to the blood of cloned calves.<br />
The occurrence of macrosomia was observed in 20.9% of Nellore calves. It is believed that this syndrome is associated with<br />
disorders of carbohydrate metabolism in the placenta and/or fetus, leading to fetal gigantism, which mimicks excessive fetal<br />
growth seen in pregnant women with diabetes. We observed the occurrence of moderate to severe normocytic and normochromic<br />
anemia, with the anemia gradually appearing from 12 h of life onwards, reaching its maximum intensity at the end of the first N<br />
week, to start a gradual recovery to normal values from the 15 th day of life. The anemia observed in cloned calves was<br />
ferropriva, as such animals showed a significant decrease in serum iron levels associated with decreased transferrin saturation<br />
index (STI). Umbilical anomalies occurred in most clones. There was an increase in the thickness of the umbilical cord, which<br />
hindered its spontaneous rupture at birth. The umbilical arteries did not undergo retraction into the abdominal cavity, leaving<br />
them exposed in the remnant of the umbilical cord. In the first three days after birth, strong pulse of these arteries was noted in<br />
umbilical cord, making it necessary to use clamps on the arteries in order to prevent bleeding. There were also intra-abdominal<br />
hematomas involving the arteries and the urachus. Between 15 and 20 days of life, it was observed the occurrence of alopecia<br />
in about 75.0% of the calves, whose origin may be related to disturbances in the synthesis and absorption of vitamins, since the<br />
supplementation of calves with ADE vitamin complex decreased symptoms.<br />
Conclusion: The results confirm the occurrence of serious cardiopulmonary disorders characterized by tachycardia,<br />
hyperphonesis, presence of heart murmurs in the 1 st and 2 nd heart sounds, episodes of arrhythmia and bradycardia associated<br />
with dyspnea, rales, and coarse breath. As a result of the non-closure of the foramen of Botal and the Ductus Arteriosus, arterial<br />
and venous blood a mix, compromising the oxygen carrying capacity in the blood of cloned calves. There was also the<br />
occurrence of macrosomia, hypoglycemia, hypothermia, abnormal umbilical structures, anemia and alopecia.<br />
Keywords: Cloned calf, somatic cell nucleartransfer (SCNT), bovine, disease, cardiopulmonary disorders.<br />
Descritores: bezerros clonados, transferência nuclear de células somáticas (TNSC), bovinos, doença, distúrbios<br />
cardiopulmonares.<br />
1<br />
Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo (USP), Campus da USP – Pirassununga, SP, Brazil. 2 Faculdade<br />
de Medicina Veterinária e Zootecnia - USP. 3 Centro de Biotecnologia - Instituto Butantan, São Paulo, SP. CORRESPONDENCE: E.H. Birgel<br />
Junior [ehbirgel@usp.br - TEL: + 55 (19) 3565-4000]. Faculdade de Zootecnia e Engenharia de Alimentos - Campus da Universidade de São<br />
Paulo (USP), Av. Duque de Caxias Norte n. 225. CEP 13635-900 Pirassununga, SP, Brazil.<br />
s243
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
I. INTRODUÇÃO<br />
II. DISTÚRBIOS PLACENTÁRIOS E OCORRÊNCIA DA<br />
HIPÓXIA INTRA-UTERINA<br />
III. DISTÚRBIOS DA ADAPTAÇÃO NEONATAL DO BE-<br />
ZERRO CLONADO<br />
IV. DISTÚRBIOS CARDIOPULMONARES NOS BEZER-<br />
ROS CLONADOS<br />
V. MACROSSOMIA / SÍNDROME DO BEZERRO GRAN-<br />
DE<br />
VI. OCORRÊNCIA DE ANEMIA<br />
VII. ANOMALIAS NAS ESTRUTURAS UMBILICAIS,<br />
DEFORMIDADES FLEXURAIS E ALOPECIA<br />
VIII. CONCLUSÕES<br />
I. INTRODUÇÃO<br />
O advento da clonagem de animais a partir<br />
de embriões reconstruídos com núcleos provenientes<br />
de células em diferentes estágios de diferenciação<br />
tem sido responsável pelo aparecimento de diversas<br />
anomalias nos bezerros, sendo algumas alterações<br />
diagnosticadas durante a gestação e outras<br />
após o nascimento de animais clonados [29].<br />
Uma grande mortalidade pré e peri-natal de<br />
bezerros foram demonstradas como resultado da produção<br />
in vitro [18,39], da transferência nuclear de<br />
células embrionárias [7,26,44] e da clonagem a partir<br />
de células somáticas [2,10,23,29].<br />
Farin et al. [14] propôs sistema de classificação<br />
das alterações observadas durante a gestação de<br />
clones apresentado a seguir:<br />
TIPO 1- Desenvolvimento anormal com morte<br />
do embrião ou do concepto (reabsorção embrionária)<br />
antes da organogênese completa (até aproximadamente<br />
42 dias de gestação).<br />
TIPO 2 - Desenvolvimento anormal de membranas<br />
placentárias e do feto; com morte fetal e<br />
abortamento entre a fase de completa diferenciação<br />
de órgãos e o término da gestação (de 42 a 280 dias<br />
de gestação).<br />
TIPO 3 - A gestação chega ao termo, o feto e/<br />
ou a placenta possuem severas anormalidades de desenvolvimento<br />
e não há evidência de resposta compensatória<br />
do feto ou placenta. A saúde dos neonatos<br />
está severamente comprometida, com alterações clínicas,<br />
de parâmetros hematológicos e bioquímicos;<br />
a morte ocorre entre a hora do parto ou durante o<br />
período neonatal.<br />
TIPO 4 - A gestação chega ao termo e tanto o<br />
feto quanto a placenta possuem alterações moderadas;<br />
entretanto ocorrem compensações fetoplacentárias<br />
e adaptações que fazem com que o animal<br />
sobreviva apesar dos distúrbios genéticos e fisiológicos.<br />
Os neonatos podem ter tamanho normal<br />
ou serem maiores do que a média da raça, podendo<br />
ter alterações clínicas e de parâmetros hematológicos<br />
e bioquímicos.<br />
Apesar dos esforços realizados nos últimos<br />
cinco anos, a taxa de mortalidade pós-nascimento<br />
dos bezerros clonados é ainda grande, cerca de 50%,<br />
tornando necessários estudos mais aprofundados em<br />
neonatologia dos bovinos. O objetivo desses estudos<br />
deve ser esclarecer a patogenia e desenvolver<br />
tratamentos eficientes para essas enfermidades, sendo<br />
fundamental o desenvolvimento de metodologia<br />
de diagnóstico que permita uma melhor avaliação<br />
das alterações observadas no sistema cardiopulmonar<br />
dos bezerros clonados, bem como o estabelecimento<br />
de valores de referência que possam ser utilizados<br />
na interpretação dos resultados obtidos. A seguir será<br />
relatada a experiência acumulada em pesquisas realizadas<br />
na Universidade de São Paulo, no período de<br />
2005 a 2010, relacionadas ao nascimento de bezerros<br />
clonados da raça Nelore.<br />
II. DISTÚRBIOS PLACENTÁRIOS E OCORRÊNCIA DA<br />
HIPÓXIA<br />
Apesar dos relatos sobre perdas fetais precoces<br />
durante a clonagem devido a alterações no ambiente<br />
uterino materno, falhas na interação maternofetal<br />
ou a anormalidade do embrião [2,20] aceita-se,<br />
atualmente, que o desenvolvimento e sobrevivência<br />
fetal, durante a gestação de clones, dependem da<br />
morfologia apropriada e desenvolvimento funcional<br />
da placenta. Estudos indicam que a causa dessas perdas<br />
está associada a um desenvolvimento anormal<br />
da placenta, possivelmente devido ao crescimento<br />
inapropriado da membrana alantóide em decorrência<br />
da falta de vascularização nos estágios iniciais do<br />
desenvolvimento [43]. Distúrbios na expressão da<br />
VEGF – fator responsável pela permeabilidade<br />
vascular e neo-vascularização da placenta foram descritos<br />
em placentas de clones [8]. Estruturalmente<br />
existe desorganização das “árvores vilosas” fetais,<br />
presença de mais de uma artéria-tronco por vilo, ramificação<br />
vascular deficiente e dilatações anormais<br />
das criptas endometriais [32]. Durante a gestação as<br />
seguintes disfunções placentárias foram relatadas:<br />
diminuição do número de placentônios, presença de<br />
s244
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
placentônios gigantes, presença de microcotiledones<br />
acessórios com diâmetro menor do que 1,0 cm, extensas<br />
áreas na membrana corioalantóide desprovidas<br />
de placentônios, edema das membranas<br />
placentárias, aumento na espessura do cordão umbilical<br />
e formação de hidroalantóide [9,23,26,32]. A<br />
insuficiência placentária culmina na ocorrência de<br />
perdas fetais durante a fase gestacional, alterações<br />
congênitas nos neonatos, complicações peri e pós<br />
natais, sofrimento fetal crônico e agudo e alta taxa de<br />
mortalidade neonatal. Em conseqüência a esse sofrimento<br />
fetal/ hipóxia intra-uterina, nota-se com freqüência<br />
o tingimento dos fetos por mecônio [4,5].<br />
Nunes [33] observou hipóxia/ sofrimento fetal em<br />
55,5% (5/9) dos animais clonados estudados, sendo<br />
que Batchelder et al. [2] correlacionaram a ocorrência<br />
de bezerros clonados tingidos de mecônio com a<br />
maior incidência de mortalidade nas primeiras horas<br />
de vida.<br />
Apesar da avaliação da vitalidade fetal por<br />
meio da carditocografia, já serem utilizados há muito<br />
tempo na medicina, como método de rotina, verifica-se<br />
que na veterinária o seu uso ainda é limitado,<br />
havendo poucas pesquisas sobre o tema<br />
[12,19,21,22]. Esse exame possibilita a avaliação do<br />
bem estar fetal e/ou avaliação da vitalidade fetal e é<br />
extremamente importante no seguimento das gestações<br />
de alto risco, principalmente nas que cursam<br />
com insuficiência placentária [17], sendo o seu principal<br />
objetivo a detecção precoce da hipoxemia fetal.<br />
Ao avaliar as características que compõem a<br />
cardiotocografia (linha de base, variabilidade, presença<br />
de acelerações transitórias e desacelerações),<br />
separadamente, Nunes [33] constatou que os clones<br />
tingidos de mecônio e que morreram nas primeiras<br />
horas de vida apresentavam com mais freqüência<br />
bradicardia e o número de acelerações transitórias<br />
era significativamente menor do que o observado nos<br />
grupos que não estavam em sofrimento fetal. A presença<br />
de bradicardia fetal é sinal de sofrimento fetal<br />
em ruminantes [12], enquanto a presença de acelerações<br />
transitórias é um dos mais importantes indicadores<br />
de bem-estar fetal para humano [28,31] e ruminantes<br />
[21,22]. Nunes [33] relacionou o menor<br />
número de acelerações transitórias da frequência cardíaca,<br />
observadas no grupo de clones que morreram<br />
nas primeiras 36 horas de vida, à diminuição do bem<br />
estar fetal decorrente da má oxigenação do feto.<br />
Segundo Nunes [33] os fetos do grupo de<br />
clones mortos tiveram uma maior ocorrência de momentos<br />
de hipoatividade e inatividade fetal do que<br />
os fetos do grupo de clones vivos e fetos do grupo<br />
controle. A estimulação dos fetos hipoativos por meio<br />
de beliscamento das extremidades, durante exame<br />
de palpação retal, evidenciou que os fetos do grupo<br />
de clone mortos foram classificados com maior freqüência<br />
como não reativos do que os fetos do grupo<br />
controle.<br />
Os resultados obtidos na Dissertação de<br />
Mestrado de Nunes [33] são compatíveis com a ocorrência<br />
de sofrimento fetal/ hipóxia intra-uterina, de<br />
grau variável nos últimos 90 dias de gestação. A gravidade<br />
da hipóxia intra-uterina explicaria a evolução<br />
do quadro clínico dos bezerros:<br />
- hipóxia intra-uterina grave nos clones tingidos<br />
de mecônio com índice APGAR menor do que 3<br />
ao nascimento e morte nas primeiras 36 h de vida<br />
decorrentes a alterações cardiopulmonares incompatíveis<br />
com a vida extra uterina;<br />
- hipóxia intra-uterina de pequena intensidade<br />
ou oxigenação intra-uterina normal nos clones que<br />
apresentaram índice APGAR entre 7 e 8 ao nascimento<br />
e que sobreviveram.<br />
N<br />
Face as disfunções placentárias, tem-se recomendado,<br />
freqüentemente, que o nascimento dos<br />
clones seja feita por meio de cesariana. Em gestações<br />
de bezerros clonados da raça Nelore tem-se<br />
optado pela realização de cesariana entre 290 a 292<br />
dias de gestação (gestação do Nelore dura, em média<br />
10 dias a mais do que na raça Holandesa), sendo<br />
utilizado o seguinte protocolo de indução: 8 mg de<br />
triancinolona aplicada por via intra-muscular 7 dias<br />
antes da cesariana, 20 a 25 mg de dexametasona<br />
aplicada por via intra-venosa 36 h antes da cesariana<br />
associado a 500 ug de prostaglandina F2α aplicada<br />
por via intra-muscular 24 h antes da cesariana. Apesar<br />
de realizar-se a cesariana antes do início da parição<br />
ou nas primeiras horas da fase de dilatação do parto,<br />
recomendou-se o uso de relaxante uterino (50 mg de<br />
cloridrato de isoxsuprine aplicado por via intra-venosa<br />
no início da intervenção cirúrgica), pois os bezerros<br />
clonados estavam mais predispostos a asfixia<br />
neonatal precoce uma vez que as disfunções<br />
placentárias podem determinar importantes variações<br />
s245
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
de oxigenação no feto mesmo que as contrações<br />
uterinas não sejam intensas [5,30]. Após pesquisa<br />
realizada por Nunes [33], na qual foi demonstrado<br />
que nas últimas 24 h antes da cesariana havia maior<br />
ocorrência de hipoatividade fetal, tem-se recomendado<br />
que a indução seja feita sem o uso da<br />
prostaglandina F2α, pois esta droga determina que o<br />
parto ocorra nas entre 24 e 28 h após o seu uso, ou<br />
seja, no momento da realização da cesariana já existiriam<br />
contrações uterinas que poderiam diminuir a<br />
oxigenação do feto.<br />
III. DISTÚRBIOS DA ADAPTAÇÃO NEONATAL DO<br />
BEZERRO CLONADO<br />
Durante o processo de nascimento, no momento<br />
da separação do cordão umbilical do feto,<br />
várias funções fisiológicas anteriormente suportadas<br />
pela mãe através da placenta devem ser assumidas<br />
pelo neonato a fim de assegurar a sua sobrevivência<br />
[3]. Dentre essas funções cabe particular destaque as<br />
desempenhadas pelo sistema cardiopulmonar, pois<br />
imediatamente após o delivramento, ocorrem profundas<br />
modificações na circulação sangüínea, passando<br />
a ser dos vasos pulmonares a responsabilidade<br />
pela adequada oxigenação do sangue. Esse aumento<br />
da oxigenação sangüínea é responsável pelo<br />
fechamento do duto arterioso. Afora o aumento na<br />
oxigenação sangüínea, ao inflar seus pulmões, o<br />
neonato diminui a resistência na rede capilar pulmonar<br />
determinando o aumento do fluxo de sangue para<br />
os pulmões, aumento do volume de sangue que retoma<br />
para o átrio esquerdo e, como conseqüência,<br />
aumento da pressão no lado esquerdo do coração. A<br />
maior pressão do sangue no átrio esquerdo faz compressão<br />
sobre o forâmen oval e promove o seu fechamento<br />
impedindo a mistura de sangue arterial e<br />
venoso [35]. Falhas nessa adaptação neonatal a vida<br />
extra-uterina estão freqüentemente associadas a grandes<br />
taxas de morbidade e mortalidade em bezerros<br />
[3,40]. Verifica-se, em bezerros clonados, que essa<br />
adaptação está particularmente afetada, sendo<br />
comumente observado prostração, letargia, ausência<br />
do reflexo de sucção e eliminação do mecônio<br />
nos primeiros 15 minutos de vida. Em decorrência<br />
aos distúrbios de oxigenação nesses animais, observa-se<br />
alterações como diminuição da pO 2<br />
e aumento<br />
da pCO 2<br />
, circulação preferencial para órgãos vitais<br />
e glicólise anaeróbica em órgãos não vitais, aumento<br />
da lactacidemia e acidose metabólica, em quadro<br />
similar ao descrito na asfixia neonatal [3].<br />
Baseado no exame clínico, nas avaliações do<br />
APGAR, realizadas ao nascimento e aos 5 minutos<br />
de vida, nos resultados da hemogasometria e nos<br />
achados de necropsia foi possível caracterizar quatro<br />
tipos de quadro clínicos nos bezerro clonados<br />
[4,5]:<br />
Grupo 1 - bezerros que apresentavam<br />
APGAR ruim (0 a 3 pontos), sinais de acidose na<br />
hemogasometria e morte na primeira hora de vida;<br />
durante a necropsia constatava-se que esses distúrbios<br />
estavam associados a má-formação do coração<br />
ou outros órgãos (fígado e rim), sendo essa condição<br />
incompatível com a vida;<br />
Grupo 2 - bezerros que apresentavam<br />
APGAR ruim (0 a 3 pontos) sinais de acidose respiratória<br />
e mista na hemogasometria, sendo nesses animais<br />
diagnosticado asfixia neonatal precoce; alguns<br />
sobreviviam e outros morriam;<br />
Grupo 3 - bezerros que apresentavam<br />
APGAR bom (7 ou 8 pontos), mamavam colostro,<br />
permaneciam em estação e a partir de 12 a 24 h após<br />
o parto desenvolviam quadro de hipóxia que culminava<br />
com a morte entre 24 e 48 h de vida;<br />
Grupo 4 - bezerros que nasciam com APGAR<br />
bom (7 ou 8 pontos), apresentavam hemogasometria<br />
compatível com o de animal saudável e permaneciam<br />
vivos.<br />
As falhas na adaptação neonatal à vida extrauterina<br />
observada nos animais que desenvolveram<br />
quadro de hipóxia entre 12 e 24 h de vida (grupo 3)<br />
apresentam características diferentes daquelas descritas<br />
na asfixia neonatal precoce ou tardia. As altas<br />
taxas de mortalidade nos bezerros do grupo 3, bem<br />
como os distúrbios cardiopulmonares diagnosticados<br />
nesses bezerros parecem estar associadas a existência<br />
de sofrimento fetal/ hipóxia crônica durante a vida<br />
intra-uterina e não somente a sofrimento fetal durante<br />
o processo de parição e/ou a falta primaria de<br />
surfactante .<br />
IV. DISTÚRBIOS CARDIOPULMONARES NOS BEZERROS<br />
CLONADOS<br />
Recentemente, descreveu-se a ocorrência de<br />
hipertensão pulmonar persistente em bezerros<br />
clonados a partir da transferência nuclear de células<br />
somáticas adultas [9,41]. A hipertensão pulmonar é<br />
uma condição patológica observada em associação<br />
à existência de doenças cardíacas, sistêmicas, do<br />
s246
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
parênquima pulmonar ou da vasculatura pulmonar<br />
que determinam surgimento de disfunção do endotélio<br />
pulmonar expresso por vasoconstricção, inflamação<br />
e trombose [38]. Entre os diversos fatores que podem<br />
estar relacionados à ocorrência de hipertensão<br />
pulmonar devem ser, ainda, destacados a aspiração<br />
de mecônio durante o parto e os quadros de hipóxia<br />
persistentes observados em animais criados em altitudes<br />
elevadas [35].<br />
Associado a essa hipertensão pulmonar, pesquisas<br />
realizadas na Universidade de São Paulo evidenciaram,<br />
em bezerros clonados, distúrbios cardiopulmonares<br />
caracterizados por taquicardia com episódios<br />
de arritmia com bradicardia, reforço de 1ª<br />
bulha, presença ou não de sopros cardíacos na 1ª e<br />
2ª bulha associados a dispnéias, respiração rude e<br />
estertores secos, úmidos e crepitantes [4,5,34]. Em<br />
nenhum dos animais examinados foi observado sinais<br />
de insuficiência cardíaca (prova de repleção da<br />
jugular negativa, ausência pulso venoso patológico<br />
e/ou edema na entrada do peito) ou aumento da área<br />
de projeção do coração detectáveis por meio da percussão<br />
do tórax. As alterações cardiopulmonares foram<br />
confirmadas nos exames anatomopatológicos,<br />
pois encontrou-se persistência do forâmen ovale<br />
(forâmen de Botal) em 46,1%, hipertrofia excêntrica<br />
de miocárdio ventricular em 15,3%, persistência do<br />
duto arterioso em 7,6%, congestão pulmonar em 23%,<br />
enfisema pulmonar, atelectasia e edema em 100 %<br />
dos bezerros necropsiados [37].<br />
Em bezerros clonados que morreram nos primeiros<br />
dias de vida verificou-se por meio de exame<br />
ultra-sonográficos realizado nas primeiras horas de<br />
vida, aumento da espessura da parede ventricular não<br />
associada ao aumento de volume da câmara, e conclui<br />
que os bezerros apresentavam hipertrofia congênita<br />
concêntrica de miocárdio [34]. Nesses bezerros<br />
constatou-se fluxo turbulento sistólico no interior<br />
do átrio direito, indicando a existência de insuficiência<br />
da valva tricúspide, sendo essas alterações compatíveis<br />
com a ocorrência de hipertensão da artéria<br />
pulmonar. Essa hipertensão deve ser considerada<br />
como secundária a ocorrência da miocardiopatia<br />
hipertrofia concêntrica do ventrículo esquerdo [34].<br />
Após o nascimento o quadro de hipertensão seria<br />
agravado pela presença de dispnéia/ insuficiência<br />
respiratória e pela produção inadequada e/ou consumo<br />
do surfactante pulmonar. Afora isso as avaliações<br />
ultra-sonográficas dos bezerros clonados tem<br />
evidenciado, nas primeiras 24 horas, fluxo turbulento<br />
no forâmen de botal no sentido do coração direito<br />
para o coração esquerdo, indicando reversão da circulação<br />
sangüínea para padrão observado na vida<br />
fetal. Em conseqüência desse processo ocorre mistura<br />
de sangue arterial e venoso, comprometendo a<br />
capacidade de oxigenação do sangue dos bezerros<br />
clonados [5,30]. Em amostras colhidas no 3º e 4º dia<br />
de vida, observou-se valores de PO 2<br />
no sangue arterial<br />
que variaram entre 50 e 70 mmHg.<br />
No grupo de bezerros clonados que sobreviveram<br />
observou-se que o fechamento funcional do<br />
forâmen oval em bezerros nelore demorava de 7 a<br />
10 dias nos clones enquanto nos animais controle<br />
esse fechamento ocorria entre 4 e 7 dias [34]. Afora<br />
isso, foram encontradas alterações que permitem supor<br />
que o coração dos clones durante o primeiro mês<br />
de vida passa por processo de crescente sobrecarga,<br />
com aumento de débito cardíaco que determinaria<br />
na evolução clínica dos casos uma hipertrofia excêntrica<br />
do miocárdio e dilatação do átrio direito.<br />
Adaptações orgânicas as disfunções cardíacas como<br />
a dilatação do átrio direito seriam compatíveis com a<br />
existência de hipertensão da artéria pulmonar [1], sendo<br />
esta alteração no grupo dos clones vivos indicativa<br />
de aumento da pressão da artéria pulmonar. N<br />
Para o tratamento da hipóxia recomenda-se a<br />
oxigenioterapia, fornecido por meio de cateter intranasal<br />
no volume de 5 litros de O 2<br />
por minuto durante<br />
a primeira semana de vida do bezerro [5,30]. A<br />
oxigenioterapia deve ser instituída imediatamente<br />
após o nascimento. Além do benefício direto na redução<br />
dos efeitos relacionados à hipóxia como<br />
glicólise anaeróbica, aumento da lactacidemia e<br />
acidose metabólica [3], a oxigenioterapia seria importante<br />
estímulo para fechamento do duto arterioso,<br />
melhorando a condição geral do bezerro. O fechamento<br />
funcional do canal arterial no recém-nascido<br />
humano a termo ocorre com 12 a 15 h de vida, sendo<br />
o aumento da elevação da PO 2<br />
no sangue arterial<br />
considerado importante fator para a constrição do duto<br />
arterioso [27]. Após a instituição de oxigenioterapia<br />
adequada nos bezerros clonados e a sua manutenção<br />
nos primeiros sete dias de vida, a incidência de<br />
sopros cardíacos de 1ª e 2ª bulha, relacionados a persistência<br />
do duto arterioso, foram menos frequentemente<br />
por nós diagnosticados.<br />
Apesar do distúrbio cardiopulmonar do clone<br />
não ser primariamente associado deficiência de<br />
s247
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
surfactante decorrente da imaturidade pulmonar,<br />
existe a possibilidade de ocorrer consumo do<br />
surfactante ou produção inadequada durante a vida<br />
neonatal. A administração de 300 a 1000 mg<br />
surfactante por via intra-traqueal, nas primeiras horas<br />
de vida, utilizando uma agulha de 26 G determinou<br />
um significativo aumento da PO 2<br />
do sangue arterial.<br />
Seis h após a aplicação do surfactante observou-se<br />
nos animais tratados PO 2<br />
do sangue arterial<br />
igual a 127±11.06 mmHg e nos animais não tratados<br />
valores iguais a 57.2 ± 5.77 mmHg. Entretanto,<br />
não foi observado qualquer efeito da administração<br />
de surfactante nos valores da PO 2<br />
do sangue arterial<br />
quando o início do tratamento ocorreu com mais de<br />
24 h de vida [30]. Atualmente, temos preconizado<br />
que sejam aplicados doses de 400 mg de surfactante<br />
a cada 15 min na primeira hora de vida. A seguir<br />
aplica-se 400 mg de surfactante nos seguinte momentos:<br />
2, 4, 6, 12 e 24 h de vida.<br />
O aumento resistência na rede capilar pulmonar<br />
determinada pela hipertensão da artéria pulmonar<br />
[13,24] impede a expansão pulmonar adequada<br />
e estão associadas às alterações respiratórias<br />
descritas nos clones [5,37]. O uso de drogas<br />
vasoativas inibidoras das fosfodiesterases poderia<br />
apresentar efeitos benéficos. Drogas inibidoras das<br />
fosfodiesterases como o sildenafil prolongam a ação<br />
da guanosina monofosfato cíclica (cGMP), o principal<br />
mediador da vasodilatação [6]. A inibição da degradação<br />
da cGMP pode ser particularmente eficaz<br />
na vasodilatação das artérias pulmonares, sendo descrito<br />
que o sildenafil diminui a pressão da artéria<br />
pulmonar, dependendo da dose usada [42]. Em nossa<br />
experiência, o uso do sildenafil, nas primeiras 24<br />
h de vida, com aplicação da medicação por via oral<br />
a cada 8 h na dose de 20 a 25 mg aumentou a<br />
sobrevida dos bezerros clonados tratados e observou-se,<br />
por meio de exame ecocardiográfico, que<br />
após o seu uso ocorria reversão de padrão de circulação<br />
fetal em decorrência a diminuição da pressão<br />
do lado direito do coração (Birgel Junior et al., dados<br />
não publicados). Dados preliminares obtidos em<br />
bezerro Nelore prematuro que recebeu nas primeiras<br />
12 h de vida com 20 mg de sildenafil por via<br />
oral, indicam que os inibidores da fosfodiesterases<br />
determinam significativa redução dos valores da pressão<br />
média da artéria pulmonar (de 60 mmHg para<br />
40 mmHg), sendo esses efeitos notados a partir de<br />
15 min da aplicação do medicamento (Birgel Junior<br />
et al., dados não publicados).<br />
Para o tratamento das bradicardias e arritmias<br />
observadas nos bezerros clonados temos recomendado<br />
o uso de sulfato de atropina, na dose 0,1 mg /<br />
Kg peso vivo por via intra-venosa, devendo esta<br />
medicação ser a de eleição nesse tratamento, pois<br />
associou-se a ocorrência desses episódios de<br />
bradicardia e arritmia a presença de estímulos do sistema<br />
nervoso parassimpático. Bezerros clonados estão<br />
predispostos a vagotonia em virtude de dilatação<br />
do abomaso por liquido amniótico espesso e viscoso<br />
durante a vida intra-uterina ou em conseqüência da<br />
irritação de vias aéreas e da presença de<br />
broncoespamos decorrentes dos problemas<br />
cardiopulmonares (Birgel Junior et al., dados não publicados).<br />
V. MACROSSOMIA / SÍNDROME DO BEZERRO GIGANTE<br />
Desde o início do desenvolvimento da técnica<br />
de fertilização in vitro de embriões e da clonagem<br />
de bovinos e ovinos, tem sido descrita uma nova<br />
síndrome: a Síndrome do bezerro gigante [2,11]. A<br />
síndrome do neonato gigante tem sido comumente<br />
relacionada a gestações prolongadas e a falhas de<br />
sinalização do parto [9]. A ocorrência de macrossomia<br />
foi observada em 20,9 % (9/4) dos bezerros da raça<br />
Nelore, ao passo que 69,8 % (30/43) apresentavam<br />
peso dentro da normalidade e 9,3 % (4/43) apresentavam<br />
peso significativamente menor do que o esperado<br />
para a raça Nelore [30]. A média de peso dos<br />
animais da raça Nelore ao nascimento foi de 38 kg,<br />
com amplitude de variação entre 14 e 62 kg [30].<br />
Acredita-se que essa síndrome esteja associada<br />
a distúrbios do metabolismo de carboidratos na<br />
placenta e/ou do feto, mimetizando quadro de<br />
gigantismo fetal descrito na gestação de mulheres com<br />
diabetes [4,5]. Alguns pesquisadores descreveram<br />
alterações hormonais em bezerros clonados recémnascidos<br />
caracterizados por aumento dos teores<br />
plasmáticos de insulina [16] e leptina [10] e diminuição<br />
dos teores plasmáticos de tiroxina [10, 16],<br />
triiodotirosina [16] e IGF-2 [10] e interpretaram esses<br />
achados como sendo um indício de distúrbio na<br />
regulação de energia intra-útero responsável pela<br />
macrossomia e pelos distúrbios de saúde observados<br />
nos clones [16].<br />
s248
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
Em 23,0% dos bezerros clonados foi observado<br />
hipoglicemia e/ou distúrbios de termoregulação<br />
nas primeiras 24 h de vida. Esses distúrbios podem<br />
estar relacionados a síndrome que mimetiza a diabetes<br />
gestacional da mulher ou a distúrbios decorrentes<br />
da hipóxia durante a vida perinatal com consumo<br />
das reservas de glicogênio hepático [4,5].<br />
Para minimizar ou evitar a ocorrência de<br />
hipoglicemia/ hipotermia, tem-se optado por realizar<br />
o parto no período vespertino (temperatura corpórea<br />
materna entre 0,5 e 1,0ºC maior do que no período<br />
matutino devido as variações nictemerais da temperatura),<br />
secagem do bezerro utilizando-se toalhas e<br />
secadores de cabelo. Os animais tingidos de mecônio<br />
foram lavados com detergente para retirar a gordurosa<br />
crosta de mecônio que dificultava a secagem<br />
dos bezerros. Imediatamente após o parto os bezerros<br />
eram transferidos para sala na qual a temperatura<br />
ambiente era de 30°C. A mamada do colostro nas<br />
primeiras horas de vida, além da transferência de<br />
imunidade passiva, é importante para o fornecimento<br />
de energia a recém-nascido. A glicemia e temperatura<br />
corpórea foram controlados às 1, 2, 3, 4, 6, 8,<br />
12, 16, 24, 36 e 48 h de vida, sendo que nos animais<br />
nos quais a glicemia fosse menor do que 50 mg /dL<br />
aplicavam-se por via intra-venosa 0,2 g/dL de glicose<br />
por via intravenosa. Com essa dosagem os animais<br />
retornavam à normoglicemia (80 a 120 mg/dL) e<br />
evitava-se a ocorrência de quadros de hiperglicemia<br />
[4,5,30].<br />
VI. OCORRÊNCIA DE ANEMIA<br />
Apesar de encontrar-se na literatura poucas<br />
referências sobre distúrbios do hemograma em bezerros<br />
clonados a partir da transferência nuclear de<br />
células somáticas [2,36], pesquisa realizada na Universidade<br />
de São Paulo, Komninou [25] observou a<br />
ocorrência de anemia de grau moderado a intensa,<br />
do tipo normocítico e normocrômico nos clones.<br />
Evidenciou-se nessa pesquisa [25] que a anemia instalava-se<br />
gradualmente a partir das 12 h de vida atingindo<br />
ao final da primeira semana, a sua intensidade<br />
máxima. Os valores médios obtidos para o eritrograma<br />
dos bezerros no 7° dia de vida foram os seguintes:<br />
Hemácias - 4,33X10 6 /mm³; Volume Globular - 23<br />
%, Taxa de Hemoglobina - 7,25 g/dl; VCM - 52,89<br />
µ³; HCM - 16,65 pg; CHCM - 31,47%. A anemia<br />
observada era de origem ferropriva, pois evidenciouse<br />
nesses animais uma significativa diminuição dos<br />
teores séricos de ferro associada à diminuição do índice<br />
de saturação da transferrina (IST), enquanto os<br />
valores da capacidade total de ligação do ferro<br />
(CTLF) não sofreram influência durante o período<br />
[25]. Durante o estudo dos tipos de hemoglobinas,<br />
utilizando-se técnica de eletroforese foram identificados<br />
três fenótipos de Hemoblogina adulta (Hb-A;<br />
Hb-B e Hb-AB) e a presença de hemoglobina fetal<br />
(Hb-fetal), não sendo observadas anomalias que pudessem<br />
sugerir a ocorrência de hemoglobinopatias<br />
hereditárias e/ ou congênitas [25]. Durante a avaliação<br />
da dinâmica da hemoglobina do tipo fetal (Hbfetal)<br />
no primeiro mês de vida observou-se, que todos<br />
os grupos animais apresentaram comportamento<br />
similar, caracterizado por sua diminuição com o<br />
desenvolvimento etário. A recuperação gradativa dos<br />
valores eritrograma ocorreu a partir do 15°dia de vida<br />
[25].<br />
Nos casos de anemia a principal recomendação<br />
terapêutica foi o tratamento dos bezerros com<br />
sais de ferro nos primeiros dias de vida e como medida<br />
preventiva a suplementação das receptoras com<br />
sais de ferro nos últimos meses de gestação.<br />
VII. ANOMALIAS NAS ESTRUTURAS UMBILICAIS,<br />
DEFORMIDADES FLEXURAIS E ALOPECIA<br />
Anomalias umbilicais com infecção e persistência<br />
do úraco; devido ao aumento na espessura do<br />
cordão umbilical em decorrência de edema na gelatina<br />
de Warthon e alteração na estrutura dos vasos<br />
umbilicais tem sido descrita em bezerros clonados<br />
[2,30,32]. (Observou-se aumento na espessura do<br />
cordão umbilical que dificultava a sua ruptura espontânea.<br />
As artérias umbilicais não sofreram retração<br />
para a cavidade abdominal, ficando expostas no resquício<br />
do cordão umbilical e nos primeiros três dias<br />
após o nascimento notou-se no umbigo presença de<br />
forte pulsação dessas artérias, tornando necessário o<br />
uso de clamps nas artérias com o intuito de evitar<br />
hemorragias. Observaram-se, ainda, hematomas<br />
intra-abominais envolvendo o úraco e as artérias [4,5].<br />
Apesar dessas alterações, a desinfecção do cordão<br />
umbilical utilizando-se tintura de iodo a 2% mostrouse<br />
eficiente no combate de complicações, pois a ocorrência<br />
de onfalites, onfaloflebites, onfaloarterites,<br />
onfalouraquites ou persistência do úraco foi observada<br />
em menos de 5,0% dos clones [4,5,30].<br />
N<br />
s249
E.H.<br />
Bir<br />
irgel Junior<br />
unior, F.V<br />
.V. Meir<br />
eirelles<br />
elles, E.R.<br />
Komninou<br />
omninou, et al. <strong>2011</strong>. Clinical disorders observed during the first 30 days<br />
of life of cloned Zebu calf. Acta Scientiae Veterinariae. 39(Suppl 1): s243 - s252.<br />
Deformidades flexurais e articulares foram relatadas<br />
freqüentemente em bezerros clonados, podem<br />
acometer membros anteriores ou posteriores,<br />
serem unilaterais ou bilaterais e atingir tendões<br />
flexores ou extensores [15,16]. Em bezerros clonados<br />
da raça Nelore foi observado com freqUência deformidades<br />
das extremidades dos membros anteriores<br />
e posteriores, caracterizadas hiperextensão da articulação<br />
metacarpo/metatarsofalangena e lassidão<br />
(frouxidão) dos tendões flexores [5,30]. Essas alterações<br />
tiveram bom prognóstico e desapareciam gradualmente<br />
sem que qualquer tratamento fosse realizado.<br />
Entre 15 e 20 dias de vida observou-se a ocorrência<br />
de alopecia em cerca de 75,0 % dos bezerros.<br />
cuja origem pode estar relacionada com distúrbios<br />
na síntese e absorção de vitaminas, pois a<br />
suplementação dos bezerros com complexo de vitamina<br />
ADE diminuiu os sintomas [4,5,30].<br />
VIII. CONCLUSÃO<br />
Apesar dos esforços realizados nos últimos<br />
cinco anos a taxa de mortalidade pós-nascimento dos<br />
bezerros clonados é ainda grande, cerca de 50%. Demonstrou-se<br />
a ocorrência de graves distúrbios cardiopulmonares<br />
caracterizados por taquicardia,<br />
hiperfonese, presença de sopros cardíacos na 1ª e 2ª<br />
bulha, episódios de arritmia e bradicardia associados<br />
a dispnéias, respiração rude e estertores. Em conseqüência<br />
ao não fechamento do Forâmen de Botal e<br />
do Duto Arterioso há mistura de sangue arterial e venoso<br />
comprometendo a capacidade de oxigenação<br />
do sangue dos bezerros clonados. Observou-se, ainda,<br />
a ocorrência de macrossomia, hipoglicemia,<br />
hipotermia, anomalias das estruturas umbilicais, anemia<br />
e alopecia<br />
Acknowledgements. We thank FAPESP (grant 2005/<br />
04636-0; 2006/ 07141-5 2007/ 05785-8; 2009/ 08703-5)<br />
for financial support.<br />
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www.ufrgs.br/actavet<br />
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P. Humblot. <strong>2011</strong>. Reproductive Technologies and Epigenetics: their Implications for Genomic Selection in Cattle. sss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s253 - s262.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s253 - s262.<br />
ISSN 1679-9216 (Online)<br />
Repr<br />
eproduc<br />
ductiv<br />
tive Technolo<br />
echnologies and Epigenetics:<br />
their Implications for Genomic Selec<br />
election<br />
in Cattle<br />
Patrice Humblot<br />
ABSTRACT<br />
Background: The development of genomic selection allowing a better selection for multiple traits (both for production and<br />
functional traits) induces dramatic changes in the way selection schemes are to be conducted. The associated needs for genomic<br />
selection which is to produce a large number of genotyped candidates as quick as possible may/will influence the way reproductive<br />
techniques are used to produce them. As the effect of the environment is no more integrated in the evaluation of performances<br />
based on genotype, there is also a need to better understand epigenetic effects and their possible implications while implementing<br />
genomic selection.<br />
Review: Information brought by reproductive physiology through access to powerful research tools in the fields of genomics,<br />
transcriptomics, proteomics and metabolomics will provide new genetic markers and will contribute to improve the precision of<br />
phenotypes. The combination of the two types of information is susceptible to increase considerably the efficiency of selection<br />
for reproductive traits. As better reproduction may facilitate the way to run selection schemes (more choice among candidates<br />
and production of those candidates at a young age), this knowledge can be profitable also to increase the efficiency of multiple<br />
trait selection. In this context, and depending on population characteristics, the interest of the reproductive techniques including<br />
assisted embryo based reproductive technologies (Multiple Ovulation Embryo Transfer (MOET) and Ovum pick up associated<br />
to in vitro Fertilization (OPU-IVF)) should be also revisited. The efficiency of systems based on scenarios involving several<br />
reproductive techniques taken in combination should be tested. The recent results obtained with embryo typing, which are<br />
N<br />
compatible with the use of the last generation of chips for genotype analysis may lead to very promising applications for the<br />
breeding industry. The combined use of several embryo based reproductive technologies will probably be more important in the<br />
near future for selection purposes to satisfy the needs of genomic selection by increasing the number of candidates and to<br />
preserve at the same time genetic variability. Since several years, genotyping has been used more or less intensively by breeding<br />
companies to genotype males and females within nucleus herds As any farmer will get access to the genotype of the females<br />
present in their herd, an increased use of embryo based reproductive technologies may result also from the demand of individual<br />
farmers who may wish to valorize as well and as quick as possible the genetic potential of their best heifers following genotyping.<br />
In the near future, a better knowledge on epigenetics will allow to estimate the interactions between genotype and environment<br />
and their impact on performances of present or future generations. This represents a critical information when evaluating<br />
performances and when selecting future sires on genomic based information especially with the objective of implementing<br />
sustainable breeding schemes.<br />
Conclusion: The manuscript describes the new context and corresponding needs for genomic selection and how reproductive<br />
technologies and additional knowledge on epigenetics can be used to meet those needs.<br />
Keywords: genomic selection, gene expression, transcriptomics, epigenetics, phenotype, reproduction, embryo, biotechnology.<br />
Division of Reproduction, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of<br />
Agricultural Sciences, Uppsala, Sweden. CORRESPONDENCE: P. Humblot [Patrice.Humblot@slu.se]. Faculty of Veterinary Medicine and<br />
Animal Science, Swedish University of Agricultural Sciences, SLU, Po Box 7054, SE 750 07, Uppsala, Sweden.<br />
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I. INTRODUCTION<br />
II. THE NEW CONTEXT AND NEEDS FOR GENOMIC-<br />
BASED SELECTION SCHEMES<br />
III. HOW REPRODUCTIVE PHYSIOLOGY AND<br />
TECHNOLOGIES CAN HELP TO MEET THE NEEDS<br />
OF GENOMIC SELECTION AND COMMERCIAL<br />
PRODUCTION ?<br />
3.1 Reproductive physiology<br />
3.2 Use of embryo based biotechnologies<br />
3.3 Embryo Typing<br />
3.4 Other reproductive techniques<br />
IV. IMPACT OF GENOMIC SELECTION ON REPRO-<br />
DUCTIVE TECHNIQUES<br />
4.1 Genetic Schemes<br />
4.2 Commercial activity<br />
V. EPIGENETICS: BASICS AND PERSPECTIVES<br />
VI. CONCLUSIONS<br />
I. INTRODUCTION<br />
During recent decades, advancement in our<br />
knowledge of reproductive physiology and regarding<br />
improvements in embryo-based reproductive biotechnologies<br />
have facilitated the development of a rather complete<br />
“tool box” including reproductive techniques used<br />
either for commercial purposes and/or in the frame work<br />
of breeding schemes. These techniques currently have<br />
varying degrees of efficiency [52] and for most of them<br />
continuous improvements may be expected in the future.<br />
Used alone or in combination, their development is<br />
influenced in many different ways including ethics and<br />
general acceptance, consumer demand for specific<br />
products, regulatory changes and also changes related<br />
to the evolution of breeding strategies.<br />
The recent development of genomic selection<br />
has lead to dramatic changes in the way genetic selection<br />
schemes are to be conducted [7,30]. Due to the present<br />
and expected evolution in the organisation of selection<br />
strategies and associated requirements, the value of the<br />
various reproductive techniques used today for<br />
commercial purposes and in genetic schemes will be<br />
revisited. The need to better understand epigenetic effects<br />
and their possible implications while implementing genomic<br />
selection will also be discussed.<br />
II. THE NEW CONTEXT AND NEEDS FOR GENOMIC-<br />
BASED SELECTION SCHEMES<br />
In genetic selection the expression for a given<br />
trait is the phenotype that integrates the effect of genes<br />
and the effect of environmental factors. In the past the<br />
effect of the genetic component was evaluated from<br />
genealogy and by measuring performances/phenotyping<br />
of candidates or of their progeny. Today, in association<br />
with information issued from genealogy, genomic selection,<br />
while linking from previous experience, the presence of<br />
genes and/or polymorphism of those genes to<br />
performances, allows to predict the genetic value of a<br />
candidate which is revealed by the presence of pertinent<br />
markers indicative of its genotype.<br />
Marker Assisted Selection (MAS) has first been<br />
developed and was based initially on a limited number of<br />
micro satellite analyses for a few Quantitative Trait Loci<br />
(QTL). Selection was performed by combining this first<br />
generation of genomic information with conventional<br />
indexes arising from quantitative genetics. Further<br />
developments of genomic selection were made when it<br />
was shown [48] that it was possible to predict the total<br />
genetic value of animals or plants by using genome-wide<br />
dense marker maps. The progress of the knowledge of<br />
the bovine genome and of DNA analyses has made dense<br />
marker maps available in this species and the position of<br />
markers in relation to genes of interest has been refined.<br />
This allows animal breeding companies to use today sets<br />
of thousands of genetic markers to select animals<br />
[8,14,19,25,50,58].<br />
The development of genomic techniques will<br />
probably make available the use of the complete genome<br />
information for selection purposes in a few years [15].<br />
Different types of chips based on the use of Single<br />
Nucleotide Polymorphism (SNP i.e a single base<br />
difference on the DNA between individuals or groups of<br />
individuals) can be used to achieve different objectives.<br />
The bovine 50K SNP chip is today the standard tool for<br />
breeding industries in dairy cattle and all the traits<br />
previously selected through quantitative genetics can now<br />
be evaluated from genomic information [20].<br />
In parallel with those technological changes the<br />
value of the genomic information is reinforced by including<br />
more and more animals in the evaluation and selection<br />
process [8,14]. Consequently, more reliable estimates can<br />
be obtained for the desired traits while genetic variability<br />
is better preserved. Candidates will have to be produced<br />
from parents of different pedigree’s (maximum of families<br />
within a breed) and at the same time breeding should be<br />
organised in a way to maximize the variability of the next<br />
generation [9]. Due to its costs and to the fact that the<br />
genetic value of a given future sire is known with enough<br />
precision from genomic analyses, the need for progeny<br />
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testing will be considerably reduced or even<br />
removed [7].<br />
For some traits, such as those related to fertility,<br />
the precision associated with genomic indexes is or will<br />
be much better than with classical selection [4,20]. Efforts<br />
are also made to build a common reference basis in<br />
different populations to optimize the evaluation process<br />
and evaluate the changes induced by genomic selection<br />
[11]. Research is made also on computational<br />
methodologies to define the best way to analyse and use<br />
the huge quantity of information arising from genomic<br />
analyses of a very large number of animals [14].<br />
For all traits of interest, these changes highlight<br />
the importance of the phenotypic information that must<br />
be unified from large number of animals in the reference<br />
base and which becomes one of the main bottlenecks in<br />
the process.<br />
III. HOW REPRODUCTIVE PHYSIOLOGY AND<br />
TECHNOLOGIES CAN HELP TO MEET THE NEEDS OF<br />
GENOMIC SELECTION AND COMMERCIAL<br />
PRODUCTION?<br />
3.1 Reproductive physiology<br />
In an attempt to improve numerous traits by<br />
genomic selection knowledge of the relationships between<br />
genome information and phenotypic criteria is of crucial<br />
importance. Initially, microarrays were used to characterise<br />
the relationships between genotype and<br />
phenotype [1,2,16,36]. More recently, high throughput<br />
technologies for DNA sequencing and RNA<br />
analysis are now currently used to study relationships<br />
between genotype and phenotype and gene expression.<br />
With such objectives, phenotyping (animal models,<br />
precise criteria and methods) becomes the main<br />
bottleneck to achieve this goal. As a consequence,<br />
research is needed and performed to phenotype new<br />
critical traits and/or to improve the precision of the<br />
phenotypes for existing traits such as fertility and<br />
reproductive traits [10,27,31,32]. For this purpose,<br />
proteomics, lipidomics and metabolomics may be<br />
particularly appropriate to find new markers for fertility<br />
[10].<br />
3.2 Use of embryo based biotechnologies<br />
One of the most important features of the new<br />
selection procedures will be to considerably increase the<br />
number of candidates submitted to genomic selection to<br />
maximize the chances of getting interesting<br />
individuals that will be positively evaluated for a large<br />
number of traits. As mentioned before, this will allow<br />
an increase in the selection pressure for those traits.<br />
Also, it will be possible to use bulls for AI at a younger<br />
age, thereby lowering the generation interval. Finally,<br />
the use of groups of bulls with a favourable genomic<br />
index will improve the precision of indexes when<br />
compared to the use of a very limited number of older<br />
sires as was the case in the past. This may be also<br />
favourable to genetic variability if adequate and wise<br />
breeding schemes are implemented; otherwise<br />
shortening the generation interval may also lead to<br />
an increased inbreeding rate.<br />
The way to produce these large numbers of<br />
animals becomes critical. In this context, AI alone may<br />
be inadequate to generate sufficient animals in a given<br />
period of time and the efficiency of MOET and OPU-<br />
IVP looks more and more critical to produce these large<br />
numbers of animals to be genotyped.<br />
With these “intensive” embryo-based<br />
reproductive techniques it is relatively easy to increase<br />
the number of candidates by increasing the number of<br />
flushes in MOET schemes. When compared to MOET,<br />
the number of embryos produced in a given period of<br />
time can even be multiplied by 2 or 3 [46,52] by the use N<br />
of repeated OPU-IVF sessions. This method presents<br />
also advantages to preserve genetic variability. A lot of<br />
research has been done to improve in vitro culture<br />
systems. This allows most teams working with IVF to<br />
obtain overall development rates up to the blastocyst stage<br />
between 30 and 40%.<br />
The effect of a previous superovulation on<br />
fertilisation and subsequent embryonic development is<br />
still controversial [52,56]. It has been shown very<br />
clearly from most studies that there is a significant<br />
decrease in embryo production when oocytes are matured<br />
in vitro in standard medium compared to in vivo<br />
conditions [12,26,28,56]. This emphasizes the roles<br />
of the final steps of oocyte growth and maturation in<br />
subsequent embryo development which have also<br />
been illustrated by epidemiological studies showing<br />
relationships between some factors influencing these<br />
steps and embryonic mortality [32].<br />
There is probably a lot of progress that can be<br />
achieved in vivo and in vitro embryo production by<br />
optimizing the conditions under which the oocytes are<br />
growing within follicles in donor females. Handling at the<br />
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time of collection and thereafter as well as in vitro<br />
maturation are also critical steps to be optimized since<br />
dramatic metabolic changes occur very quickly after<br />
oocyte recovery [21].<br />
Despite the above mentioned limitations and<br />
potential margins for progress, the work that has been<br />
done in the past 15 years to improve oocyte collection<br />
and in vitro embryo production systems has made those<br />
systems used by the most advanced breeding companies<br />
to produce more embryos in their genetic schemes<br />
[46,59].<br />
However, i) to mis-manage the use of these<br />
techniques may lead to increase inbreeding significantly<br />
especially if bull dams are overexploited (Colleau 2010,<br />
personal communication) and ii) due to the new<br />
requirements in relation to the implementation of genomic<br />
selection (increased number of candidates), additional<br />
strong limitations exist for giving birth to a very large<br />
number of calves that would be genotyped after birth.<br />
Effectively, one of the main bottlenecks experienced by<br />
breeding organisations working in Europe with dairy cattle<br />
is the limited availability of female recipients. This is<br />
reinforced by the fact that, due to lower pregnancy rates<br />
when using cows instead of heifers as recipients, the<br />
efficiency of embryo transfer is much lower if the heifers<br />
are used mainly as donors and not as recipients [52].<br />
In addition to this, high costs will be induced by<br />
the transfer of a very large number of embryos into<br />
recipients that must be maintained pregnant until birth of<br />
progeny and the economic potential of the non selected<br />
calves will be low. When producing these candidate<br />
animals on farm, the amount of field work in relation to<br />
embryo transfer and in vitro production will be even<br />
greater than today and will generate high logistical costs.<br />
Finally, this process may increase the contractual<br />
cost with individual farmers especially due to the<br />
potential existence of very interesting candidates<br />
identified by genomics. For these reasons, genotyping<br />
the embryos and selecting them before transfer appears<br />
to be an attractive scenario to maximize the chances to<br />
finding interesting individuals for multiple traits while<br />
transferring a “reasonable” number of embryos.<br />
3.3 Embryo Typing<br />
The interest of embryo typing for breeding<br />
companies was discussed even before the emergence<br />
of the new techniques for genomic selection that includes<br />
today thousands of markers [46]. Doing typing and<br />
selection early in life was expected to be a solution to<br />
shorten the generation interval and to limit the costs<br />
s256<br />
of producing the high number of calves and of<br />
progeny testing to achieve multi character selection.<br />
Today the potential advantages of combining intensive<br />
embryo production and genotyping are even higher.<br />
Results reported initially in the literature for<br />
ruminants were based on the typing for a limited number<br />
of markers [23,29,39,40,51]. Field studies with in vivo<br />
produced, biopsied embryos either fresh or frozen have<br />
shown that pregnancy rates following the transfer on farm<br />
were compatible with field use [22,37,53] (Table 1).<br />
Initially typing was envisaged from a large<br />
number of cells issued from reconstituted embryos<br />
following cloning of blastomeres [39] (Figure 1). As<br />
preliminary studies from limited numbers of biopsies and<br />
typing have shown that the use of pre amplified DNA is<br />
possible [22] and compatible with the typing from high<br />
density markers chips [41] it may be useful to perform<br />
economic and genetic simulations to precisely evaluate<br />
the costs and advantages for the genetic schemes of such<br />
procedures based on embryo typing.<br />
3.4 Other reproductive techniques<br />
To a certain extent, sperm sexing can help to<br />
limit the number of embryos to be produced for this<br />
purpose and may be used in combination with in vitro<br />
fertilisation - in vitro production (IVF-IVP) procedures.<br />
Use of semen sexing in association with IVF-IVP may<br />
also avoid some of the present limitations of the use of<br />
semen sexing in selection schemes in relation to the high<br />
number of sperm that must be discarded and the large<br />
individual variation associated with the sexing process<br />
by flow cytometry [54].<br />
Finally, considering the need to maximize genetic<br />
variability and due to strong limitations in reproductive<br />
efficiency, cloning is unlikely, at least at present, to<br />
represent a useful tool in the frame work of selection<br />
schemes. However, besides selection schemes driven<br />
by breeding associations / companies, individual farmers<br />
that may get access to genomic selection, may be<br />
interested in the duplication of their best animals with the<br />
help of cloning for commercial purposes in countries<br />
allowing the use of this process.<br />
IV. IMPACT OF GENOMIC SELECTION ON<br />
REPRODUCTIVE TECHNIQUES<br />
4.1 Genetic Schemes<br />
Artificial Insemination (AI), Multiple Ovulation<br />
and Embryo Transfer (MOET) and Ovum Pick Up<br />
associated with in vitro Embryo Production (OPU–<br />
IVP) have been used more or less intensively to
P. Humblot. <strong>2011</strong>. Reproductive Technologies and Epigenetics: their Implications for Genomic Selection in Cattle. sss<br />
ssssssss Acta Scientiae Veterinariae. 39(Suppl 1): s253 - s262.<br />
Table 1. Pregnancy rates (day 90 post transfer) following transfer of biopsied embryos<br />
either fresh or frozen. Results from (*) and 1 were obtained after embryo sexing. Results<br />
from series (2) were obtained after biopsy and typing ffor 45 microsatellites (detection rate<br />
98%), from [22].<br />
Type Frozen Fresh<br />
Location No % Preg. (D90) No % Preg. (D90)<br />
Total farm (*)<br />
61/116<br />
52.3<br />
109/171 63.7<br />
Total station (1)<br />
46/83<br />
55.4<br />
Total station 28/54 52<br />
Total 135/253 53 109/171 63.7<br />
N<br />
Figure 1. Typing the embryo either from cloning blastomeres issued from biopsies (1) or following<br />
preamplification of DNA issued from few cells [22, 41].<br />
generate the future sires following selection through<br />
highly effective and very costly progeny testing<br />
programmes. The changes in breeding strategies and<br />
use of reproductive techniques associated with the<br />
needs of genomic selection will make the use and<br />
efficiency of embryo transfer, OPU and IVF very<br />
critical and these techniques will be probably more<br />
used than in the past to increase the number of candidates<br />
[24,47].<br />
On top of this, the potential value of the genotyped<br />
animals will probably lead breeding associations/<br />
companies to adopt strategies allowing them to control<br />
the production of genome-selected animals through<br />
use of embryo-based reproductive techniques (MOET<br />
and IVP) in nucleus herds to give birth to previously<br />
(pre) selected animals within a given structure /<br />
company and not on farm.<br />
4.2 Commercial activity<br />
As soon as genotyping will be extended,<br />
farmers will have access to the corresponding<br />
information in females. This will probably induce a<br />
strong rise in the demand for ET and even OPU and<br />
IVF from farmers wishing to optimize the value of<br />
their best females within their herds and/or for<br />
commercial purposes.<br />
V. EPIGENETICS: BASICS AND PERSPECTIVES<br />
Epigenetic changes can be defined as stable<br />
alterations of DNA associated molecules but that do<br />
not involve changes of the DNA sequence itself. As a<br />
consequence to those changes, the expression of<br />
genes can be altered due to modifications of active<br />
regulatory sequences inducing alterations of the<br />
cellular phenotype which can lead in turn to<br />
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modifications of animal phenotype. Epigenetic<br />
alterations involve changes in DNA methylation<br />
(including global hypomethylation and more locus<br />
specific hypermethylation) and methylation or<br />
acetylation of histones [34,49] (Figure 2).<br />
Epigenetic alterations happen in all cells in the<br />
body. If they occur in egg cells or sperm they can be<br />
passed on to the next generation. The epigenome is<br />
especially vulnerable during embryogenesis, fetal and<br />
neonatal life and at puberty [3,18,33]. Effectively,<br />
the epigenome undergoes extensive reprogramming<br />
when the gametes (eggs and sperms) are formed at<br />
fertilization (during the final stages of meiosis and<br />
around fertilization due to chromosomal<br />
decondensation and intense remodelling) and during<br />
the preimplantation period. These epigenetic changes<br />
are necessary for normal embryonic development and<br />
survival [33]. The modification of epigenetic marks<br />
that occurs during pre implantation development<br />
correlates with the activation of the embryonic<br />
genome [38,45].<br />
After fertilization, different patterns of<br />
methylation exist for some epigenetic markers<br />
followed by more or less early demethylation which<br />
allows transcriptional activity of the embryonic<br />
genome [6]. Later, at the morula stage, DNA<br />
Figure 2. The 2 major components of the epigenetic code [34, 49]. 1) Methylation of histones influences<br />
chromatin structure and nucleosomal remodelling and 2) Methylation of DNA bases. Changes in methylation<br />
are associated with chromosomal instabilities due to repression of repair DNA genes in the case of<br />
hypermethylation whereas hypomethylation leads generally to up regulation and over expression of some<br />
genes. Histone modifications leads to either a more condensed state of chromatin structure associated with<br />
transcriptional repression (methylation) or to a more relaxed state associated with transcriptional activation<br />
(acetylation). These different modifications are closely interconnected as the methylation status influences<br />
histone status and some regulators of nucleosome remodelling controls also methylation and histone<br />
modifications.<br />
methylation at specific loci influences differential<br />
gene expression patterns of the cells at the periphery<br />
of the embryo [57], whereas cells at the center of the<br />
embryo do not receive the same environmental<br />
influence and instead conserve their totipotency.<br />
Reproductive technologies, such as in vitro<br />
fertilization, cloning by nuclear transfer in domestic<br />
animals and assisted reproduction technologies<br />
(ARTs) in humans, are used during stages of<br />
fertilization and early embryo development, when a<br />
potential window of vulnerability exists. These<br />
techniques have been used as experimental models<br />
for the role of epigenetic effects on embryonic<br />
development and a large number of studies<br />
demonstrate the impact of ART on gene expression<br />
in the mouse [13,17,35,42,43,55]. In the mouse and<br />
cow, epigenetic modifications induced by ART were<br />
associated with impaired early embryonic survival but<br />
also with deleterious effects on further postimplantation,<br />
fetal, placental and postnatal<br />
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development [5,17,44]. In those species, as well as<br />
in humans, some of the alterations may be due to<br />
changes related to the epigenetic regulation of<br />
endometrial function [49].<br />
In brief, epigenetic effects can influence i)<br />
reproductive efficiency (through alterations in the viability<br />
of embryos, foetus and new born and control of<br />
endometrial gene expression which may alter<br />
implantation) ii) health (especially occurrence of cancer<br />
through regulation of proto oncogenes and suppression<br />
of tumor suppressor genes) and iii) phenotypic<br />
performance for a variety of functions/traits. Some<br />
epigenetic modifications can be even transmitted to next<br />
generations leading to remanent alterations of the<br />
phenotype within families.<br />
Numerous factors can induce epigenetic effects<br />
and for instance evidence has been shown for the role<br />
of i) nutrition (either overnutrition increasing the rates of<br />
diabetes and obesity or undernutrition) as nutritional<br />
challenges to the established germ cells can determine<br />
the chromatin structure leading to metabolic responses<br />
throughout life in an individual, ii) endocrine disruptors<br />
(which can induce durable changes in receptor sensitivity<br />
to steroids (androgens and oestrogens) that may be<br />
involved in cancer occurrence) and iii) for various<br />
pollutants in inducing such effects.<br />
This information shows that epigenetic regulation<br />
of gene function is a key factor to understand the<br />
interactions between environment and genome function.<br />
This has clear implications in selection especially today<br />
when using genomics based on DNA sequence<br />
characteristics to predict future performance instead of<br />
observed phenotypic performances of offspring through<br />
progeny testing which were integrating potential epigenetic<br />
effects in the evaluation.<br />
Considering the information given above,<br />
when using Assisted Reproductive Technologies, a<br />
better knowledge of epigenetic effects will help also<br />
to better define culture conditions for oocytes and<br />
embryos that will not impair subsequent embryo,<br />
foetus, new born development and health of<br />
offsprings. More generally this knowledge may help<br />
to define preventive measures which may be<br />
favourable to fertility and health for a variety of species<br />
including man.<br />
VI. CONCLUSIONS<br />
In the new context of genomic selection, there<br />
is still a lot of work for the reproductive physiologist<br />
to study gene expression and identify markers and<br />
networks of genes associated with fertility. As far as<br />
selection for fertility is concerned, more precise<br />
phenotyping is needed for particular reproductive<br />
events and more especially for precocity of<br />
reproductive traits that has not been well<br />
characterized so far.<br />
More generally, for all production traits and<br />
functional traits, in the present context showing very<br />
impressive improvements induced by the intensive use<br />
of MAS, it is likely that the use of a set of intensive<br />
reproductive techniques together with embryo typing will<br />
bring very significant advantages to breeding organisations<br />
capable of monitoring all those techniques with efficiency<br />
and to implement them in selection schemes. However,<br />
strategies must be developed to use all these techniques<br />
in such a way that they contribute to maintain genetic<br />
variability.<br />
There will probably also be some changes in<br />
relation to commercial activity due to valuable genomic N<br />
information becoming available in females that may lead<br />
individual farmers/companies to make a larger use of<br />
semen sexing and embryo related technologies.<br />
Knowledge about epigenetic effects on various<br />
functions will help in many ways to better integrate<br />
environmental effects in the evaluation system when using<br />
genomic selection in farm animal species and more<br />
generally to define preventive measures to optimize<br />
reproductive efficiency and health. To achieve this last<br />
objective, a comparative approach involving many species<br />
as different models to investigate such epigenetic effects<br />
within different tissues (which are more or less accessible<br />
depending on species), will be probably profitable.<br />
Acknowledgements. Some of the results presented have<br />
been obtained through the programme “TYPAGENAE”<br />
conducted with the financial support of ANR/FRT (French<br />
call GENANIMAL) and Apis-Genes.<br />
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www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl1): s263 - s272.<br />
ISSN 1679-9216 (Online)<br />
Application of gene expression profiling to the study of placental and fetal<br />
function<br />
Jorge A. Piedrahita<br />
ABSTRACT<br />
Background: One of the key biological principles that differentiate mammals from other phyla is the presence of the placenta.<br />
This is a unique and complex tissue that plays an essential role in the development of the fetus and has both short terms effects<br />
in terms of reproductive efficiency, and long term effects in terms of adult onset diseases exacerbated by an inadequate fetal<br />
environment. Yet in spite of decades of intense study there is still a large number of unanswered questions regarding the<br />
function of this organ. This is true for both humans and domestic animals species.<br />
Review: In the past, studies focused on placental function and placental/fetal interactions have examined at most a few genes/<br />
proteins at a time; what is commonly referred to as the candidate gene approach. While this approach has been quite successful,<br />
and has helped develop the knowledge base that we have at present, it is also limiting in scope. New genomic technologies<br />
allow simultaneous analysis of most, if not all, of the genes expressed in placental and fetal tissues. However these technologies<br />
also present significant challenges due to the massive amount of information that is generated. This entails not just how to<br />
handle the generated data, but also how to properly analyze it and interpret it. Fortunately, a large number of computer<br />
algorithms have been, and continued to be, developed that permit not just the identification of which genes are disregulated<br />
in the system being studied, but perhaps more importantly, permits the identification of which biological pathways are<br />
affected. This in effect convert a list of genes that in most cases defy interpretation, into a more biologically-oriented set of<br />
N<br />
results that provide a better understanding of the system. This knowledge can then be used to design direct biochemical and<br />
physiological experiments that, in essence, allow the investigator to move from the gene level to the system level. This review<br />
will cover some of the fundamental aspects of gene expression profile data capture, and the different approaches that are used<br />
to analyze the data generated, including brief descriptions of some of the most commonly used web-based gene expression<br />
profiling analysis programs. In addition, methods for searching and downloading dataset of interest that can help complement<br />
your own data will be reviewed. In particular, focus will be placed on how this technology can be utilized to study placental<br />
and fetal growth abnormalities in humans, with an emphasis on fetal growth restriction and preeclampsia. Additionally, similar<br />
approaches will be described that helped to elucidate the conservation and possible function of imprinted genes in swine.<br />
Conclusion: This manuscript describes methods for capturing and analyzing gene expression profiles with an emphasis of<br />
how this technology was applied to the study of placental function in humans and swine. In addition, it provides a description<br />
of web-based systems that can be used to analyze data generated by your own studies as well as method for searching and<br />
downloading data generated by others.<br />
Keywords: Epigenetics, placenta, transcriptome, imprinting.<br />
Center for Comparative Medicine and Translational Research and Department of Molecular Biomedical Sciences, College of Veterinary<br />
Medicine, North Carolina State University, Raleigh, NC, U.S.A. CORRESPONDENCE: J.A. Piedrahita [jorge_piedrahita@ncsu.edu]. Department<br />
of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., ZIP CODE:<br />
27606, Raleigh, NC, U.S.A.<br />
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I. INTRODUCTION<br />
II. GENE EXPRESSION PROFILES; HOW ARE THE<br />
DATA GENERATED?<br />
III. HOW TO ANALYZE GENE EXPRESSION<br />
PROFILING/MICROARAY DATA<br />
IV. APPLICATION OF FUNCTIONAL GENOMIC<br />
APPROACHES TO THE STUDY OF PLACENTAL<br />
FUNCTION<br />
4.1 Application to human placental disorders; preeclampsia<br />
4.2 Application to domestic species; pig placenta<br />
V. CONCLUSION<br />
I. INTRODUCTION<br />
“Functional genomics” can be defined as the<br />
application of genomic technologies to the<br />
understanding of biological function both at the<br />
cellular and organismal level. It can cover areas such<br />
as the application of gene expression profiling as a<br />
tool to understand biological function, and the<br />
generation of genetically modified animals as a way<br />
of understanding the function of a gene in the context<br />
of the whole animal. For the purpose of this review<br />
functional genetics refers to the application of gene<br />
expression profiling to enhance our knowledge of<br />
the function of the placenta in both humans and<br />
mammals. In the most basic sense there are three areas<br />
that are key to this technology: a) how to collect the<br />
data; b) how to analyze the data and; c) how to apply<br />
the results generated.<br />
II. GENE EXPRESSION PROFILES; HOW ARE THE DATA<br />
GENERATED<br />
Scientists have been doing gene profiling for<br />
decades by low output technologies such as<br />
Northern’s and RT-PCR (Reverse transcriptionpolymerase<br />
chain reaction). These techniques can be<br />
highly accurate but are limiting in scope as only a<br />
few genes can be analyzed simultaneously. As a result,<br />
most scientists relied on developing a hypothesis,<br />
selecting a few candidate genes thought to be<br />
involved in the process being studied, and generating<br />
the required information about those few genes; what<br />
is commonly referred to as the candidate gene<br />
approach. The candidate gene approach is still in use<br />
today and while it remains highly valid, and was the<br />
basis for most of the knowledge on gene expression<br />
generated to date, it is limiting in scope. In the last<br />
fifteen years a new alternative was developed. This<br />
approach has been referred to as the RNA microarray<br />
system, gene expression profiling, or transcriptome<br />
analysis. The initial microarray versions consisted of<br />
partial or complete sDNA to the gene of interest being<br />
“printed” into small glass slides. The capacity of these<br />
initial slides were in the hundreds of genes and most,<br />
if not all, were custom made by different groups interested<br />
in addressing specific systems - see [58] for<br />
comprehensive review in this area. While useful, this<br />
approach was limiting due to the time and effort<br />
required to build and test these arrays, as well as by<br />
technical issues related to early glass slide printing<br />
methods. A significant advance in this technology<br />
was the development of the oligonucleaotide-based<br />
arrays. Now instead of whole or partial cDNAs, genes<br />
could be measured by using carefully designed<br />
oligonucleotides ranging is size from 21 to<br />
approximately 60 base pairs [58]. The lower costs of<br />
oligonucleotide synthesis, combined with the completion<br />
of a wide range of genomic sequencing<br />
projects, including humans, pigs, horses, cattle, and<br />
dogs to name a few, allowed the development of<br />
species-specific commercial microarrays targeting<br />
most if not all of the transcripts being made by a particular<br />
species. In addition, printing variability issues<br />
were reduced by either avoiding the use of the glass<br />
slide completely or by printing the oligonucleotides<br />
directly into a solid matrix. Many groups including<br />
us have examined the utility of these commercial<br />
arrays. My group, specifically compared the utility<br />
of glass arrays versus commercial oligonucleotide<br />
arrays (Affymetrix) to determine which system was<br />
better adapted to address questions related to gene<br />
expression changes in swine [53]. While both<br />
methods were capable of detecting changes between<br />
two samples tissues, the commercial platform was<br />
found to be considerably better at detecting significant<br />
differentially expressed genes. In a side by side<br />
comparison using the same test cDNA samples, glass<br />
spotted arrays representing 12,000 genes were able<br />
to identify 3 differentially expressed genes while<br />
commercial microarrays, representing approximately<br />
24,000 genes, identified 210 differentially expressed<br />
genes [53]. Since then, arrays continue to increase in<br />
coverage and in technical reproducibility further<br />
increasing their usefulness. However, while still the<br />
main option available for many laboratories<br />
worldwide there is new technology that has an even<br />
greater capacity for capturing transcriptome data than<br />
the microarrays. This is the so called deep sequencing,<br />
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massive parallel sequencing, next generation<br />
sequencing, or whole genome sequencing technology<br />
[36]. This technology can generate sequence over<br />
55 billion (55 Gb) bases in a single day [62]. To put<br />
that number in perspective, the complete human<br />
genome is 3.4 billion bp, thus, in a single day this<br />
technology has the potential to sequence with a 10-<br />
15 fold coverage the genome of a single individual;<br />
A task that required several years and hundreds of<br />
millions of dollars just a few years ago. In essence,<br />
this new sequencing technology allows one to capture<br />
and sequence every transcript that is being made<br />
in a cell. It is highly accurate and quantitative, as it<br />
will calculate the relative proportion of each transcript<br />
within your population. It measures both mRNA and<br />
microRNAs and will examine all transcripts, even those<br />
that have not previously been described. Thus, in<br />
terms of data collection, this approach is<br />
comprehensive and unbiased and thus has many<br />
advantages over oligonucleotide-based arrays, even<br />
those arrays designed to cover the complete<br />
transcriptome. At present the application of this new<br />
approach is limited due to costs, which at presents<br />
can be 2-5x greater than microarrays (on a per-sample<br />
costs basis), and equally important by the computing<br />
and bioinformatic resources required to compile and<br />
analyze the massive amounts of data that are<br />
generated. Moreover, while implementation of deep<br />
sequencing to commonly studied species such as<br />
humans and mice is relatively straightforward due to<br />
the high quality of the whole genome sequencing<br />
information available, the same cannot be stated for<br />
species such as swine. While some groups have been<br />
able to implement this technology in swine<br />
successfully [1], it is not a project that most<br />
laboratories can manage on their own. Until cost of<br />
generating the data are reduced, and methods for rapid<br />
extraction of the expression data from the massive<br />
amounts of sequence information are developed, the<br />
broad applicability of this new approach will remain<br />
limited in scope. However, it is likely that this method<br />
will eventually become the method of choice for most,<br />
if not all, investigators interested in gene expression<br />
profiling.<br />
Finally, an often overlooked source for data<br />
collection is data repositories. At present most journals<br />
require that the original raw data used to generate the<br />
results included in any manuscript has to be deposited<br />
in free-access databases such as the Gene Expression<br />
Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/)<br />
[4]. In May <strong>2011</strong>, GEO contained information on<br />
2,800 experiments covering over 563,000 samples.<br />
In domestic species, GEO had information from 158<br />
pig projects representing over 3332 samples, 169<br />
cattle projects representing over 3309 samples, 29<br />
sheep projects representing 444 samples, and 9 goat<br />
projects representing 118 samples. When examining<br />
data from GEO it is important, however, to determine<br />
whether the data was generated using cross-species<br />
hybridization approaches. That is, in species such as<br />
goats where no goat-specific arrays are available<br />
investigators have used cattle specific arrays. While<br />
cross-species hybridization approaches can generate<br />
some valuable information it has drawbacks that need<br />
to be taken into consideration [52]. Even with this<br />
caveat, GEO and similar databases are a highly<br />
underutilized resource that could be of tremendous<br />
use to investigators and students to generate unique<br />
hypotheses to be tested and to become familiar with<br />
data analysis programs before generating their own<br />
datasets.<br />
III. HOW TO ANALYZE GENE EXPRESSION PROFILING/<br />
MICROARAY DATA<br />
N<br />
This section is not intended as a complete<br />
review of this field that combines complex statistical<br />
approaches with computing problems related to the<br />
large amounts of data that need to be processed. There<br />
is in addition to issues of quality control and data<br />
normalization that are beyond the scope of this review.<br />
And lastly, there are dozens if not hundreds of<br />
programs available for transcriptome analysis some<br />
with significant overlaps and others with highly<br />
specialized goals. The Gene Ontology consortium<br />
alone has over fifty different optional programs listed<br />
in their website (http://www.geneontology.org/<br />
GO.tools.microarray.shtml). What is described here<br />
is a single method that illustrates how one can go<br />
from a large microarray dataset to a more defined<br />
and biologically relevant set of results- See [43] for a<br />
more detailed review of this topic.<br />
In general, when comparing two or more<br />
treatments, the following broad questions are asked:<br />
1. Which genes are differentially expressed<br />
between the different treatments? This is a simple gene<br />
list that should contain fold change as well as si-<br />
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gnificance (p values). It is usually generated by either<br />
commercially available software or by using free webbased<br />
statistical programs.<br />
2. What does the data collectively tell you<br />
about what biological pathways or processes are<br />
affected by the treatment(s)? This area, usually<br />
referred to as functional analysis, is where gene lists<br />
are converted into more biologically relevant<br />
information that can lead you to identify the<br />
biologically-relevant process that is (are) affected.<br />
Without this type of analysis, moving forward from a<br />
gene list it fraught with difficulties as, in some cases,<br />
several hundred gene are found to be statistically<br />
different.<br />
So what exactly is functional analysis In essence,<br />
investigators have compiled lists of genes that<br />
are involved in different biologically processes, or<br />
that work in certain cellular compartments. For<br />
instance, there is a curated list of genes that are known<br />
to be involved in apoptosis. What functional analysis<br />
does, is compare your microarray data to that curated<br />
list of apoptosis-related genes and ask whether there<br />
is any evidence that the genes that are affected by<br />
your treatment are enriched for genes belonging to<br />
the apoptosis group. It does this through statistical<br />
methods and gives you a probability associated with<br />
the likelihood that your data indicates that genes in<br />
the apoptosis pathway are being affected. The end<br />
result being that you have just gone from a gene list<br />
to a biological function that can be tested experimentally<br />
in vitro or in vivo.<br />
Thus, the value of this analysis is that it moves<br />
from single genes to a biological function that<br />
can be tested in vitro or in vivo depending on your<br />
system. And as the field matures, more and more of<br />
these groupings or categories of genes are available,<br />
thus increasing the value and usefulness of the<br />
information generated. One caveat for those working<br />
in domestic species is that the groupings or categories<br />
are developed using data generated in species such<br />
as Drosophila, Saccharomyces, and mice. While this<br />
is not likely to affect results in a highly conserved<br />
category such as apoptosis, it could have significant<br />
drawbacks if the phenotype of interested is more<br />
species-specific.<br />
One of the better known system used to<br />
categorize gene function is known as the Gene Ontology<br />
or GO (http://www.geneontology.org) [2].<br />
This system assigns each gene properties at three<br />
levels; cellular compartment (i.e. nucleus, membrane,<br />
mitochondria); molecular function (i.e. kinase, transcription<br />
factor, phosphatase); and biological function<br />
(i.e. apoptosis, signal transduction, cholesterol<br />
transport). This system is hierarchical in structure and<br />
you can go from a very broad to a highly specific<br />
category. A more comprehensive system that<br />
encompasses GO but also adds additional gene<br />
datasets is the Molecular Signatures Database or<br />
MSigDB (http://www.broadinstitute.org/gsea/msigdb/<br />
collections.jsp#C2) [45]. This database contains close<br />
to 7,000 annotated gene sets divided onto five major<br />
collections; each collection targeted to a particular<br />
goal. The different collections are:<br />
1) C1 or positional gene set which can be used<br />
to determine if there are certain chromosomal<br />
locations that are affected into the system under study.<br />
In other words, does the data suggest that a particular<br />
chromosomal region is showing either a hotspot of<br />
gene activation, or conversely, gene down-regulation?<br />
2) C2 a collection of gene sets compiled from<br />
online pathway databases such as Biocarta and<br />
KEGG, published literature, and experts in the field.<br />
In compares the experimental data to these known<br />
groups and determine whether there is a probability<br />
that the data is enriched for genes in one of these<br />
groupings. This collection can tell whether the dataset<br />
suggests that a particular biochemical pathway is<br />
affected (i.e. cholesterol biosynthesis) or whether the<br />
gene expression profile it is similar to that found in<br />
specific systems such as in cancer or stem cells.<br />
3) C3 a motif gene collection that can help<br />
identify microRNA and transcription factors that are<br />
involved in the system under study. It analyses the<br />
data and determines whether the genes that are<br />
affected in the system are enriched for genes<br />
containing certain transcription factor binding sites;<br />
suggesting that that transcription factors are coregulating<br />
all those genes. Similarly, it determines<br />
whether genes affected in the dataset are enriched<br />
for targets of one of more microRNA suggesting that<br />
those microRNAs may be behind the effect being<br />
seen.<br />
4) C4 is a cancer related collection that groups<br />
known cancer-related molecular signatures and compares<br />
the data to those signatures to see if there are<br />
commonalities.<br />
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5) C5 is the Gene Ontology (GO) compilation<br />
that can be used to examine the data for enrichment<br />
into any of the tree GO categories mentioned above;<br />
cellular location, molecular function, and biological<br />
function.<br />
The MsigDB forms the backbone of a webbased<br />
analysis program known as Gene Set<br />
Enrichment Analysis (GSEA). GSEA will not give a<br />
list of genes per se but will give processes, pathways,<br />
and a host of additional information about the data. It<br />
is an excellent way to mine the data for biologically<br />
relevant information and is very comprehensive as<br />
described above. A detailed set of instructions on how<br />
to use this program can be found at http://<br />
www.broadinstitute.org/gsea/index.jsp. As mentioned<br />
previously, this is not intended as a comprehensive<br />
review of this field but as a brief description of the<br />
type of analysis that lead to the information that will<br />
be presented in the following section.<br />
IV. APPLICATION OF FUNCTIONAL GENOMIC<br />
APPROACHES TO THE STUDY OF PLACENTAL<br />
FUNCTION<br />
One of the most fascinating and poorly<br />
understood organs in the mammalian system is the<br />
placenta. It is critical for normal fetal development<br />
and plays a crucial role in the flow of nutrients from<br />
the mother to the fetus. In humans, it plays a central<br />
role in diseases such as preeclampsia and in domestic<br />
species it has been postulated to affect litter size in<br />
species such as swine. While the candidate gene<br />
approach has been useful for elucidating some of the<br />
basic mechanisms behind these phenomena there are<br />
still may questions remaining to be answered. As a<br />
result, we applied a functional genomics approach to<br />
this question in order to determine of this approach<br />
was useful in helping unravel the complexity of each<br />
of these two phenotypes.<br />
4.1 Application to human placental disorders;<br />
preeclampsia<br />
Up to 8% of human pregnancies results in<br />
maternal high blood pressure [38] most due to<br />
preeclampsia. Preeclampsia is a pregnancy-associated<br />
disorder triggered by placental dysfunction and<br />
characterized by maternal hypertension and protein<br />
in the urine [37]. It also increases the incidence of<br />
preterm labor and about 15% of preterm births are<br />
due to preeclampsia [15]. Risk factors for preeclampsia<br />
include: first pregnancy, family history of<br />
preeclampsia, diabetes, and multiple pregnancies [32].<br />
redmanPreeclampsia is usually separated into two<br />
distinct stages: 1) An initial stage characterized by<br />
abnormalities in placentation due to inadequate uterine<br />
remodeling and placental invasion and 2) a later stage<br />
where the maternal symptoms such as high blood<br />
pressure, associated with factors secreted by the<br />
affected placenta [35], are observed. Some of these<br />
factors include Activin-A and Inhibin A (INHA) [30,<br />
21], Leptin (LEP) [28], soluble Endoglin (sENG) [55],<br />
soluble fms-like tyrosine kinase-1 (sFlt-1) [26], and<br />
placental growth factor (PGF) [51].<br />
While the pathophysiology of the placenta<br />
and its downstream effects are well understood, it<br />
has been more difficult to identify potential triggers<br />
that lead to the defective placenta. Recently, we utilized<br />
a microarray-based approach and compared<br />
gene expression profiles of preeclamptic and normal<br />
term human placentas [52]. The data was analyzed<br />
for differentially expressed gene as well as pathways<br />
affected. Our data confirmed overexpression of the<br />
ENG, FLT1, and INHA as had been previously<br />
reported. In addition, pathway analysis identified<br />
several immune-regulated pathways as being affected<br />
including Fc Receptor mediated phagocytocis in N<br />
macrophages and monocytes, CXCR4 signaling, and<br />
leukocyte extravasation signaling. CXCR4 is a<br />
chemokine receptor specific for stromal-derived<br />
factor-1 (SDF-1) and ubiquitin and has been implicated<br />
in inflammation as well as the process of<br />
implantation [39,23,40,13]. Thus, the pathway<br />
analysis was supportive of a role for immune system<br />
involvement in preeclampsia. This has been<br />
previously reported [13] but the cause of the immune<br />
activation was not clear. What our results suggest is<br />
that the sialic acid pathways, a pathway central to<br />
self-recognition by the immune system [48] is<br />
disregulated in preeclamptic placentas. This allowed<br />
us to propose a model whereby preeclampsia is<br />
initiated by an abnormal sialic acid pathway resulting<br />
in a form of autoimmune disease where the placenta<br />
is not recognized as self. This results in abnormal<br />
branching and shallow trophoblast invasion with the<br />
end result being preeclampsia. While there is still<br />
considerable work to be done to fully understand the<br />
disease, the gene profiling approach allowed us to<br />
uncover the previously unknown role of sialic acid<br />
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in the process of autoimmune-related placentas<br />
abnormalities. It is highly unlikely that we could have<br />
reached this point, this rapidly, using a candidate gene<br />
approach, as there are hundreds if not thousands of<br />
molecules that could play a role in immune responses.<br />
Thus, the combination of the large amount of data<br />
generated and the ability to examine that data in<br />
multiple ways led us to make a crucial observation<br />
regarding this disorder.<br />
4.2 Application to domestic species; pig placenta.<br />
As mentioned earlier, there are commercial<br />
arrays available for use in species such as swine, cattle,<br />
and chicken. Additionally, with the completion of the<br />
genomic sequencing projects for these species it is<br />
now also possible to utilize the deep sequencing<br />
technology. In cattle, gene expression profiling has<br />
been utilized as a method for increasing our understanding<br />
of early embryonic development under a<br />
variety of experimental system [48, 8], milk production<br />
[7], oocyte quality [24], adipose tissue in<br />
dairy cattle during lactation [46], spermatozoa [9],<br />
IVF embryos [14], endometrial changes during the<br />
estrus cycle [10], muscle marbling [17], and meat<br />
tenderness [61], among others. In swine, this<br />
approach has been used to examine muscle growth,<br />
and regulation of muscle growth [50,34,22,49]<br />
regulation of the immune system and response to<br />
infections [54,16,12,11], factors controlling ovulation<br />
[56], and gene expression in embryos [59] and placentas<br />
[44, 6].<br />
We have utilized this technology as a way of<br />
understanding the family of imprinted genes in swine<br />
[5]. The imprinted gene family is particularly relevant<br />
as it play a major role in placental and fetal<br />
development and function. While 99% of genes in<br />
mammalian species are expressed from both the paternal<br />
and maternal allele (biallelic expression),<br />
imprinted genes are mono-allelically expressed. That<br />
is, either the maternal or paternal allele is expressed,<br />
but not both. This results in a form of dosage<br />
compensation and is controlled by epigenetic<br />
modifications including DNA methylation and histone<br />
modifications [19, 18].<br />
Of particular interest to my laboratory is the<br />
concept of parental conflict put forward by Moore<br />
and Haig [29]. This hypothesis states that imprinted<br />
genes expressed from the maternal allele evolved as<br />
a mechanism of resource conservation for the mother<br />
(at the expense of the fetus), while imprinted genes<br />
expressed from the paternal alleles increase the<br />
extraction of resources from the mother (favoring the<br />
fetus). In a normal pregnancy these two competing<br />
forces balance each other resulting in normal fetal<br />
growth. However, if the system is unbalanced it can<br />
result in either intra uterine growth restriction or large<br />
for gestational age fetuses (too small or too large).<br />
One experimental approach to study imprinted genes<br />
and their role in fetal and placental development is<br />
the use of uniparental embryos. Uniparental embryos<br />
are composed only of maternal DNA (two haploid<br />
maternal genomes; known as gynogenotes or<br />
parthenotes) or of paternal DNA (two haploid paternal<br />
genomes; known as androgenotes) [25, 47]. Litters<br />
from gynogenotes or parthenotes yield intrauterine<br />
growth-restricted (IUGR) conceptuses, and a small<br />
hypo-vascular placenta [25, 47]. Androgenotes, in<br />
contrast, develop into a very large abnormal placenta<br />
but lack a fetus proper [27]. Both phenotypes<br />
support the main idea behind the parental conflict<br />
hypothesis.<br />
Further evidence for the conflict theory<br />
between maternal and paternally imprinted genes<br />
comes from transgenic mouse studies. In the mouse,<br />
the insulin-like growth factor Igf2 is paternally<br />
expressed and increases placental and fetal weights<br />
as well as nutrient flow, while its receptor is expressed<br />
maternally and sequesters the function of Igf2 by<br />
binding and, subsequently, trafficking to the lysosome<br />
[3]. Also, inactivation of Peg10 or Peg3, two paternally<br />
expressed genes, result in smaller fetuses and placentas<br />
[33, 20]. Thus, the evidence for the opposing<br />
roles of maternally and paternally imprinted genes is<br />
strong.<br />
Unfortunately, while there are a limited<br />
number of imprinted genes with known functions in<br />
placental development in mice and humans, there<br />
are a limited number of studies of their role in domestic<br />
species (see [60]for a more comprehensive review in<br />
this area). In order the gain a greater understanding<br />
of this gene family in an agriculturally relevant<br />
species we utilized a microarray based approach,<br />
combined with uniparental pregnancies, to a) determine<br />
the degree of conservation of imprinted genes<br />
among different mammalian species and b) to explore<br />
the feasibility of using this approach to understand<br />
the physiological role of imprinted genes.<br />
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To accomplish this we developed porcine parthenotes.<br />
In pigs, the parthenogenetic fetus develops as a small<br />
fetus with a small placenta and dies at approximately<br />
day 32 of gestation. Parthenogenetic and control<br />
fetuses were allowed to develop to days 28-30 and<br />
samples collected for microarray analysis. Data was<br />
analyzed to indentify individually affected genes as<br />
well as biological processes/pathways affected. The<br />
expectation was that uniparental embryos containing<br />
2 doses of maternal DNA and no paternal DNA would<br />
have a double dose of maternally expressed imprinted<br />
genes and lack expression of paternally expressed<br />
genes. Comparison of this profile with the control<br />
fetuses carrying one dose of each, the maternally and<br />
the paternally imprinted genes, would allow the<br />
identification of which genes were imprinted in swine.<br />
In mice, this approach had been used successfully<br />
by others [42, 31]. As expected, we were able to<br />
identify a large number of imprinted genes using this<br />
approach, and found a high conservation of<br />
imprinting with the mouse and humans [5]. Equally<br />
important we were able to identify the following<br />
pathways affected in the parthenotes 1) phosphatidylinositol<br />
binding, 2) microtubule associated<br />
complex, 3) lipid transporter activity, 4) prothoracicotrophic<br />
hormone activity, 5) transmembrane<br />
receptor protein serine/threonine kinase signaling<br />
pathway and 6) double-stranded DNA binding.<br />
Overall, there were differences in proliferation,<br />
biogenesis and biosynthesis pathways. This is consistent<br />
with the role of imprinted genes as regulators<br />
of energy/nutrient flow between the mother and the<br />
fetus.<br />
Additionally, using GSEA, we were able to<br />
identify several microRNAs affected in the partenote<br />
samples. Two of these microRNAs have been implicated<br />
as a target of angiogenesis receptor II type 1<br />
AGTR1, a gene responsible for angiogenesis [41]<br />
vasoconstriction and increased pregnancy complication<br />
by preeclampsia [57]. Gross morphological<br />
examination of swine parthenote placentas shows<br />
reduced number of blood vessels, and this observation<br />
is also supported by differential expression of<br />
AGTR1 in placental tissues (p
J.A. Piedrahita. <strong>2011</strong>. Application of gene expression profiling to the study of placental and fetal function. aaaaaa<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s273 - s283.<br />
ISSN 1679-9216 (Online)<br />
Derivation and Potential Applications of Pluripotent Stem Cells for<br />
Regenerative Medicine in Horses<br />
Olivia Eilers Smith, Bruce Douglas Murphy & Lawrence Charles Smith<br />
ABSTRACT<br />
Background: The ability to create tissues using pluripotent stem cells to repair or replace tissue lost due to damage, i.e.<br />
regenerative medicine, is developing very rapidly in many fields of human medicine. For veterinarians, regenerative medicine<br />
has focused mainly in the use of stem cells for arthritis and tendon ligament repair, indicating a need for treating musculoskeletal<br />
injuries. Our objective is to review the available approaches being used to derive pluripotent stem cells and discuss<br />
their potential use for regenerative medicine in the horse.<br />
Review: Adult adipose- and bone marrow-derived mesenchymal stem cells (MSC) are being used in practice to treat injuries in<br />
horses. However, there is scarce scientific evidence of their effectiveness and little is known of the mechanisms by which such<br />
cell preparations improve the healing process. For instance, although early healing response of articular cartilage injury was<br />
improved by treatement with injection of MSC, they did not enhance the long-term tissue response, indicating that cell<br />
proliferation was attenuated. Better protocols for the isolation and clinical testing of equine MSC are required to confirm<br />
healing properties. In contrast to MSC, embryonic stem cells (ESC) derived from the inner-cell- mass (ICM) of blastocyst stage<br />
embryos carry the ability to proliferate indefinitely in vitro and, given appropriate and favorable conditions, can differentiate<br />
into any tissue in the body. Parthenogenesis (PG) and somatic cell nuclear transfer (SCNT) are used to obtain a genetic match<br />
to the host animal and, thereby, eliminate the risk of inducing immune rejection of the grafted tissue. However, apart from the<br />
typical markers of pluripotency, equine ESC also express markers of trophoblastic tissues, indicating that they are different and<br />
N<br />
possibly less able to differentiate than the ESC lines obtained in other species. Consequently, further studies are underway to<br />
identify conditions to obtain fully pluripotent ESC lines from equine SCNT embryos. To overcome the limitations of ESC<br />
lines derived from equine embryos, induced pluripotent stem cells (iPSC) were derived using a piggyBac transposon-based<br />
method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally<br />
controlled fashion. Our established fetal-derived iPSC lines express hallmark pluripotency markers, display a stable karyotype<br />
after prolonged culture, and are able to form teratomas in immunodeficient mice containing tissues from all three embryonic<br />
layers. By establishing a protocol for deriving stable iPSC lines in the horse, we expect that new opportunities will be shortly<br />
developed for regenerative therapies in this species.<br />
Conclusion:It is possible to derive autologous pluripotent stem cells in horses by using both ESC and iPSC-derived approaches.<br />
Although ESC lines are generally the gold standard of pluripotency, further research is required to improve the proliferative<br />
and pluripotency characteristics for clinical applications. On the other hand, equine iPSC show excellent stability during<br />
prolonged in vitro culture and have the capacity to differentiate into the three germ layers in vivo, suggesting that they could<br />
soon be used in pre-clinical trials. Therefore, further studies need to be performed to establish reliable protocols for assessing<br />
the regenerative properties of iPS and ESC for equine muscle-skeletal injuries.<br />
Keywords: Pluripotent, Stem Cells, ESC, iPSC, MSC, Regeneration, Horse.<br />
Centre de Recherche en Reproduction Animale (CRRA), Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC,<br />
Canada J2S 7C6. CORRESPONDENCE: L.C. Smith [smithl@medvet.umontreal.ca - TEL: +1 (450) 7738521, ext.: 8463]. O.E. Smith is<br />
supported by a scholarship from the Morris Animal Foundation.<br />
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I. INTRODUCTION<br />
II. ADULT STEM CELLS<br />
2.1 Adult adipose tissue-derived stem cells<br />
2.2 Bone marrow-derived stem cells<br />
III. EXTRA-EMBRYONIC STEM CELLS<br />
3.1 Umbilical cord blood<br />
3.2 Umbilical cord matrix<br />
IV. EMBRYO-DERIVED STEM CELLS<br />
V. INDUCED REPROGRAMMING OF ADULT CELLS<br />
VI. CONCLUSION<br />
I. INTRODUCTION<br />
Regenerative medicine is an emerging field<br />
of medicine focused on repairing and replacing<br />
damaged cells and tissues either by tissue engineering<br />
(producing organs in vitro and implanting them in a<br />
living organism), or by cell therapy (injecting<br />
undifferentiated cells to help stimulate restoration of<br />
diseased organs). In the last 20 years the new science<br />
consisting of molecular imaging and biotechnology<br />
have led to an explosion in the knowledge of the<br />
biological processes of the human body. The field of<br />
regenerative medicine has grown tremendously.<br />
Often, this involves harnessing the properties of stem<br />
cells, which are capable of self-renewal and<br />
differentiation into many other cell types. Stem cell<br />
research provides the basis for the development of<br />
future medical procedures in a broad range of human<br />
diseases enabling the regeneration of many tissues<br />
and organs, including muscles, bone, heart and nerve.<br />
When compared to human applications, the<br />
progress of regenerative medicine in veterinary<br />
medicine is in its infancy. For instance, one of the<br />
chief current therapies of human stem cell based<br />
regenerative medicine is to treat leukemia and other<br />
types of blood related cancer, requiring myeloablative<br />
chemotherapy followed by hematopoietic stem cell<br />
induced recovery of the immune system [26]. As<br />
mentioned above, these applications rarely apply to<br />
animals and they usually remain untreated for the<br />
sake of the animal’s welfare, as well as monetary<br />
reasons. However, being a relatively young and<br />
emerging field, stem cell research for human and<br />
veterinary medicine remain fundamentally attached<br />
to one another. For instance, animal models are used<br />
to study the properties and potential of stem cells for<br />
future human medicine therapies. Moreover, various<br />
stem cell treatments for animal patients are currently<br />
being developed and some, like the treatment of equine<br />
tendinopathies with mesenchymal stem cells<br />
(MSC), have successfully entered the market for this<br />
purpose [32].<br />
Stem cells are generically defined as undifferentiated<br />
cells that are capable of self-renewal<br />
through replication as well as differentiation into<br />
specific cell lineages from at least one of the three<br />
germ layers [36]. Depending on the developmental<br />
stage and tissue from which they are obtained, they<br />
can be classified as embryonic, extra-embryonic or<br />
adult. There are three measures of potency used to<br />
describe levels of plasticity associated with the various<br />
kinds of stem cells. Totipotency is used for cells that<br />
can form all cells or tissues that contribute to the<br />
formation of an organism (ex: the fertilized egg or<br />
zygote). Pluripotency is for cells that can differentiate<br />
into most but not all cells lines of an organism (ex:<br />
embryonic stem cells). Lastly, multipotency can form<br />
a small number of cells/tissues that are usually<br />
restricted to a particular germ layer (e.g. hematopoietic<br />
or mesenchymal stem cells) [29].<br />
Many sources of stem cells exist and when<br />
choosing the appropriate one for the effective, stable<br />
and long-lasting repair of damaged tissue, a few<br />
common criteria should be considered. First a<br />
sufficient number of cells must be generated in order<br />
to fulfill the treatment. Such cells must also be capable<br />
of differentiation towards the right phenotype, and<br />
remain in that state. Also, they should be structurally<br />
and mechanically compliant with the native tissue<br />
and successfully avoid immunological rejection.<br />
Finally, they should adopt the appropriate cellular<br />
organization with extracellular matrix production,<br />
with or without the presence of structural support,<br />
and be able to integrate completely with the damaged<br />
tissue.<br />
Adult-derived stem cells are somewhat easily<br />
accessible, are found in various tissues of the living<br />
organism, and when used autologously do not require<br />
treatment to lower the risks of graft rejection.<br />
However, MSC have a low proliferative potential,<br />
making the production of a large number of cells<br />
difficult, and their differentiation is often restricted to<br />
a specific cell lineage. Embryonic stem (ES) cells, on<br />
the other hand, have unlimited self-renewing abilities<br />
as well as multilineage differentiation potential but<br />
their derivation is more complicated, requiring the<br />
production and destruction of embryos. Their clinical<br />
use is also risky since high plasticity may lead to<br />
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uncontrolled teratoma formation. Currently, adultderived<br />
stem cells are the most commonly used cells<br />
in the clinical field, and scientists are still conducting<br />
experimental transplantation therapies in animal<br />
models to assess the safety and long-term stable<br />
functioning of transplanted cells.<br />
Horses are pioneering the application of<br />
regenerative medicine in several veterinary fields. Not<br />
only do horses hold enormous potential as a model<br />
for a various of medical conditions found in humans,<br />
such as injuries or diseases related to muscles,<br />
tendons, ligaments and joints, they also represent<br />
substantial commercial value in sport and recreational<br />
fields. One of the most common injuries in these large<br />
animals involves the musculoskeletal system causing<br />
serious consequences due to poor response to standard<br />
treatment used successfully in other species. In<br />
the case of bone fracture, casting and long-term<br />
immobilization is either impossible or accompanied<br />
by high risks of devastating secondary complications,<br />
such as damaged cartilage, tendons and ligaments<br />
that have a low capacity to heal. Similar complications<br />
are commonly observed in other species, including<br />
humans. Successful grafting therapies have recently<br />
been developed in the horse using autologous MSCs<br />
[10]. Although equine MSCs show improvement in<br />
the early healing response of articular cartilage lesions,<br />
they do not enhance long-term tissue repair and<br />
clinical treatments have yet to do so.<br />
The objective of this review is to highlight<br />
our current understanding of stem cell biology, with<br />
particular emphasis on equine studies, by comparing<br />
the various sources of stem cells (Figure 1). Current<br />
and potential applications of equine regenerative<br />
medicine will also be reviewed.<br />
N<br />
Figure 1. Overview of the pluripotent stem cells used in equine regenerative medicine. Examples of adult<br />
(bone marrow), extra-embryonic (allantois), embryonic (inner cell mass-derived) and induced pluripotent<br />
stem (iPS) cells show specific morphology in vitro and have multilineage differentiation potential for<br />
healing several injuries in horses.<br />
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II. ADULT STEM CELLS<br />
Mesenchymal stem cells (MSCs) are an adultderived<br />
stem cell population that can be isolated from<br />
multiple body tissues. These multipotent cells have<br />
the capacity to differentiate into lineages of<br />
mesenchymal origins, including osteoblasts (bone),<br />
chondrocytes (cartilage) and adipocytes (adipose<br />
tissue) [34]. Some prefer to refer to MSCs as<br />
multipotent stromal cells or mesenchymal progenitor<br />
cells, observing that the term “stem” might<br />
attribute more biological properties than the MSCs<br />
actually hold [9]. Only cells that have shown selfrenewal<br />
ability, in-vivo long-term survival, and tissue<br />
repopulation with multilineage differentiation should<br />
be identified as MSCs [16].<br />
Equine MSCs are of particular interest both<br />
for basic research and for the therapeutic approach<br />
to musculoskeletal diseases in the horse. Their<br />
multilineage differentiation potential gives them the<br />
capability to contribute to the repair of tendon,<br />
ligament and bone damage. Yet, enthusiasm for the<br />
use of MSCs for therapeutic use is tempered by their<br />
age-dependent decline in absolute numbers and the<br />
invasive nature of their harvest [28].<br />
In humans, adult MSCs have been detected<br />
in various tissues such as the dermis, blood, muscle<br />
and the trabecular bone [35]. The cell population<br />
from some of these sources may have a more limited<br />
capacity for differentiation, containing monopotent<br />
or bipotent cells that have differentiation potentials<br />
developmentally adapted and restricted to the tissues<br />
in which they were found. This could lead to issues<br />
involving ease of isolation, cell yield, and donor site<br />
complications, suggesting that certain sources may<br />
be more suitable than others [3].<br />
In equine medicine, which centers generally<br />
on musculoskeletal repair, the bone marrow is the<br />
most common source for isolation of multipotent<br />
MSCs and adipose tissues as well, due to the low<br />
morbidity associated with their harvest and their<br />
renewable nature. Herein we compare the advances<br />
made in both domains.<br />
2.1 Adult adipose tissue-derived stem cells<br />
Adult adipose tissue (AT) originates from the<br />
embryonic mesenchyme and consists mainly of<br />
adipocytes and a supportive stroma, composed of<br />
fibroblast-like precursor cells known as preadipocytes<br />
[5]. The latter were initially believed only be able to<br />
differentiate into cells of its tissue of origin, but recent<br />
studies have shown that these stromal cells are actually<br />
capable of differentiation into multiple other cell-lines<br />
[41]. This supportive stroma represents an important<br />
source of adult MSCs since its cells can be easily<br />
isolated in large quantities. AT-MSC can be isolated<br />
from its tissue when digested in collagenase type I<br />
[7], expanded in vitro and then inoculated into the<br />
damaged tissue.<br />
There are many advantages to using adipose<br />
tissue as a source of MSCs in equine regenerative<br />
medicine. First, the presence of adipose tissue in<br />
horses is quite substantial and its harvest is much<br />
simpler and less prone to complications than bone<br />
marrow extraction [10]. Most horses have enough fat<br />
around their tail head to obtain the required amount<br />
for stem cell injection into their damaged tissue. The<br />
adipose tissue is collected either under sedation and<br />
local anesthesia or under a quick general anesthesia.<br />
In humans it has been reported that AT-MSCs can be<br />
quickly isolated from adipose tissue and the resulting<br />
stromal vascular cell fraction (SVF) contains a greater<br />
proportion of stromal/stem cells per unit volume in<br />
comparison to bone marrow [14]. In horses, it has<br />
been shown that for the same quantity of tissue sample,<br />
the total quantity of AT-MSCs attained after 21 days<br />
in culture is significantly larger that for bone marrow<br />
MSCs [36]. This represents an important advantage<br />
since tissue lesions in horses will usually require a<br />
large quantity of cells to ensure successful and long<br />
lasting therapeutic repair. Adipose tissue seems to be<br />
an accessible and abundant source of adult derived<br />
stem cells.<br />
Some disadvantages of AT-MSCs are the<br />
slightly lower osteogenic capability than that of BM-<br />
MSCs, the non-sterile conditions and risk of pathogen<br />
agents at the collection site and the difficulty to obtain<br />
fat from highly fit athletes [3]. Also, although the use<br />
of autologous AT-MSCs allows a lower risk of<br />
immunosuppression, it also requires a longer wait<br />
before treatment, due to time period required for tissue<br />
collection, stem cell isolation, culture and characterization.<br />
Although there is a higher possibility of<br />
rejection and risk of disease transmission, allogenic<br />
AT-MSCs present certain advantages as well for<br />
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allowing rapid treatment to injured horses using cells<br />
with optimal proliferation and differentiation<br />
potential. Moreover, the treated animal will also be<br />
exempt of the anesthesia and surgery required for<br />
AT-MSC harvesting [6].<br />
2.2 Bone marrow-derived stem cells<br />
The bone marrow (BM) stroma is formed of<br />
hematopoietic, for the most part, and mesenchymal<br />
multipotent stem cells. It has been reported that MSCs<br />
populate 0.001 to 0.1% of the total population of the<br />
human bone marrow [26], and a similar assumption<br />
may be valid for the equine species. Currently, equine<br />
BM-MSC are harvested from the horse’s sternum and<br />
isolated by Percoll density gradient separation [2]<br />
followed by in vitro proliferation and characterization<br />
by adipogenic, osteogenic, and chondrogenic in vitro<br />
differentiation analysis [1] and finally grafted in the<br />
damaged tissue. Since the preparations of the BM-<br />
MSCs takes 2-4 weeks, the wounded animals’ bone<br />
marrow is aspirated as promptly as possible.<br />
Optimally, stem cell clinical implantation is therefore<br />
performed within one to two months of the injury.<br />
The cells are supported by the granulation bed formed<br />
and strategy avoids substantial fibrosis of the site.<br />
BM-MSCs are the most commonly chosen<br />
source of stem cells because of their easy accessibility<br />
and for their capacity to produce large numbers of<br />
MSCs. Much like AT-MSCs, an important advantage<br />
linked to BM-MSCs is that when recovered from the<br />
injured animal itself they avoid the risks of immune<br />
rejection. Osteogenic gene expression and mineral<br />
deposition of the BM-MSCs, before and after<br />
induction with osteogenic culture conditions, show<br />
that the bone marrow contains the largest quantity of<br />
osteoprogenitors and these cells possess the highest<br />
osteogenic potential in vitro [3].<br />
The presence of these precursor cells,<br />
however, show a lower cell plasticity for the BM-<br />
MSCs, making differentiation into other cell lines more<br />
difficult. Also, the production of large numbers of<br />
autologous BM-MSCs is lengthy and costly, requiring<br />
bone marrow extraction, MSC isolation and<br />
expansion, testing for contaminants and, finally,<br />
transplantation. The bone marrow collection method<br />
commonly used exposes the horses to possible<br />
complications such as pneumothorax and pneumopericadium.<br />
[10]<br />
Currently the most utilized source of stem cells<br />
for clinical purposes, BM-MSCs are used to treat<br />
bone, ligament, tendon and cartilage lesions. More<br />
specifically, BM-MSCs have been reported to<br />
successfully repair superficial digital flexor tendon<br />
lesions as well as soft palate defects in horses [32].<br />
Equine MSCs improve the early healing response in<br />
articular cartilage lesions, but long-term tissue repair<br />
not does seem to be enhanced.<br />
III. EXTRA-EMBRYONIC STEM CELLS<br />
In addition to stem cells derived from adult<br />
tissues, stem cells can be found in extra-embryonic<br />
tissues including umbilical cord blood, umbilical cord<br />
matrix, and amnioticuid. Stem cells from extra-embryonic<br />
sources have the advantages of being<br />
obtained by non-invasive procedures and allowing<br />
treatment with low immunogenicity. The three sources<br />
of extra-embryonic tissue are harvested immediately<br />
after foaling and stored frozen for potential future<br />
transplantation; thereby enabling the horse to have a<br />
supply of autologous stem cells in case of future injuries<br />
or disease.<br />
3.1 Umbilical cord blood<br />
A few ml of cord blood can be collected from<br />
intact umbilicus at birth without complication to the N<br />
foal or the mare. The umbilical cord blood (UCB)<br />
stem cells have a fibroblast-like morphology and have<br />
been found to express stem cell markers such as Oct-<br />
4, Tra1-60 and Tra1-81 and SSEA-1 [28]. An<br />
important factor is that the stem cells isolated from<br />
UCB have better proliferative and plasticity potency<br />
than that of adult derived MSCs, but not as good as<br />
embryonic stem cells, suggesting UCB-MSCs are the<br />
primitive cell type of the two. Adult-derived MSCs<br />
show better differentiation capacity when limited to<br />
their tissue of origin cell line. Predominance for the<br />
chondrogenic and osteogenic pathways is observed<br />
for BM-MSCs and the adipogenic pathway for AT-<br />
MSCs. However, once in tissue-specific culture with<br />
presence of growth factors, UCB stem cells are<br />
capable of differentiation towards osteogenic, chondrogenic<br />
and adipogenic pathways as well as cell<br />
types of hepatocytes and endodermal origin [3].<br />
Equine UCB-MSCs have superior immune tolerance,<br />
proliferative potential and less senescence occurrence<br />
in later passages than other adult-derived equine<br />
MSCs [19].<br />
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3.2 Umbilical cord matrix<br />
The umbilical cord matrix (UCM) is a<br />
gelatinous connective tissue composed of<br />
myo?broblast-like stromal cells, collagen ?bers, and<br />
proteoglycans [18]. It is reported to be rich in young<br />
MSCs with high proliferation ability. The UCM are<br />
harvested identically as UCB, except that it is the<br />
blood vessel free umbilical cord tissue that is<br />
collected. UCM stem cells are isolated by collagenase<br />
digestion of the cord tissue and expanded in culture<br />
until an abundant quantity of cells becomes available<br />
for characterization and di?erentiation procedures<br />
[17]. Much like UCB stem cells, UCM stem cells have<br />
the capacity to differentiate into the three major<br />
mesenchymal cell lineages (bone, cartilage and fat).<br />
They also express the embryonic markers Oct-4 and<br />
SSEA-4 and can adopt neuron-like morphology,<br />
implying they are also situated between adult-derived<br />
MSCs and embryonic stem cells in pluripotency [25].<br />
These characteristics play an important role in<br />
successful cell-based therapies in the horse. One<br />
downside for both UCB and UBM MSCs is the lack<br />
of sterilization of the tissue and the environment<br />
during the harvest, causing higher risks of<br />
contamination. In cell culture, the presence of larger<br />
amounts of antibiotics is a means to reduce this risks<br />
in clinical experiments.<br />
Experiments with equine amniotic<br />
membranes showed possible presence of stem-like<br />
cells, due to pluripotency marker expression and<br />
osteogenic differenciation potential, giving rise to a<br />
plausible new source of MSCs. However,<br />
immunohistochemical studies, preclinical<br />
experimentation, and immunological evaluation must<br />
be performed before more can be said about this<br />
potential new source of pluripotent stem cells [20].<br />
IV. EMBRYO-DERIVED STEM CELLS<br />
Before implantation in the uterus, embryos<br />
are at the blastocyst stage and are composed of an<br />
outer layer of cells called the trophectoderm, a fluidfilled<br />
cavity named the blastocoele and finally the<br />
inner cell mass (ICM), also called epiblast. The<br />
trophectoderm cells contribute to the placental<br />
chorion, whereas the ICM contains pluripotent<br />
embryonic stem (ES) cells that possess the ability to<br />
develop into any cell type of the organism [4]. It was<br />
found that, when cultured in a media containing<br />
leukemia inhibitory factor (LIF) or in presence of<br />
embryonic fibroblast as feeder cells, ES cells<br />
proliferate, replicate and can be maintained in an<br />
undifferentiated pluripotent state providing a<br />
potentially unlimited source of stem cells. When<br />
withdrawn from these culture conditions, ES cells will<br />
spontaneously differentiate into cells of the three germ<br />
layers; ectoderm, mesoderm and endoderm [31]. It<br />
is thus possible to control the differentiation pathway<br />
taken by the stem cells by adjusting the culture<br />
conditions and generating tissue-specific precursors<br />
[38]. Unlike adult or extra-embryonic derived MSCs<br />
that reach senescence after a certain number of<br />
passages, the ES cells ability to remain pluripotent<br />
throughout extended culture periods is an important<br />
advantage in choosing blastocyst derived stem cells<br />
for therapeutic and research applications. In contrast,<br />
adult stem cells replicative life span is influenced by<br />
the cell type, donor age and donor species [12].<br />
Equine ES cells could represent a significant<br />
source of stem cells in the field of regenerative<br />
medicine. Using the somatic cell nuclear transfer<br />
(SCNT), an enucleated oocyte can be reconstructed<br />
with any cell provided by the injured animal to<br />
produce an embryo. The oocyte’s cytoplasm is<br />
capable of reprogramming the donor cell’s nucleus<br />
to re-establish embryonic gene expression patterns.<br />
Nuclear transfer embryonic stem (NTES) cells can<br />
then be isolated from the inner cell mass (ICM) once<br />
the embryo has developed to the blastocyst stage<br />
[8][22]. Such NTES cell line differentiation would<br />
then be induced to produce the desired cell type for<br />
transplantation in the injured horse (Fig. 2). These<br />
autologous cells, except for the mtDNA inherited from<br />
the oocyte, would avoid the risk of immune rejection<br />
often caused by use of allogenic MSCs. ES cells also<br />
have a higher plasticity than MSCs, which could allow<br />
better long-term repair in damaged tissues.<br />
Unfortunately, a large number of oocytes is<br />
necessary for equine NTES cell production, since both<br />
SCNT and NTES cell line isolation have low<br />
efficiencies. Horses are seasonal breeders and single<br />
ovulatory species and slaughterhouses are scarce,<br />
making the harvest of multiple oocytes complicated<br />
[13]. Another potential obstacle when using ES cells<br />
for therapeutic treatments is their potential for<br />
uncontrolled proliferation and predisposition towards<br />
teratoma formation in vivo.<br />
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N<br />
Figure 2. Comparison of embryonic stem (ES) and induced pluripotent stem (iPS) cell production for regenerative<br />
medicine in horses. Somatic cells harvested from an injured horse are fused to an enucleated oocyte (1a) and activated<br />
to develop to the blastocyst stage (1b). The inner cell mass (ICM) is isolated to form immune-compatible ES cells (1c).<br />
In contrast, to produce iPS cells fibroblasts are transfected (2a) with a PiggyBac transposon that contains the<br />
reprogramming factors c-Myc, Klf-4, Oct-4 and Sox-2 that proliferate (2b) and eventually become iPS colonies (2c). The<br />
pluripotent and autologous nature of ES and iPS cells enable tissue specific differentiation and grafting to heal the<br />
original injured horse.<br />
However, unlike mouse and human ES<br />
derivation, previous attempts to derive equine ES line<br />
have been unable to confirm true pluripotency. The<br />
first ES-like cells produced, using in vivo derived<br />
equine embryos, expressed a few characteristic<br />
murine and human pluripotency cell markers, and<br />
were capable of in vitro differentiation in vitro into<br />
hematopoietic and neural precursor cells [30].<br />
Another study showed the successful spontaneous<br />
in vitro differentiation of ES-like cells into the three<br />
germ layer once removed from the feeder layer [21].<br />
Parthenogenic-derived equine embryos have been<br />
successfully used to obtain ES-like lines, indicating<br />
that in vitro culture conditions are not detrimental to<br />
the quality of the ICM cells utilized for ES cell line<br />
derivation [8]. These ES-like cells could not, however,<br />
form teratomas when injected in vivo, nor produce<br />
chimeric horses, two important ES cell line chara-<br />
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cterization tests. On the other hand, this inability to<br />
form teratomas may be advantageous for therapeutic<br />
use by lowering the risk of uncontrolled<br />
differentiation once the ES-like cells are injected in<br />
the horse’s injury site. There has yet to be a clinical<br />
experiment done on horses to see if these cells would<br />
help regenerate cells on a damaged tissue without<br />
tumor formation.<br />
V. INDUCED REPROGRAMMING OF ADULT CELLS<br />
A landmark in stem cell research was the<br />
establishment of a protocol to reprogram differentiated<br />
cells back to their initial stage of pluripotency.<br />
Although it was possible to reprogram differentiated<br />
cells to an embryonic-like state by transfer of nuclear<br />
contents into oocytes or by fusion with ES cells,<br />
little was known about the factors that induce<br />
reprogramming (Figure 2). Induced pluripotent stem<br />
(iPS) cells were generated by the expression of a set<br />
of typical stem cell transcription factors (Oct4, Sox2,<br />
Klf4, c-Myc, NANOG and Lin28) in adult somatic<br />
cells [34]. These iPS cells show great potential for<br />
regenerative therapy as they can differentiate into all<br />
three embryonic germ layers and can be expanded<br />
to large quantities in vitro. Apart from murine rodents,<br />
iPS cells have been produced in a few species such<br />
as humans [40] and porcine [39] and most recently<br />
in equine [24]. The later was achieved by<br />
incorporating via piggyBac transposon-based method<br />
that, once the transposon electroporated into the<br />
equine fetal fibroblast, delivers transient transgenes<br />
containing the reprogramming factors Oct4, Sox2,<br />
Klf4 and c-Myc. The ectopic expression of these<br />
factors is doxycycline-dependant and the presence<br />
of this antibiotic in the culture media initiates the<br />
somatic cells’ reprogramming. Equine iPS cells show<br />
a stable karyotype after extended culture, express<br />
pluripotent properties, and are able to form teratomas<br />
composed of all three embryonic germ layers in<br />
immunodeficient mice [24]. Once iPS cells are<br />
confirmed to be safe for therapeutic application, they<br />
could have a major impact in regenerative medicine<br />
especially since they can be produced for specific<br />
patients without raising the ethical issue that ES cells<br />
tend to.<br />
This new discovery represents a new way of<br />
approaching musculoskeletal injury treatment in<br />
horses, providing stem cells with control over the<br />
expression of the pluripotent factors. Injecting these<br />
cells in the injury site of a horse being treated with<br />
doxycyclin allows the cells to proliferate and expand<br />
in the damaged tissue. Once the animal’s doxycyclin<br />
treatment is withdrawn, the iPS cells will differentiate<br />
into the intended tissue that requires repair. Cell<br />
specification could either happen naturally, because<br />
of the tissue surrounding factors inducing the right<br />
differentiation, or by outside influence via incorporation<br />
of specific factors for the iPS cells. This<br />
control over the expression of the pluripotency<br />
markers could also reduce the chances of unwanted<br />
teratoma formation. iPS cells could represent a better<br />
source for long-term tissue repair because of its higher<br />
pluripotent state than MSCs and, for equine, ES cells.<br />
Their capacity to expand in large quantities in vitro is<br />
an advantage towards ES cells, and their painless and<br />
easy accessibility, requires only a skin biopsy, is more<br />
appealing than the methods used for adult MSC<br />
harvest.<br />
To date only fetal fibroblasts have successfully<br />
been used to derive equine iPS, which is<br />
somewhat disadvantageous for clinical applications.<br />
Allogenic iPS cells could have a higher risk of<br />
rejection if implanted in an injured horse. Ideally, one<br />
would use the injured animal’s own adult fibroblast<br />
to produce and proliferate iPS cells that would then<br />
be transplanted to the damaged tissue. However, adult<br />
cells might be a bigger challenge to derive, their<br />
reprogramming efficiency being affected by age,<br />
origin and cell type [15], which will most likely also<br />
have an impact on the resulting iPS cell quality.<br />
VI. CONCLUSION<br />
Presently, equine MSCs are more commonly<br />
used in regenerative therapies due to their autologous<br />
and tissue specific differentiation properties. For<br />
musculoskeletal damages, bone marrow MSCs seem<br />
to be the most effective cells to improve early healing<br />
response, but their low plasticity hinders long-term<br />
tissue restoration, which can lead to facilitated re-injuries.<br />
Extra-embryonic stem cells could be a valid<br />
option of stem cell source because of their wider<br />
plasticity and their less advanced cell state, compared<br />
to adult-derived stem cells. Studies have recently<br />
shown that UCB-MSC transplantation into fractured<br />
mouse femurs allowed the accelerated the repair of<br />
the tissues and bone substitution [23]. However, no<br />
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clinical applications have yet been conducted on the<br />
horse.<br />
It is also possible to derive autologous<br />
pluripotent stem cells in horses by using both ES and<br />
iPS cell-derived approaches. Although ES cell lines<br />
are generally the main source of stem cells used for<br />
regenerative medicine in other species, due to its<br />
pluripotency and easy proliferation, a real equine ES<br />
cell line has yet to be derived. The risk of<br />
uncontrollable division once implanted in the animal<br />
is also an important factor against ES cells for clinical<br />
applications. Alternatively, equine iPS cells have<br />
effectively been produced and show excellent stability<br />
during prolonged in vitro culture. They additionally<br />
have the ability to differentiate into the three germ<br />
layers in vivo, suggesting that they could soon be<br />
used in pre-clinical trials. Since they are antibiotic<br />
dependent, it is possible that their proliferative power<br />
could be controlled and unwanted tumor formation<br />
avoided. Although further studies need to be<br />
performed to assess their regenerative properties, iPS<br />
cells will most possibly become one of the most<br />
important sources of stem cells for future clinical<br />
applications.<br />
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N<br />
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39(Suppl 1)<br />
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alamone, R. Bevacqua,<br />
F.P<br />
.P. Bonnet<br />
onnet, et al. <strong>2011</strong>. Recent advances in micromanipulation and transgenesis in domestic<br />
mammals. Acta Scientiae Veterinariae. 39(Suppl 1): s285 - s293.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s285-s293.<br />
ISSN 1679-9216 (Online)<br />
Recent advances in micromanipulation and transgenesis in domestic mammals.<br />
Daniel Salamone, Romina Bevacqua, Federico Pereyra Bonnet, Andrés Gambini, Natalia Canel, M.<br />
Inés Hiriart, Gabriel Vichera, Lucia Moro & Javier Jarazo<br />
ABSTRACT<br />
Background: Intracytoplasmic sperm injection (ICSI) involves mechanical transfer of a single sperm cell into ooplasm. A new<br />
application has been recently found for ICSI, the production of transgenic animals. Since the birth of ‘‘Dolly’’, the first adult<br />
somatic cloned mammal, viable offspring has been produced by nuclear transfer in many species including cattle. The present<br />
review briefly summarizes our experience with ICSI and somatic cell nuclear transfer mainly to produce transgenic embryos,<br />
as well as for the generation of new micromanipulation technique.<br />
Review: We have evaluated different factors that affect SCNT and transgenesis including the chemical activator, the transfection<br />
event and the effect of recloning. Also, we included a brief description of the ICSI technique, which we used in five different<br />
species, examining its potential to produce transgenic embryos. Finally different strategies to produce transgenic animals were<br />
analyzed: ICSI- mediated gen transfer (ICSI-MGT), Injection of cumulus cell and ooplasmic vesicle incubated for 5 min with<br />
the transgene or injection of the plasmid alone. All of them were very efficient in exogenous DNA expression at embryo stages<br />
but resulted in mosaic embryos. We demonstrated that “ICSI-MGT” assisted by chemical activation is the only treatment of<br />
sperm mediated gen transfer capable to generated transgenic embryos in ovine. Besides, after ICSI-MGT, it is possible to<br />
obtain enhanced green fluorescent protein (EGFP)-expressing embryos in five diferent species: ovine, porcine, feline, bovine<br />
and equine. Our studies also established for the first time that short term transgene co-incubation with somatic cells can<br />
produce transgene-expressing mammalian SCNT embryos, and also that parthenogenic, eDNA- expressing embryos can be<br />
obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic N<br />
injection of vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique. We tried a further<br />
simplification of the technique in bovine oocytes and zygotes, by intracytoplasmically injecting them with eDNA-liposomes<br />
complexes. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp–liposome was<br />
injected 16 h post-fertilization. Different approaches were assayed to reverse the mosaicism including a novel technique of<br />
gamete cloning. Our first approach consisted of the production of transgenic IVF embryos by vesicle microinjection to<br />
generate transgenic blastomeres to be used as donor cells for cloning. A high efficiency in mosaicism reversal and multiplication<br />
of transgenic embryos was attaineded. Other technique assayed was the separation of transgenic blastomeres followed by the<br />
aggregation of two-cell fused embryos or by the asynchronous younger blastomere successfully multiplied transgenic embryos,<br />
and theoretically reduces mosaicism rates in future offspring [15]. This technology can also be used to multiply embryos from<br />
animals with high genetic value. We demonstrated that a sperm and oocyte can be efficiently cloned. Green haploid androgenic<br />
blastomeres produced with the injection of a single sperm by egfp ICSI-MGT could be used to fertilized oocytes resulting in<br />
several homogeneous expressing embryos. This approach shows great potential because it allows for determination of the sex<br />
of the sperm nucleus prior to fertilization. It is also possible to clone previously transfected oocytes followed by the reconstruction<br />
of biparental bovine embryos to generate homogeneous transgene-expressing embryos. This review summarizes recent<br />
experiments in micromanipulation and gene transfer in domestic animals. The objective is not to exhaustedly describe the<br />
research done in this field but to present the promising methods recently developed or evaluated in our lab.<br />
Conclusion: Significant advancements have been made in the course of the recent years in micromanipulation and transgenesis<br />
techniques. In our lab we have been evaluating ICSI and Nuclear transfer mainly to produce transgenic embryos. We used also<br />
transgensis to apply or developed new micromanipulation technique in domestic animals linke sperm and oocyte cloning.<br />
Keywords: Micromanipulation, transgenesis, ICSI, cloning, nuclear transfer.<br />
CORRESPONDENCE: D. Salamone [salamone@agro.uba.ar]. Laboratorio de Biotecnología Animal, Departamento de producción animal.<br />
Facultad de Agronomía, Universidad de Buenos Aires, Argentina. Avenida San Martin 4453, 1417 Buenos Aires, Argentina.<br />
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.P. Bonnet<br />
onnet, et al. <strong>2011</strong>. Recent advances in micromanipulation and transgenesis in domestic<br />
mammals. Acta Scientiae Veterinariae. 39(Suppl 1): s285 - s293.<br />
I. INTRODUCTION<br />
II. CLONING BY NUCLEAR TRANSFER<br />
III. CLONING BY NUCLEAR TRANSFER USING A<br />
TRANSFECTED CELL LINE<br />
IV. INTRACYTOPLASMIC SPERM INJECTION (ICSI)<br />
V. ICSI MEDIATED GENE TRANSFER<br />
VI. GENE TRANSFER BY NUCLEAR MICROINJECTION<br />
VII. GENE TRANSFER BY CYTOPLASMIC<br />
MICROINJECTION<br />
VIII. DIFFERENT APPROACHES FOR EMBRYO<br />
MULTIPLICATION AND MOSAICISM REVERSION<br />
IX. CONCLUSIONS<br />
I. INTRODUCTION<br />
II. CLONING BY NUCLEAR TRANSFER<br />
Cloning by somatic cell nuclear transfer has raised<br />
enormous interest; mainly it has made possible the<br />
propagation of elite domestic animals and the enginering<br />
of transgenic animals for agricultural and biomedical<br />
purposes. Briefly, nuclear transfer (NT) involves the<br />
enucleation of a recipient oocyte, followed by the transfer<br />
of a donor cell to the perivitelline space in close apposition<br />
of the recipient cytoplast, and their fusion. Development<br />
is induced artificially by chemical or physical activation.<br />
Production of cloned offspring by somatic cell nuclear<br />
transfer has been successfully attained in sheep [5,50,53],<br />
goats [1] and cows [8,17,50].<br />
There are several factors that influence the<br />
results of NT including the methods of enucleation [26],<br />
fusion, activation and donor-recipient cell cycle synchrony.<br />
High efficiencies in enucleation of recipient oocytes have<br />
been achieved using DNA specific vital dyes to visualize<br />
chromatin [51,41]. Fusion of the donor cell with the<br />
recipient oocyte depends on the accuracy of cell alignment<br />
in the pulse field, contact of the donor cell with the<br />
recipient oocyte and size of the donor cells [9]. Nucleus<br />
can also be injected [6]. Activation of NT reconstructed<br />
embryos has been refined and rates of development into<br />
blastocysts are equivalent to in vitro fertilized oocytes<br />
[21].<br />
Successful development of NT embryos has been<br />
accomplished using mature oocytes [52], zygote [24] and<br />
cleavage-stage embryos [44] as recipient cytoplast.<br />
However, this is dependent on the source of the donor<br />
nucleus. Compatibility of the cell cycle between the<br />
recipient cytoplasts and the donor cell is one of the<br />
important factors that influence development of NT<br />
embryos. Appropriate synchronization is necessary to<br />
preserve the ploidy of the reconstituted embryo.<br />
A problem with existing nuclear transfer systems<br />
is that the survival of cloned embryos and fetuses is low.<br />
One possible explanation is that the donor nucleus may<br />
not be competent to support normal development. Somatic<br />
cells have been programmed to differentiate into a particular<br />
cell; its nucleus is programmed to transcribe a<br />
specific set of RNAs that are translated into a set of<br />
proteins that results in the cell having all the characteristics<br />
of its type. After nuclear transfer the nuclei condense,<br />
and many proteins that bind to chromatin and regulate<br />
transcription must be released. Other proteins may bind<br />
at the time of activation to induce decondensation.<br />
Reconstructed embryos can also present errors in genomic<br />
imprinting. The modification or disruption of genomic<br />
imprinting in early development may cause genetic<br />
diseases. Embryo imprinting was evaluated by several<br />
authors and may represent a problem for embryo or fetal<br />
survival during reconstruction [14,33].<br />
A new compound Dehydroleucodine (DhL) was<br />
evaluated as a chemical activator to produce SCNT<br />
reconstructed embryos [46]. Results showed that DhL<br />
induces pronuclear formation dynamics more similar to<br />
IVF than 6-dimethylaminopurine (6-DMAP). Blastocyst<br />
development was higher after activation with this new<br />
compound combined with Citochalasyn B. Moreover, all<br />
DhL treatments induced polyploidy rates lower than<br />
Ionomycin followed by 6-DMAP, but cloned blastocyst<br />
rates statistically similar [6].<br />
One of the main applications of SCNT is the<br />
cloning of animals of genetic value. We assessed a new<br />
alternative cloning technique, previously described in the<br />
bovine [34], which consists of aggregating zone free<br />
genetically identical cloned embryos as a strategy to<br />
improve in vitro and in vivo embryo development in the<br />
equine. Embryo aggregation improved the quality of in<br />
vitro equine cloned embryos at day 7, and pregnancy<br />
rates were higher. The sizes of vesicles and embryos in<br />
vivo were normal for all groups, and in vitro development<br />
of aggregated embryos beyond day 7 resulted in the<br />
highest embryo viability. Cloning equines by means of<br />
aggregation of two or three embryos does not imply extra<br />
oocytes, and it is a good strategy to improve in vitro<br />
embryo development without alterations in in vivo<br />
progress. The first living cloned foal obtained by this<br />
method was born from two aggregated embryos On<br />
August 4, 2010 [11].<br />
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.P. Bonnet<br />
onnet, et al. <strong>2011</strong>. Recent advances in micromanipulation and transgenesis in domestic<br />
mammals. Acta Scientiae Veterinariae. 39(Suppl 1): s285 - s293.<br />
III. CLONING BY NUCLEAR TRANSFER USING A<br />
TRANSFECTED CELL LINE<br />
After nuclear transfer was developed, microinjection<br />
tended to be replaced by nuclear transfer using<br />
genetically modified somatic donor cells [39,8]. A cow<br />
capable of expressing human growth hormone (hGH)<br />
was delivered in South America in September of 2002.<br />
The hGH production in milk from this animal was<br />
evaluated and results were published by Salamone et al.<br />
[38] in 2006. It was demonstrated that hGH can be<br />
produced at a large scale in the milk of transgenic cows.<br />
Only about 15 animals would be necessary to meet the<br />
worldwide requirements of this protein for the treatment<br />
of dwarfism in GH deficient children [38]. However,<br />
nuclear transfer as was described previously using<br />
genetically modified somatic donor cells presents a low<br />
overall efficiency, explained partly by epigenetic<br />
reprogramming failure [36].<br />
Although, cloning is one of the most powerful<br />
techniques available to generate transgenic animals,<br />
several additional problems appear during clone<br />
production. For example, different transfection events of<br />
the same somatic cell line can affect embryo and/or fetal<br />
survival. In one experiment performed for a<br />
Biotechnology company, a fetal cell line was established<br />
from a 75-day-old Jersey female fetus, which was used<br />
as control and was also transfected 3 times with the same<br />
protocol. They were named Transfection 1, 2, and 3.<br />
Genetically modified cells were produced and isolated<br />
after selection with geneticin for 10–15 days following<br />
liposome transfection with a DNA construct containing<br />
a selectable neomycin resistance gene.<br />
Results of embryo and fetal development are<br />
presented in table 1. One birth was obtained from the<br />
control. Four and 7 births were obtained from<br />
Transfections 1 and 3, respectively. Although Transfection<br />
Table 1. Effect off different transfection events on same line in embryo and fetal survival.<br />
Treatment n Blastocyst (%) Implanted Recipients Preg. 30d (%) Birth<br />
Control 197 122 (62) 33 (18) 4 1<br />
Transfection 1 130 106 (82) 28 (22) 5 4<br />
Transfection 2 137 96 (70) 34 (24) 0 0<br />
N<br />
Transfection 3 470 282 (60) 71 (15) 12 7<br />
Percentages within columns with different superscripts are different (P < 0.05)<br />
2 had good in vitro development, this treatment did<br />
not produce any pregnancies. This fact demonstrated<br />
that the transfection event provides an additional<br />
source of variability in obtaining live transgenic<br />
animals. Our results pointed out the necessity to<br />
monitor fetal survival by ultrasonography in order to<br />
detect any deficiencies in development introduced<br />
by transfection as soon as possible.<br />
In a large scale cloning program destined to obtain<br />
transgenic animals, it is very important to produce wellcharacterized<br />
transgene integration and gene expression.<br />
However, after non-homologous transfections a wide<br />
variety of transgene copy numbers are introduced in<br />
different chromosome locations. Recloning a selected<br />
first-generation of transgenic calves offers the opportunity<br />
to increase the homogeneity among transgenic animals.<br />
Calf recloning was performed in an experiment (n=<br />
1739 cloning procedure) in which the survival rate<br />
was evaluated after a second round of cloning from<br />
transgenic umbilical cord and ear calf fibroblast cell<br />
cultures. Seven births were obtained from the original<br />
fetal cell line, one birth was obtained from recloned<br />
umbilical cord cells and two calves from recloned<br />
ear fibroblasts. Development to blastocysts was<br />
different between transfected fetal fibroblasts and<br />
both recloned treatment groups. Differences were<br />
observed in pregnancy rates between blastocysts<br />
generated by the different sources of donor cells.<br />
Although it results in lower blastocyst production,<br />
our results suggest that recloning provides an additional<br />
method to obtain transgenic animals, where fibroblasts<br />
from umbilical cord tissue could give better results for<br />
recloning than those obtained from young calf ear cells.<br />
IV. INTRACYTOPLASMIC SPERM INJECTION (ICSI)<br />
ICSI has been used in humans and mice [28,18].<br />
In these species, sperm cell injection causes oocyte<br />
activation [27]. However, after ICSI most domestic<br />
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alamone, R. Bevacqua,<br />
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.P. Bonnet<br />
onnet, et al. <strong>2011</strong>. Recent advances in micromanipulation and transgenesis in domestic<br />
mammals. Acta Scientiae Veterinariae. 39(Suppl 1): s285 - s293.<br />
animals do not develop appropriately [23,22].<br />
Chemical activation protocols for ICSI frequently use<br />
ionomycin, a calcium ionophore, followed by 3 h of<br />
incubation previous to 6-DMAP treatment [35].<br />
Recently, we produced a high rate of ICSI blastocysts<br />
in a domestic cat without chemical assisted activation,<br />
simplifying the procedure, and allowing for its<br />
application in the wild cat [25]. Most groups perform<br />
ICSI using a piezo-drill, but we recently obtained<br />
good blastocyst rates using chemical activation<br />
protocols, producing a live lamb Fig. 2 [31]. In<br />
equines this technique has great potential, because<br />
although oocytes can be recovered in vivo via<br />
ultrasound guided transvaginal aspiration, they are<br />
impossible to fertilize by regular in vitro protocols.<br />
ICSI appears to bypass this problem and we recently<br />
generated good blastocyst rates in the equine.<br />
V. ICSI MEDIATED GENE TRANSFER<br />
Several authors published techniques<br />
alternative to somatic cell nuclear transfer and<br />
pronuclear microinjection to obtain transgenic<br />
embryos and offspring including: Laparoscopic<br />
Insemination (LI) [19], In Vitro Fertilization (IVF) [20]<br />
and ICSI [32]. By LI and IVF, the possibility of<br />
generating transgenic animals is simple. However the<br />
use of these techniques has been in substantial debate<br />
[20,4]. On the other hand, ICSI is an efficient<br />
technique to produce offspring in the murine [32].<br />
One of the limitations of ICSI-mediated gene<br />
transfer is that it results in a high frequency of mosaic<br />
expression of the transgene. A possible explanation for<br />
this is that the transgene is not integrated in the embryo<br />
genome before the first cell divisions [40,32,43,16].<br />
Moreover, the transgene could remain extra-chromosomal<br />
and be lost during successive mitotic divisions, as was<br />
already shown in two previous reports [7].<br />
In one experiment, we used “ICSI-MGT”<br />
assisted by chemical activation in five species, ovine,<br />
porcine, feline, bovine and equine, demonstrating that it<br />
is possible to obtain enhanced green fluorescent protein<br />
(EGFP)-expressing embryos in all of them. The<br />
spermatozoa of the five species were coincubated with<br />
pCX-EGFP plasmid and injected into the MII oocyte.<br />
The chemical activation protocol was ionomycin followed<br />
3 hours later by 3 hours of treatment with 6-DMAP. We<br />
detected high proportions of the fluorescent EGFP<br />
embryos in all five species (23 to 60%) at day four and<br />
produced green blastocysts in bovine, ovine and cat [30].<br />
In cattle, ICSI- mediated gene transfer was not<br />
evaluated until a report by Pereyra et al. [30] in 2008.<br />
The main reason for this is the poor outcome after<br />
conventional ICSI in this species. In another study we<br />
looked at the best conditions for intracytoplasmic ICSI-<br />
MGT in cattle. Various aspects of fertilization and<br />
embryonic development were assessed after five<br />
activation treatments. Spermatozoa were co-incubated<br />
with pCX-EGFP plasmid and injected into metaphase II<br />
oocytes, which were then treated with ionomycin (Io)<br />
before further activation with the following agents: 6-<br />
DMAP (Io-DMAP), additional Io plus 6-DMAP (2Io-<br />
DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium<br />
chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI,<br />
presence of a condensed spermatozoon head on Day 4,<br />
and blastocyst and EGFP expression rates on Day 7 were<br />
evaluated. Fertilization rates did not differ significantly<br />
among treatments. All (100%) of EGFP-positive embryos<br />
resulted from ICSI fertilization, whereas at least 60% of<br />
EGFP-negative embryos had a condensed sperm head.<br />
Blastocyst rates after 2Io-DMAP were not significantly<br />
different from Io-DMAP or Io-EtOH, but they were<br />
higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7,<br />
9.4, and 10.9% respectively). In bovine, ICSI-MGT<br />
proved to be a powerful technique because over 80% of<br />
the blastocysts expressed EGFP protein [2]. We evaluated<br />
LI, IVF and ICSI to produce egfp-expressing ovine<br />
embryos, using spermatozoa previously exposed to pCX-<br />
EGFP plasmid. High cleavage and morulae/blastocysts<br />
rates were obtained with LI and IVF, but no egfpexpressing<br />
embryos resulted. In contrast, 91.6% egfpexpressing<br />
morulae and blastocysts were generated by<br />
ICSI [29].<br />
VI. GENE TRANSFER BY NUCLEAR MICROINJECTION<br />
The first method reported to produce<br />
transgenic animals was the microinjection of foreign<br />
DNA into the male pronuclei of zygotes [12]. Transgenesis<br />
by microinjection is still used mainly for<br />
research purposes in mice. This procedure has proven<br />
to be efficient in species such as rabbits, sheep and<br />
pigs [13], but it is quite dependent on proper male<br />
pronuclei visualization and in some species, like the<br />
bovine, the efficiency is very low [10].<br />
VII. GENE TRANSFER BY CYTOPLASMIC<br />
MICROINJECTION<br />
Based in our results with ICSI, we tested if<br />
the sperm cell was a special cell to transfer the<br />
transgene, or if other cell types could also act as<br />
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.P. Bonnet<br />
onnet, et al. <strong>2011</strong>. Recent advances in micromanipulation and transgenesis in domestic<br />
mammals. Acta Scientiae Veterinariae. 39(Suppl 1): s285 - s293.<br />
vectors. Surprisingly, when we injected cumulus cells<br />
or oolemma vesicles previously incubated with<br />
eDNA, and naked eDNA into the cytoplasm of MII<br />
oocytes we observed expression of eDNA [29]. Using<br />
confocal microscopy interaction, an interaction of<br />
FITC- labeled eDNA with cumulus cells and vesicles<br />
was demonstrated. In contrast, oocytes injected with<br />
DNA alone did not show signs of transgene<br />
accumulation, and their eDNA expression rates were<br />
lower. In a further experiment, we evaluated if<br />
transgene-expressing embryos could be produced by<br />
means of vesicle injection followed by IVF.<br />
Preliminary FISH analysis indicated detectable<br />
integration events in 1/5 of SCNT blastocysts treated.<br />
Our studies demonstrate for the first time that short<br />
term transgene co-incubation with somatic cells can<br />
produce transgene-expressing mammalian SCNT<br />
embryos, and also that parthenogenic, eDNAexpressing<br />
embryos can be obtained by injection of<br />
vesicles or eDNA alone. Moreover, eDNA-expressing<br />
embryos can be also obtained by cytoplasmic<br />
injection of vesicles in IVF zygotes, simplifying the<br />
traditional IVF pronuclear injection technique.<br />
In other experiments different approaches were<br />
studied to improve transgenesis efficiency and to avoid<br />
mosaic expression patterns of transgene-expressing<br />
embryos. Circular and linear plasmid structures and cell<br />
cycle inhibitors (6-DMAP, and DhL) were tested to this<br />
aim [3]. Egfp expression was higher for linear than circular<br />
pCX-EGFP, and green blastocyst rates were higher<br />
for the groups inoculated with linear transgene incubated<br />
with vesicles than for free linear plasmid alone. FISH<br />
analysis showed integration evidence in green embryos.<br />
The cell cycle inhibitor 6-DMAP increased phosphorylated<br />
histone H2AX foci area, which mark DNA double<br />
stranded breaks and reduced mosaic expression.<br />
We tried a further simplification of the techniqe<br />
in bovine oocytes and zygotes, by intracytoplasmically<br />
injecting them with eDNA-liposomes complexes.<br />
Approximately 70% of the cleaved embryos and 50% of<br />
the blastocysts expressed EGFP, when egfp–liposome<br />
was injected 16 h post-fertilization [48].<br />
The percentage of integration of all these<br />
methods remains to be confirmed.<br />
VIII. DIFFERENT APPROACHES FOR EMBRYO<br />
MULTIPLICATION AND MOSAICISM REVERSION<br />
Our first approach consisted of the production<br />
of transgenic IVF embryos by vesicle microinjection to<br />
generate transgenic blastomeres to be used as donor<br />
cells for cloning. A high efficiency in mosaicism<br />
reversal and multiplication of transgenic embryos was<br />
attaineded [3].<br />
The separation of transgenic blastomeres<br />
followed by the aggregation of two-cell fused embryos<br />
or by the asynchronous younger blastomere successfully<br />
multiplied transgenic embryos, and theoretically reduces<br />
mosaicism rates in future offspring [15]. This technology<br />
can also be used to multiply embryos from animals with<br />
high genetic value.<br />
Another technique that we assessed was<br />
reducing mosaicism through gamete cloning. We<br />
demonstrated that a sperm and oocyte can be efficiently<br />
cloned [47,45,49]. Green haploid androgenic blastomeres<br />
produced with the injection of a single sperm by egfp<br />
ICSI-MGT could be used to fertilized oocytes resulting<br />
in several homogeneous expressing embryos. This<br />
approach shows great potential because it allows for<br />
determination of the sex of the sperm nucleus prior to<br />
fertilization. It is also possible to clone previously<br />
transfected oocytes [49] followed by the reconstruction<br />
of biparental bovine embryos to generate homogeneous<br />
transgene-expressing embryos. This opens the possibility<br />
for sperm or oocyte genome cloning by multiplying the<br />
gamete in a haploid line. This would have the potential Nto<br />
generate an unlimited number of biparental embryos by<br />
combining these haploid cells with haploid hemizygotes<br />
of the opposite sex.<br />
The applications of these technologies are subject<br />
to our capacity for imagination and innovation. Animals<br />
that we have generated with some of these<br />
methodologies (Figure 1, 2 and 3) have formed a kind of<br />
postcard into the future that demonstrates the possibility<br />
to use these technologies in our countries.<br />
IX. CONCLUSIONS<br />
Significant advancements have been made in<br />
the course of the recent years in micromanipulation<br />
and transgenesis techniques. In our lab we have been<br />
evaluating ICSI and Nuclear transfer mainly to produce<br />
transgenic embryos. We used also transgenesis<br />
developed to mark with flurorecence protein expression<br />
and develop new micromanipulation technique in domestic<br />
animals like gamete cloning. All these approach has<br />
enormous potential for use in livestock production. One<br />
of the main applications is introducing genes to modify<br />
the genome for biomedicine or agriculture.<br />
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alamone, R. Bevacqua,<br />
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.P. Bonnet<br />
onnet, et al. <strong>2011</strong>. Recent advances in micromanipulation and transgenesis in domestic<br />
mammals. Acta Scientiae Veterinariae. 39(Suppl 1): s285 - s293.<br />
Figure 1. BS Ñandubay Bicentenario cloned horse produce in 2010.<br />
Figure 2. Pampa dynasty: clones produced from a Growth Hormone<br />
transfected cell line in 2002.<br />
Figure 3. Esperanza lamb generated by ICSI produced in 2008.<br />
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.P. Bonnet<br />
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N<br />
www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
s293
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Maga & J.D. Mur<br />
urray. <strong>2011</strong>. Genetic engineering of livestock to improve human health: The human lysozyme<br />
transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s295 - s300.<br />
ISSN 1679-9216 (Online)<br />
Genetic engineering ing of livest<br />
estock to improve human health: The human lyso<br />
sozyme<br />
transgenic goat model<br />
Elizabeth A. Maga 1 & James D. Murray 1,2<br />
ABSTRACT<br />
Background: Transgenic animals have been generated for a variety of purposes including research tools, medical models,<br />
bioreactors (dairy animals producing human pharmaceuticals in their milk or in the egg whites of chickens) and for production<br />
agriculture (animals with increased growth, decreased environmental pollution, disease resistance), which includes the generation<br />
of animals designed to benefit human health. For example, transgenic goats expressing human lysozyme in their milk are being<br />
used as a model method to supply milk with antibacterial properties to help fight diarrheal illnesses in children. Lysozyme is a<br />
naturally occurring antimicrobial found in human milk at much higher levels than in the milk of dairy goats and cows. Lysozyme<br />
serves as part of the natural defense system against infection and also helps establish a healthy gut microbiota in the infant. We<br />
hypothesized that the presence of increased levels of lysozyme in the milk of dairy goats could offer several benefits that affect<br />
human health, including the promotion of a healthy gut microbiota and associated benefits such as improved growth and<br />
resistance to intestinal infections.<br />
Review: Research with this line of transgenic goats over the last 12 years has demonstrated that the presence and expression of<br />
the human lysozyme transgene is not detrimental to the animals themselves and that the milk can indeed act in an antimicrobial<br />
fashion when consumed by pigs, a model animal for human health, and impact the state of the intestine in a positive manner. Pigs<br />
consuming milk from human lysozyme transgenic goats had significantly lower levels of coliforms and E. coli in their intestine<br />
than did pigs consuming milk from non-transgenic control animals. In addition to bacterial changes, intestinal tissue of pigs<br />
N<br />
consuming milk from lysozyme transgenic goats had a significantly larger surface area with significantly fewer intraepithelial<br />
lymphocytes and an elevated level of expression of the anti-inflammatory cytokine TGF-â1 compared to control-fed animals, all<br />
indicators of a healthier intestinal tract. Metabolite profile analysis demonstrated significant differences in the levels of 18<br />
metabolites in the serum of pigs fed lysozyme milk with the direction of changes beneficial to the health of the animal. Finally, pigs<br />
consuming milk from lysozyme transgenic goats were more resistant to infection when challenged with an enteropathogenic E.<br />
coli, indicating a protective effect of lysozyme milk. In the semi-arid northeast of Brazil, 89 of 1000 children die before they reach<br />
the age of 2 years and 17% of these deaths can be attributed to diarrhea. The use of genetically engineered animals containing<br />
increased levels of lysozyme in their milk is a novel and simple approach to fight this problem. Work will be presented outlining<br />
the characterization of these animals and the impact of consuming the milk with the goal of one day having the milk available as<br />
a preventative or treatment agent for diarrheal illnesses.<br />
Conclusions: Genetic engineering is a viable approach to produce animal food products that can be used to improve human<br />
health. Brazil’s acceptance of this technology has positioned itself at the forefront to demonstrate to the world a new tool to help<br />
fight common diarrhea and its impact on the growth and development of children.<br />
Keywords: Genetic engineering, transgenic, lysozyme, diarrhea, goats.<br />
1<br />
Department of Animal Science and 2 Department of Population Health and Reproduction, University of California. CORRESPONDENCE: E.<br />
A. Maga [eamaga@ucdavis.edu]. One Shields Avenue, Davis, CA 95616, U.S.A.<br />
s295
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Maga & J.D. Mur<br />
urray. <strong>2011</strong>. Genetic engineering of livestock to improve human health: The human lysozyme<br />
transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300.<br />
I. INTRODUCTION<br />
II. HUMAN LYSOZYME AND THE PROTECTIVE<br />
PROPERTIES OF HUMAN MILK<br />
III. THE HUMAN LYSOZYME (HLZ) TRANSGENIC LINE<br />
IV. ANTIMICROBIAL ACTIVITY OF HLZ TRANSGENIC<br />
GOAT MILK<br />
V. IMPACT OF CONSUMING HLZ TRANSGENIC GOAT<br />
MILK<br />
VI. CONCLUSIONS<br />
I. INTRODUCTION<br />
The goal of genetic engineering (GE) of<br />
domesticated livestock is the same as breeding and<br />
selection: the introduction and propagation of a desired<br />
trait. In this approach, the genetic change is introduced in<br />
the form of a transgene consisting of the DNA encoding<br />
the gene of interest coupled to regulatory elements which<br />
are designed to express a specific protein in a tissue- and<br />
temporally-specific fashion to give the animal the desired<br />
trait. The first demonstration of an altered phenotype in<br />
an animal via transgenesis came in 1982 when increased<br />
growth was reported in transgenic mice expressing a rat<br />
growth hormone (GH) transgene [24]. The first reports<br />
of GE livestock soon followed in 1985 [13]. Since that<br />
time, the use of GE food animals has been focused on<br />
two main areas, namely the development of improved<br />
animals for production purposes including growth [1,7,13],<br />
decreasing the environmental footprint by reducing<br />
phosphorous pollution [12] and engineering disease<br />
resistance [28,35] and specialized non-agricultural<br />
purposes such as using dairy animals and chickens as<br />
bioreactors to produce human pharmaceuticals [8,9,15,26]<br />
or pigs to produce compatible organs for human transplant<br />
[25,34]. Also included is work designed to improve human<br />
health through the GE of animal food products including<br />
increased protein levels in milk [2], healthier fat<br />
composition of milk and meat [16,27], and the expression<br />
of antimicrobials in milk [18,19]. We have generated a<br />
line of transgenic dairy goats expressing increased levels<br />
of the antimicrobial human lysozyme in their milk with<br />
the intent of using the milk as a treatment or preventative<br />
agent against debilitating childhood diarrhea. Here, we<br />
will review work with our human lysozyme transgenic<br />
goat model with respect to the health and well-being of<br />
the transgenic line as well as work demonstrating the<br />
potential of the milk to improve intestinal health.<br />
II. HUMAN LYSOZYME AND THE PROTECTIVE<br />
PROPERTIES OF HUMAN MILK<br />
Lysozyme is a naturally occurring antimicrobial<br />
protein found in avian egg whites and the tears, saliva<br />
and milk of all mammals [reviewed by 22]. Lysozymes<br />
are part of the natural defense mechanism against bacterial<br />
infection and assist in digestion of intestinal bacteria.<br />
Lysozyme specifically catalyzes the cleavage of the<br />
glycosidic linkage between the C-1 of N-acetylmuramic<br />
acid and the C-4 of N-acetylglucoseamine that make up<br />
the peptidoglycan component of bacterial cell walls.<br />
Cleavage of the protective peptidoglycan layer by<br />
lysozyme causes leakage of the cell’s interior<br />
components and results in cell lysis. Lysozyme is<br />
more effective against gram positive bacteria but also<br />
has been demonstrated to kill gram negative bacteria.<br />
Lysozyme is naturally present in human milk at<br />
concentrations 1600 times greater than in goat milk<br />
[5]. Human milk maintains high levels of lysozyme<br />
(400 µg/mL) throughout lactation, as opposed to the<br />
milk of dairy animals which has high levels of<br />
lysozyme at parturition and involution, corresponding<br />
to when the animal is most susceptible to infection,<br />
thereby offering protection, with bovine milk<br />
averaging only 0.130 µg/mL and goat milk 0.250<br />
µg/ml of lysozyme.<br />
Lysozyme, along with lactoferrin and secretory<br />
IgA, are considered to be responsible for the passive<br />
immunity of human milk and play an important role for<br />
the infant by offering defense against bacterial infection<br />
by pathogenic organisms, promoting the development and<br />
maturation of the intestinal tract, acting as antiinflammatory<br />
agents, and stimulating a beneficial gut<br />
microbiota [reviewed by 11,17]. Breast-fed human infants<br />
tend to have a more simple gut microbiota comprised<br />
primarily of Bifidobacteria, along with Lactobacilli and<br />
Staphylococci, while the fecal flora of formula-fed infants<br />
is more complex, with Coliforms, Enterococci,<br />
Bacteroides, Clostridia and Streptococci all being<br />
prevalent [29]. One reason for the growth of fewer<br />
facultative anaerobes in breast-fed infants is believed to<br />
be the antimicrobial factors in human milk such as<br />
lysozyme and lactoferrin [23]. A probiotic intestinal<br />
microbiota is thought to confer a number of positive<br />
benefits including growth, protection against diarrhea and<br />
other gastrointestinal illnesses [reviewed by 32] and<br />
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Maga & J.D. Mur<br />
urray. <strong>2011</strong>. Genetic engineering of livestock to improve human health: The human lysozyme<br />
transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300.<br />
indeed, breast-fed infants are less afflicted by acute and<br />
chronic diseases, including infections of the<br />
gastrointestinal, respiratory, and urinary tract than are<br />
formula-fed infants [30].<br />
Diarrhea is one of the leading causes of death<br />
of children worldwide. According to the WHO, two<br />
million children under the age of five die each year<br />
from common diarrheal illnesses. In addition, multiple<br />
episodes of acute and persistent diarrhea can leave<br />
lasting nutritional and cognitive shortfalls. In the semiarid<br />
Northeast region of Brazil, childhood mortality<br />
rates are 3 times higher than the rest of the country<br />
(89 deaths/1000 births by the age of 2) with death<br />
due to diarrhea accounting for 15-20% of the overall<br />
deaths. Breastfeeding is the recommended<br />
intervention for prevention of diarrhea in young children.<br />
A series of studies documented the reduction of diarrhea<br />
episodes and also a fast recovery in breast-fed children.<br />
If the amounts of important human antimicrobial proteins<br />
could be increased in the milk of common dairy animals<br />
such as the goat, there would be a continuous supply of<br />
milk that mimics the antimicrobial function of human milk.<br />
One means of providing the beneficial properties<br />
of lysozyme to human consumers is to GE dairy animals<br />
to produce milk with higher levels of lysozyme throughout<br />
lactation. The consumption of increased amounts<br />
lysozyme would pose little risk to human consumers of<br />
the milk as lysozyme is naturally present in saliva and<br />
thus already consumed. By producing the human form<br />
of lysozyme, no allergic reactions would be anticipated<br />
and lysozymes in general are not known to be related to<br />
any known allergens or toxins. Indeed, lysozyme from<br />
hen egg whites is currently used with Generally<br />
Recognized As Safe (GRAS) status as a preservative in<br />
the food industry (sprayed on cheeses and meats, in edible<br />
films and in cosmetics) to prevent product spoilage caused<br />
by bacteria [22]. Lysozyme is also able to retain activity<br />
at pasteurization temperatures [31], survive transit, and<br />
be active in the environment of the gastrointestinal tract<br />
[10].<br />
If lysozyme were expressed at a higher level in<br />
a goat mammary gland throughout the duration of<br />
lactation, we hypothesized that several benefits could be<br />
considered that affect both animal and human health while<br />
posing little risk. Because of its antimicrobial nature,<br />
lysozyme in milk could reduce the growth of bacterial<br />
contaminants in milk that cause disease in humans<br />
(Listeria) making for a safer product for consumption<br />
and also reduce the growth of bacteria that case the<br />
spoilage of milk thereby increasing the shelf-life of milk<br />
and thus the availability of the nutrients. The presence of<br />
an antimicrobial in the udder could also decrease the<br />
incidence and severity of mastitis, thereby improving animal<br />
health and welfare. Furthermore, due to its<br />
purported role in human breast milk, consumption<br />
of lysozyme-rich milk could promote a healthy gut<br />
microbiota in individuals consuming the milk, thereby<br />
imparting health benefits such as improved growth,<br />
reduction or cessation of illness, and resistance to new<br />
infections. We therefore propose a strategy to improve<br />
human health based on local agriculture, whereby<br />
milk from GE dairy goats producing increased levels<br />
of human lysozyme can be used to treat and/or<br />
prevent diarrhea in children of all ages. The work<br />
described below directly addresses these possibilities.<br />
III. THE HUMAN LYSOZYME (HLZ) TRANSGENIC LINE<br />
A line of transgenic dairy goats of Alpine and<br />
Toggenburg origins was generated using standard<br />
pronuclear microinjection with a bovine α s1<br />
-casein-HLZ<br />
cDNA transgene [18]. To date we have produced, by<br />
natural breeding, a total of 82 (37 female and 45 male)<br />
hemizygous transgenic goats through the 5 th generation<br />
carrying and expressing this transgene. This line of animals<br />
transmits the transgene in a Mendelian fashion, stably N<br />
expresses the transgene at the mRNA and protein level<br />
across generations, and the HLZ in milk is biologically<br />
active [19]. HLZ protein expression in milk of these HLZ<br />
transgenic goats averages 270 ± 84 µg/mL [19]. This<br />
represents a 1000 fold increase over the mean level of<br />
lysozyme normally present in goat milk and is<br />
approximately 68% of that normally found in human milk.<br />
The percentage of milk yield that represents total fat and<br />
protein was the same range as the means for our nontransgenic<br />
control dairy goat herd [19], indicating that<br />
expression of the transgene did not disrupt the gross<br />
composition of milk. Basic functions such as growth and<br />
reproduction of the transgenic animals themselves were<br />
not adversely impacted by either the presence or<br />
expression of the transgene, or by consumption of the<br />
HLZ-containing milk [14].<br />
IV. ANTIMICROBIAL ACTIVITY OF HLZ TRANSGENIC<br />
GOAT MILK<br />
Several strains of bacteria important to animal<br />
health and food safety were susceptible to HLZ<br />
transgenic goat milk in vitro [20]. When incubated<br />
with various bacterial isolates, milk from HLZ transgenic<br />
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Maga & J.D. Mur<br />
urray. <strong>2011</strong>. Genetic engineering of livestock to improve human health: The human lysozyme<br />
transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300.<br />
animals significantly slowed the growth of S. aureus, E.<br />
coli and P. fragi as demonstrated by an overall lower<br />
mean number of colony forming units (CFU)/mL after<br />
incubation than milk from non-transgenic controls. The<br />
growth of a lactic acid bacteria (L. lactis) was not<br />
affected by the presence of HLZ in milk. Milk from<br />
HLZ transgenic animals was also capable of slowing<br />
the growth of bacteria in vivo. Milk from HLZ transgenic<br />
animals was found to have a different bacterial population<br />
corresponding to a longer shelf life [20]. Fewer bacteria<br />
grew in milk of transgenic animals and milk survived at<br />
room temperature for longer periods than control milk<br />
before bacterial growth occurred. The differential growth<br />
of bacteria in milk from transgenic animals indicates that<br />
HLZ expressed in milk is able to act in an antimicrobial<br />
fashion to alter the growth of bacteria in vivo.<br />
V. IMPACT OF CONSUMING HLZ TRANSGENIC GOAT<br />
MILK<br />
As the efficacy of HLZ in milk was confirmed<br />
as described above, the biological action of HLZ milk at<br />
the level of the intestine after consumption was evaluated<br />
by assessing the growth of coliform bacteria in the small<br />
intestine in two animal models, the goat and the pig. Pigs<br />
represent a monogastric animal with a digestive tract<br />
similar to humans. The use of pigs as a relevant human<br />
medical model is well documented [33] as pigs are<br />
frequently used in cardiovascular and nutritional research.<br />
Due to the antimicrobial properties of HLZ and evidence<br />
that natural components of milk result in different intestinal<br />
microbiota and overall intestinal development, it is<br />
our hypothesis that the consumption of milk containing<br />
active HLZ will impact intestinal microbiota, and thus<br />
intestinal health, and resistance to intestinal illness. The<br />
first part of this hypothesis was shown to be correct as<br />
pasteurized milk from HLZ transgenic animals was<br />
capable of modulating intestinal bacteria in both ruminant<br />
and non-ruminant animal models [21]. In the more humanrelevant<br />
model, weanling pigs receiving pasteurized milk<br />
from HLZ transgenic animals for 16 days had significantly<br />
lower numbers of coliforms and E. coli in their intestine<br />
than did pigs fed milk from non-transgenic control animals<br />
[21]. These data indicate that HLZ expressed in the milk<br />
of dairy animals can indeed be biologically active in the<br />
intestine and modulate gut microbiota, much like human<br />
milk.<br />
Further work in pigs confirmed the second<br />
part of our hypothesis by demonstrating that<br />
consumption of pasteurized milk from HLZ transgenic<br />
goats resulted in beneficial changes in gut histology and<br />
protection against intestinal infection [3]. Pigs fed HLZ<br />
milk had fewer numbers of coliforms and E. coli in both<br />
the duodeneum and ileum than did pigs fed milk from<br />
non-transgenic controls, repeating the findings of our first<br />
study. Animals receiving HLZ milk had significantly wider<br />
villi in the duodenum indicating a healthier gut with<br />
increased absorptive area. The number of intraepithelial<br />
lymphyocytes per micron of villi height was significantly<br />
decreased in the duodenum of HLZ-fed animals, an<br />
additional indicator of increased gastrointestinal tract<br />
health. Animals fed HLZ milk for a period of 4 weeks<br />
and then challenged with a porcine-specific<br />
enteropathogenic Escherichia coli (EPEC) had<br />
significantly lower levels of coliforms and E. coli in their<br />
ileum than did those receiving milk from non-transgenic<br />
control animals, indicating a protective effect of HLZ<br />
milk against EPEC infection. In addition, standard CBC<br />
analysis indicated that no allergic response was occurring<br />
upon consumption of HLZ milk. Furthermore, there was<br />
no significant difference in the expression of key proinflammatory<br />
cytokines (TNF-α and IL-8) in intestinal<br />
tissue of pigs consuming HLZ or control milk, indicating<br />
that an inflammatory response is not induced upon<br />
consumption of HLZ milk [6].<br />
Serum from non-challenged pigs was also<br />
subjected to a metabolite profiling analysis [4]. A total of<br />
234 metabolites were quantified (178 known, 56 unknown)<br />
with levels of 18 known metabolites and 4 unknown<br />
metabolites being significantly different in pigs reared on<br />
HLZ milk compared to pigs that received control milk.<br />
These differences could be broken down into effects of<br />
bacteria, increased growth, healthier gastrointestinal tract,<br />
and modulation of the immune system with the direction<br />
of changes indicative of a healthier gut. In addition,<br />
consumption of HLZ milk significantly increased the<br />
expression of the anti-inflammatory cytokine TGF-â1 in<br />
the small intestine [6], again indicative of a healthier gut.<br />
Taken together, these data strongly suggest that milk from<br />
HLZ transgenic goats can be used to improve human<br />
health by fighting the high childhood mortality rates<br />
associated with common diarrheal illnesses.<br />
VI. CONCLUSIONS<br />
Data collected over the years on most<br />
applications of transgenic animals for agriculture<br />
indicate that the implementation of GE animals can<br />
indeed have a positive impact on animal productivity<br />
and sustainability as well as animal and human health.<br />
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urray. <strong>2011</strong>. Genetic engineering of livestock to improve human health: The human lysozyme<br />
transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300.<br />
The HLZ transgenic goat model has produced convincing<br />
data that the adoption of GE livestock has the potential to<br />
benefit human health with little risk. Future work with<br />
this approach will revolve around translating the use of<br />
HLZ milk to treat/prevent diarrhea by developing a pig<br />
model of infection and generating lactoferrin transgenic<br />
goats. Our hope is to contribute a new solution to an old<br />
problem and prevent the suffering and deaths associated<br />
with common diarrhea in Brazil and throughout the world.<br />
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transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300.<br />
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20 Maga E.A., Cullor J.S., Smith W., Anderson G.B. & Murray J.D. 2006. Human lysozyme expressed in the mammary gland<br />
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www.ufrgs.br/actavet<br />
39(Suppl 1)<br />
s300
M.M. Franc<br />
anco, A. Pellegr<br />
ellegrin,<br />
R.A. Figueir<br />
igueiredo<br />
edo, et al. <strong>2011</strong>. The Innovation Network in Animal Reproduction: EMBRAPA’s<br />
Experience in Organizing a Research Project on Reprodutive... Acta Scientiae Veterinariae. 39(Supl 1): s1 - s5.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s301.<br />
ISSN 1679-9216 (Online)<br />
Use of Color-D<br />
olor-Doppler oppler Ultrasono<br />
asonogr<br />
graph<br />
aphy to Monit<br />
onitor or Follicle Dynamics in Horses<br />
Eduardo Leite Gastal<br />
ABSTRACT<br />
Background: Recently, transrectal Doppler ultrasonography has been utilized increasingly for research and clinical studies of<br />
ovarian and follicle hemodynamics in large farm animals. High-resolution ultrasonographic machines with B-mode (grayscale)<br />
and color-, power-, and spectral-Doppler modes have brought a powerful dimension to the evaluation of the equine<br />
preovulatory follicle during recent years. These technologies have permitted the development of more in depth scientific and<br />
clinical studies with regard to the characteristics of the preovulatory follicle and the ovulation process. The striking similarities<br />
between mares and women in follicle dynamics and hormonal changes during the interovulatory interval and the ovulatory<br />
follicular wave, in ultrasonographic changes of the preovulatory follicle before ovulation, and in reproductive aging processes<br />
highlight the relevance of the mare as an important experimental model for the study of folliculogenesis in women.<br />
Review: The use of color-Doppler technology to evaluate the vascularity of the follicle wall in mares started in 2004. Follicle<br />
blood-flow assessment by Doppler ultrasonography has been used in mares to study: (a) follicle selection, (b) anovulation<br />
during transitional seasons, (c) first versus later ovulations of the year, (d) follicle maturity and proximity to ovulation, (e)<br />
oocyte recovery rate, maturity, and quality, (f) normal vs. abnormal ovulation (septated evacuation) or anovulation (hemorrhagic<br />
anovulatory follicles -HAFs or luteinized unruptured follicles -LUFs), (g) the relationship of circulatory hCG antibodies on<br />
follicle vascularity, maturity and oocyte qualities, (h) age-related effects, and (i) potential for pregnancy establishment. Greater<br />
vascularity of the preovulatory follicle has been associated with greater follicle diameter (women, mares, heifers), retrieval rate<br />
of oocytes (women, mares, heifers), retrieval rate of mature oocytes (mares), in vitro fertilization rate (women, heifers), pregnancy<br />
rate (women, mares, heifers), and a lower incidence of triploidy (women). In addition, follicles with greater blood flow have N<br />
resulted in better embryos and more pregnancies after embryo transfer in women. This presentation will focus on the main<br />
findings of our experiments that used B-mode and color-Doppler ultrasonography during the preovulatory period to study the<br />
morphological and blood flow/perfusion changes of the preovulatory follicle in mares. The topics to be addressed in this<br />
presentation will be: ultrasonographic characteristics of the preovulatory follicle; B-mode echotextural changes of the follicle<br />
wall; blood flow and perfusion changes of the follicle wall; signs of impending ovulation; prediction of impending ovulation;<br />
types of preovulatory follicle outcomes (ovulation, septated evacuation, HAF, atresia); follicle blood flow during evacuation;<br />
entry of follicular fluid into the abdomen; infundibular fluid; early corpus luteum blood flow; vascularity of the preovulatory<br />
follicle versus fertility; and the influence of hCG antibodies on follicle blood flow and oocyte quality.<br />
Conclusion: Results of recent studies have demonstrated the potential of Doppler ultrasonography for providing clinical<br />
information on the status and future success of a follicle to ovulate and its oocyte to become fertilized and to generate an<br />
embryo/pregnancy. The equine model allows hypothesis testing using the three Doppler technologies for examining the<br />
ovaries and may provide additional information that can also be considered useful for other farm animal species and in human<br />
clinical medicine. This presentation is directed to equine theriogenologists and scientists who are involved in monitoring,<br />
managing, and manipulating ovarian function in mares.<br />
Keywords: color-Doppler, equine, follicle, ovary, ultrasonography.<br />
Department of Animal Science, Food and Nutrition, Southern Illinois University Carbondale, IL, USA. CORRESPONDENCE: E.L. Gastal<br />
[egastal@siu.edu]. Department of Animal Science, Food and Nutrition, Southern Illinois University Carbondale, 1205 Lincoln Drive, MC<br />
4417, Carbondale, IL 62901, USA.<br />
s301
R.C. Uliani, L.A. Silv<br />
ilva,<br />
M.A. Alv<br />
lvar<br />
arenga.<br />
<strong>2011</strong>. Mare’s Folliculogenesis: Assessment of ovarian and perifollicular<br />
vascular perfusion by Doppler ultrasound. Acta Scientiae Veterinariae. 39(Suppl 1): s113 - s116.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s303.<br />
ISSN 1679-9216 (Online)<br />
Biotechnology of reproduction in the canine species: where do we go?<br />
John Verstegen¹, Karine Verstegen-Onclin¹ & Karine Reynaud 2<br />
ABSTRACT<br />
Background: In most species significant advancement in biotechnology of reproduction have occurred over the last 30 to 40<br />
years. Bovine reproduction has known a development similar or significantly larger than human reproduction with nowadays<br />
the routine use of semen or embryos in vitro technologies including IVF, IVM, ICSI as well as transgenesis, cloning, or the most<br />
advanced proteomic and genomic developments. In the canine although semen technologies have known a similar development<br />
as in other species, only a few significant progresses have been published in terms of oocytes and embryos. A basic, in other<br />
species, technology like embryo transfer, is still unfortunately not yet available.<br />
Review: If the dog is nowadays considered as a significant model for human diseases , environmental toxicology including<br />
endocrine disruptors, or genetic evaluation, the funding of research to allow a better understanding of its reproductive<br />
biology as well as improvement of the in vitro efficiency have been limited. Oocytes and embryo developments are essentially<br />
limited to in vivo while in vitro technologies have up to now failed to provide the resources needed to allow for a significant<br />
commercial development of artificial reproduction and all the associated benefits for the canine species or as a comparative<br />
model. Besides the recent successes related to dog cloning, unfortunately limited by exclusive rights and licensing agreement<br />
limiting the overall extension of NT in the species, only one aborted pregnancy using in vitro fertilized oocytes has up to now<br />
been presented almost 10 years ago. The limiting factors to the penetration of these technologies in dogs are among others<br />
limited knowledge of basic physiology, poor availabilities of tissues (oocytes or ovaries) , specific biology of oocytes<br />
maturation, ovulation and development, limited treatment and therapeutic approaches, costs of the procedures and limited<br />
fund’s availability while fighting against the lobby of animal protectionism and spay and neuter campaigns developed Nto<br />
control the pet overpopulation problem present worldwide. However, despite all these constraints and limiting factors, on a<br />
limited scientific basis or with commercial objectives, progresses are made and presented every year associated with hope and<br />
expectative: genetic identification of diseases, sexing technologies or stem cells initiatives are encouraging.<br />
Conclusions: The specificities of dog reproductive anatomy, physiology and biology require numerous adaptations to the<br />
other species worldwide developed technologies. Most techniques are either poorly efficient or rendered unavailable by<br />
commercial regulations and licensing restrictions preventing their development to occur. However, the significant interest for<br />
the dog as companion animals as well as the advantages associated with this large animal model for human diseases or as a<br />
model for the protection of endangered species of carnivores should be associated during the coming 5 or 10 years with a<br />
marked development of the most advanced procedures like in human or bovine and lead to significant progress in canine<br />
reproductive biotechnologies.<br />
Keywords: canine, assisted reproduction, biotechnology, biology, development, embryos, ET, cloning.<br />
1<br />
Minitube of America, Venture Court 419, Verona, WI, USA. 2 Ecole Nationale Vétérinaire d’ Alfort (ENVA), UMR 198 Biologie du development<br />
et Reproduction, 7 Avenue du Général de Gaulle, 94700 Maisons-Alfort, France. CORRESPONDENCE: J.P. Verstegen<br />
[Jverstegen@Minitube.com - FAX: +1 (608) 845-1522]. Minitube of America, Venture Court 419. Verona, 53593 Wisconsin, USA.<br />
s303
C.G. Gutier<br />
utierrez,<br />
S. Fer<br />
errar<br />
aro, V. Mar<br />
artine<br />
tinez,<br />
et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s305 - s316.<br />
ISSN 1679-9216 (Online)<br />
Increasing ovulation quota: more than a matter of energy<br />
Carlos G. Gutierrez, Silene Ferraro, Victor Martinez, Adriana Saharrea, Clarisa Cortez, Arantzatzu Lassala,<br />
Héctor Basurto & Joel Hernandez<br />
ABSTRACT<br />
Background: Nutritional supplementation before breeding (Flushing) has become a common practice and is a reliable method<br />
to improve lambing and twining rates in sheep. The improvement of the body condition of a ewe is reflected in a higher number<br />
of ovulatory follicles and is termed “static effect of nutrition”. Shorter periods of nutritional supplementation can also affect<br />
follicular development in the absence of changes in the body condition and weight of the animal, which is known as “acute effect<br />
of nutrition”. Studies of follicular development in small ruminants have shown that 4 to 5 follicular waves occur during the<br />
estrous cycle, and that waves emerge every 5 to 7 days. However, the selection phase of the follicular wave occurs within 3 to<br />
4 days, thus the length of flushing could last merely the time needed to push follicles over the selection threshold.<br />
Review: Here we examined the evidence produced by our research on the minimum length and appropriate timing of nutrient<br />
supplementation needed to enhance ovulation rate and prolificacy in sheep. Ewes have follicles ready to reach ovulatory size at<br />
any time of the estrous cycle and, when a follicular phase is induced, most show estrus and ovulate within 60 to 80 h. Hence,<br />
ovulatory follicles should commit for ovulation shortly after the decline in progesterone if they are to achieve ovulatory<br />
competence. We showed that an ultrashort flushing (USF) given as a single administration of a glycogenic substance at the time<br />
of prostaglandin-induced luteolysis (Control =1.6 ± 0.06 vs. USF= 2.08± 0.06) or progestin withdrawal (Control =1.64 ± 0.07 vs.<br />
USF= 2.41± 0.09) increased ovulation rate (P < 0.01). This increase was associated with elevated glucose and insulin concentrations<br />
for 12 h after USF (P < 0.01). However, the diameter of the three largest follicles did not change between the day of flushing and<br />
the day of estrus and did not differ between the control and the USF (P > 0.10). The USF could act either by advancing follicle N<br />
maturation, or by affecting the feedback loop between the ovaries and gonadotrophin secretion. Therefore, we measured mRNA<br />
abundance for LH receptor (LHr), 3β-hydroxysteroid dehydrogenase (3β-HSD) and P450 aromatase at 0, 12, 24 and 48 h after the<br />
start of luteolysis in ewes treated or not with USF. Aromatase mRNA decreased in large follicles 12 h after USF (P < 0.01), with no<br />
changes on mRNA for LHr or 3β-HSD. Further, we observed that the in vitro rumen fermentation and the in vivo glucose plasma<br />
concentrations in response to an isoenergetic (1470.83 kcal) single oral drench of glycerol, propylene glycol or molasses differed.<br />
The longer time to begin fermentation of glycerol allows for its absorption and its direct use for glucose production in the liver.<br />
Molasses was preferably fermented to butyrate, whereas propylene glycol was preferably fermented to propionic acid thus<br />
serving as glycogenic substrate. Glycerol and propylene glycol increased glucose and insulin concentrations in vivo, whereas<br />
molasses did not, thus the later may not be suitable for the USF. An increase in ovulation rate and prolificacy was also obtained<br />
with treatments that cause nutrient redistribution in the animal such as beta adrenergic receptor agonists and bovine somatotropin.<br />
Conclusions: We have developed an ultrashort flushing with glycogenic solutions that when applied at luteolysis will increase<br />
ovulation rate in sheep. This hyper-acute effect of nutrition caused a reduction in mRNA for P450 aromatase 12 hours after the<br />
glycogenic drench. In addition, from our results we can infer that ovine follicles can develop ovulatory capacity within 48 h after<br />
being selected when coinciding with the follicular phase.<br />
Keywords: flushing, ovulation rate, nutrition, sheep.<br />
Facultad de Medicina Veterinaria y Zootecnia. Universidad Nacional Autónoma de México. CORRESPONDENCE: C.G. Gutierrez<br />
[carlos_gutierreza@hotmail.com]. Av. Universidad 3000, 04510, México, DF, México. Organ that provided funding for the cited studies:<br />
CONACYT (Research Grant CB103801).<br />
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C.G. Gutier<br />
utierrez,<br />
S. Fer<br />
errar<br />
aro, V. Mar<br />
artine<br />
tinez,<br />
et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
I. Introduction<br />
II. Length and timing of the flushing treatment<br />
III. Mechanisms by which the ultra short flushing enhances<br />
the ovulation rate<br />
IV. Are all glycogenic substances equal for the ultrashort<br />
flushing?<br />
V. What is the minimum dose needed to increase ovulation<br />
rate with an ultrashort flusing?<br />
VI. Other manipulations to increase ovulation rate<br />
VII. Concluding remarks<br />
I. INTRODUCTION<br />
Flushing is one of the oldest reproductive<br />
technologies known to improve reproductive performance<br />
and to enhance the lambing rate of sheep and goat flocks<br />
[20,34]. The first scientific report of flushing in 1899 [11],<br />
describes that sheep that are heavier or with a higher<br />
nutritional intake have a higher proportion of twin lambings.<br />
Subsequent studies have confirmed that as body weight<br />
of the ewe increases, there is also an increase in the<br />
twinning rate [6] and in the ovulatory quota [24]. This<br />
effect of nutrition on ovulation rate that is associated with<br />
elevated body weight is termed “static effect of nutrition”<br />
[33,39]. Traditionally, the recommendation for animal<br />
husbandry is to improve the feeding in quantity and/or<br />
quality of the sheep somewhat around 2 months to 3<br />
weeks prior to the breeding season, in an attempt to<br />
improve the body condition of the sheep and thus achieve<br />
higher lambing rates and prolificacy. In the tropics, where<br />
high quality pastures are not available, sheep need to be<br />
fed nutritional supplements containing proteic or energy<br />
loaded components to improve these parameters.<br />
However, this approach is rather time consuming and in<br />
some instances the cost could make it impractical.<br />
Food supplementation may acutely increase<br />
ovulation rate without affecting the body condition or the<br />
weight of the animal. This effect is termed “acute effect<br />
of nutrition” where supplementation given for only 4 to 9<br />
days can increase ovulation rate [19,36]. Studies of<br />
follicular development in small ruminants have shown<br />
that 4 to 5 follicular waves occur during the estrous cycle,<br />
and that waves emerge every 5 to 7 days [1,18]. The<br />
selection phase of the follicular wave lasts only 3 to 4<br />
days, thus, if ovulatory follicles are to be chosen from the<br />
pool of growing follicles, flushing may not need to last<br />
more than the length of a follicular wave.<br />
To get an insight into the mechanism governing<br />
the selection of ovulatory follicles and ovulation rate as<br />
affected by nutritional influences, we have used a model<br />
where flushing is given via glycogenic solutions. Glycerol<br />
and propylene glycol are substances often used as energy<br />
additives in ruminant nutrition for their glycogenic and<br />
anti-ketogenic properties [17,31]. In dairy cows, their<br />
addition in early lactation diets decreased free fatty acidβ<br />
and β-hydroxybutyrate whilst increasing blood glucose<br />
concentrations [5,27].<br />
In this manuscript we will examine the evidence<br />
produced by our research regarding the minimum length<br />
of the nutritional supplementation needed to enhance<br />
ovulation rate and prolificacy, and the appropriate timing<br />
for nutrient supplementation.<br />
II. LENGTH AND TIMING OF THE FLUSHING TREATMENT<br />
Recent studies have shortened the length of<br />
flushing establishing a span of supplementation between<br />
nine and four days prior to breeding. However, the<br />
minimum length of flushing needed to increase ovulation<br />
rate is still to be determined. The answer to this question<br />
could depend on the time that a follicle needs to grow<br />
and mature to reach an ovulatory status. In small<br />
ruminants, the follicular wave is no more than 6 to 7 days<br />
long, and the ovulatory follicle is selected only a few days<br />
before ovulation [32,35] Thus, it appears that in these<br />
species, an ovulatory follicle could grow and develop to<br />
reach ovulatory capacity within a short period of time.<br />
We therefore tested whether a short flushing, starting<br />
three days prior to the end of the luteal phase, would<br />
suffice to induce an increase in ovulation rate. Following<br />
this protocol, we administered glycerol (100 mL, tid) as<br />
a glucogenic substrate and found that glycerol drench<br />
increased both glucose and insulin concentrations, and<br />
the number of ovulating follicles form 1.2 to 1.6 [30].<br />
Similar results have been obtained by other investigators<br />
after short flushing treatments with oral glycogenic<br />
substances [19], intravenous infusions of glucose [13,25]<br />
or glucosamine [25], or by adding lupin to the diet [8,26].<br />
It appears that the increase in ovulation rate is related to<br />
an increase in glucose, insulin and leptin concentrations<br />
without detectable changes in IGF-I or FSH [38].<br />
Although the follicular waves in sheep and goats,<br />
as for other ruminants, have a recruitment phase where<br />
a pool of follicles respond to FSH, and further a selection<br />
and dominant phases, their waves are not as clearly<br />
spaced as in cattle and seem to overlap in time. In<br />
addition, sheep and goats appear to have follicles prepared<br />
to reach ovulatory size at any time of the estrous cycle,<br />
since when a follicular phase is induced by prostaglandin<br />
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errar<br />
aro, V. Mar<br />
artine<br />
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et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
administration or by the withdrawal of a progestin<br />
treatment, most sheep show estrus and ovulate within 60<br />
to 80 h respectively. Furthermore, the ovulatory follicles<br />
could originate from the last, second last or both waves<br />
of follicular development [18]. In addition, when follicles<br />
that ovulated derived from the last and second last<br />
follicular waves, the second follicle was selected within<br />
the previous two days [18]. Therefore, this evidence<br />
allows us to speculate that in small ruminants: 1) The<br />
dominance effected by the largest follicle does not<br />
completely suppress the development of new follicles up<br />
to a gonadotrophin dependent stage; 2) Regardless of<br />
the stage of the oestrous cycle, sheep have follicles well<br />
advanced in the developmental process, capable to grow<br />
and mature within a very short period of time and ovulate<br />
if the adequate endocrine stimuli is encountered and 3) A<br />
second ovulatory follicle could be drawn from the latest<br />
follicular wave even in the presence of a dominant follicle.<br />
These observations led us to believe that the period<br />
needed to achieve an increase in ovulation rate by<br />
flushing could be further shortened if given at a time where<br />
follicles predestined for ovulation are selected. The precise<br />
moment for this stimulus seems to be the time where<br />
the LH pulse frequency increases after the decline in<br />
progesterone concentrations (i.e. the start of luteolysis).<br />
However, the strength and duration of the stimulus<br />
remains to be determined. Ovulatory follicles should<br />
commit for ovulation shortly after the decline in<br />
progesterone if they are to achieve ovulatory<br />
competence. Consequently, we hypothesized that the<br />
window of time where the follicle is selected for ovulation<br />
occurs within the first 12 hours after the initiation of<br />
luteolysis.<br />
A short nutritional stimulus that effectively<br />
enhances ovulation rate would necessarily be acting either<br />
directly at the ovarian level or through hormones and<br />
factors other than gonadotropins, whose concentrations<br />
would be directly affected by the particular given nutrient.<br />
We chose to use oral glycerol as the flushing stimulus<br />
since it can be administered as a drench, it provides a<br />
reliable source of quickly available energy, and is short<br />
lived, ensuring that the stimulus is focused in a limited<br />
and defined period of time. We characterized the<br />
peripheral concentrations of glycerol, glucose and insulin<br />
and the ovulation rate in response to this ultrashort flushing<br />
(USF) in sheep whose estrous cycles were synchronized<br />
either with prostaglandin F2α or progestin protocols. The<br />
ovaries of these sheep were scanned by ultrasonography,<br />
and animals bearing a corpus luteum were treated with<br />
PGF2α and assigned randomly either to a control group<br />
or to a group where the USF was given as a single drench<br />
of 300 mL glycerol (90% glycerol: 10% water) at the<br />
time of PGF2α injection. Estrus was detected by a<br />
vasectomized ram and ovulation was estimated by<br />
counting the number of corpora lutea by ultrasound<br />
scanning seven days later. Ultrashort flushing, at the time<br />
of luteolysis, increased the ovulation rate of the sheep<br />
with 89% of ewes having multiple ovulations, and 17.5%<br />
with three or more ovulations (P < 0.01; Table 1) [22].<br />
Progestin containing devices are a common<br />
method for the synchronization of estrus in sheep.<br />
However, treatment with progestins to synchronize the<br />
estrous cycle, when in the absence of the CL as a natural<br />
source of progesterone, modifies follicular development N<br />
in cattle [3], and sheep [10], inducing the formation of<br />
persistent dominant follicles. Therefore, we tested the<br />
effect of the USF in sheep synchronized with<br />
fluorogestone acetate containing intravaginal sponges [22].<br />
Ultrashort flushing at the time of pessary withdrawal<br />
increased the proportion of ewes with multiple ovulations<br />
(P < 0.01), 43% ovulating three or more follicles, 51%<br />
with double ovulations and only 6% with single ovulations<br />
(Table 2). In addition, blood concentrations of glycerol<br />
and glucose increased (P < 0.01) immediately after USF<br />
and remained elevated for at least 10h post-treatment<br />
(Figure 1). However, there were no differences in<br />
Table 1. Ovulation type and ovulation rate in Pelibuey ewes following ultrashort flushing (USF)<br />
with a single oral administration of 300 ml of a glycogenic solution (glycerol: water; 90:10 v/v) at<br />
estrous synchronization with PGF2α.<br />
Treatment Group n Ovulation type (%) Ovulation rate<br />
Single Double Triple Quadruple<br />
Control a 58 39.66 60.34 0 0 1.6 ± 0.06 a<br />
USF b 74 10.81 71.62 16.22 1.35 2.08 ± 0.06 b<br />
a,b Different superscripts within a column differ (P < 0.01).<br />
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C.G. Gutier<br />
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errar<br />
aro, V. Mar<br />
artine<br />
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et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
glucose, insulin and IGF-I concentrations 24 h after<br />
flushing compared to their respective concentrations<br />
before drenching [22].<br />
In a further study, the follicular dynamics were<br />
assessed in 12 sheep by ultrasound scanning. On day 10<br />
of the estrous cycle ewes were injected with PGF2α<br />
and treated with USF as previously described. Ultrasound<br />
scanning of the ovaries was carried out from day one of<br />
the previous cycle and continued until the next estrus<br />
occurred. The number of small (4mm)<br />
follicles was not affected by USF. Similarly, the diameter<br />
of the three largest follicles present in the ovaries at the<br />
time of estrus was not different between treatments and<br />
there were no changes in the diameters of these follicles<br />
between the day of flushing and the day of estrus [22].<br />
This hyper-acute effect of flushing demonstrates<br />
that the time of luteolysis is a period where follicles could<br />
be selected for ovulation concomitant with a transient<br />
increase in glucose lasting for at least 10 h, but less than<br />
24 h, that suffice to stimulate the selection of more than<br />
Table 2. Ovulation type and ovulation rate in Pelibuey ewes following ultrashort flushing (USF)<br />
with a single oral administration of 300 ml of a glycogenic solution (glycerol: water; 90:10 v/v) after<br />
estrous synchronization with FGA+ PGF2α.<br />
Treatment Group n Ovulation type (%) Ovulation rate<br />
a,b Different superscripts within a column differ (P < 0.01).<br />
Single Double Triple Quadruple<br />
Control a 55 39.62 56.60 3.77 0 1.64 ± 0.07 a<br />
USF b 53 5.66 50.94 39.62 3.77 2.41 ± 0.09 b<br />
TIME (min)<br />
Figure 1. Blood concentrations of glycerol and glucose in control and USF (300 mL<br />
of a 90% glycerol drench) treated ewes, from 30 min before to 600 min after<br />
treatment. *Values over the detection limit of the assay.<br />
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et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
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one follicle and increase the ovulation rate.<br />
III. MECHANISMS BY WHICH THE ULTRA SHORT<br />
FLUSHING ENHANCES THE OVULATION RATE<br />
Scarramuzzi et al. [33] revised the possible<br />
mechanisms of action of the acute effect of flushing on<br />
ovulation rate and proposed a feedback loop between<br />
the ovaries and gonadotrophin secretion, where nutrition<br />
causes a direct inhibition of follicular estradiol production<br />
leading to compensatory secretion of FSH that stimulates<br />
folliculogenesis. Alternatively, insulin could advance the<br />
maturation of the follicle that would be reflected in<br />
increased expression of LH receptor (LHr) and 3βhydroxysteroid<br />
dehydrogenase (3β-HSD) mRNA<br />
expression [9].<br />
To test these hypotheses the estrus cycle of 30<br />
ewes was synchronized with progestin intravaginal<br />
sponges and prostaglandins. Animals were given the USF<br />
on the day of progestin withdrawal. The ovaries were<br />
removed surgically before treatment (time 0; n=6), or at<br />
12, 24 and 48 h after being treated with either glycerol or<br />
water (n=4 per treatment by time category). The ovarian<br />
follicles were dissected out and counted. Follicles larger<br />
than 3mm in diameter were pooled and the mRNA<br />
extracted for specific P450aromatase, 3β-hydroxysteroid<br />
dehydrogenase (3β-HSD) and LH receptor (LHr)<br />
determination. Ultrashort flushing increased the number<br />
of follicles larger than 3mm at 48 h after treatment. No<br />
effect was observed in small follicles. In large follicles,<br />
aromatase mRNA abundance decreased (P < 0.01) 12<br />
h after treatment (Figure 2). There was no effect of<br />
treatment on mRNA for LH receptor or 3β-HSD (Figure<br />
2) [9].<br />
These results demonstrate a reduction in<br />
aromatase expression in potential ovulatory follicles twelve<br />
hours after flushing. The decrease in aromatase may<br />
favour a transient increase in FSH concentrations that<br />
would allow the stimulation and selection of<br />
supplementary ovulatory follicles. In addition, it shows<br />
that the twelve hours that follow luteolysis are fundamental<br />
in the selection of ovulatory follicles and in the<br />
determination of the ovulatory quota in sheep.<br />
converted to glucose in vivo [2,12]. Propylene glycol<br />
can be obtained from glycerol [37] and it is frequently<br />
used as feeding additive in dairy cows during early<br />
lactation in order to increase blood concentrations of<br />
propionic acid, glucose and insulin [5,27]. Molasses, is a<br />
byproduct of the sugar cane industry, which is widely<br />
used in ruminants as a source of soluble carbohydrates<br />
rapidly fermentable in the rumen [40]. The effects of<br />
molasses on VFA production is related to its level in the<br />
diet. Low dietary inclusion of molasses (
C.G. Gutier<br />
utierrez,<br />
S. Fer<br />
errar<br />
aro, V. Mar<br />
artine<br />
tinez,<br />
et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
and the fatty acid preferably produced after fermentation<br />
may alter the glycogenic capacity of the substrate. We<br />
studied the in vitro rumen fermentation and the in vivo<br />
response of sheep to an isoenergetic (1470.83 kcal) single<br />
oral drench of glycerol, propylene glycol or molasses.<br />
We observed that glycerol and propylene glycol increased<br />
(P < 0.01) the proportion of propionate production in<br />
vitro [9]. Glycerol remained unfermented for the longest<br />
time as shown by the greater lag time (10h; P < 0.05)<br />
and molasses showed a fastest rate of gas production<br />
and (P < 0.05) the shortest time to be fermented (Table<br />
3). In vivo, glycerol and propylene glycol increased<br />
glucose plasma concentration for up to 12 h after<br />
treatment (Figure 3). Insulin secretion increased for a<br />
period of 12 h in the glycerol and propylene glycol groups<br />
(P < 0.01). In contrast, molasses caused a mild increase<br />
in both glucose and insulin that lasted for less than 60<br />
and 12 min respectively (Figure 3) [9].<br />
Hence, the results of this trial showed important<br />
differences in the fermentation and glycogenic responses<br />
to these three energetic substrates. The longer lag time<br />
of glycerol allows for its absorption and its direct use for<br />
glucose production by the liver. Propylene glycol has a<br />
similar lag time than molasses, but it is preferably fermented<br />
to propionic acid thus serving as glycogenic substrate.<br />
Thus, glycerol and propylene glycol cause a large and<br />
sustained increase in insulin concentrations that together<br />
with glucose may enhance ovulation rate.<br />
V. WHAT IS THE MINIMUM DOSE NEEDED TO INCREASE<br />
OVULATION RATE WITH AN ULTRASHORT FLUSING?<br />
So far, we have established that USF with a<br />
single drench of 300 ml of a 90% glycerol solution<br />
coinciding with the initiation of luteolysis augments the<br />
number of ovulations and is associated with a 12h increase<br />
in glucose and insulin. In addition, this increase in ovulation<br />
rate is related to reduced aromatase mRNA abundance<br />
in the ovulatory follicles at 12 h after treatment. Thus, we<br />
have limited the time frame where follicles are selected<br />
to add to the ovulation quota within a 12h period after<br />
luteolysis. However, this period of time could actually be<br />
shorter than the period indicated by the results already<br />
shown. In all cases, the ultrashort flushing does not alter<br />
the body condition score or weight of the ewe and seems<br />
to act by its effects on insulin and glucose concentrations<br />
rather than the net amount of energy given to the animal.<br />
Although flushing can be limited to a single<br />
glycogenic drench at the time of luteolysis, we observed<br />
as much as 43% of superovulated animals with the 300<br />
mL glycerol dose (Table 2). Thus, the determination of a<br />
minimum flushing dose could help reduce the incidence<br />
of triple and quadruple ovulations. One hundred and<br />
twenty sheep were scanned by ultrasonography to determine<br />
their ovulation rate, and were randomly divided<br />
into four groups of 30 ewes each that received 300, 200,<br />
100 or 50 mL of glycerol at the time of PGF2α injection.<br />
Flushing at all the doses tested increased ovulation rate<br />
(P< 0.01). A hundred milliliters of glycerol solution caused<br />
the highest ovulation rate. However, doses of 100 mL or<br />
above superovulated between 17 and 25% of the sheep<br />
(Table 4). Thus, it appears that ultrashort flushing with 50<br />
mL dose is sufficient to cause an increase in ovulation<br />
rate without the hindrance of superovulation in sheep [22].<br />
VI. OTHER MANIPULATIONS TO INCREASE OVULATION<br />
RATE<br />
The static effect of flushing on ovulation quota is<br />
apparently due to an increase in the number of<br />
Table 3. Cumulative gas production and estimated kinetic parameter model for liquid energetic sources.<br />
Substrate Maximum volume (Y), mL<br />
-1<br />
Rate of gas production (s), h Lag time (L), h<br />
Glycerol 320 µL 178.4 b 0.024 e 10.23 b<br />
Propylene glycol 320µL 22.6 d 0.062 c 1.81 d<br />
Molasses 320 µL 161.5 cb 0.085 a 1.86 d<br />
SEM 4.03 0.001 0.12<br />
R 2 0.98 0.98 0.99<br />
P value of regression model 0.0001 0.0001 0.0001<br />
abc Means within column with different superscript differ (P < 0.05).<br />
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aro, V. Mar<br />
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et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
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N<br />
Figure 3. Mean blood concentrations of insulin and glucose at different times after an oral drench (1470.8 Kcal) of glycerol, propylene glycol<br />
or molasses in ewes. Values are means ± S.E.D. of logarithmic transformation of insulin and glucose concentrations. Mean values with different<br />
letters differ (P < 0.05).<br />
gonadotropin responsive follicles. It appears that the<br />
increase of glucose and insulin is responsible for the<br />
increase in follicular development and, consequently, for<br />
the number of available follicles for ovulation. But then,<br />
could any nutritional of hormonal treatment that increases<br />
insulin cause an increase in ovulation rate<br />
We conducted a series of studies to test the<br />
effect of treatments that enhance glucose redistribution<br />
on the number of small antral follicles growing in the<br />
ovaries. However, an increase in the number of<br />
gonadotropin responsive follicles is difficult to evaluate<br />
in sheep, as the diameter of the gonadotropin sensitive<br />
follicles (2 mm) is close to the lower range of sensitivity<br />
of the ultrasound scanner. To circumvent this<br />
inconvenience, we carried out the first study in cattle,<br />
where the gonadotropin sensitive follicles grow up to 4<br />
mm in diameter and can be easily counted by transrectal<br />
ultrasound scanning. In addition, we used the gonadotropin<br />
inhibited model [16], where cattle are treated chronically<br />
with a GnRH agonist for a long period of time, with a<br />
consequent inhibition of the pulsatile release of LH and<br />
FSH to their basal concentrations, and the arrest of follicle<br />
development at 4 mm in diameter. Since dominant follicles<br />
do not develop, the gonadotropin sensitive follicles grow<br />
without the restrain and the fluctuating inhibition of<br />
dominant follicles.<br />
Nineteen cows were treated with a continuous<br />
infusion of buserelin (37.5 ng/h) for six months after which<br />
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errar<br />
aro, V. Mar<br />
artine<br />
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et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
Table 4. Ovulation type and ovulation rate in Pelibuey ewes following ultrashort flushing (USF)<br />
with oral administration of 50, 100, 200 or 300 mL of a glycogenic solution (glycerol: water; 90:10<br />
v/v) at the time of estrus synchronization with PGF2α.<br />
Treatment Group Ovulation type (%) Ovulation rate<br />
Single Double Triple Quadruple<br />
Control a 38.60 54.39 7.02 0 1.68 ± 0.05<br />
50 mL b 10.71 78.57 10.71 0 2.00 ± 0.08<br />
100 mL b 3.85 76.92 3.85 15.38 2.30 ± 0.15<br />
200 mL b 14.29 60.71 14.29 10.71 2.21 ± 0.15<br />
300 mL b 17.24 65.52 13.79 3.45 2.03 ± 0.12<br />
a,b Different superscripts within a column differ (P < 0.0001).<br />
the emergence of large follicles did not occur. Sequential<br />
treatments with glycerol (lt of a 90% glycerol:water<br />
solution) for 4 days, followed by a Beta-agonist receptor<br />
treatment (2.2g zilpaterol daily) for 4 days and a final<br />
treatment of a single bolus injection of recombinant<br />
growth hormone (BST, 500 mg) were given to all animals.<br />
Between treatment periods cows were left untreated<br />
for a period of a minimum of 5 days to avoid the carry<br />
over effect of one treatment to the next. The numbers<br />
of follicles in the ovaries were counted 4 days before<br />
treatment and 4 days after treatment, with exception of<br />
BST where the scanning was performed for 6 days after<br />
treatment.<br />
Glycerol and zilpaterol increased (P < 0.05) the<br />
number of antral follicles 4 days after the start of treatment<br />
by 38 and 23% respectively (Table 5). In contrast, BST<br />
took longer to increase the number of antral follicles as<br />
at 4 days after treatment there were no changes in the<br />
number of follicles. However, by day 5 and 6 after<br />
treatment the number of follicles increased 27 and 45%<br />
respectively (P< 0.05) (Table 5). In all cases, the increase<br />
in follicle number was associated with an increase in<br />
glucose, insulin and IGF-I plasma concentrations.<br />
We further tested if these treatments, when<br />
applied to sheep, would increase the number of follicles<br />
ovulating. Sheep were synchronized with progestin<br />
intravaginal sponges for 13 days and were simultaneously<br />
treated with a Beta-agonist (zilpaterol, 0.15 mg/kg/day)<br />
or served as control. There was no difference in food<br />
intake between experimental groups. Glucose and insulin<br />
blood concentrations were increased in sheep consuming<br />
the Beta-agonist. The ovulation rate was increased<br />
Table 5. Follicle number in sheep before and after oral daily administration of either 300 mL of a<br />
glycogenic solution (glycerol:water; 90:10 v/v daily) for six days, 2.2 g of zilpaterol/cow/day for five<br />
days, or a single subcutaneous injection of slow-release recombinant bST.<br />
Treatment group Follicle Number P value<br />
Pretreatment<br />
(n=19)<br />
Post-treatment<br />
(n=19)<br />
Glycerol 25.4 ± 0.3 Day 4 35.12 ± 1.8
C.G. Gutier<br />
utierrez,<br />
S. Fer<br />
errar<br />
aro, V. Mar<br />
artine<br />
tinez,<br />
et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
twofold from 1.37 in the control group to 1.72 in the<br />
treated group (P < 0.01) (Table 6) [7]. Fertility was similar<br />
in both groups, however the treatment with zilpaterol<br />
increased the proportion of ewes lambing twins as<br />
compared to the control (P < 0.05) (Table 7) [7].<br />
As for growth hormone, studies by other<br />
researchers have shown that it increases the number of<br />
follicles growing in the ovaries, and that these follicles<br />
respond to exogenous gonadotropins by augmenting the<br />
response to superovulatory treatments in heifers [14,15].<br />
If a similar increase in follicle number is achieved in sheep,<br />
the low strength of the dominant follicle may permit by<br />
itself an increase in the number of follicles and ovulations.<br />
We synchronized the estrous of 92 ewes and treated<br />
them with 125 mg of bST five days before the end of<br />
progestin synchronization. Treatment with bST increased<br />
Table 6. Distribution of ovulation type in Pelibuey and F1 Pelibuey-<br />
Dorper ewes following administration of zilpaterol hydrochloride<br />
(0.15 mg/kg/day) for 13 days mixed in the feed from the start of<br />
estrous synchronization with FGA until mating.<br />
Treatment Group n Ovulation type (%)<br />
Single Double Triple<br />
Control a 52 63 37 0<br />
Zilpaterol b 50 34 60 6<br />
a,b Different superscripts within a column differ (P < 0.005).<br />
Table 7. Distribution of type of parturition in Pelibuey and F1<br />
Pelibuey-Dorper ewes following administration of zilpaterol<br />
hydrochloride (0.15 mg/kg/day) for 13 days mixed in the feed<br />
from the start of estrous synchronization with FGA until mating.<br />
Treatment Group n Type of parturition (%)<br />
Single<br />
Multiple<br />
N<br />
Control a 61 85 15<br />
Zilpaterol b 68 71 29<br />
a,b Different superscripts within a column differ (P < 0.05).<br />
(P < 0.01) the proportion of ewes lambing twins or triplets<br />
and the number of lambs born per ewe (Table 8) [4].<br />
Taken together, the latter results show that<br />
treatment with (non-gonadotropin) hormones that affect<br />
nutrient partitioning may affect folliculogenesis and<br />
increase ovulation rate without changes in nutrient intake.<br />
VII. CONCLUDING REMARKS<br />
Nutrition enhances ovulation rate in sheep by<br />
either an increase in body weight (static effect), or by<br />
changes in metabolic hormones (acute effect). Both of<br />
these effects enhance the ovulation rate by increasing<br />
the number of gonadotropin responsive follicles. Herein,<br />
we described a series of studies that demonstrate a<br />
hyper-acute effect of nutrition that works at the time of<br />
luteolysis to select supplementary follicles for ovulation.<br />
The hyper-acute effect of nutrition seems to work by the<br />
already proposed [33] reduction in estradiol production<br />
as the abundance of mRNA for P450 aromatase declined<br />
12 h after the glycogenic drench. These studies also show<br />
that ovine follicles can develop ovulatory capacity within<br />
48 h after being selected when coinciding with the<br />
follicular phase.<br />
We have developed an ultrashort flushing method<br />
with glycogenic solutions that could be administered<br />
together with other managements (e.g. internal<br />
deworming) and that will increase the ovulation rate in<br />
the flock. However, we believe that not all glycogenic<br />
substances will work equally and that only those that<br />
increase insulin concentrations have the capacity to<br />
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utierrez,<br />
S. Fer<br />
errar<br />
aro, V. Mar<br />
artine<br />
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et al. <strong>2011</strong>. Increasing ovulation quota: more than a matter of energy. sssssss<br />
sss Acta Scientiae Veterinariae. 39(Suppl 1): s305 - s316.<br />
Table 8. Proportion of ewes lambing singletons, twins or triplets from ewes treated or not with bST (125 mg) five<br />
days before progestin sponge withdrawal.<br />
Groups n Percent of ewes lambing Lambs born per ewe lambing<br />
Singletons Twins Triplets<br />
bST 47 43.5 48.7 7.7 1.64<br />
Control 45 74.3 25.7 0 1.25<br />
The lambing pattern differ between groups (P < 0.01).<br />
augment the number of ovulations, although this remains<br />
to be tested.<br />
Finally, hormonal flushing can be achieved<br />
without changes in the energy intake of the sheep. Both<br />
BST and Zilpaterol increased ovulation rate and prolificacy<br />
and could be adapted as a management practice to<br />
increase the profitability of the farm.<br />
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jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335.<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): s317 - s335.<br />
ISSN 1679-9216 (Online)<br />
Ovum pick-up and in vitro embryo production in buffalo species: an update<br />
Bianca Gasparrini<br />
ABSTRACT<br />
Background: In the last few years, there has been an increasing interest in the in vitro embryo production (IVEP) technologies<br />
for faster propagation of superior germplasm in buffalo, due to the low efficiency of MOET programs. Early attempts to produce<br />
buffalo embryos in vitro have been made by using procedures that were proven effective in cattle. However, the acquisition of<br />
specific information on oocyte and embryo culture requirements in vitro in this species has resulted in an improved efficiency<br />
over the years. Although the IVEP efficiency has enhanced, as indicated by competitive embryo yields, pregnancy rate and<br />
development to term are still poor.<br />
Review: The present review intends to describe the state of the art of IVEP in buffalo species, emphasizing the advances<br />
achieved in the sequential steps of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC), and the<br />
limitations still to overcome. The IVM system has been greatly improved over the years, leading to 90 % maturation rate. In<br />
particular, the enrichment of the IVM medium with thiol compounds, known to act as antioxidants factors, has highly improved<br />
the IVEP efficiency. The important role of antioxidants factors during IVM has been confirmed by the improved blastocyst<br />
development recorded by supplementing the IVM medium with taurine and melatonin. A major factor impairing IVF in buffalo is<br />
the bull effect, consisting in the high degree of variation between buffalo bulls in the fertilizing capability in vitro. It has also been<br />
demonstrated that marked differences in the kinetics of sperm penetration exist among buffalo bulls and that this parameter can<br />
be a useful marker to predict the in vitro fertilizing ability. Although heparin is the in vitro capacitating agent most widely used,<br />
a significant improvement of sperm capacitation has been obtained by incubating sperm with biological fluids, such as buffalo N<br />
estrus serum and the follicular fluid recovered from a pool of dominant follicle. We have recently demonstrated that osteopontin<br />
(OPN), a protein present in the oviduct fluid and in the semen, improves capacitation and enhances both cleavage rate and<br />
blastocyst yields. The great improvement of blastocyst yields (35-40%) achieved in the following years is, according to our<br />
experience, due to the optimization of the IVM and the IVF systems rather than to modifications applied to the IVC system. In<br />
fact, at present the original version of SOF remains the most suitable medium for embryo culture in buffalo. Nevertheless, we<br />
have demonstrated that energy requirements of buffalo embryos are different from those of sheep and cattle. These findings<br />
highlight the importance of identifying species-specific requirements in order to optimize the in vitro culture conditions for<br />
buffalo embryos. The major limitation to the diffusion of IVEP in buffalo is the low number of recruitable oocytes, arising from<br />
peculiarities of the reproductive physiology of the species. Another limitation is given by the high susceptibility of buffalo IVP<br />
embryos to cryopreservation, in part due to their greater lipid content, but also to their poor viability, likely determined by<br />
suboptimal in vitro culture conditions.<br />
Conclusions: The IVEP efficiency in buffalo species has greatly improved over the years. However, the optimization of culture<br />
conditions is fundamental to increase the viability of IVEP embryos for advanced reproductive strategies to become a routine<br />
procedure in buffalo breeding. At the same time efforts should be addressed to identify the best management of the recipients,<br />
in order to increase pregnancy maintenance after ET of cryopreserved IVP embryos.<br />
Keywords: buffalo, ovum pick-up, oocyte, embryo, in vitro embryo production.<br />
DISCIZIA, Faculty of Veterinary Medicine, Federico II University, Napoli, Italy. CORRESPONDENCE: B. Gasparrini [bgasparr@unina.it -<br />
PHONE: +39 (081) 2536494; Fax: +39 (081) 292981]. Federico II University, Via F. Delpino 1, 80137 Napoli, Italy.<br />
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I. INTRODUCTION<br />
II. OOCYTES SOURCE AND QUALITY<br />
III. IN VITRO MATURATION (IVM)<br />
IV. IN VITRO FERTILIZATION (IVF)<br />
V. IN VITRO CULTURE (IVC)<br />
VI. EMBRYO CRYOPRESERVATION<br />
V. CONCLUSION<br />
I. INTRODUCTION<br />
In the last two decades interest in buffalo breeding<br />
has tremendously increased worldwide, due to the critical<br />
role that this species plays in many climatically disadvantaged<br />
agricultural systems. It is worth pointing out that<br />
environmental conditions, especially climate, make the<br />
River buffalo an irreplaceable dairy producer for developing<br />
countries situated in the tropics north of the equator.<br />
In these countries, the perfect interaction between the<br />
reproductive seasonality, environment, and forage<br />
availability during the year ensures that the buffalo<br />
compensates for the lack of bovine milk during the rainy<br />
season (winter-spring), and produces animal protein at<br />
competitive costs. The crucial role played by this species<br />
is evident when the increase in the world buffalo<br />
population during the past 40 years is compared to the<br />
increase in cattle population (86% vs 34 %, respectively).<br />
In the current scenario, the competitiveness of<br />
buffalo breeding highly depends on genetic improvement.<br />
Therefore, the reproductive biotechnologies are extremely<br />
important, allowing to plan selective programs and their<br />
accomplishment in a shorter time. Furthermore, the use<br />
of these technologies is crucially important to meet the<br />
genetic requests from developing countries, for the<br />
grading up, i.e. the replacement of the local working buffalos<br />
(Swamp type) with the milk producing buffaloes<br />
(River type), in order to increase animal proteins and<br />
meet human needs.<br />
With regard to the reproductive technologies<br />
aimed to speed up the genetic progress through the maternal<br />
lineage, in buffalo, due to the low and inconsistent<br />
response to multiple ovulation and embryo transfer<br />
(MOET) programs [79,119], the interest has shifted to<br />
the in vitro embryo production (IVEP) technology.<br />
The ovum pick-up (OPU) technique combined<br />
with IVEP has greater potential than MOET to enhance<br />
the maternal contribution to the genetic improvement,<br />
allowing the production of more embryos on long term<br />
basis. In addition to the limited embryo output obtainable<br />
by MOET in this species [79,119], it is worth emphasizing<br />
that OPU can be carried out on a wider typology of<br />
donors, such as non-cyclic animals, pregnant cows,<br />
subjects with patent oviducts or genital tract infections,<br />
and animals that are not responsive to hormonal<br />
stimulation, the last representing a high proportion in<br />
buffalo.<br />
This technique was used in buffalo for the first<br />
time in 1994 on deep anoestrous animals under ovarian<br />
hypotrophic conditions [11]. Since then, OPU has been<br />
carried out in buffalo several other times in our country<br />
[12,14,15,86,87] and, subsequently in Brazil [100, 101],<br />
China [55,62], Argentina [95] and India [66,70]. Due to<br />
the improvements of the in vitro system, the IVEP<br />
efficiency has greatly increased throughout the years<br />
[48,86].<br />
Ovum pick-up has been performed under a<br />
continuous regime for up to 6 months in non-lactating<br />
buffalo cows at the end of their productive and reproductive<br />
career with high embryo production eficiency<br />
[89]. However, due to a slowing down of the follicular<br />
turnover occurring approximately at day 70 of the trial, it<br />
was necessary to increase the OPU sampling interval<br />
from 3–4 to 7 days. Interestingly, after the first 6 months<br />
of oocyte collection, in addition to the decline of the number<br />
of follicles and oocytes, the oocyte developmental<br />
competence was compromised, as indicated by the failure<br />
in blastocyst development.<br />
The combined OPU and IVEP technology is<br />
currently the most promising tool for increasing the number<br />
of transferable embryos (TE) obtainable per donor over<br />
the long term in most species. In buffalo, this technology<br />
is even more competitive in terms of embryo yields<br />
compared to MOET: 11 TE were produced on average<br />
in a 6 month trial vs 5 TE theoretically obtainable with<br />
MOET [44]. It should be specified that the number of<br />
TE theoretically obtained by MOET is overestimated since<br />
multiple ovulations can be induced only in cyclic animals,<br />
and it is unlikely that buffalo cows are cyclic for 6 months<br />
because they tend to go into seasonal anoestrus. In<br />
addition, if donors are selected on the basis of their<br />
folliculogenetic potentials, the limitation due to the high<br />
variability of follicular recruitment, oocyte retrieval, and<br />
hence, blastocyst production (1-37 TE in 6 months, [44]),<br />
can be overcome, thus resulting in a further increase of<br />
the embryo yield. This selection only needs two weeks<br />
screening, as it has been observed that the potential of<br />
animals to recruit follicles can successfully be predicted<br />
after the first 4 oocyte collection days [44].<br />
Although the buffalo IVEP system has greatly<br />
improved over the years, leading to high blastocyst yields<br />
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[48] and to the production of offspring [55,56,87,100],<br />
this technology is still far from being commercially viable.<br />
The major limitation to the diffusion of IVEP in<br />
buffalo is the low number of recruitable oocytes, arising<br />
from peculiarities of the reproductive physiology of the<br />
species, and as such, not easily modifiable.<br />
Another limitation is given by the high susceptibility<br />
of buffalo IVP embryos to cryopreservation,<br />
related in part to their greater lipid content [16] but also<br />
to their poor viability, likely determined by suboptimal in<br />
vitro culture conditions. This results in the overall poor<br />
pregnancy to term recorded following transfer of IVP<br />
cryopreserved embryos (8-25%). However, it is worth<br />
specifying that the development to term is strongly<br />
reduced because of the high incidence of embryonic<br />
mortality occurring between 25 and 50 days; in our recent<br />
experience pregnancy rate was 50% on day 30, to lower<br />
down to 10% pregnancy to term, with the embryo loss<br />
recorded before day 50. At our latitudes during the longday<br />
length period a high incidence of embryonic mortality<br />
is also observed during natural mating, but it increases<br />
further when reproductive biotechnologies, such as AI<br />
and embryo transfer (ET) of in vitro produced embryos<br />
(IVP) are utilized [17]. It has been hypothesized that this<br />
phenomenon is due to both a poor quality of the oocytes<br />
and to the reduced function of corpus luteum (CL), leading<br />
to lower progesterone (P4) secretion [17-19]. The<br />
problem of embryonic mortality is even greater when<br />
IVEP technology is employed because in this case another<br />
factor plays a role, i.e. the poorer viability of in vitro<br />
produced embryos. It is known, in fact, that suboptimal<br />
culture conditions affect all post-implantation events [74].<br />
It is worth reminding that scientific improvement<br />
in this species has been hampered by contingent factors.<br />
The major factor that has delayed scientific advances in<br />
buffalo IVEP is the scarcity of experimental material in<br />
all the countries in which the species is bred, due to either<br />
the heads consistency, the low culling rate and/or the<br />
breeding systems. Furthermore, the economic importance<br />
of the species has only been recently appreciated, and as<br />
a consequence, the first studies of advanced reproductive<br />
strategies in this species date back only to the 1990s.<br />
Finally, the majority of buffaloes is bred in developing<br />
countries where scientists often have to deal with poor<br />
resources and lack of facilities. The scarcity of experimental<br />
material for buffalo, together with the assumption<br />
that the reproductive biology in all ruminants is similar,<br />
led in the early attempts, to use the IVEP system in buffalo<br />
based solely on information acquired in cattle, with the<br />
consequent result of low IVEP efficiency. On the contrary,<br />
it has been demonstrated that improvements in IVEP<br />
are possible through the optimization of each procedural<br />
step, especially when taking into account species-specific<br />
differences, as shown by the higher blastocyst rates<br />
reported over recent years [48].<br />
Therefore, the aim of this review is to report the<br />
major breakthroughs in the OPU and IVP technologies<br />
in buffalo in the past 20 years, the current state of the art<br />
and expected future improvements. The review will be<br />
structured in separate subsections, that will highlight the<br />
most important limitations and progresses achieved in the<br />
different sequential steps of in vitro maturation (IVM),<br />
in vitro fertilization (IVF) and in vitro culture (IVC), as<br />
well as in embryo cryopreservation.<br />
II. OOCYTE SOURCE AND QUALITY<br />
The major intrinsic limitation of IVEP technology<br />
in buffalo lies in the low number of immature oocytes<br />
that can be recovered per donor. With controlled follicular<br />
aspiration of abattoir-collected ovaries, the average<br />
number of total oocytes per ovary varies between 0.7<br />
and 4.3 [10, 27, 63, 111]. In addition, because of the high<br />
incidence of atresia, the mean recovery of good quality<br />
oocytes per ovary is further reduced: 0.4 [63,111], 0.9<br />
[27], 1.76 [102], and 2.4 [51]. In our setting, controlled N<br />
follicular aspiration of abattoir-collected ovaries allows<br />
the retrieval of 4.3 total oocytes [10] and 2.4 good quality<br />
oocytes per ovary on average [51]. The slightly lower<br />
oocyte recovery reported by Indian authors may be due<br />
to differences in breed, older age at slaughter, management<br />
and nutritional status [54], as the plane of nutrition<br />
is known to affect the follicular dynamics [105].<br />
Among factors affecting oocyte recovery, the<br />
ovarian status plays a role, as it has been reported that<br />
the number of oocytes is further decreased when ovaries<br />
have a corpus luteum [84]. Furthermore, results obtained<br />
in OPU trials carried out on buffalo cows at different<br />
days in milk [12,14,15] suggest that the number of follicles<br />
and hence that of the oocytes decrease at increasing<br />
postpartum period (>500 d). Indian authors reported that<br />
the number of COCs is reduced during summer [70],<br />
suggesting an influence of season on the follicular<br />
population. In contrast, no variation in the follicular and<br />
oocyte population has been recorded among seasons in<br />
temperate climate [32].<br />
However, despite small differences, it is<br />
overwhelming evident that the recovery rate in buffalo is<br />
much lower than in cattle, in which 10 good quality oocytes<br />
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are obtained on average per ovary [53]. Similarly, when<br />
OPU is carried out the number of oocytes collected per<br />
donor is much lower in buffalo than in cattle (4.5 vs<br />
approximately 10 respectively [41]).<br />
This is not surprising if we consider some<br />
physiological features, peculiar of this species: 1) lower<br />
number of primordial follicles in the buffalo ovary, varying<br />
from 10,000 to 19,000 [26,103] compared with 150,000<br />
in cattle [39]; 2) lower number of antral follicles<br />
throughout the whole estrous cycle [61]; 3) high incidence<br />
of follicular atresia, i.e., 82% [90] or 92% [91] and 4)<br />
smaller and lighter gonad compared with that of cattle<br />
(2.5 cm vs. 3.7 cm of length; 3.9 g vs. 8.5 g of weight<br />
respectively; [116]). It results that this limitation is currently<br />
the major impediment for the diffusion of IVEP in the<br />
field, arising from physiological peculiarities of the species,<br />
and as such, it is not easy to improve.<br />
However, as previously stated, it is possible to<br />
increase the number of competent oocytes by operating<br />
the selection of the donors on the basis of their follicular<br />
population [44,100]. An earlier trial carried out on deep<br />
anoestrus buffalos demonstrated that it is possible to<br />
increase the number of aspirated follicles (6.75 vs 4.17),<br />
recovered oocytes (3.00 vs 1.33), and the oocyte quality<br />
(55.6% vs 31.3%) by priming OPU donors with FSH-P<br />
[11]. However, the oocyte yield was low also in the<br />
primed-donors, because of the poor starting ovarian status.<br />
More recently, it has been shown that rBST pretreatment<br />
of buffalo donors [100] promotes follicular growth (12.2<br />
vs 8.7 total follicles punctured) and tends to increase the<br />
number of oocyte recovered per session (5.2 vs. 4.1; P<br />
= 0.07), as well as the percentage of good quality oocytes<br />
(48.8% vs. 40.6%; P = 0.07). Nevertheless, in agreement<br />
with results previously reported in cattle [9,112], the<br />
treatment failed to increase the blastocyst production rate<br />
and the number of blastocyst produced per buffalo per<br />
session. Similar results were also obtained in a subsequent<br />
trial in which the rbST treatment was tested on donors<br />
undergone OPU once per week [2]. Therefore, these<br />
approaches are successful to increase the number of<br />
gametes but only to a limited extend. Furthermore, it is<br />
worth underlining that one of the advantages of OPU<br />
over MOET is exactly the possibility to avoid hormonal<br />
treatment of the donors and subsequent side effects.<br />
However, for a complete review it is worth to<br />
cite that recently Baruselli et al. [2] reported a number<br />
of oocytes recovered per buffalo per OPU session much<br />
higher than the average values reported in literature, in a<br />
trial in which the interval between sessions was extended<br />
to one week (8.5 and 6.1 total and viable oocytes,<br />
respectively) and two weeks (10 and 7.2 total and viable<br />
oocytes, respectively). It is known that many factors may<br />
affect the recovery rate, such as the tractability of the<br />
animals, the skill of the operator, the efficiency of the<br />
aspiration set, etc but the extraordinary high number of<br />
follicles observed is difficult to explain as this may only<br />
depend on the donors. We may speculate that the evident<br />
difference in the follicular population may be due to the<br />
genetics of the animal, the age (heifers vs adults), the<br />
type of breeding and environment, as well as the longer<br />
interval between sessions. The latter factor may account<br />
for both the greater number of follicles and the lower<br />
developmental competence of the oocytes, indicated by<br />
the poor blastocyst rate (9 %); indeed, it is known that an<br />
extension of the interval increases the number of follicles<br />
but also the heterogeneity of the oocyte source, as the<br />
phenomena of dominance and atresia in this case occur.<br />
Oocyte quality, that is known to be a major factor<br />
affecting the IVEP efficiency in most species, plays a<br />
determining role in buffalo, further decreasing the<br />
availability of the oocytes suitable for IVEP. Indeed, it is<br />
possible to increase the IVEP efficiency by selecting the<br />
oocytes but this in turn reduces the number of oocytes<br />
that can be processed, that is the real limiting factor in<br />
this species.<br />
It is known that the oocyte morphology can<br />
reliably predict the gamete developmental competence;<br />
according to our classification, a progressive decrease<br />
of efficiency is recorded from Grade A to Grade D<br />
oocytes [86], with Grade A and B considered suitable for<br />
IVEP. It is worth pointing out that the percentage of good<br />
quality oocytes (Grade A and B), is lower in this species<br />
compared to others, not exceeding, in our experience 50<br />
% of the total oocytes recovered. An analysis of the data<br />
collected over a 4-years period in our lab showed that<br />
from a total number of 35.286 abattoir-derived oocytes<br />
(over 158 replicates) 47.8% were Grade A + B, 6.2 %<br />
were Grade C and 46.7 % were unsuitable for IVEP<br />
(Gasparrini, personal communication, 2010). A recent trial<br />
has shown an even lower proportion of good quality<br />
oocytes (33.7 % of Grade A+B), together with a higher<br />
incidence (37.9 %) of Grade C [78].<br />
In alternative to morphological criteria, the non<br />
invasive brilliant cresyl blue (BCB) staining before IVM<br />
can be used to select developmentally competent abattoirderived<br />
oocytes [65]. This method allows the selection<br />
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of a slightly higher percentage of competent oocytes<br />
(57%), without improving, though, the average blastocyst<br />
yields.<br />
The oocyte quality may be affected by several<br />
factors, such as the aspiration pressure during collection,<br />
the source of gametes, the time between collection and<br />
processing, the temperature during transportation, season,<br />
etc.<br />
In our experience, the oocyte morphology varies<br />
with the source of gametes, with an apparent worse<br />
quality of OPU-derived oocytes, characterized by fewer<br />
layers of granulosa cells, compared to abattoir-derived<br />
ones. A different distribution of COCs classes in relation<br />
to the oocyte source was also recorded in cattle, with a<br />
higher incidence of better quality oocytes for abattoirderived<br />
compared to OPU-derived COCs [76]. As this<br />
difference was not due to the OPU equipment or to the<br />
collection medium, it was hypothesized that, due to a postmortem<br />
effect, the COCs becomes less tightly connected<br />
to the follicle wall and therefore are collected with a more<br />
complete morphology. This is even more likely to occur<br />
in buffalo because of the poor adhesion of cumulus cells<br />
[44]. This feature led us to speculate that also technical<br />
factors during OPU, such as the length of the needle, as<br />
well as that of the line connected to the suction unit, may<br />
result in a greater loss of granulosa cells in buffalo oocytes<br />
and, hence in an underestimated evaluation of their quality.<br />
In fact, in contrast to what reported in cattle [76],<br />
despite their worse morphological appearance, OPUderived<br />
buffalo oocytes have a higher developmental<br />
competence compared to abattoir-derived ones [86]. Similar<br />
results have been obtained by Indian authors who<br />
reported both higher blastocyst yields (30.6±4.3 vs<br />
18.5±1.8) and higher blastocyst hatching rates (52.8±4.2<br />
versus 40.2±4.4) following embryo vitrification from<br />
OPU-derived compared to abattoir-derived buffalo<br />
oocytes [66].<br />
The improved embryo yield may be accounted<br />
for by the OPU-induced modification of the follicular<br />
dynamics; resetting the follicular population twice weekly<br />
results in increased follicular wave frequency and, hence<br />
in the aspiration of follicles before they become atretic,<br />
with an improved oocyte “quality”. On the contrary, an<br />
heterogeneous oocyte population is recovered from pooled<br />
ovaries of slaughtered buffaloes. In addition, it is worth<br />
reminding that buffaloes are usually slaughtered when<br />
they are old and/or hypo fertile. It has been suggested<br />
that the better embryo yields recorded in cattle for<br />
slaughterhouse compared to OPU-derived oocytes are<br />
due to early atresia occurring in the post-mortem which<br />
positively affects oocyte developmental competence [3].<br />
This does not seem to be the case in buffalo, suggesting<br />
that further studies are needed to elucidate the occurrence<br />
of atresia and its effect on the oocyte.<br />
It has been speculated that the better<br />
developmental competence of OPU-derived vs abattoirderived<br />
oocytes is related to the shorter exposure to<br />
environmental stress. Indeed, abattoir-derived oocytes<br />
spend a longer time between excision of ovaries from<br />
the peritoneal cavity and laboratory processing and are<br />
probably affected by cellular damages due to autolytic<br />
processes, especially when they reside in excised ovaries<br />
for prolonged periods.<br />
Furthermore, when OPU is carried out in the<br />
field, i.e. in farms distant from the laboratory, a significant<br />
improvement of blastocyst yield can be achieved by<br />
reducing the time between oocyte collection and their<br />
maturation. In these situations, an increased efficiency is<br />
recorded with oocytes searched directly in the farm and<br />
immediately transferred in a hepes-buffered in vitro<br />
maturation (IVM) medium in a portable incubator,<br />
compared with those searched in the laboratory, after<br />
many hours of their permanence in the follicular fluid N<br />
(Gasparrini, personal communication, 2006).<br />
The oocyte competence also depends on the<br />
morphofunctional state of ovaries, with an improved<br />
development recorded with oocytes recovered from<br />
ovaries bearing either a corpus hemorragicum or a CL,<br />
in the absence of a dominant follicle [68]. Furthermore,<br />
when a dominant follicle and a CL are present on the<br />
ovary, cleavage rate and transferable embryo yield are<br />
higher when oocytes are collected from the dominant<br />
follicle compared to the largest subordinate and<br />
subordinate follicles.<br />
Buffalo oocytes are very sensitive to shock<br />
temperature so it is important to monitor the temperature<br />
carefully during collection as fluctuations can easily occur.<br />
When OPU is carried out temperature is controlled by<br />
holding the collection tubes in suitable warm boxes. When<br />
abattoir-derived oocytes are utilized for in vitro embryo<br />
production, the ovaries are usually kept in physiological<br />
saline, under controlled temperature (30-37°C), during<br />
collection and transportation to the laboratory. It has been<br />
recently observed that oocyte developmental competence,<br />
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evaluated in terms of cleavage and blastocyst rates<br />
following IVF, is improved by lowering the temperature<br />
range during transportation to 25-29.5°C [33].<br />
Finally, it is known that buffalo is a short-day<br />
breeder and that the efficiency of reproductive<br />
technologies such as A.I. and MOET is significantly<br />
affected by season [19]. The analysis of data collected<br />
in our IVEP laboratory over 3 years has shown that,<br />
although the number and the quality of the oocytes are<br />
not affected, the oocyte developmental competence is<br />
improved during the autumn months compared to spring,<br />
as indicated by better cleavage and blastocyst rates [32].<br />
Furthermore, the effect of season on the oocyte<br />
developmental competence was recently confirmed<br />
during an OPU trial carried out on buffalo donors bred in<br />
temperate climate, in which, despite similar proportions<br />
of Grade A+B COCs, the embryo yields doubled in<br />
autumn compared to mid-winter and spring-summer (Di<br />
Francesco, personal communication, <strong>2011</strong>). These<br />
findings are in agreement with the seasonality pattern<br />
exhibited by the species at our latitudes, with the fertility<br />
improved during short-day months. These results strongly<br />
suggest to restrict the gametes collection during autumn<br />
when planning OPU trials at our latitudes, in order to<br />
save resources and make the benefit/costs ratio more<br />
favorable.<br />
The overall results of the present study are<br />
different from those obtained in a recent trial carried out<br />
in India [70]. These authors also reported a seasonal<br />
effect on IVEP efficiency that was, though, mainly due<br />
to the reduction of follicular population. In fact, the oocyte<br />
developmental competence, indicated by blastocyst<br />
production rate, was not affected by season, although<br />
the numbers of aspirated follicles, recovered oocytes,<br />
and blastocysts produced per animal per session<br />
decreased during the unfavorable season<br />
III. IN VITRO MATURATION (IVM)<br />
A fundamental requirement for a successful<br />
fertilization is undoubtedly the appropriate maturational<br />
status of the oocytes at the time they encounter the sperm.<br />
The oocyte maturation process, that involves both the<br />
nuclear and cytoplasmic compartments, is fundamental<br />
for the acquisition of full developmental competence.<br />
Buffalo oocytes can be matured in vitro in<br />
complex media, such as Tissue Culture Medium 199 (the<br />
most widely employed) and Ham’s F-10, supplemented<br />
with sera, hormones and other additives. Different sources<br />
of serum have been utilized as supplements of IVM<br />
medium [24,102,110]. Although it has been observed that<br />
buffalo oocytes can reach the maturation status even in<br />
the absence of hormones [63], higher maturation and<br />
fertilization rates have been recorded when oocytes are<br />
matured in the presence of gonadotrophins and 17âestradiol<br />
[110,111]. It is known that hormones interact<br />
with receptors located on the follicular cells, and that the<br />
signals are transduced into the oocyte through gap<br />
junctions or extracellular mechanisms. It results that the<br />
presence of cumulus cells is critical for the acquisition of<br />
developmental competence during IVM, as confirmed<br />
by the significantly reduced cleavage and embryo<br />
development of denuded vs cumulus-enclosed oocytes<br />
following IVF [45,83]. This aspect is particularly important<br />
in buffalo because of the high proportion of totally or<br />
partially denuded oocytes usually recovered in this species.<br />
In order to rescue germinal material, it has been proposed<br />
to provide poor quality oocytes with the somatic support<br />
by performing IVM on a cumulus cells monolayer,<br />
obtaining improved maturation and fertilization rates [94].<br />
In order to reduce IVEP costs, buffalo follicular<br />
fluid, a waste product of oocyte collection, has been used<br />
in replacement of expensive hormones and serum<br />
additives, obtaining comparable maturation, fertilization,<br />
and blastocysts rates [22].<br />
The beneficial effects of several ovarian-derived<br />
growth factors, such as IGF-l, IGF-2 and insulin on oocyte<br />
maturation, fertilization and development to the blastocyst<br />
stage [93] have been also reported in this species. It has<br />
also been observed that supplementation of the IVM<br />
medium with EGF improves cumulus expansion, nuclear<br />
maturation, and cleavage rate of cumulus-enclosed<br />
buffalo oocytes without affecting the post-fertilization<br />
embryonic development [25]. An improvement of<br />
blastocyst yields has been, however, reported by enriching<br />
the IVM medium with EGF, either alone or in combination<br />
with fibroblast growth factor (FGF) and vasoactive intestinal<br />
peptide (VIP), only when the zygotes were cultured<br />
in a complex co-culture system [85].<br />
Based on the assumptions that buffalo oocytes<br />
and embryos, because of their high lipid content [16], are<br />
particularly sensitive to oxidative damages, the IVM<br />
medium has been enriched by thiol compounds, known<br />
to act as antioxidants factors, by stimulating glutathione<br />
(GSH) synthesis. It is known that GSH plays a critical<br />
role in protecting mammalian cells from oxidative stress,<br />
that is a major factor affecting in vitro mammalian<br />
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embryo development. It has been demonstrated in other<br />
species [43] that the GSH reservoir formed during IVM<br />
is the only source of reducing power for the embryos<br />
before genomic activation occurs. It has been previously<br />
demonstrated that cysteamine supplementation during<br />
IVM improves blastocyst yield in buffalo [51], by<br />
increasing intracytoplasmic glutathione concentration [52],<br />
without nevertheless affecting cleavage rate.<br />
Subsequently, the addition of cystine, in the presence of<br />
cysteamine, to the IVM medium [48] has been proven to<br />
increase the proportion of oocytes showing normal<br />
synchronous pronuclei post fertilization (81%), cleavage<br />
rate (78%) and blastocyst yield (30%), via a further<br />
increase of the GSH reservoir of the oocytes. Improved<br />
blastocyst development has also been recorded by<br />
enriching the IVM medium with other antioxidant factors,<br />
such as taurine and melatonin [64].<br />
It has been recently demonstrated [92] that<br />
incorporation of mitogenic lectin in the IVM medium<br />
improves maturation of buffalo oocytes, as indicated by<br />
increased cumulus expansion and cleavage rate following<br />
IVF, as well as by the up regulation of the transcripts<br />
levels of genes involved in different physiological activities<br />
like gap junction and cell communication protein (Cx43),<br />
cumulus-expansion enabling factor and cell cycle protein<br />
(GDF-9), basic growth factor (FGF-4) and cell membrane<br />
protein (Fibronectin).<br />
Among factors affecting mammalian embryo<br />
development in vitro, the duration of IVM plays a critical<br />
role, since an inappropriate timing of maturation results<br />
in abnormal chromatin [37], oocyte aging [57] and<br />
reduced development [73]. We have investigated both<br />
the kinetics of oocyte maturation and the influence of the<br />
duration of IVM on subsequent embryo development. In<br />
this study the attainment of the MII stage commenced<br />
after 18-19 h maturation and the majority of oocytes<br />
completed nuclear maturation between 20 and 24 h;<br />
furthermore, it was demonstrated that the duration of<br />
IVM affects buffalo oocyte developmental competence,<br />
with a progressive decrease of fertilization capability and<br />
embryo development as the IVM duration increases from<br />
18 to 30 h [49]. Therefore, the optimal time for IVF in<br />
buffalo appears to be at 18 h post-IVM or, in any case,<br />
not later than 24 h; in fact, delaying IVF over 24 h has<br />
resulted in a significant deterioration of oocyte<br />
developmental competence that could be predicted by<br />
the poor morphological appearance of oocytes matured<br />
for prolonged periods. An earlier aging of buffalo oocytes<br />
had been previously hypothesized based on the anticipated<br />
accomplishment of maturation, together with the<br />
increased incidence of degenerated oocytes at increasing<br />
times post-IVM [88]. The importance of oocyte aging in<br />
this species is also confirmed by activation studies that<br />
showed, in contrast to most other species, a deterioration<br />
of post-parthenogenetic embryo development at<br />
increasing times post-maturation [46]. This aspect is<br />
important to be considered when OPU is carried out<br />
because usually, to improve cost efficiency, all oocytes<br />
batches collected the previous OPU day are fertilized in<br />
one session.<br />
IV. IN VITRO FERTILIZATION (IVF)<br />
Fertilization has often been considered the most<br />
critical step of the IVEP procedures in buffalo, as cleavage<br />
rates lower than those obtained in other domestic species<br />
have been widely reported [41,47,86]. In our earlier<br />
studies, despite similar maturation rates (87% vs 94%<br />
respectively in buffalo vs cattle) significantly lower<br />
cleavage rates (65% vs 84%) were observed [86]. The<br />
overall lower IVEP efficiency recorded in buffalo<br />
compared to cattle (26 vs 34%, respectively) was mainly<br />
related to the poor cleavage rate; in fact similar blastocyst<br />
yields were obtained in buffalo and cattle (40%) when<br />
the percentages were calculated in relation to the zygotes. N<br />
Many factors may affect the in vitro fertilization<br />
efficiency, such as the adequate in vitro environment for<br />
gametes survival, the sperm viability and capability, the<br />
appropriate time of insemination, the duration of gametes<br />
co-incubation, the presence of cumulus cells and also the<br />
acquisition of the oocyte developmental competence<br />
during the complex process of cytoplasmic maturation.<br />
In fact, it is likely that the fertilization failure is related to<br />
inadequacies of the IVF system, but a previous<br />
inappropriate maturation of the egg should not be ruled<br />
out.<br />
The media commonly utilized for buffalo IVF<br />
are Tyrode’s modified medium (TALP) and Brackett<br />
Oliphant (BO), supplemented by sperm motility inducing<br />
factors, such as combined hypotaurine-penicillamine or<br />
caffeine. However, significantly higher cleavage and<br />
blastocysts rates have been obtained, in a direct<br />
comparison trial, by using TALP medium, supplemented<br />
with heparin, hypotaurine and penicillamine [47]. High<br />
sperm motility is required to accomplish fertilization, and<br />
this aspect is particularly important when frozen-thawed<br />
sperm is employed. A preliminary selection of motile<br />
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spermatozoa can be carried out by 1-hr swim-up method<br />
or a percoll density gradient. A direct comparison trial<br />
[75] recently reported that swim-up separated sperm give<br />
higher cleavage rate (66.8 vs 55.6%) and cleavage index<br />
than percoll gradient. However, sperm separated by<br />
swim-up showed significant differences among the bulls<br />
in cleavage rate and cleavage index (P < 0.05), that were<br />
not observed with the Percoll gradient method. In our<br />
experience a marked bull effect is also observed with<br />
swim-up: in particular, there are bulls that do not respond<br />
well to this separation approach, giving low concentrated<br />
sperm and, hence, requiring more straws/IVF. As a<br />
consequence, the choice of the method depends on the<br />
bull enrolled in the IVF program.<br />
In earlier times the quality of the frozen semen<br />
was considered the major factor impairing in vitro<br />
fertilization (IVF), based on the demonstration of several<br />
damages of the male gamete occurring following<br />
cryopreservation [77], together with the drastic reduction<br />
of cleavage rate reported with frozen compared to fresh<br />
semen [111]. Currently, the quality of frozen semen has<br />
improved, as indicated by similar fertility parameters,<br />
recorded for fresh compared to frozen semen [117],<br />
suggesting that other factors may negatively affect<br />
fertilization.<br />
However, the overall improvement of the quality<br />
of cryopreserved sperm has not eliminated another<br />
serious impediment, the so-called “bull effect”, consisting<br />
in the high degree of variation between buffalo bulls in<br />
the fertilizing capability in vitro [110]. It follows that,<br />
because only few bulls are characterized by good<br />
fertilizing capability in vitro (approximately 10 %), an<br />
accurate screening of sperm of several bulls is required<br />
in order to identify a suitable semen for IVF programs.<br />
Despite the availability of several fertility tests, it is known<br />
that currently the most accurate screening still goes<br />
through IVF trials, with different bulls tested on the same<br />
batch of eggs, that, in this species, because of the poor<br />
number of oocytes usable, are very time-consuming.<br />
Interestingly, it has been recently found that an easy, quick<br />
double staining technique with Trypan-blue/Giemsa [60]<br />
can be used to predict the fertilizing capability in vitro of<br />
buffalo bulls, as shown by the correlation existent between<br />
the percentages of acrosome-intact viable sperm cells at<br />
thawing and the blastocyst yields [7].<br />
Sperm need to undergo capacitation to acquire<br />
the fertilizing ability; this process, that in vivo occurs within<br />
the female genital tract, must be induced in vitro. Although<br />
several agents have been proven to induce sperm<br />
capacitation in vitro, heparin is still the most efficient<br />
method in the majority of the domestic species. In order<br />
to investigate whether the capacitation process in vitro<br />
can be improved by agents different than heparin, buffalo<br />
sperm have been incubated under different conditions<br />
and capacitation has been indirectly assessed by evaluating<br />
the capability of sperm to acrosome react following<br />
incubation with lysophosphatidilcholine, a fusogenic lipid,<br />
known to induce acrosome reaction in capacitated sperm<br />
without affecting motility. Doing so it has been<br />
demonstrated that progesterone [5], sodium nitroprusside<br />
[4] and melatonin [35] induce buffalo sperm capacitation<br />
in vitro and may be considered as alternative capacitating<br />
agents for buffalo IVF. However, the most interesting<br />
results have been obtained by incubating sperm with some<br />
biological fluids, such as buffalo estrus serum (BES) and<br />
the follicular fluid (FF) recovered from a pool of dominant<br />
follicles [6]. In fact, sperm treatment with both BES and<br />
FF resulted in a significantly higher incidence of<br />
acrosome-reacted sperm than with heparin treatment<br />
(84.3, 94.5 vs 50.1% respectively). It is likely that factors<br />
derived by BES and FF, present in the oviduct<br />
environment around fertilization, play a critical role in<br />
processing the male gamete in vivo.<br />
Therefore, we investigated whether mimicking<br />
the oviduct environment could improve the IVF efficiency<br />
in this species. It was demonstrated that both the<br />
capacitation process [104] and the oocyte penetration<br />
rate after heterologous IVF [71], are enhanced by<br />
incubating buffalo sperm on a 6-day bovine oviduct<br />
epithelial cells (BOEC) monolayer. This suggests to carry<br />
out IVF of buffalo oocytes on BOEC monolayer to<br />
improve the IVF efficiency in buffalo.<br />
A more practical approach to mimic the oviduct<br />
environment for optimizing fertilization is the identification<br />
of key molecules and their subsequent incorporation in<br />
the in vitro system. Osteopontin (OPN), an acidic singlechain<br />
phosphorylated glycoprotein, is one of the proteins<br />
present in both the oviductal fluid and epithelium in cattle,<br />
proven to improve IVEP efficiency and facilitate sperm<br />
capacitation [81]. In cattle OPN was proposed as a male<br />
fertility marker, as its expression in seminal plasma was<br />
highly correlated with bull fertility [20]. Osteopontin has<br />
been also detected in buffalo semen, at greater<br />
concentration in the seminal plasma than in sperm cells<br />
[96], suggesting that OPN is produced by the ampullae<br />
and seminal vesicles, similar to what was reported for<br />
cattle [20]. Interestingly, buffalo semen frozen by standard<br />
procedures showed a reduction in amount of OPN by up<br />
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to 50% [96]. Recently, we demonstrated that OPN<br />
improves the efficiency of capacitation in vitro [72] and<br />
that the supplementation of IVF medium with OPN<br />
significantly enhances cleavage rate (71.6 vs 59%) and<br />
blastocyst yields (29.9 vs 17.4%; [34]).<br />
It is known that proteolytic enzymes appear to<br />
have an essential role in multiple phases of mammalian<br />
fertilization. We recently reported that the addition of<br />
plasmin, the active enzyme of the plasminogen activation<br />
system, to heparin-capacitated buffalo sperm increases<br />
the percentage of acrosome reacted spermatozoa and<br />
stimulates motility [115]. Our results suggest that plasmin<br />
may play a role in events surrounding fertilization and<br />
suggest to evaluate in further studies whether the addition<br />
of plasmin during IVF improves the efficiency in buffalo.<br />
A positive effect of cumulus cells at the time of<br />
IVF has been observed in buffalo, as in cattle [118],<br />
demonstrated by the higher cleavage rate and embryo<br />
development obtained with cumulus-enclosed oocytes vs<br />
oocytes that were freed of their cumulus investment [45].<br />
Recently, we demonstrated that co-culture of buffalo<br />
oocytes deprived of their cumulus at the end of IVM<br />
with bovine intact COCs in a 1:1 ratio completely restores<br />
their fertilizing capability and post-fertilization development<br />
[28], suggesting that this approach can be used for<br />
technologies requiring cumulus removal, such as oocyte<br />
vitrification.<br />
Another factor that may affect embryo<br />
development is the duration of gamete co-incubation<br />
during IVF. It has been suggested that prolonged gamete<br />
co-incubation under the conditions of IVF, in which high<br />
concentrations of spermatozoa are incubated in small<br />
volumes of medium, results in the production of high levels<br />
of hydrolytic enzymes [98] and free radicals [1] that<br />
damage the oocytes. It was demonstrated that the optimal<br />
sperm-oocyte co-incubation time for maximizing the<br />
blastocyst yield in buffalo is 16 h [49]. Interestingly, the<br />
lower blastocyst development recorded at the shorter<br />
durations of sperm-oocyte co-incubation tested were<br />
mainly due to the lower cleavage rates, as suggested by<br />
the fact that the oocytes that had cleaved developed<br />
further and as fast as those in the 16 h group. On the<br />
contrary, extending gamete co-incubation to 20 h was<br />
deleterious because, despite similar cleavage rates, the<br />
blastocyst production was reduced. Furthermore,<br />
increasing the sperm-oocyte incubation time to 20 h was<br />
found to be correlated to a higher incidence of polyspermy.<br />
It is worth pointing out that these results were obtained<br />
using semen from a single bull previously tested for IVF.<br />
Subsequently, it was demonstrated that marked<br />
differences in the kinetics of sperm penetration exist<br />
among buffalo bulls and that this parameter is correlated<br />
to the blastocyst rate [99], and, hence, can be a useful<br />
marker to predict the in vitro fertilizing ability of buffalo<br />
bulls. The great variability in the penetration speed<br />
suggests to insert this assessment in the preliminary<br />
screening of bulls before their utilization in IVF programs.<br />
It is worth noting that among the 6 bulls tested the bull<br />
with the fastest penetration rate (64% penetration at 3 h<br />
post-insemination with the maximum rate – 72-77% –<br />
between 6 and 9 h) showed a significant increase in<br />
polispermy as early as at 12 h post-insemination. This<br />
finding strongly suggests to adapt the gametes coincubation<br />
time during IVF in relation to the bull used.<br />
As previously mentioned, the poor cleavage rate<br />
may also be due to the lack of oocyte developmental<br />
competence, normally acquired during the maturation<br />
process. In order to investigate these aspects oocytes<br />
were parthenogenetically activated. A significant<br />
improvement of cleavage (71 % vs 56 %, respectively)<br />
and blastocyst yield (33 vs 23 %, respectively) was<br />
obtained with ethanol-induced activation vs IVF, indirectly<br />
suggesting that buffalo oocytes had acquired the<br />
developmental competence during IVM [46]. It has more<br />
recently reported that activation with different methods N<br />
give significantly higher cleavage and blastocyst rates<br />
compared to IVF, suggesting that the problem has paternal<br />
rather than maternal origin [78].<br />
Nevertheless, it is worth pointing out that, after<br />
many fruitless attempts to increase cleavage rate in this<br />
species, the fertilization efficiency has at last improved,<br />
reaching approximately 80 % of cleavage rate, by<br />
enriching the IVM medium with thiol compounds. This<br />
improvement has been proven to be related to enhanced<br />
intracytoplasmic GSH levels [48]. This interesting finding<br />
would indicate that the poor cleavage rate of this species<br />
so far recorded was in part related to an inappropriate<br />
maturation of the female gamete. It has been, in fact,<br />
suggested that the GSH production is critical for the<br />
acquisition of developmental competence of oocytes at a<br />
cytoplasmic level and that the measurement of GSH at<br />
the end of IVM can be a reliable indicator of the<br />
cytoplasmic maturation [29].<br />
V. IN VITRO CULTURE (IVC)<br />
The development of in vitro culture systems for<br />
buffalo embryos has followed that for other ruminant<br />
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species. Thus, the IVC started from in vivo culture in an<br />
intermediate host, such as ligated oviduct of sheep [42],<br />
which was replaced by the co-cultural system, which<br />
was in turn substituted by defined media in the absence<br />
of serum and feeder cells. Buffalo embryos have been<br />
co-cultured with cumulus and oviductal cells[63,111] or<br />
with established cell lines such as BRL [13]. Although<br />
many authors still prefer the co-culture system for embryo<br />
production in this species, the utilization of defined media<br />
for embryo culture has become necessary to comprehend<br />
the requirements of buffalo embryos in vitro which, in<br />
turn, would allow the formulation of an optimal speciesspecific<br />
culture system. A well known defined medium,<br />
such as the Synthetic Oviduct Fluid (SOF) has been<br />
utilized for embryo culture in this species since 1999 [13],<br />
obtaining higher blastocyst rates compared to the coculture<br />
system with BRL cells (13.5% vs 7%).<br />
Subsequently, buffalo zygotes/embryos have been<br />
successfully cultured either in SOF and in another defined<br />
cell-free system, known as Potassium Simplex Optimized<br />
Medium (KSOM) with similar embryo development [21].<br />
The great improvement of blastocyst yields (35<br />
- 40%) achieved in the following years is, according to<br />
our experience, due to the optimization of the IVM and<br />
in part of the IVF systems rather than to modifications<br />
applied to the IVC system. In fact, at present the original<br />
version of SOF remains the most suitable medium for<br />
embryo culture in buffalo. Despite high blastocysts rates,<br />
pregnancy to term following ET of cryopreserved buffalo<br />
embryos is still very poor. This is likely due to poor viability<br />
of IVP embryos, resulting from suboptimal culture<br />
conditions.<br />
It has been demonstrated that buffalo oocytes<br />
and embryos undergo DNA damage during IVC,<br />
determined by oxidative stress, and that the incidence of<br />
DNA damage progressively increases throughout culture<br />
and, hence as development proceeds [82]. It has also<br />
been found that this damage can be partly ameliorated<br />
by supplementation of culture media with the antioxidant<br />
cysteamine [82].<br />
Currently, despite many efforts to improve<br />
embryo culture systems in different species, the oviduct<br />
remains irreplaceable for embryo development. The<br />
analysis of buffalo oviduct fluid composition indicated<br />
species-specific differences that may suggest the<br />
modification of the composition of the media used for<br />
buffalo IVEP [114]. Interestingly, both the protein<br />
concentration and the total quantity of proteins secreted<br />
in 24 h in buffalo ODF were 5 and 3.5 times lower<br />
respectively than those reported in cattle [59]. Notably,<br />
the protein concentration in buffalo ODF was<br />
approximately 4 times lower than the BSA concentration<br />
usually employed in media for both buffalo in vitro<br />
fertilization and embryo culture. This interesting finding<br />
suggests that a reduction in the protein concentration of<br />
media used for buffalo IVEP may be warranted. It is<br />
known that one of the major problems affecting viability<br />
of IVP embryos is the ammonium generated by protein<br />
metabolism and that has been associated to the large<br />
offspring syndrome in ruminants [40,74].<br />
The easy expedient to change the medium more<br />
frequently during culture for reducing the accumulation<br />
of free radicals, ammonium and other catabolites that<br />
may affect embryo development, successful in other<br />
species, did not exert any positive influence in buffalo<br />
[8]. It is likely that this is related to the higher sensitivity<br />
of buffalo embryos to fluctuations of temperature and/or<br />
pH that normally occur during a culture change even if<br />
to limited extents; it results that it is advisable “not to<br />
disturb” buffalo embryos during culture.<br />
In the attempt to modify the IVC system for<br />
buffalo embryos we demonstrated that energy<br />
requirements of buffalo embryos are different from those<br />
of sheep and cattle, that show a significant rise in glucose<br />
uptake just around compaction, i.e. during late culture<br />
[107,108]. Indeed, it has been demonstrated that relatively<br />
high concentrations (1.5 mM) of glucose are required<br />
for in vitro culture of buffalo embryos, particularly during<br />
the early embryonic development (up to Day 4). In fact,<br />
both the removal of glucose [80] in the IVC medium and<br />
the decreased concentration of the sugar [106] for the<br />
entire duration of culture or limitedly to early culture (up<br />
to Day 4) seriously compromised blastocyst development<br />
in buffalo. On the contrary, when glucose was provided<br />
during early culture and removed/reduced during late<br />
culture, blastocyst yields have been high and comparable<br />
to the control (SOF), in which glucose was present<br />
throughout culture. These findings highlight the<br />
importance of identifying species-specific requirements<br />
in order to optimize the in vitro culture conditions for<br />
buffalo embryos.<br />
Buffalo embryos in vitro develop approximately<br />
12 - 24 h earlier than cattle embryos [41] and this pattern<br />
of development reflects that observed in vivo, with most<br />
of the blastocysts collected by uterine flushing in the<br />
hatched stage at 6.5 days after the onset of oestrus [38].<br />
On Day 6 (Day = IVF) it is possible to find embryos in<br />
advanced stages of development, including hatched<br />
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blastocysts but most embryos reach the blastocyst stage<br />
on Day 7. A small proportion of embryos are delayed,<br />
reaching the blastocyst stage on Day 8 but their quality<br />
and viability is poor, as demonstrated by their lower<br />
resistance to cryopreservation [50]. Totey et al. [109]<br />
reported a higher embryo production efficiency along with<br />
faster blastocyst production and a higher cell number in<br />
case of early cleaved buffalo embryos. The poor viability<br />
of slower-developing buffalo embryos has been recently<br />
confirmed by their reduced total cell number and by the<br />
altered expression profile of developmentally important<br />
genes such as HSP-70.1 and GLUT-1 [97]. This finding<br />
implies that although blastocyst rate can be increased,<br />
prolonging the incubation time, it is at the cost of embryo<br />
viability.<br />
Hatching is also a good indicator of embryo quality.<br />
Supplementation of the IVC medium with FBS, BSA,<br />
and insulin can increase the hatching rate of Day 7 late<br />
morulae/early blastocysts [23]. We do not believe it is<br />
advisable though to extend the culture time to increase<br />
the hatching rate. In our in vitro standard system, hatched<br />
blastocysts are observed since Day 6 post-insemination,<br />
and the final embryo output is assessed not later than<br />
Day 7.<br />
VI. EMBRYO CRYOPRESERVATION<br />
Embryo cryopreservation allows to overcome the<br />
major problem affecting the commercial application of<br />
embryo transfer (ET) procedures, i.e. the limited number<br />
of suitable recipients, that is particularly accentuated in<br />
buffalo because of the lower response to hormonal<br />
stimulation and hence to synchronization treatments.<br />
Furthermore, because of the seasonality of the species,<br />
it is advisable to carry out the transfers in the most<br />
favourable period for reproductive activity. Unfortunately,<br />
buffalo IVP embryos seem very sensitive to<br />
cryopreservation, due to their high lipid content [16]. The<br />
tolerance to cryoprotectants may be increased by in vivo<br />
culture of the cleaved embryos in surrogate sheep oviducts<br />
[42], as demonstrated by improved development to term<br />
following transfers of embryos, which were frozen in 10<br />
% glycerol with the slow-freezing method. Nevertheless,<br />
although this system yields embryos of quality comparable<br />
to MOET, it requires appropriate facilities and is more<br />
unpractical and expensive.<br />
Buffalo embryos that were entirely produced in<br />
vitro have been successfully cryopreserved by<br />
vitrification, as demonstrated by their survival following<br />
in vitro culture [50] and development to term after ET<br />
[56,86,100]. Although development to term has been<br />
obtained (8-25%), the efficiency still needs to be improved<br />
for the diffusion of OPU-IVEP technologies in the field.<br />
In vitro produced embryos are less resistant to<br />
cryopreservation compared to their in vivo counterparts<br />
because of their lower viability resulting from suboptimal<br />
culture conditions. Among factors affecting the efficiency,<br />
the culture conditions play an important role, as<br />
demonstrated by the higher survival rate of frozen-thawed<br />
blastocysts recorded when cleaved embryos were<br />
cultured in serum-free defined medium such as mSOF<br />
than in complex co-culture system [85], similar to what<br />
reported in other species. In fact, it is known that bovine<br />
embryos cultured in the presence of cells and serum are<br />
darker because they uptake lipids and that the intracellular<br />
lipid content is negatively correlated to the efficiency of<br />
cryopreservation. Another factor affecting the efficiency<br />
of cryopreservation is the source of the oocytes, as<br />
indicated by the higher hatching rate after culture of<br />
vitrified-warmed blastocysts obtained from OPU-derived<br />
compared to abattoir-derived oocytes [66].<br />
In an earlier study, buffalo IVEP embryos have<br />
been cryopreserved by in straw vitrification, using a threestep<br />
dilution up to 3.4 M glycerol and 4.6 M ethylene<br />
glycol (EG) and warming into decreasing concentrations<br />
of sucrose [50]. It was demonstrated that the survival N<br />
rate after cryopreservation, assessed at 24 and 48 hrs<br />
culture post-warming, was greatly enhanced in the case<br />
of faster developing embryos, i.e. those that reached the<br />
blastocyst stage by day 6. Subsequently, three pregnancies<br />
were obtained after transfer of 8 buffalo embryos<br />
produced in vitro from OPU-derived oocytes (pregnancy<br />
rate of 37.5%) and vitrified with the same method, two<br />
of which developed to term (25 %).<br />
Hufana-Duran et al. [56] reported high hatching<br />
rate (83%) after 72 h in vitro culture post-warming and<br />
pregnancy to term (11%) after ET of cryopreserved IVP<br />
embryos that were vitrified in pointed-shape open straws<br />
using the EFS40 vitrification solution (ethylene glycol, 40%,<br />
v/v; ficoll, 18%, w/v; sucrose, 0.3 M). Interestingly, in<br />
this study, five of the six calves were born from recipients<br />
that received embryos following natural estrus while only<br />
one calf was conceived from estrus-synchronized<br />
recipient. It was speculated that the lower efficiency in<br />
synchronized recipients may be due to the higher<br />
incidence of a short luteal phase, resulting from a<br />
precocious luteolysis, as well as to an inappropriate timing<br />
of ET. This may derive from the greater difficulty of<br />
estrus detection in synchronized animals, in which estrus<br />
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signs are even weaker. The embryo-recipient synchrony<br />
is known to play a critical role for embryo survival after<br />
ET. In fact, it has been shown that buffalo in vivoproduced<br />
cryopreserved embryos can only tolerate<br />
asynchrony of 12 h [58]. The identification of the optimal<br />
time for ET may even be more critical for in vitro<br />
produced embryos because they are delayed in<br />
development and less viable.<br />
A significant improvement of embryo survival<br />
rate following IVC of vitrified-warmed buffalo IVP<br />
embryos has been recently obtained by using minimum<br />
volume vitrification methods, such as Open Pulled Straw<br />
[31] and Cryotop [30] vitrification. The first time OPS<br />
was used, the vitrification and warming solutions<br />
previously utilized to vitrify buffalo embryos in French<br />
traditional straws [50] were compared with those<br />
previously utilized for OPS vitrification of cattle embryos<br />
[113], and the overall embryo survival rate recorded at<br />
24 h was not significantly different between the two<br />
methods (70% vs 62% respectively).<br />
Subsequently, the cryotop was utilized for the<br />
vitrification of IVP buffalo embryos in 16.5 % EG and<br />
16.5 % DMSO and 0.5 M sucrose, with very promising<br />
results [30]. In these studies it was clearly demonstrated<br />
that the stage of development affects freezability of IVP<br />
embryos, with increased in vitro survival for the advanced<br />
embryo stages [30,31], such as the expanded blastocysts<br />
and hatched blastocysts (approximately 75%), which are,<br />
in any case, better quality embryos since they develop<br />
faster in vitro. Unfortunately, only few IVP embryos<br />
vitrified by CTV have been transferred so far into<br />
recipients (n =10; unpublished data) and, despite a high<br />
pregnancy rate (50%) diagnosed on day 30, the<br />
development to term was still poor (10%).<br />
In a recent study IVP buffalo embryos have<br />
been cryopreserved by a two-step vitrification method in<br />
straws, to examine the effects of different vitrification<br />
medium compositions (40% EG; 25% glycerol + 25%<br />
EG, and 25% EG + 25% DMSO) and exposure times (2,<br />
4 and 6 min in the equilibration solution) on the post-thaw<br />
development of buffalo IVP embryos [67]. The higher<br />
expansion (72%) and hatching rates (46%) after postwarming<br />
culture were found for blastocysts vitrified in<br />
25% EG + 25% DMSO with an exposure time of 4 min<br />
compared with the other combinations.<br />
It is known that cryoprotectants might cause<br />
irreparable damage to cytoskeletal components.<br />
Therefore, maintaining the integrity of the cytoarchitecture<br />
within an embryo during cryopreservation is of utmost<br />
importance. In porcines, treatment of embryos with<br />
cytochalasin-B prior cryopreservation was found to<br />
improve development [36]. In buffalo, cytoskeletal<br />
stabilization with cytochalasin-B did not improve the postwarming<br />
in vitro developmental competence of vitrified<br />
buffalo blastocysts [69] but favoured that of morulae.<br />
We may conclude that up to know there is not<br />
an overwhelming evidence of the superiority of a method<br />
vs the others for buffalo embryo cryopreservation.<br />
Different labs use different tools, and combinations of<br />
cryoprotectants/exposure times and hence it is also<br />
difficult to compare the results. The scenario is also<br />
complicated by the utilization of different culture systems,<br />
known to affect embryo viability. Regarding the tool, the<br />
choice may depend also on the future destination of the<br />
cryopreserved embryos, as according to the rules for<br />
transportation of embryos among different countries, it is<br />
not allowed to use methods based on the direct contact<br />
of the embryos with the liquid nitrogen because of the<br />
potential sanitary risks. However, in case the superiority<br />
of the minimum volume vitrification methods is clearly<br />
demonstrated also in terms of development to term after<br />
transfer, strategies may be developed to reduce these<br />
risks, that should take into account utilization of sterile<br />
filtered liquid nitrogen and a safe sealing system of the<br />
tools before storage in the tank.<br />
It is worth pointing out that the main factor<br />
affecting the efficiency of cryopreservation of<br />
mammalian embryos is undoubtedly the quality of the<br />
embryos and that it is current practice in cattle and other<br />
domestic species to operate a strict selection of the<br />
embryos prior to cryopreservation. This is not the case in<br />
buffalo because the low number of oocytes and, hence,<br />
of the embryos produced imposes the cryopreservation<br />
of all the embryos obtained, regardless of their quality.<br />
A strict selection of only Grade 1 embryos would<br />
result in improved cryopreservation efficiency further<br />
limiting, though, the number of embryos and, hence<br />
rendering the cost-benefit ratio of IVEP more<br />
unfavorable. Furthermore, as the embryo quality is in part<br />
due to the oocyte quality but is also affected by the culture<br />
conditions, the optimization of the culture system is<br />
compulsory to improve the efficiency of cryopreservation.<br />
VII. CONCLUSIONS<br />
The acquisition of more specific information on<br />
buffalo oocytes and embryo requirements in vitro, has<br />
led to a significant improvement of the IVEP efficiency<br />
in this species. However, although blastocyst yield has<br />
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jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335.<br />
greatly increased, pregnancy rates are still low and only<br />
few calves have been produced after transfer of<br />
cryopreserved embryos from live animals. In addition to<br />
the low number of oocytes recruitable, that seems to be<br />
a feature intrinsic to the species, an important limiting<br />
factor is the low resistance to cryopreservation of buffalo<br />
in vitro derived embryos, that can be also considered a<br />
result of poor viability due to suboptimal culture conditions.<br />
In fact, in contrast to the significant improvements of<br />
both the IVM and IVF systems for buffalo achieved<br />
throughout the years, leading to high blastocyst yields,<br />
the progress in the optimization of the IVC system is still<br />
far behind.<br />
Currently, the IVEP efficiency is highly<br />
competitive, with 90% maturation rate, 75-80% cleavage<br />
and 30-35% blastocyst rates. Operating a preliminary<br />
selection of the donors so that 3 viable oocytes are<br />
recovered on average per animal, it can be predicted<br />
that in 6 months approximately 50 embryos are produced/<br />
buffalo. This yield is much higher than with MOET<br />
programs. However, pregnancy rate after ET of<br />
cryopreserved embryos is still poor, ranging from 8 to 25<br />
%. If we consider an average pregnancy rate of 15%, 7<br />
calves can be obtained on average per buffalo after 6<br />
months OPU. It follows that, to make cost-benefits ratio<br />
more favourable, the only suitable options at present are<br />
to transfer either fresh embryos or frozen embryos<br />
previously cultured in vivo, i.e. in an intermediate host.<br />
It is likely that the poor pregnancy rate is mainly<br />
due to the scarce viability of IVP buffalo embryos,<br />
determined by suboptimal culture conditions. This can in<br />
part be also related to an inappropriate cryopreservation<br />
method. Furthermore, it is possible that the status of the<br />
recipient and the perfect synchrony between the age of<br />
the embryo and that of the recipient uterus play a role.<br />
With this regard, it is worth reminding that the percentage<br />
of buffaloes responding to synchronization treatments is<br />
low and inconsistent and that a high incidence of<br />
embryonic mortality is recorded in the unfavourable<br />
season also after natural mating.<br />
In conclusion, the optimization of culture<br />
conditions is fundamental to increase the viability of IVEP<br />
embryos for advanced reproductive strategies to become<br />
a routine procedure in buffalo breeding. At the same time<br />
efforts should be addressed to identify the best<br />
management of the recipients, in order to increase<br />
pregnancy maintenance after ET of cryopreserved IVP<br />
embryos.<br />
N<br />
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91 Palta P., Banzai N., Prakash B.S., Manik R.S. & Madan M.L. 1998. Endocrinological observation of atresia in individual<br />
buffalo ovarian follicles. Indian Journal of Animal Science. 68: 444-447.<br />
92 Pandey A., Gupta N. & Gupta S.C. 2009. Improvement of in vitro oocyte maturation with lectin supplementation and<br />
expression analysis of Cx43, GDF-9, FGF-4 and Fibronectin mRNA transcripts in Buffalo (Bubalus bubalis). Journal of<br />
Assisted Reproduction and Genetics. 26(6): 365-371.<br />
93 Pawshe C.H., Appa Rao K.B.C. & Totey S.M. 1998. Effect of insulin-like growth factor I and its interaction with gonadotrophins<br />
on in vitro maturation and embryonic development, cell proliferation, and biosynthetic activity of cumulus-oocytes complexes<br />
and granulosa cells in buffalo. Molecular Reproduction and Development. 49(3): 277-285.<br />
94 Pawshe C.H. & Totey SM. 1993. Effects of cumulus cells monolayer on in vitro maturation of denuded oocytes of buffalo<br />
(Bubalus bubalis). Theriogenology. 39(1): 281.<br />
95 Pellerano G., Gasparrini B., Crudeli G., Zicarelli L., Fontana S., Brandan N., Husulak A., Taboada C. & Cerdera A. 2007.<br />
Ovum pick-up and in vitro embryo production technology in field conditions in the North East of Argentina. In: <strong>Proceedings</strong><br />
of the 8th World Buffalo Congress (Caserta, Italy). Italian Journal of Animal Science. 6(2): 743-746.<br />
96 Pero M.E., Killian G.J., Lombardi P., Zicarelli L., Avallone L. & Gasparrini B. 2007. Identification of osteopontin in water<br />
buffalo semen. Reproduction, Fertility and Development. 19(1): 279.<br />
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jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335.<br />
97 Rajhans R., Kumar G.S., Dubey P.K. & Sharma G.T. 2010. Effect of timing of development on total cell number and<br />
expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in<br />
vitro. Cell Biology <strong>International</strong>. 34(5): 463-468.<br />
98 Rehman N., Collins A.R., Suh T.K. & Wright R.W. 1994. Effect of sperm exposure time on in vitro fertilization and embryo<br />
development of bovine oocytes matured in vitro. Theriogenology. 41(7): 1447-52.<br />
99 Rubessa M., Di Fenza M., Mariotti E., Di Francesco S., De Dilectis C., Di Palo R., Zicarelli L. & Gasparrini B. 2009.<br />
Kinetics of sperm penetration is correlated with in vitro fertility of buffalo (Bubalus bubalis) bulls. Reproduction, Fertility<br />
and Development. 21(1): 206-207.<br />
100 Sá Filho M.F., Carvalho N.A.T., Gimenes L.U., Torres-Junior J.R., Ferriera C.R., Perecin F., Perini A.P., Tetzner T.A.D.,<br />
Vantini R., Soria G.F., Garcia J.M., Tonhati H., Zicarelli L., Gasparrini B. & Baruselli P.S. 2005. Birth of the first buffalo<br />
calves after transfer of vitrified embryos produced in vitro in America. In: <strong>Proceedings</strong> of the 3° Congresso Nazionale<br />
sull’Allevamento del Bufalo - 1° Buffalo Symposium of Europe and the Americas (Capaccio-Paestum, Italy). p. 250.<br />
101 Sá Filho M.F., Carvalho N.A., Gimenes L.U., Torres-Júnior J.R., Nasser L.F., Tonhati H., Garcia J.M., Gasparrini B.,<br />
Zicarelli L. & Baruselli P.S. 2009. Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro<br />
buffalo embryo production. Animal Reproduction Science. 113(1-4): 51-59.<br />
102 Samad H.A., Khan I.Q., Rehman N.U. & Ahmed N. 1998. The recovery, in vitro maturation and fertilization of Nilli-Ravi<br />
buffalo follicular oocytes. Asian-Australasian Journal of Animal Science. 11: 491-497.<br />
103 Samad H.A. & Nasseri A.A. 1979. A quantitative study of primordial follicles in buffalo heifer ovaries. Compendium 13<br />
FAO/SIDA Int. Course Anim. Reprod.<br />
104 Siniscalchi C., Mariotti E., Boccia L., Albero G., Di Francesco S., Suárez M.V., Di Palo R. & Gasparrini B. 2010. Co-culture<br />
with oviduct epithelial cells promotes capacitation of buffalo (Bubalus bubalis) sperm. Reproduction, Fertility and<br />
Development. 22(1): 316.<br />
105 Smith J.F. 1984. Protein, energy and ovulation rate. In: Land RB, Robinson, DW (Ed). Genetics of Reproduction in Sheep<br />
(London,Butterworth). p. 349-359.<br />
106 Suárez Novoa M.V., Di Francesco S., Rubessa M., Boccia L., Longobardi V., De Blasi M. & Gasparrini B. <strong>2011</strong>. Effect of<br />
reducing glucose concentration during in vitro embryo culture in buffalo (Bubalus bubalis). Reproduction, Fertility and<br />
Development. 23(1): 168.<br />
107 Thompson J.G., Partridge R.J., Houghton F.D., Cox C.I. & Leese H.J. 1996. Oxygen uptake and carbohydrate metabolism<br />
by in vitro derived bovine embryos. Journal of Reproduction and Fertility. 106(2): 299-306.<br />
108 Thompson J.G., Simpson A.C., Pugh P.A., Wright Jr R.W. & Tervit H.R. 1991. Glucose utilization by sheep embryos<br />
derived in vivo and in vitro. Reproduction, Fertility and Development. 3(5): 571-576.<br />
109 Totey S.M., Daliri M., Appa Rae K.B.C., Pawshe C.H., Taneja M. & Chillar R.S. 1996. Differential cleavage and developmental<br />
rates and their correlation with cell number and sex ratios in buffalo embryos generated in vitro. Theriogenology. 45(2): 521-<br />
533.<br />
110 Totey S.M., Pawshe C.H. & Singh G.P. 1993. In vitro maturation and fertilization of buffalo oocytes (Bubalus Bubalis):<br />
Effects of media, hormones and sera. Theriogenology. 39(5): 1153-1171.<br />
111 Totey S.M., Singh G., Taneja M., Pawshe C.H. & Talwar G.P. 1992. In vitro maturation, fertilization and development of<br />
follicular oocytes from buffalo (Bubalus Bubalis). Journal of Reproduction and Ferti1ity. 95(2): 597-607.<br />
112 Tripp M.W., Ju J.C., Hoagland T.A., Riesen J.W., Yang X. & Zinn S.A. 2000. Influence of somatrotopin and nutrition on<br />
bovine oocyte retrieval and in vitro development. Theriogenology. 53(8): 1581–1590.<br />
113 Vajta G., Holm P., Kuwayama M., Booth P.J., Jacobsen H., Greve T. & Callesen H. 1998. Open Pulled Straw (OPS)<br />
vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development.<br />
51(1): 53-58.<br />
114 Vecchio D., Neglia G., Di Palo R., Campanile G., Balestrieri M., Giovane A., Killian G., Zicarelli L. & Gasparrini B. 2010.<br />
Ion, protein, phospholipid and energy substrate content of oviduct fluid during the oestrous cycle of buffalo (Bubalus<br />
bubalis). Reproduction in Domestic Animals. 45(5): 32-39.<br />
115 Venditto I., Mariotti E., Boccia L., Rubessa M., De Blasi M., Sattar A. & Gasparrini B. 2010. Effect of plasmin on acrosome<br />
reaction of buffalo (Bubalus bubalis) spermatozoa in vitro. Reproduction, Fertility and Development. 22(1): 317.<br />
116 Vittoria A. 1997. Anatomy of the female genital tract in the buffalo. In: <strong>Proceedings</strong> of the Third Course on Biotechnology<br />
of Reproduction in Buffaloes (Caserta, Italy). pp.15-20.<br />
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jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335.<br />
117 Wilding M., Gasparrini B., Neglia G., Dale B. & Zicarelli L. 2003. Mitochondrial activity and fertilization potential of fresh<br />
and cryopreserved buffalo sperm. Theriogenology. 59(1): 466.<br />
118 Zhang L., Jiang S., Wozniak P.J., Yang X. & Godke R.A. 1995. Cumulus cell function during bovine oocyte maturation,<br />
fertilization and embryo development in vitro. Molecular Reproduction and Development. 40(3): 338-344.<br />
119 Zicarelli L. 1997. Superovulatory response in buffaloes bred in Italy. In: <strong>Proceedings</strong> of the Third Course on Biotechnology<br />
of Reproduction in buffaloes (Caserta, Italy). pp. 167-188.<br />
N<br />
www.ufrgs.br/actavet<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A001 STUDENT COMPETITION<br />
CHANGES ON OOCYTE QUALIT<br />
ALITY OF REPEAT BREEDER HOLSTEIN COWS MAY EXPLAIN<br />
THEIR REDUCED FERTILIT<br />
TILITY<br />
Rob<br />
ober<br />
erta Machado Fer<br />
erreir<br />
eira 1 , Mar<br />
arcos Rob<br />
ober<br />
erto Chiar<br />
hiaratti<br />
2 , Henderson Ayr<br />
yres<br />
1 , Márcio Leão Fer<br />
erraz<br />
3 , Car<br />
arlos Alb<br />
lber<br />
erto Rodr<br />
drigues<br />
4 , Yeda Fumie<br />
Watanab<br />
tanabe 5 , Walt<br />
Yamazak<br />
amazaki 6 , August<br />
ugusto De Castr<br />
astro Nett<br />
etto 6 , Flávio<br />
Vieir<br />
ieira Meir<br />
eirelles<br />
elles 2 & Pietr<br />
ietro Sampaio Bar<br />
aruselli<br />
1<br />
1<br />
FMVZ-USP, SÃO PAULO, SP, BRAZIL. 2 FZEA-USP, PIRASSUNUNGA, SP, BRAZIL. 3 VIDA REPRODUTIVA, CRAVINHOS, SP, BRAZIL. 4 CLÍNICA VETERINÁRIA SAMVET DE SÃO CARLOS,<br />
SÃO CARLOS, SP, BRAZIL. 5 VITROGEN PESQUISA E DESENVOLVIMENTO EM BIOTECNOLOGIA DA REPRODUÇÃO S/C LTDA, CRAVINHOS, SP, BRAZIL. 6 BIOEMBRYO, BAURU, SP,<br />
BRAZIL.<br />
The aim of this study was to evaluate the relationship among mitochondrial DNA (mtDNA) copy number, expression of some<br />
genes and oocytes competence to develop into blastocysts in repeat breeder (RB) Holstein cattle. This study was conducted in 2 dairy farms in<br />
2009 (Exp 1) and <strong>2011</strong> (Exp 2). Heifers (H; n = 74), peak lactation (PL; n = 73) and RB (n = 71) cows were submitted to OPU after<br />
synchronization of follicular wave emergence. Exp 1 was conducted during summer and winter. Part of the oocytes (n = 100) was used to<br />
determine mtDNA copy number and gene expression. Part (n = 1.493) was used to IVP, and to determine the total number of cells and percentage<br />
of nuclear fragmentation of the resulting blastocysts. Exp 2 (47 oocytes) was conducted during summer to confirm data related to mtDNA<br />
quantification previously obtained. In Exp 1, mtDNA and gene expression were assayed in relation to H during winter. In Exp 2, a quantitative<br />
assay was employed to determine mtDNA copy number. At IVP, blastocyst rate (D7) was affected by category and season [winter: H=30.3%a<br />
(74/244) vs.. PL=22.0%b (42/191) vs.. RB=22.5%b (93/413); summer: H=23.3%a (35/150) vs.. PL=14.6%b (15/103) vs.. RB=7.9%c (14/<br />
177); P < 0.01). Blastocyst cell number was greater in H (253 ± 12) than in PL (203 ± 10) and RB cows (207 ± 8; P < 0.01). RB blastocysts<br />
had higher (P < 0.01) nuclear fragmentation rate during summer (3.9%) than H (1.8%) and PL (2.5%). In Exp 1, the number of copies of<br />
mtDNA was reduced by ~4 folds (P < 0.01) in oocytes from RB than H and PL during summer; however, this difference was not observed<br />
during the winter. In Exp 2, the number of copies of mtDNA was also reduced in RB (244.875 ± 39.314) compared to H (399.162 ± 65.969),<br />
but it was similar to PL (278.411 ± 53.661; P < 0.05). Regardless of season, RB oocytes had higher expression of PPARG, POLG, POLG2,<br />
TFAM, HSP90 e ITM2B compared to H and PL (P < 0.05). Also, higher expression of BAX/BCL2 ratio was observed in RB than PL cows<br />
(P < 0.05). However, during the summer, the expression of BCL2 was not detected in most of the H oocytes. These results suggest that the worse<br />
reproductive performance of RB, especially during heat stress, can be related to reduced oocyte competence to develop into blastocyst. The<br />
decreased of mtDNA copies and the increase in the expression of genes related to its replication may be stimulating the activation of compensatory<br />
mechanisms by RB. Also, it seems like that pro-apoptotic mechanisms were activated (i.e., increase of BAX/BCL2 ratio), which is reinforced<br />
by the occurrence of greater nuclear fragmentation rate in blastocysts from RB cows exposed to heat stress. [Acknowledgment: Farms Sta. Rita<br />
and Campestre, OuroFino, CAPES and FAPESP (proc09.00938-3)].<br />
N<br />
Keywords: oocyte, repeat breeder, mitochondrial DNA.<br />
A002 STUDENT COMPETITION<br />
EFFECT OF ZONA PELUCIDA DEFICIENCY ON EARLY DEVEL<br />
VELOPMENT OF BOVINE EMBRYOS<br />
Alejandra Estela Velasquez, , José Reinaldo Manriquez, Fidel Ovidio Castro & Lleretny Rodriguez<br />
UNIVERSIDAD DE CONCEPCIÓN, CHILLÁN, CHILE.<br />
During early embryo development the zona pellucida (ZP) first acts as a barrier against polyspermia and then guarantees communication<br />
between blastomeres before and during compaction. However, the development of new technologies for embryo production such as “hand made<br />
cloning” demands removal of this membrane to facilitate oocyte manipulation. At the moment very little is known about the effect of the absence<br />
of the ZP on early embryo development. We hypothesize that the quality of pre-implantated bovine embryos will be compromise because of the<br />
lack of ZP. In order to test our hypothesis we used as a model embryos produced by parthenogenesis and IVF. To assess the developmental<br />
potential of zona free embryos we analysed the rate of the first cleave and of blastocysts, total cell number as well as the expression of genes<br />
related with apoptosis and cell-cell communication (BAX, BCL2, Casp-3 and Ecad). In vitro matured oocytes were parthenogenetic activated<br />
using 7% ethanol followed by 5h incubation in cycloheximide/cytochalasin B (10µg/mL) or in vitro fertilized. The ZP was removed after<br />
parthenogenesis or IVF by incubation in 0,5 mg/mL pronase and embryos were assigned in two groups: zona included (ZI) and zona free (ZF).<br />
Embryos were cultured for 7d in SOFaci in sealed aluminium foiled bags. ZF embryos were cultured using the well of the well system (WOW).<br />
For gene expression analysis, Day-7 blastocysts were pooled in tens for RNA extraction, converted to cDNA and amplified with specific<br />
primers in real time PCR reactions. Data were analyzed with non-parametric tests using Infostat (Buenos Aires, Argentina). For IVF embryos,<br />
no significantly differences were found for the percentage of first cleavage after fertilization (ZF:80,4%; ZI:74,9%), or of Day-7 blastocysts<br />
(ZF:19,3%; ZI:21%) or the total cell number (ZF:132; ZI:146) among ZF and ZI embryos. The figure for parthenogenetic embryos followed the<br />
same general pattern: percentage of first cleavage (ZF:78,3%; ZI:51,3%), percentage of Day-7 (ZF:25,6%; ZI:28,8%) and total cell number<br />
(ZF:69; ZI:82). However the percentage of embryos that cleaved at 24h was significantly higher in ZF parthenogenetic embryos but this did not<br />
impact upon further development to blastocyst stage. Furthermore, no significantly differences in gene expression were obtained between ZF<br />
and ZI embryos for the studied genes. We concluded that the lack of ZP does not affect the early development when WOW system is used for<br />
embryo culture to ensure blastomere contact and normal compaction. This work was partially supported by Fondecyt grant No. 11100082 from<br />
the Ministry of Education of Chile.<br />
Keywords: zona pellucida; bovine embryos; embryo development.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A003 STUDENT COMPETITION<br />
EFFECT OF ETHANOL EXTR<br />
TRACT OF AZADIRACHT<br />
CHTA INDICA IN THE SYNCHRONIZA<br />
ONIZATION OF THE CELL CYCLE CLE OF BOVINE<br />
FIBROBLASTS<br />
Natana Chaves Rabelo 1 , Carolina Capobiango R. Quintão 2 , Ana Paula Moreira 3 , Savana Giacomini Brito 4 , Michele Munk Pereira 3 , Juliane Dornellas Nunes 2 ,<br />
Ana Luisa Sousa Azevedo<br />
edo 2 , Nadia Barb<br />
arbosa Rezende ende Rap<br />
aposo<br />
2,3 , Lilian Tam<br />
amy Iguma 2 , João Henr<br />
enrique Mor<br />
oreir<br />
eira Viana<br />
2 & Luiz Sérgio Almeida Camar<br />
amargo<br />
2<br />
1<br />
CENTRO DE ENSINO SUPERIOR DE JUIZ DE FORA, JUIZ DE FORA, MG, BRAZIL. 2 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 3 UNIVERSIDADE FEDERAL DE JUIZ DE<br />
FORA, JUIZ DE FORA, MG, BRAZIL. 4 UNIVERSIDADE PRESIDENTE ANTONIO CARLOS, JUIZ DE FORA, MG, BRAZIL.<br />
The success of somatic cell nuclear transfer (SCNT) depends initially on the cell cycle synchrony between the donor nucleus and<br />
the recipient cytoplasm. Many agents have been tested for cell cycle synchronization, but with limited success rate. Components of the<br />
Azadirachta indica A. Juss plant (popularly known as Neem) may be an alternative for cell cycle synchronization of donor cell for SCNT. The<br />
aim of the present study was to evaluate the effect of different concentrations of ethanol extract of Neem on synchronization at G0/G1 phases of<br />
the cell cycle of bovine fibroblasts. The extract was obtained by dynamic maceration and rotary evaporation. Bovine fibroblast cells collected<br />
from a Gyr cow were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity<br />
in air. After cells reaching about 70% confluence, the extract was at the following concentrations: 0 µg/mL, 50 µg /mL, 100 µg /mL, 200 µg /mL<br />
and 300 µg /mL, with exposure times of 12 and 24 h. Simultaneously, a control group with serum starvation (cells cultured in DMEM plus 0.5%<br />
FCS for three days) was prepared. To examine the cell cycle, we performed flow cytometry analysis (FacsCallibur, Becton Dickinson, San Jose,<br />
CA, USA), and DNA histograms were analyzed with the WinMDI software to determine the percentage of cells at G0/G1, S and G2 phases.<br />
Three repetitions were performed in triplicate for each concentration. Results were analyzed by analysis of variance and means compared by<br />
Student Newman Keuls. Concentrations of 100 µg/mL (88.6 ± 0.3%) e 200 µg/mL (88.4±0.4%), both at 24 h of exposure, resulted in more (P<br />
< 0.05) cells arrested in G0/G1 phase than the other concentrations, but they were similar to the serum starvation group (89.6 ± 0.3%). In<br />
conclusion, ethanol extract from Azadirachta indica A. Juss is able to synchronize cell cycle of bovine fibroblast, arresting in G0/G1 phase.<br />
Further studies are needed to evaluate the reversibility of such arrest and the cell viability for SCNT. [Financial Support. CNPq, FAPEMIG and<br />
Innovation Network Project on Animal Reproduction (01.07.01.002)].<br />
Keywords: cell cycle, flow cytometry, nuclear transfer.<br />
A004 STUDENT COMPETITION<br />
INFLUENCE OF HIGH OR LOW INTAKE OF DRY MAT TER/ENERGY ON IN VITRO PRODUCTION OF BOVINE<br />
EMBRYOS<br />
Alexandre Barbieri Prata 1 , Ricardo Silva Surjus 1 , Marta Borsato 1 , Mariana Curci Martins da Silveira 1 , Maria Clara Costa Mattos 1,2 , Gerson Barreto Mourão 3 ,<br />
Flávio Augusto Portela Santos 1 , Andrea Cristina Basso 1,2 , José Henrique Fortes Pontes 1 & Roberto Sartori 1<br />
1<br />
ESALQ/USP, PIRACICABA, SP, BRAZIL. 2 IN VITRO BRAZIL, MOGI MIRIM, SP, BRAZIL. 3 ESALQ/USP, PIRACICABA, SP, BRAZIL.<br />
The aim of this study was to evaluate the influence of high or low dry matter (DM) intake and/or energy on oocyte quality and<br />
embryo production in vitro. Nonlactating Nellore cows (n = 32, 4 to 10 years old) weighing 489.5 ± 11.3 kg and with a BCS of 3.25 (scale from<br />
1 to 5) were used. The cows were confined without access to pasture, being two animals per stall. Mineral salt was provided in the diet and water<br />
was offered ad libitum. After 15 days on the adaptation diet, groups of cows were blocked by initial body weight (BW) and randomly divided<br />
in four experimental groups. The maintenance group (M) received a diet to provide 1.2% of DM per kg of BW. The restriction group (0.7M)<br />
received the equivalent of 70% of the Group M diet (0.84% of DM per kg of BW). The high intake group (1.5M) received the equivalent of 150%<br />
of the M group (1.8% of DM per kg of BW). The energy group (E) received a diet with the DM similar to the M group, however, with an energy<br />
level equivalent to the 1.5M group. The cows were offered all diets in a crossover design study. There were four sessions of ovum pick up<br />
(OPU), 42 days apart. Recovered oocytes were classified and taken to the In vitro Brazil laboratory, where all procedures for in vitro embryo<br />
production were performed. Data were analyzed by PROC GLIMMIX of SAS and the results are presented as least squares means ± SE, always<br />
following the order of treatments M, 0.7M, 1.5M and E. More total (20.2 ± 2.0b; 23.0 ± 2.3a; 21.5 ± 2.2ab and 20.1 ± 2.0b; P = 0.02) and viable<br />
(14.4 ± 1.6b; 17.0 ± 1.9a; 15.7 ± 1.7ab and 14.1 ± 1.6b; P = 0.006) oocytes were recovered per cow per OPU session in Group 0.7M than in<br />
Groups M and E. However, there was no difference in the number of atretic and degenerate oocytes per treatment (5.7 ± 0.6; 5.7 ± 0.6; 5.6 ± 0.6<br />
and 5.7 ± 0.6; P = 0.99). Although Group 0.7M had a higher number of cleaved oocytes than Group M (10.7 ± 1.4b; 13.4 ± 1.7a; 12.6 ± 1.6ab<br />
and 11.7 ± 1.5ab; P = 0.04), the number of blastocysts was similar among treatments (5.4 ± 0.8; 6.9 ± 0.9; 5.9 ± 0.8 and 6.6 ± 0.9; P = 0.15).<br />
There was also no difference among groups regarding the percentage of viable oocytes (72.8, 75.4, 72.9 and 71.7%; P = 0.41) or blastocysts<br />
(31.9; 30.6; 31.1 and 34.0%; P = 0.67) per total oocytes, or percentage of blastocysts per cleaved oocytes (52.7; 53.2; 46.5 and 56.4%; P = 0.14).<br />
Although there was an apparent advantage of food restriction on the number of total, viable, and cleaved oocytes compared to other groups, such<br />
feature was not translated into blastocyst production. Likewise, contrary to our initial hypothesis, high DM or energy intake for a period of 42<br />
days did not seem to compromise the in vitro embryo production from Nellore cows. [Acknowledgment. Fapesp and CNPq].<br />
Keywords: nutrition, in vitro production, embryo.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A005 STUDENT COMPETITION<br />
OOCYTE RECOVER<br />
OVERY IN QUEENS AFTER CONTR<br />
ONTRACEPTIVE<br />
TREATMENT<br />
WITH DESLORELIN ACET<br />
CETATE TE (SUPRELORIN<br />
®)<br />
Camila Louise Ackermann, , Eduardo Trevisol, Rodrigo Volpato, Ana Augusta Pagnano Derussi, Carlos Renato De Freitas Guatolini, Flávia Caroline Destro,<br />
Nicole Ruas Sousa,<br />
Tatiana Da Silv<br />
ilva Rasc<br />
ascado ado & Mar<br />
aria Denise Lop<br />
opes<br />
UNESP, BOTUCATU, SP, BRAZIL.<br />
GnRH agonists are used as reversible contraceptives in wildlife as an alternative to chemical contraceptives that cause adverse<br />
effects and surgical contraception. However, the GnRH agonists reversibility is not completely understood, few studies describe spontaneous<br />
estrus after use this contraceptive, and only one described induced ovulation (ACKERMANN et al., <strong>2011</strong>; Anais XIX CBRA, <strong>2011</strong>.). This<br />
study aimed to evaluate the reversibility of deslorelin acetate in female cats through the induction of estrus, ovulation and subsequent oocyte<br />
recovery. Ten pubescent female cats underwent vaginal cytology (CV) three times a week to identify the stages of the estrous cycle. When the<br />
females presented CV characteristic of interestrus, evidenced by a percentage lower than 70% of superficial cells, the animals were sedated,<br />
an implant of deslorelin acetate (4.7 mg Suprelorin ® ) was introduced in the subcutaneous tissue interscapular region. After insertion of the<br />
implants, CV were performed every 48 h, and on day 90 the implants were removed. Ten days after implant removal, estrus and ovulation<br />
were induced using 100 IU eCG and 84 h later, 100 IU hCG, respectively. Three days after hormone treatment, females were submitted to<br />
ovariohysterectomy. The oviducts were washed with PBS at 38°C and the liquid recovered was observed under stereomicroscopic magnifying<br />
glass for identification and enumeration of potential oocytes. The oocytes were isolated and stained with Hoechst 33342 and propidium iodide<br />
for viability assessment in a fluorescent microscope Leica DM LB ® - blue filter (535 and 617 nm); oocytes were considered viable when<br />
fluoresced blue. The corpora lutea (CL) were quantified, and to calculate the oocyte recovery rate the following formula was used: number of<br />
oocytes recovered/number of CL x 100. The results were described as mean and standard deviation. After application of the implant, three<br />
females showed CV characteristic of estrus during 4.3 ± 2.2 days. After this period, all 10 females showed CV characteristic of ovarian<br />
quiescence. All females responded the induction of estrus and the ovulation protocol presenting CV and estrus behavior. On average, 13.1 ±<br />
5.5 CL and 8.1 ± 5.5 oocytes were quantified per cat. The oocyte recovery rate was 56.8% ± 25.4 and all the stained oocytes were viable. We<br />
conclude that there is reversibility of ovarian activity after implant of deslorelin acetate in domestic cats since estrus and ovulation were<br />
induced successfully, allowing the recovery of viable oocytes.<br />
Keywords: domestic cats, GnRH agonists, contraception.<br />
A006 STUDENT COMPETITION<br />
THE METHYLATION TION PATTERNS TERNS OF THE IGF2 AND IGF2R GENES IN BOVINE SPERMATOZ<br />
OZOA ARE NOT AFFECTED BY FLOW<br />
CYTOMETR<br />
OMETRY SEX SORTING<br />
José De Oliveira Carvalho Neto 1 , Valquíria Alice Michalczechen-Lacerda 2 , Roberto Sartori 3 , Fernanda Castro Rodrigues 4 , Otávio Bravim 5 , Maurício Machaim<br />
Franco 6 & Margot Alves Nunes Dode 7<br />
1,3,5,8<br />
UNIVERSIDADE DE SÃO PAULO, PIRACICABA, SP, BRAZIL. 2,5,8 UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL. 4 UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA,<br />
MG, BRAZIL. 6,7,8 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL.<br />
The process of sexing by flow cytometry exposes sperm to factors that can cause epigenetic changes, such as the methylation pattern<br />
of the DNA. These changes may not affect development until the blastocyst stage but may explain later problems in embryo development and<br />
failures in plancentation. These problems are common in cloned embryos, with higher embryonic loss between Days 30 and 90 of gestation likely<br />
due to abnormal methylation of DNA. The objectives were to investigate the effect of sexing by flow cytometry on the methylation patterns of<br />
the IGF2 and IGF2R genes. Each ejaculate of Nellore bulls (n = 4) was collected and separated into three fractions: non-sexed (NS), sexed for<br />
X-sperm (SX), and sexed for Y-sperm (SY). Frozen-thawed sperm cells were used for genomic DNA isolation that was then treated with<br />
sodium bisulfate in three different replicates. Treated DNA was used for PCR amplification, which was cloned, transformed into E. coli and<br />
sequenced for analyses of the methylation patterns. For comparisons among groups, the Kruskal-Wallis test was performed (P = 0.05). No<br />
differences in DNA methylation were found among NS (96.4 ± 0.4 and 7.2 ± 0.6%), SX (95.4 ± 0.4 and 8.0 ± 0.6%) and SY (96.9 ± 0.4 and<br />
8.0 ± 0.6%) groups for IGF2 (n = 195 sequences) and IGF2R (n = 150 sequences) genes, respectively. However, for the IGF2R gene, a very<br />
specific methylation pattern was observed in the <strong>25th</strong> and 26th CpG sites, which were highly methylated in the NS (86.4 and 84.1%), SX (86.2<br />
and 81.0%) and SY (89.6 and 62.5%) groups, differently from the pattern expected for this region in sperm. Furthermore, the percentage of<br />
methylation on the 26th CpG site was lower in the SY group than NS and SX groups. In addition, after DNA sequencing, we identified 28 CpG<br />
sites in each clone of the IGF2 gene, which was different from the 27 CpGs reported by other studies in Bos taurus. When the methylation pattern<br />
between treatments was compared for each bull, bull 3 had a higher percentage of methylation in the SY group than in the SX, for the IGF2 gene.<br />
However, when comparing bulls for each treatment, we observed an effect of bull in the methylation of the gene IGF2R for the SY group. Bulls<br />
1 and 3 (6.1 ± 0.7 and 6.4 ± 0.6%) had a lower percentage of methylation in comparison to bulls 2 and 4 (9.9 ± 1.8 and 9.6 ± 1.3%). In conclusion,<br />
the sex-sorting procedure by flow cytometry did not affect the DNA methylation patterns of the IGF2 and IGF2R genes. However, there was<br />
a bull variation in the methylation pattern. Furthermore, a polymorphism was found in IGF2 gene, and a very specific methylation pattern was<br />
observed in the IGF2R gene, probably due to an epigenetic characteristic in Bos indicus cattle. [Financial support. CNPq, FAPESP and<br />
Embrapa].<br />
Keywords: epigenetic, methylation pattern of the DNA, sexed sperm.<br />
N<br />
s339
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A007 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
HIGH FAT DIET AND GENETIC DYSLIPIDEMIA IMPAIRED<br />
TESTICLE MORPHOMETRY IN MICE: PRELIMINARY RESULTS<br />
Paulo A B Cordeiro, Ana Cristina Silva De Figueiredo, , Miller Pereira Palhão, Iolanda Christina Souza Loyola, Marilu Martins Gioso, Carlos Antônio De<br />
Carvalho Fernandes & Jose Antonio D Garcia<br />
UNIFENAS, ALFENAS, MG, BRAZIL.<br />
High levels of cholesterol and low plasma concentrations of testosterone are associated with a high risk for atherosclerosis.<br />
Therefore, cholesterol is the major precursor for sex hormones. Thus, the mice deleted for LDL-receptor gene (LDLr-/-) are related to low tissues<br />
influx of cholesterol, which can affect the normal function of the gonads. The aim of this study was to evaluate the effects of high levels of<br />
cholesterol on circulating concentrations of insulin and glucose. Additionally, changes in testicle morphometry were evaluated in LDLr-/- mice<br />
feeding with different levels of fat. For this purpose, male mice (n = 30) aged 3 months were randomly divided in 3 groups: 1) WT (n = 10) –<br />
a wild type mice feeding regular diet; 2) S (n = 10) - gene deleted mice (LDLr-/-) and regular diet; 3) HL (n = 10) - LDLr-/- mice and diet with<br />
high levels of fat. Fifteen days after the period of feeding adaptation, blood samples were collected for plasma analysis of the lipids, insulin and<br />
glucose. The HOMA index ([Insulin*Glucose]/22,4) was calculated to establish the insulin resistance condition. After testis removal, sectional<br />
slices of the tissue were prepared for histological analysis. For each testicle, one microscope slide with 4 sections of the tissue were prepared and<br />
stained with Haematoxylin and eosin to assess morphological and morphometric changes. Data were submitted to ANOVA and differences<br />
among groups were accessed by Tukey test (5%). The plasma concentrations of HDL were high in S mice, but the insulin resistance was not<br />
detected even with moderate hyperlipidemia when compared to WT. For HL mice, very high levels of total lipids were observed (severe<br />
hyperlipidemia) and insulin resistance were associated with a decreasing levels of plasma HDL, when compared to S. Pronounced morphological<br />
changes of the testicles were observed in HL group. The morphometric analysis had shown an increase in the length and width of the testicles<br />
in HL (4.6±0.2 and 7.4±0.2 mm) compared to WT (3.0±0.1 and 5.0±0.2 mm) and S (2.6±0.2 e 5.0±0.2 mm) groups. However, no significant<br />
differences were observed for testis (mg)/body (g) weights ratio among groups (4.3±0.1, 4.4±0.1, 4.5±0.2 mg/g for WT, S and HL groups,<br />
respectively). At histologic analysis of the testis, the average thickness of the seminiferous tubules was greater in WT (10.0±0.7 µm) when<br />
compared to S (6.0±0.3 µm) and HL (6.0±0.4 µm) groups. However, the seminiferous tubule in WT (1.6±0.1 µm) group had smaller diameter<br />
of the lumen compared to S (10.7±1.6 µm) and HL (7.5±0.6 µm). In conclusion the genetic dyslipidemia associated with high fat diet induces<br />
insulin resistance in mice. Metabolic changes in insulin, glucose and HDL levels affected the testicle structure, mainly in LDLr-/- mice feeding<br />
high fat diets. Further analysis of the plasma testosterone concentrations and the seminiferous epithelium can contribute to better elucidate the<br />
testicles changes. [Support. FAPEMIG].<br />
Keywords: testoterone, insulin, lipids.<br />
A008 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
RECOMMENDED ANTIBIOTIC TIC FOR RAM SEMEN CRYOPRESER<br />
OPRESERVATION EXTENDER<br />
Elisângela Mirapalheta Madeira, Karina Lemos Goularte, Jorgea Pradieé, Ivan Bianchi, Fábio Pereira Leivas Leite, Rafael Gianella<br />
Mondadori, , Arnaldo Diniz Vieira & Thomaz Lucia Jr<br />
UFPEL, PELOTAS, RS, BRAZIL.<br />
Antibiotics used in ram semen cryopreservation were only tested in bovines, without an assessment of effectiveness in controlling<br />
bacterial and the possible impact on ovine spermatozoa. In consequence, this study aimed to determine the bacterial control ability and the<br />
influence on sperm viability of different antibiotics for use in freezing extender. Treatments were established using a pool 20x10 9 sperm/mL<br />
obtained from the combination of the ejaculates of five Crioula lanada breed rams. In each routine (n = 5), microbiological evaluation and<br />
determination of sperm viability were performed before and after freezing. The base extender used was tris-egg-yolk-glycerol without antibiotics<br />
(control) or supplemented with antibiotics: gentamicin (500 µg/mL) + tylosin (100 µg/mL) + lincomycin (300 µg/mL) + spectinomycin (600 µg/<br />
mL) = GTLS, penicillin (500 µg/mL) + streptomycin (100 µg/mL) = PENSTREP; sodic ceftiofur (50 µg / mL) = CEFT and enrofloxacin (1000<br />
µg / mL) = ENRO in total dose or concentration 50 and 25% lower (-50 and -25) and higher (+25 and +50) to form the treatments (n = 21). The<br />
diluted semen was packaged in 0.25 mL straws, cooled and stabilized at 5° C, frozen in LN2 vapor and stored in a cryogenic container. Sperm<br />
viability was determined by evaluating the motility and morphology under phase microscopy and plasma membrane integrity, acrosome and<br />
DNA under epi-fluorescence microscopy. Semen samples from each treatment were plated by spreading on agar and brain heart infusion (BHI)<br />
and incubated at 37°C/48 h, for colony forming units (CFU) counting. Comparisons between means were made by Tukey test. Plasma<br />
membrane integrity, acrosome and sperm DNA, were not affected by treatment (P> 0.05), however, sperm motility was negatively impacted in<br />
a dose-dependent manner in ENRO group. The number of CFU in the control group (no antibiotics) was higher (P < 0.05) than in other<br />
treatments. The groups PENSTREP and CEFT (in all concentrations used) were less efficient (P < 0.05) than the groups GTLS and ENRO,<br />
especially in treatments GTLS+50, ENRO+25 and ENRO+50 which promoted a reduction in the number of CFU. Therefore it is concluded that,<br />
despite being effective in reducing the number of CFU, enrofloxacin is not recommended for use in ovine semen extender. The GTLS<br />
association, 25% more concentrated than the base, is highly recommended for ram semen extender since controlling bacterial growth without<br />
compromising viability.<br />
Keywords: viability sperm, antibiotics, ram.<br />
s340
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A009 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
QUALIT<br />
ALITATIVE TIVE ANALISIS OF THE ESTABLISHED SPERMATOGENESIS IN CAVIA PORCELL<br />
CELLUS<br />
(LINNAEUS 1758)<br />
Adr<br />
driana Gradela<br />
1 , Amanda Kar<br />
aroline Rodr<br />
drigues Nunes<br />
1 , Laura a Flávia<br />
Teix<br />
eixeir<br />
eira Mar<br />
artins<br />
1 , Juliana Muniz San<br />
antos<br />
1 , Bruna Bor<br />
ortolini olini Gouv<br />
ouveia<br />
1 , Vanessa Sobue<br />
Franzo 2 & Marcelo Domingues Faria 1<br />
1<br />
UNIVASF, PETROLINA, PE, BRAZIL. 2 UFMT, CUIABÁ, MT, BRAZIL.<br />
Knowledge of testicular physiology and spermatogenesis are essential for the study of germ line stem cells, which are of great<br />
practical value in neoplasms or endangered animals, to safeguard their germ line. In this sense, the Cavia porcellus can be of great importance<br />
in animal experiments (Snitkoff. In: Gennaro, A.R.. Remington: a ciência e a prática da farmácia. 20.ed. RJ: G. Koogan, 2004. p.556-68). Our<br />
objective was to investigate the development of testicular parenchyma of C. porcellus, evaluating the progress of the process of lumination of the<br />
testicular cords. Thirty males from the UNIVASF vivarium – Petrolina- PE were used and divided into six experimental groups with five animals<br />
each, within the age of three, five, six, seven, eight and eleven weeks. After fixation and subsequent histological processing of the testicles, were<br />
analyzed: the process of seminiferous tubule lumination, the presence of primary spermatocytes, spermatids and the formation of the early stages<br />
of the seminiferous epithelium cycle (SEC) according to the tubular morphology method. Animals were classified in stages of testicular<br />
development (Courot et al. In: Johnson et al. (Eds.) The testis. NY and London: Acad. Press, 1970. v.1, p.339-432). With three weeks formed<br />
gaps of varying sizes even though many testicular cords had persisted with vacuolization in the central cytoplasmic mass and had appeared early<br />
primary spermatocytes and round spermatids, featuring the pre-pubertal testicular development. Within five weeks there was seminiferous<br />
tubules formed with elongated spermatids, indicating the puberty stage. However this process occurred asynchronously, verifying testicular<br />
cords in different stages of vacuolization between the seminiferous tubules already with lumen. After six to seven weeks the tubular lumen was<br />
extensive and after eight weeks there was the expansion of the tubular lumen associated with cell proliferation in the seminiferous epithelium and<br />
the increase of testicular volume and weight, indicating the post-pubertal stage, with spermatogenesis showed itself established and the presence<br />
of associations of the stages of the (SEC). We conclude that in the C. porcellus the period of three weeks of age corresponded to the pre-puberty<br />
stage, five to seven weeks of the puberty stage, and from 8 to 11 weeks of post-puberty.<br />
Keywords: Cavia porcellus, spematogenesis, process of lumination.<br />
A010 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
GLYCER<br />
CEROL AND DIMETHYLFORMAMIDE CRYOPRETECT<br />
OPRETECTANS ASSOCIATION FOR THE OVINE SEMEN<br />
CRYOPRESER<br />
OPRESERVATION<br />
Rodrigo Freitas Bittencourt 1 , Antônio De Lisboa Ribeiro Filho 2 , Gabriel Felipe Oliveira De Menezes 3 , Renata Cardoso Andrade 4 ,<br />
Lais Oliveira Mascarenhas 5 , Priscila Assis Ferraz 6 , Alexandra Soares Rodrigues 7 , Marta Vasconcelos Bittencourt 8 & Marcos<br />
Chalhoub 9<br />
1,3,4,5,8<br />
FACULDADE DE CIÊNCIAS AGRÁRIAS E DA SAÚDE - UNIME, LAURO DE FREITAS, BA, BRAZIL. 2,6,7,9 ESCOLA DE MEDICINA VETERINÁRIA - UFBA, SALVADOR, BA, BRAZIL.<br />
N<br />
Ten semen samples of five rams of the Santa Ines breed were cryopreserved with the objective of verifying the use of cryoprotectants<br />
glycerol and dimethyl formamide alone (GL6% e DF3%) or in different levels of association (GL5%+DMF1%, GL4%+DMF2%,<br />
GL3%+DMF3%, GL2%+DMF4% e GL1%+DMF5%). The base extender was the Tris-egg yolk (TRIS). After evaluation, the semen was<br />
diluted in the different extenders, cooled to 5°C and afterward the samples were frozen in liquid nitrogen vapour. After the thawing, the kinetic<br />
sperm parameters were analyzed (total motility-TM, progressive motility-PM and sperm vigor-VIG). Aliquots were collected for the supravital<br />
test dye eosin (EOS); the sperm morphology was analyzed and the percentage of bent tails (BT) calculated. The functional integrity of the<br />
plasmatic membrane was studied by osmotic shock (OS) followed the dilution: One part semen to 10 (OS 10) parts of the final solution with<br />
deionized water. After the diluted semen incubation for five minutes at 37°C, it was fixed with 10 µL of formaldehyde buffered saline. The<br />
percentage of reactive spermatozoa to OS was determined by subtracting the percentage of spermatozoa with OS-induced BT, by the BT obtained<br />
after thawing. These evaluations were observed in phase contrast microscopy (1000x) and one hundred cells were analyzed per semen<br />
sample.All the statistical analyses were performed using the SAS software version 5.0 (1996) (Proceeding MEANS and GLM with P < 0.05).<br />
The means (%) of post-thaw TM, EOS and OS were, for the GL6%: 78,0 a ; 44,8 and 42,2; DF3%: 47,0 b ; 41,4 and 33,6; GL5%+DMF1%: 73,0 a ;<br />
37,8 and 37,7; GL4%+DMF2%: 73,0 a ; 48,2 and 47,4; GL3%+DMF3%: 71,0 a ; 43,4 and 44,4; GL2%+DMF4%: 52,0 b ; 40,4; 39,0 and<br />
GL1%+DMF5%: 43,0 b ; 31,8 and 47,6.It was observed greatest (P < 0,05) rates of TM and PM for the extenders GL6%, GL5%+DMF1%,<br />
GL4%+DMF2% and GL3%+DMF3% in relation to the groups with higher levels of DMF (GL2%+DF4% e GL1%+DF5%) or when this one<br />
was alone (DF3%). Despite the wide numerical variations, the rates of EOS, OS and BT did not differ (P > 0,05) among the groups. It can be<br />
concluded that the DMF and GL association was effective for the sperm viability maintenance, although high levels of DMF, or in the absence<br />
of GL, it had deleterious effect on post-thaw ovine sperm. [Financial support. FAPESB- Brazil].<br />
Keywords: ovine, semen, cryoprotectants.<br />
s341
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A011 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
ECHOGENICITY EVAL<br />
ALUATION OF TESTICULAR PARENCHYMA IN PREPUBERTAL OVINES<br />
Pedr<br />
edro Paulo Maia<br />
Teix<br />
eixeir<br />
eira, Dio<br />
iogo José Car<br />
ardilli,<br />
Luciana Cristina Padilha,<br />
Car<br />
arla Cristina D’ama<br />
’amato, Mar<br />
aria Emilia Franc<br />
anco Oliv<br />
liveir<br />
eira,<br />
Felip<br />
elipe e Far<br />
arias Per<br />
ereir<br />
eira Da<br />
Câmara Barros, Leandro Nassar Coutinho & Wilter Ricardo Russiano Vicente<br />
FCAV/UNESP, JABOTICABAL, SP, BRAZIL.<br />
The aim of this study was to establish an ultrasonographic standard of echogenicity of testicular parenchyma of prepubertal<br />
ovine correlationated to testicular biometry. Eight Santa Ines ovines, 6 months old, were evaluated in Jaboticabal, SP, Brazil. An Aquila Vet<br />
ultrasound with a 6 MHz linear transducer were used. Images of sagittal, transverse and frontal plans were analyzed of left and right testicles.<br />
Using echogenitcity image function (grey scale) of this ultrasound, predeterminated areas of testicular parenchyma were selected. The<br />
biometrics evaluations were done in according to CBRA (1998). Data were expressed in mean and standard deviation by one-way test ANOVA<br />
(non-parametric) and linear regression for correlationing, using the statistical package Grafped Prisma 4. The testicular parenchymas, in a<br />
general way, were observed as homogeneous and with an echogenicity of 53,95±6,5%, 55,70±6,4% and 55,68± 6,4% to right, left and the<br />
mean of testicles, respectively. Also, a high correlation between echogenicity and scrotal circumference (P = 0,0027, r2= 0,80), was verified.<br />
The study showed accurate and practical way a way of measuring the echogenicity of testicular parenchyma of prepubertal sheep, could be<br />
utilized as a complementary method of diagnosis, usually early, and great value for andrological evaluation.<br />
Keywords: ultrasonography, ovine, testicle.<br />
A012 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EVAL<br />
ALUATION OF PROTAMINE AND TRANSITION PROTEIN GENE EXPRESSION IN BOVINE SPERM CELLS AND TESTICLES<br />
Renata Simões 1 , Marcella Pecora Milazzotto 2 , Flavia Regina Oliveira De Barros 3 , Marcia Almeida Monteiro Melo Ferraz 4 , Marcilio Nichi 5 , José Antonio<br />
Visintin 6 & Mayra Elena Assumpção 7<br />
1,2<br />
UFABC, SANTO ANDRÉ, SP, BRAZIL. 3,4,5,6,7 FMVZ-USP, SÃO PAULO, SP, BRAZIL.<br />
Protamines are proteins found in the nucleus of the sperm that are synthesized and deposited in the DNA of the cell at the end<br />
of spermiogenesis. These proteins have the function to compact and protect the chromatin, exerting great influence on human and mice<br />
fertility. Several studies have demonstrated the positive relationship between unregulated production of protamines and infertility. In humans<br />
and mice protamine 1 and 2 (P1/P2) ratio is important to predict fertility and that 1:1 ratio is ideal. According to Aoki et al. (2005, Human<br />
Reproduction 20, 1298-1306) and Suganuma et al. (2005, Human Reproduction 20, 3101-08) when the 1:1 ratio is disrupted, the sperm DNA<br />
integrity and embryo development are altered. The inactivation of transition protein 2 (TP2) in mice leads to failure in the protamine 2<br />
transduction and a subfertile phenotype, showing less condensed sperm nuclei, and elevated level of breaks in the DNA. However, in the<br />
bovine species, the expression of these proteins is not completely understood. Literature data suggest that protamina 2, in humans, is absent<br />
in this species. Thus, this study aimed to determine the expression of protaminas 1 and 2 (P1 and P2) and transition proteins 1 and 2 (TP1<br />
and TP2) in bovine testis and sperm cells. Testis and epididymal sperm cells from post-pubertal bulls (n = 9) were obtained from slaughterhouse.<br />
The RNA extraction and cDNA synthesis were performed using commercial kits. The gene expression (P1, P2, TP1 and TP2) was determined<br />
by real time RT-PCR, using bovine specific primers and β-actin and GAPDH as endogenous controls. A relative expression software tool<br />
(REST) was used to compare all samples of each group. All transcripts were present in all animals and tissues, except P2 and TP2 that were<br />
not found in one semen sample. The quantification of mRNA relative expression, in testicles, showed that P1 was 4.5 fold higher than the<br />
relative expression of TP1 and 8.9 fold higher than P2. The relative expression of P2 was 41.8 fold lower than TP2 (P < 0.05). For sperm<br />
samples, the relative expression of P1 was 4 fold higher than TP1 and 8 fold higher than P2. The relative expression of P2 was 15.3 fold lower<br />
than TP2 (P < 0.05). Until now, this is the first work that shows the P2 gene expression in bovine sperm. According to these results, we<br />
verified that there was a pattern of expression between P1-TP1 and P1-P2 in the testis and semen samples. However, the relative expression<br />
between P2-TP2 did not show a pattern of expression between samples of testis and bovine semen. [Financial support. FAPESP (2010/<br />
09145-3)].<br />
Keywords: bovine, gene expression, protamine.<br />
s342
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A013 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
AGE AT PUBERTY IN BOARS SUBMITTED<br />
TED TO PORCINE SOMATOTR<br />
TROPIN (PST) TREATMENT<br />
Camila Piz<br />
izoni,<br />
Cláudia F. Demar<br />
emarco, Ismael Mateus Cavazini,<br />
azini, Viviane Rohr<br />
ohrig Rabassa<br />
abassa, August<br />
ugusto Schneider, Ivan Bianchi & Mar<br />
arcio N. Cor<br />
orrêa<br />
rêa<br />
UNIVERSIDADE FEDERAL DE PELOTAS, CAPÃO DO LEÃO, RS, BRAZIL.<br />
Growth hormone (GH) or somatotropin (ST) is widely used in animal production. Several studies in humans relate the use of<br />
exogenous GH to testicular development and age at puberty when there is a deficiency in endogenous GH production. In veterinary applications<br />
GH use in healthy animals has also been investigated. The aim of this work was to evaluate the effect of exogenous pST administration on age<br />
at puberty in healthy boars. Starting at 22 days of age, fifteen young boars were used (Landrace x Large White): 7 animals in Control Group, that<br />
received 0.5 mL of saline solution each 3 days and 8 animals in GH Group, that received 90 µg/Kg IM of pST (Reporcin ® , Zamira Life Sciences<br />
Pty Ltd., Australia) also each 3 days. The animals were trained to ejaculate for semen collection three times a week starting at 150 days of age.<br />
For the training a dummy sow was used and the boars were allowed to ejaculate and semen was submitted to motility analysis (%). The animals<br />
were considered as reaching puberty when motility was higher than 10%. For statistical analysis, means were compared by analysis of variance<br />
with Tukey adjustment using the SAS software. Control Group reached puberty at 177.0 (± 3.1) days of age with 47.1% (± 11.2) of motility,<br />
while GH Group reached puberty at 182.0 (± 2.0) days with 50.0% (± 8.0) motility. There was no difference for age at puberty (P = 0.19) and<br />
motility in the first ejaculate (P = 0.83). In conclusion, growth hormone had no effect on age at puberty when injected in healthy prepuberal boars.<br />
Keywords: puberty, boars, growth hormone.<br />
A014 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EVAL<br />
ALUATION OF SPERM MEMBRANE INTEGRITY OF BUFF<br />
UFFAL<br />
ALOES (<br />
(BUB<br />
UBAL<br />
ALUS BUB<br />
UBALIS<br />
ALIS) ) USING DIFFERENT<br />
LABOR<br />
ABORATORIAL<br />
ORIAL TECHNIQUES<br />
G. Rocha Silva 1 , Alexandre Rossetto Garcia 2 , Sâmia Rubielle Silva De Castro 3 , Benjamim de Souza Nahúm 2 , Alessandra Ximenes<br />
Santos 1 , Arnaldo Algaranhar Gonçalves 1 & Daniel Vale Barros 4<br />
1<br />
UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2 EMBRAPA AMAZÔNIA ORIENTAL, BELÉM, PA, BRAZIL. 3 FACULDADES INTEGRADAS DO TAPAJÓS, SANTARÉM, PA, BRAZIL.<br />
4<br />
UNIVERSIDADE FEDERAL RURAL DA AMAZÔNIA, BELÉM, PA, BRAZIL.<br />
Simple techniques, reliable and easy to execute are critical to laboratory analysis of semen. The assessment of integrity of sperm<br />
membrane is an important element for andrology, because it allows evaluate the cell functionality, even when morphological defects are not<br />
previously diagnosed. The eosin used in eosin-nigrosin (EN) seminal staining doesn’t penetrate the sperm presenting intact membrane.<br />
However, when membrane are damaged, eosin stains sperm in pink, and nigrosin gives the contrast to visualize the unstained cells (Brito 2007, N<br />
Clinical Technique Equine Practice 6, 249-264). The hypoosmotic swelling test (HOST) is able to evaluate the integrity of the cell membrane<br />
considering the liquid transportation that occurs when membrane is intact. Consequently, tails become bent when the sperm cell is subjected to<br />
hypoosmotic medium (Jeyendran et al., 1992, Archives of Andrology 29, 105-116). These research aimed was to compare the use of EN and<br />
HOST in diagnosis of sperm membrane integrity on buffalo raw semen. The experiment was executed at Embrapa Eastern Amazon, in Belém,<br />
Pará. Five buffalo bulls had semen collected by artificial vagina, and each bull provided six ejaculates (n = 30). Semen was assessed and each<br />
ejaculate was subjected to EN and HOST tests HOST (Jeyendran et al. 1984, Journal of Reproduction and Fertility 70, 219-228). The plasma<br />
membrane integrity (%) obtained with EN or HOST was correlated with progressive motility (%). Data were analyzed using t-Test and the level<br />
of association between variables was calculated by Pearson correlation (P < 0.01). The percentage of spermatozoa with intact membrane detected<br />
by the techniques EN and HOST was 85.4 ± 8.3% and 73.1 ± 10.5%, respectively (P < 0.01). Results were higher than those published by Iqbal<br />
et al (2010, Journal of Animal Science 88, 922-925) using EN (69.1 ± 0.6%) and HOST (59.4 ± 0.8%) for analysis seminal of Nili-Ravi<br />
buffaloes. The correlation of motility with EN was highly significant and of medium intensity (r=0.66, P < 0.0001), but it was not observed using<br />
HOST (r = 0.19, P = 0.31). These findings indicate superior efficiency of EN technique for raw buffalo semen evaluation, when sperm motility<br />
is used as a parameter for predicting the potential fertility of an ejaculate. Thus, it was concluded that EN and HOST techniques are reliable,<br />
especially the first, which doesn’t require sophisticated equipment to be executed, such as phase-contrast microscope. Therefore, EN is<br />
recommended for the analysis of the sperm membrane integrity of buffalo semen, that makes it profitable for laboratory routines.<br />
Keywords: eosin-nigrosin, hypoosmotic swelling test, raw semen.<br />
s343
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A015 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EVAL<br />
ALUATION OF DIFFERENT EXTENDERS IN THE CRYOPRESER<br />
OPRESERVATION OF BOVINE SPERMATOZ<br />
OZOA FROM CAUDA<br />
EPIDIDYMIDES STORED FOR 24 HOURS<br />
Ines Cristina Giometti 1 , Patrícia De Mello Papa 1 , Frederico Ozanam Papa 2 & Letícia Amélia De Oliveira 1<br />
1<br />
UNOESTE, PRESIDENTE PRUDENTE, SP, BRAZIL. 2 UNESP, BOTUCATU, SP, BRAZIL.<br />
Ejaculated sperm differ from epididymal sperm in many factors including the types of proteins bound to the plasma<br />
membrane and different motion characteristics. The cryopreservation of epididymal spermatozoa allows a more efficient and<br />
economical way to use that material, because it can be utilized anytime not just after death of an animal. The aim of this study<br />
was to evaluate the influence of two different extenders in the cryopreservation of bovine spermatozoa from cauda epididymides<br />
stored at 5 o C for 24 h. Twenty-eight testicles with epididymides were collected from a slaughterhouse and stored at 5 o C for 24<br />
h. After that, the epididymides were flushed with Botu-semen ® (Biotech Botucatu, São Paulo, Brazil), then the samples were<br />
incubated for 15 min at 37 o C and centrifuged at 2500 rpm for 15 min. The pellets were resuspended with Botu-Bov ® (Biotech<br />
Botucatu, São Paulo, Brazil) or Tris, groups BB and TRIS respectively. The samples were loaded into 0.5 mL straws, stored at<br />
5oC for 4 h, maintained at 3 cm above liquid nitrogen level for 20 min and then they are stored in liquid nitrogen. The straws<br />
were thawed at 46oC for 20 s then evaluated by CASA (HTM—IVOS 12, Hamilton Thorne Research, USA). Statistic Analyses<br />
utilized was unpaired t test with or without Welch correction, depending of samples behavior (P < 0.05). Medium values for<br />
motility parameters of epididymal sperm analyzed by CASA were: total motility (TM, %) = BB (36.3±20.6) and TRIS<br />
(32.9±25.4); progressive motility (PM, %) = BB (18.5±11.8) and TRIS (18.5±15.0); path velocity (VAP, µm/s) = BB (79.0±11.8)<br />
and TRIS (74.0±18.3); straight line velocity (VS.L, µm/s) = BB (57.1±7.6) and TRIS (55.0±13.6); curvilinear velocity (VCL,<br />
µm/s) = BB (141.6±21.3) and TRIS (133.0±33.0); lateral head displacement (ALH) = BB (6.3±0.8) and TRIS (6.1±1.6); beat<br />
cross frequency (BCF, Hz) = BB (22.6±3.2) and TRIS (23.9±6.0); straightness (STR, %) = BB (72.8±4.3) and TRIS (72.4±14.7);<br />
linearity (LIN, %) = BB (42.8±4.0) and TRIS (42.1±9.5); and percentage of rapid cells (RAP, %) = BB (30.7±18.2) and TRIS<br />
(28.0±22.5). There was no significant difference between the freezing extenders Botu-Bov ® and Tris. Based on these results,<br />
it is possible to conclude that both Botu-Bov ® and Tris are efficient for freezing bovine spermatozoa from cauda epididymides<br />
stored at 5 o C for 24 h. [Acknowledgements. financial support by FAPESP].<br />
Keywords: bulls, cryopreservation, spermatozoa.<br />
A016 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EVAL<br />
ALUATION OF T WO DIFFERENT PERCOLL GRADIENT<br />
ADIENTS FOR BOVINE SPERM SELECTION<br />
Rosana Camargo Nishimura 1 , José De Oliveira Carvalho Neto 2 & Margot Alves Nunes Dode 3<br />
1<br />
UNB, BRASILIA, DF, BRASIL. 2 USP, PIRACICABA, SP, BRAZIL. 3 EMBRAPA, BRASÍLIA, DF, BRAZIL.<br />
Percoll gradient is one of the most used techniques for sperm preparation on bovine in vitro embryo production (IVP). However,<br />
protocol modifications have been studied to use more efficiently semen doses from valuable animals or from sex-sorted sperm and, also to<br />
decrease the IVP costs. The aim of this study was to evaluate the effect of two different Percoll gradients in sperm selection, recovery rate, and<br />
in embryo production and sex rate. Semen samples from four sires were used, and all sperm quality assessment and IVF experiments were<br />
repeated three times. In each replica one semen straw was thawed, and samples were collected to evaluate total and progressive motility (by<br />
computer analysis -CASA), membrane integrity (stained with propidium iodide – PI, and 6 carboxyfluorescein diacetate – CFDA) and<br />
acrosome integrity (stained with IP and fluorescein isothiocyanate – FITC labeled peanut agglutinin – PNA). The remaining fractions were then<br />
placed in 90:45% (P90) or 60:45% Percoll gradient. After centrifugation, the resulting pellet was homogenized and used for the same pre-Percoll<br />
sperm evaluations, recovery rate and embryo production. For IVP, 1065 in vitro maturated oocytes were used. Cleavage rates (D2) as well as<br />
blastocyst rates (at D7 and D8 of culture) were evaluated. Blastocysts from D8 were frozen for later sex determination. Data was analyzed using<br />
GLIMMIX of SAS ® (P < 0.05) and, for sex proportion, ÷2 test was used (expected ratio of 50%, P < 0.05). Percoll gradient influenced several<br />
studied parameters. Samples submitted to 90:45% gradient presented higher motility (P90: 81.9±3.2% and P60: 70.4±2.9%), higher percentage<br />
of cells with intact membrane (P90: 77.9±2.5% and P60: 65.9±2.9%), lower recovery rate (P90: 28.3±2.1% and P60: 47.9±4.0%), higher<br />
blastocyst rate at D7 (P90: 29.1±3.4% and P60: 19.3±3.0%) and at D8 (P90: 34.9±3.6% and P60: 22.2±3.2%) than those submitted to 60:45%<br />
Percoll gradient. However, no gradient effect was observed in the progressive motility (P90: 40.2±3.5% and P60: 38.5±3.0%), acrosome<br />
integrity (P90: 66.0±3.2% and P60: 61.0±3.3%), cleavage rate (P90: 59.5±3.8% and P60: 53.6±3.6%), and sex proportion (P90: 43.04%:56.96%<br />
and to P60: 47.14%:52.86%). It was noticed that although the recovery rate of 90:45% gradient was lower, the recovered sperm had better quality<br />
and produced more embryos without affecting the sex ratio. [Financial Support. CNPq, Embrapa].<br />
Keywords: percoll gradient, sperm preparation, ivp.<br />
s344
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A017 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
ACTION OF NITRIC OXIDE ROUTES CGMP IN IN VITRO CAP<br />
APACIT<br />
CITATION TION OF CRYOPRESER<br />
OPRESERVED BOVINE<br />
SPERMATOZ<br />
OZOA<br />
Ana Carolina De Macedo Soares Leal 1 , Maria Clara Caldas-Bussiere 2 , Carla Sobrinho Paes De Carvalho 3 , Célia Raquel Quirino 4 &<br />
Patricia Alves Pinho Machado Silva 5<br />
1,2,3,4,5<br />
UENF, NITEROI, RJ, BRAZIL. 2 UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
The aim of the study was to evaluate the effects of nitric oxide (NO) in in vitro capacitation induced by heparin (H) of in<br />
cryopreserved spermatozoa from 3 bulls, by adding L-arginine (L-arg, precursor synthesis NO), 8-bromo-cGMP (cGMP analog) and 1H-<br />
(1,2,4) oxadiazole-(4,3-a) quinoxaline-1-one (ODQ, inhibitor of activity cGMP soluble). The control consisted of capacitated sperm in TALP<br />
medium-sp plus H (10 mg/mL). It was added different concentrations of L-arg (1, 2.5, 5, 10 and 50mM), 5 and 10mM cGMP and 0.1mM ODQ<br />
in medium capacitation (200mL). The capacitation and acrosome reaction were evaluated by chlortetracycline test (CTC) and the concentration<br />
of nitrate and nitrite (NO3-/NO2-) in the medium capacitation by the Griess method after 4 h. It was performed 6 replicates of each animal. Those<br />
data were analyzed by analysis of variance and means compared by the Tukey test, 5% probability. The addition of 1 and 10mM L-arg in the<br />
medium capacitation increased the percentage (17.3% and 4.4%) of capacitated spermatozoa (P < 0.05) compared to control. The addition of<br />
0,1mM ODQ decreased (11.1%) capacitation (P < 0.05) compared to control. The addition of 1mM L-arg + 0.1mM ODQ in the medium<br />
capacitation reversed the inhibitory effect of ODQ. Treatment with cGMP (5 and 10mM) was used to evaluate whether L-arg had a similar role.<br />
The addition of 5mM cGMP increased (11.3%) the capacitation and the addition of 10mM cGMP decresed (13.9%) the capacitation, but<br />
increased (16,8%) the AR (P < 0.05). The addition of 5mM cGMP + 0.1mM ODQ partially reversed (97.4%) the inhibitory effect of ODQ (P<br />
< 0.05). The addition of 10mM cGMP + 0.1mM ODQ inhibited capacitation and AR (P < 0,05). Increasing concentrations of L-arg showed a<br />
dose-response effect in NO synthesis (P < 0.05). The addition of 0.1mM ODQ decreased (3.9%) NO synthesis (P < 0,05) compared to control.<br />
The addition of 1mM L-arg + 0.1mM ODQ in medium capacitation reversed the inhibitory effect of ODQ (P < 0.05). The addition of 5 mM<br />
cGMP increased (3.85%) the synthesis of NO compared to control but not differed from 1 mM L-arg. Yet the addition of 10 mM cGMP<br />
increased (54.8%) NO synthesis compared with control and lower concentrations of L-arg (1-10 mM). These results indicate that: 1) NO is<br />
involved in the control of capacitation routes cGMP, 2) the state of activity of cGMP regulates the synthesis of nitric oxide by feedback.<br />
Keywords: nitric oxide, spermatozoa, bovine.<br />
A018 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
TESTIS SIZE, SEMEN CRITERIA AND SEMINAL PLASMA PROTEINS OF RAMS SUBJECTED TO SCROTAL<br />
INSULATION<br />
Arlindo Alencar Moura 1 , David Ramos Da Rocha 1 , Carlos Eduardo Azevedo Souza 1 , Airton Alencar Araújo 2 ,<br />
Maurício Fraga Van Tilburg 1 , Jorge Andre Matias Martins 1 , Veronica Gonzalez Cadavid 1 , Romulo Messias<br />
Diogenes Lima 1 , Lucas Dos Santos Fonseca 1 & Aderson Martins Viana Neto 1<br />
1<br />
UNIVERSIDADE FEDERAL DO CEARA, FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.<br />
N<br />
We studied the effects of scrotal insulation on testis biometry, semen parameters and seminal plasma of White Morada Nova rams.<br />
Six mature rams had their testes insulated for 8 days. Scrotal circumference (SC) and semen samples were taken 7 days before, during insulation<br />
and weekly until semen samples were equivalent to pre-insulation values. From semen samples, seminal plasma was obtained by centrifugation<br />
and subjected to 2-D electrophoresis. Commassie-stained gels were evaluated using PDQuest software (BioRad, USA). Variations in all<br />
parameters were evaluated by analysis of variance and Tukey statistical test (P < 0.05). During insulation, rectal temperature was 38.2 ± 0.1 o C.<br />
Testis temperature increased from 31.2 ± 0.2 o C, before insulation, to 35.2 ± 0.3 o C during insulation, returning to normal values 7 days after<br />
withdrawal of the scrotal bags. Average SC before insulation was 30.0 ± 0.4 cm, decreasing progressively after insulation, reaching the smallest<br />
size after 28 days (22.6 ± 0.6 cm), and returning to pre-insulation values after 56 days. Seminal parameters followed the same trend and 8 days<br />
after the end of the insulation, there were no motile spem and, 8 days later, rams became azoospermic. The first sperm could be detected only 63<br />
days after the end of insulation, and reached normal ranges only after 84 days. Before insulation, seminal plasma protein maps had, on average,<br />
269 ± 25 spots, with 132 consistently present on all gels. However, 24 days after insulation, when seminal quality was severely compromised,<br />
when most rams were azoospermic, only 107 ± 13 spots could be detected per gel, and 57 were present on every member of the matchset. Spots<br />
remaining in seminal plasma samples after insulation had predominantly low molecular weight and, based on our previous results, were mostly<br />
secreted by the accessory sex glands, corresponding to Ram Seminal Vesicles Proteins (RSVPs) 14 and 22 kDa and Bodhesin 2. In spite of the<br />
large number of spots that were not expressed in the seminal plasma after insulation, 8 spots (28 and 70 kDa; pIs from 4.8 to 6.0) could not be<br />
detected before insulation. Such proteins likely represent a response of the reproductive tract to thermal injury. In conclusion, extreme conditions<br />
of heat stress, induced by scrotal insulation, caused drastic alterations in testis function and expresstion of seminal plasma proteins.<br />
Keywords: proteomics, seminal plasma, scrotal insulation.<br />
s345
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A019 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
GLASS<br />
WOOL COL<br />
OLUNM<br />
VS.<br />
S.<br />
MINI-PERCOLL<br />
® GRADIENT IN BOVINE SPERM SEPAR<br />
ARATION-PRELIMINAR<br />
TION-PRELIMINARY RESULTS<br />
Stephanie Mayra Pacífico De Souza, Gina Marcela Mican, , Juli Angélica Narváez, Reginaldo Silva Fontes & Célia Raquel Quirino<br />
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
The study was aimed at evaluating two methods of sperm separation using Mini-Percoll ® gradient (Bio Siences, uppsala) and<br />
glass wool column filtration and their effects on motility, concentration, force and kinematics. Frozen semen has been used from the same<br />
source and submitted to sperm separation. Three groups were formed, a control group (C) (n = 6), a Mini-Percoll ® group (MnP) (n = 6) and<br />
a glass wool column group (GW) (n = 6). The MnP was prepared in 90 and 45% concentrations with a total volume of 800µl and centrifuged<br />
at 1400xg for 8 min. In the GW method an insulin syringe was used and glass wool was added to complete the volume of 0,1ml, later, the semen<br />
was deposited in the syringe in an upright position to facilitate the living sperm to be separated from the dead ones by gravity. All experimental<br />
groups were evaluated at two times, post-thawing time (T0) and post-spinning time in Talp-sp (Sigma, Aldrich, USA) for three minutes at<br />
200xg (T1). At time 0 total motility (TM), progressive motility (PM) and force (F) have been rated. At time 1 TM, PM, F, concentration and<br />
track speed (TS) were evaluated. The TM, PM, TS and kinematics have been evaluated by the Hamilton Thorn Research 10.8 Ceros ®<br />
computer assisted sperm analysis (CASA) program. Tukey test has been used for analysis of the data, considering a 5% significance level,<br />
comparing the groups regarding time. No significant differences (P > 0,05) regarding the experimental groups at time 0 and time 1 have been<br />
observed for PM, F and TS. It has been observed at time 1 that the GW group presented a lower concentration (spermatozoa/ml) (15,8±13),<br />
when compared to the C group (73,8±37,3) and the MnP group (63,5±34,3). There was increase in the PM (%) along time, T0=32,5±37,3<br />
and T1=42,2±13,7, probably related to the reduction of the dead cells counted by the CASA program after the separation methods. It is<br />
concluded that the GW method allows the separation of the dead and the living sperm, additionally, this method has coast of preparing a lot<br />
cheaper and also implementing a more agile. However, the sperm concentration decreases. New experiments are underway to confirm effect<br />
of the use of the GW method in sperm separation.<br />
Keywords: sperm separation, glass wool column, mini-percoll ® gradient.<br />
A020 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
COMP<br />
OMPARISON OF BI AND TRIDIMENSIONAL CULTURE SYSTEM STEM OF EQUINE TESTICLE SAMPLES<br />
Ian Martin 1 , Leandro Maia 1 , Camila Chavier Macedo 1 , Bruna de Vita 1 , Gabriel Augusto Monteiro 1 , Denise Pereira Leme 2 , Fernanda da Cruz Landim Alvarenga 1<br />
& Frederico Ozanam Papa 1<br />
1<br />
FMVZ - UNESP/BOTUCATU, BOTUCATU, SP, BRAZIL. 2 UFSC, FLORIANÓPÓLIS, SC, BRAZIL.<br />
The aim of the present study was to develop a methodology to culture testicular fragments obtained from adult horses comparing<br />
the bi and tridimensional system. So, testicles samples of approximately 1-3 mm 3 were placed on 6-well culture plates and maintain in a<br />
culture incubator for 1 hour or included in stands made of 1,5% agarose gel (Difco Ágar Noble- BD 214230). Approximately 6 testicle samples<br />
included or not in agarose gel were maintain in each well. Thereafter, each well was filled with 5 ml of culture medium which was replaced after<br />
48 hours and maintain until the end of the culture period (7 days). Culture medium was composed by DMEM high glucose (Gibco 10569-<br />
010) + F12 (Gibco 31765-035) 1:1 (vol:vol), 20% SFB (Gibco 12657-029), 50 µg/mL of gentamicine (Sigma-Aldrich G1264), 3,0 µg/mL of<br />
Amphoterecin B (Gibco 15290-018), 1,0 µg/mL of ITS (Sigma-Aldrich I1884), 50 UI/L of rFSH (Puregon, Organon), 100 µl/mL of essential<br />
amino acid (Sigma-Aldrich B6766), 100 µl/mL non-essential amino acid (Sigma-Aldrich M7145), 1 µmol/L of testosterone and 10µl/mL of LH<br />
(Lutropin - Tecnopec). The culture system was maintained with 5% carbon dioxide in air at 32 o C. The testicles samples were evaluated at 0<br />
(fresh, control), 48, 120 and 168 hours (7 days) under a light microscope and stained with haematoxylin and eosin. After 48h in bidimensional<br />
culture a substantial reduction on germ cells, degeneration of some seminiferous tubules, eosinophilia and pyknosis in Leydig cells was<br />
observed; in tridimensional culture similar alterations were observed, however the germ and Sertoli cells were in higher number with<br />
characteristic morphology, presence of higher number of cells in division and absence of degeneration. Within 120h of bidimensional culture<br />
it was possible to observe worsening of the signals previously observed, with the presence of intense eosinophilia, karyorrhexis and<br />
karyolysis in the Leydig cells. For the tridimensional culture the maintaining of the seminiferous tubules structure was observed and in spite<br />
of the presence of the alterations described for the bidimensional system, in minor intensity, there were more Sertoli cells with a normal<br />
morphology. After 7 days of culture the samples maintained in bidimensional culture presented intense degeneration and presence of few germ<br />
and Sertoli cells, however some spermatocytes with condensed chromatin and multinucleated cells could be observed; on tridimensional<br />
culture similar signs were observed, but without degeneration, higher number of Sertoli cells and the presence of a few spermatogonia.<br />
According to these findings it was possible to suggest that the tridimensional culture presents maintaining of cells important to spermatogenesis,<br />
as spermatogonia and seroli cells, although the culture medium still needs to be improved in search of better cellular viability after the culture<br />
period.<br />
Keywords: culture, testicle, equine.<br />
s346
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A021 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EFFICIENCY COMP<br />
OMPARISON OF MEDIA FOR THE EQUINE SPERM CAP<br />
APACIT<br />
CITATION<br />
TION THROUGH FLOW CYTOMETR<br />
OMETRY ANALYSIS<br />
Thayna Pantoja Gardes 1 , Rubens Paes de Arruda 2 ; Daniela Franco da Silva 2 , Rafaela Nogueira Rodrigues Cardoso 1 , Juliana Nascimento 2 , Kleber Menegon<br />
Lemes 2 , Henrique Fulaneti Carvalho 2 & André Furugen Cesar de Andrade 2<br />
1<br />
USP-FZEA, PIRASSUNUNGA, SP, BRAZIL. 2 USP-FMVZ, PIRASSUNUNGA, SP, BRAZIL.<br />
The mammalian sperm must undergo a series of changes in their metabolism before they are able to fertilize the<br />
oocyte, known as capacitation. Two mediums were described to successfully capacitate the equine sperm in laboratory, which<br />
are the medium for bovine sperm capacitation (B) (ANDRADE et al., Anim Reprod Science, v.107, p.304-5, 2008) and the<br />
medium utilized in the murine sperm capacitation (M) (MCPARTLIN et al., Theriogenology, v.69, p.639-50, 2008). The aim<br />
of this study was to compare mediums B and M, through analysis by flow cytometry, for cryopreserved equine sperm capacitation,<br />
in order to verify which of these is more efficient in promoting capacitation and the equine sperm acrosome reaction (AR)<br />
simultaneously maintaining the integrity of plasma membrane (PMI) for a period of 5 h of incubation. Three ejaculates from<br />
each of the three stallions (n=9) were cryopreserved. Post-thawed and selection at Percoll ® semen was submitted to treatment<br />
and analyzed by flow cytometry evaluating whether there is PMI, percentage of viable cells showing AR and the presence of<br />
tyrosine phosphorylation (P-Ty) at 0 (T0), 60(T60), 120(T120) and 300(T300) min incubation at 38 o C and 5 % CO2. Data was<br />
analyzed using ANOVA added to the factor repeated measures on time by the Tukey test (P < 0.05). The analysis of samples<br />
submitted in the B medium showed, in times T0, T60, T120 E T300, the respective values for AR: 6.26% ± 6.55%; 8.30% ±<br />
5.40%; 10.20% ± 5.19%; 17.67% ± 9.83%; while for the group M samples: 1.79% ± 1.64%; 1.97% ± 1.37%; 1.94% ± 1.09%;<br />
4.76% ± 4.50%. The PMI values obtained after analyses, during the five incubation times, for treatments B and M were T0<br />
(43.65% ± 22.22% and 42.23% ± 18.49%), T60 (38.19% ± 17.39% and 34.27% ± 16.40%), T120 (36.22% ± 16.63% and<br />
36.03% ± 15.73%) and T300 (36.79% ± 17.58% and 35.47% ± 20.11%). Regarding P-Ty, the following results were observed<br />
in relation to the median fluorescence intensity for B: T0 (474.55ua ± 108.45ua); T60 (463.00ua ± 120.69ua), T120 (539.33ua<br />
± 261.32ua) T300 (520.11ua ± 167.46au) and M: T0 (438.11ua ± 101.13ua) T60 (464.78ua ± 101.83ua); T120 (438.11ua ±<br />
115.56ua), T300 (543.66ua ± 140.84ua), suggesting that the P-Ty increased (P
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A023 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
CORREL<br />
ORRELATION AMONG OSMOTIC<br />
TEST AND DIFFERENT PROCEDURES OF POST-THAW SPERM VIABILITY EVAL<br />
ALUATION<br />
Gabriel Felipe Oliveira de Menezes 1 , Rodrigo Freitas Bittencourt 1 , Léia Ribeiro Alves Santos 1 , Renata Cardoso Andrade 1 , Lais Oliveira Mascarenhas 1 ,<br />
Alexandra Soares Rodrigues 2 , Priscila Assis Ferraz 2 & Antônio de Lisboa Ribeiro Filho 2<br />
1<br />
UNIME, SALVADOR, BA, BRAZIL. 2 UFBA, SALVADOR, BA, BRAZIL.<br />
The evaluation of structural and functional integrity of sperm plasma membrane has been studied and improved with the goal of<br />
increasing its reliability in assessing the potential fertility of the breeding (BITTENCOURT et al., 2005, Ciên Anim Bras, 6:213-218). This study<br />
aimed to correlate the osmotic shock with the supravital test and fluorescent probes for evaluation of ovine semen thawed. For this experiment,<br />
20 ovine frozen semen samples were used, from Santa Inês e Dorper breedS, with a straw of semen from each animal (n = 20). Straws were<br />
thawed in a water bath at 38°C for 50 s, and then placed into 2 mL microtubes, previously warmed at 38°C. Aliquots were collected for the<br />
supravital test dye eosin (EOSC); the sperm morphology was analyzed and the percentage of bent tails (BT) was calculated. For evaluation in<br />
fluorescence microscopy it was employed the combination of fluorescent probes propidium iodide, FITC-PSA and JC-1, using the methodology<br />
described by Celeghini et al. (2010, Arq Bras Med VetZoot, 62:536-543). The osmotic shock (OS) followed the dilution: One part semen to 10<br />
(OS 10) parts of the final solution, deionized water (0 mOsmol). After incubation for 5 min at 37°C, samples were fixed with 10 µL of<br />
formaldehyde buffered saline. The percentage of spermatozoa reactive to OS was determined by subtracting the percentage of spermatozoa with<br />
OS-induced BT, by the sperm morphology obtained after thawing. These evaluations were observed by phase contrast microscopy with an<br />
increase of 1000x immersion oil and made the 100 spermatozoa count. The statistical analysis was made in the package Statistical Analysis<br />
System (SAS), version 5.0 (1996) with a significance level of 5%. The OS 10 was the only one to show positive and significant correlation with<br />
EOSC (r = 0.8, P < 0.05). The absence of significant correlation between the OS and the fluorescent probes can be explained, since functional<br />
activity in some situations cannot be directly associated with membrane integrity. The membrane can be intact but not functional; or superficially<br />
injured, but with the functionality unchanged. Further studies should be made with fresh semen in an attempt to repeat the results of this study<br />
to incorporate the OS into the routine evaluation of semen for breeding sheep.<br />
Keywords: osmotic test, sperm, sheep.<br />
A024 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
CANINE SEMEN CRYOPRESER<br />
OPRESERVATION:<br />
IDENTIFICATION TION OF CRITICAL POINTS<br />
Cristina Fátima Lucio, , Fernanda Machado Regazzi, Liege Garcia Silva, Daniel Souza Ramos Angrimani, Marcílio Nichi, Camilla Motta<br />
Mendes , Mayra Elena Assumpção & Camila Infantosi Vannucchi<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL - UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.<br />
Cryopreservation of canine semen is fundamental for the preservation of genetic material, as well as considered a biotechnique for<br />
wild canids, with the domestic dog as a biological model. However, studies are still necessary to achieve the efficiency observed in other species.<br />
The objective of this experiment was to verify seminal characteristics during the cryopreservation process, seeking to identify the critical step for<br />
sperm viability. Eleven semen samples were cryopreserved in a two steps process, according to a protocol previously established (Thomassen,<br />
R et al., 2006, Theriogenology 66, 1645-50). Aliquots of the fresh semen, the chilled (after 1 h at a 5 o C extender), after glycerolization (1 hour<br />
after addition of 5% glycerol to the extender) and the thawed semen (30 s at 37°C) were evaluated for motility and vigor, computerized motility<br />
analyses (CASA), percentage of live sperm by eosin/nigrosin staining, oxidative stress by TBARS concentration, mitochondrial activity by<br />
DAB staining, mitochondrial membrane potential by JC1 probe and membrane integrity by FITC / PI probes using flow cytometry. Results<br />
show that the cryopreservation protocol promotes decrease in subjective and computerized motility and vigor. However, the decrease in motility<br />
was significantly more pronunciated in the sample that suffered exposure to the liquid nitrogen. The frozen-thawed sample showed decrease in<br />
the percentage of progressive sperm motility and increase in slow and static sperm. The analysis of mitochondrial activity demonstrates that the<br />
crioprotectant influx and the thawing process were deleterious, but an intense loss was observed for the thawed sample, corroborated by the<br />
significant decrease in the mitochondrial membrane potential. The freezing-thawing process increases the association of the plasma and<br />
acrosome membrane lesions, however, the cooling process promoted membrane stabilization with a low percentage of sperm injury. The<br />
changes detected in the thawed semen can be explained in part by the oxidative stress. In conclusion, the decrease in temperature during cooling<br />
and the change in osmolarity with the influx of glycerol in the spermatic cell did not cause deleterious changes. The crucial moment for the<br />
viability of sperm during cryopreservation is the exposure to liquid nitrogen. Despite the intracellular ice formation, the intense production of<br />
reactive oxygen species can be the explanation for these injurious factors. [FAPESP 09/52760-3].<br />
Keywords: cryopreservation, semen, canine.<br />
s348
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A025 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
DETECTION OF PATHOGENS IN BOVINE SEMEN USING FLUORESCENT MULTIPLEX PCR<br />
Francisc<br />
ancisca a Elda Fer<br />
erreir<br />
eira Dias<br />
1 , Tania<br />
Vasc<br />
asconc<br />
oncelos Cavalc<br />
alcan<br />
ante 2 , Ana Kelen Felip<br />
elipe e Lima 3 , Car<br />
aris Mar<br />
aroni Nunes<br />
4 , Juliano Franc<br />
anco De Sousa<br />
5 & José Fer<br />
ernando<br />
Garcia 6<br />
1,2,3<br />
ESCOLA DE MEDICINAVETERINÁRIA E ZOOTECNIA, UFT, ARAGUAÍNA, TO, BRAZIL. 4,6 LABORATÓRIO DE BIOQUÍMICA E BIOLOGIA MOLECULAR ANIMAL, UNESP, ARAÇATUBA,<br />
SP, BRASIL. 5 LABORATÓRIO BRIO GENÉTICA E BIOTECNOLOGIA LTDA, ARAGUAÍNA, TO, BRAZIL.<br />
The use of cryoprotectants in semen allows the survival of most infectious agents found there, contaminating herds using artificial<br />
insemination (AMIN, 2003; Veterinary Journal 166: 86-92).Thus, the PCR is a tool that has been used with the purpose of simultaneously<br />
detecting multiple infectious agents causing similar clinical syndromes and/or share similar epidemiological features (MARKOULATOS et al.,<br />
2002; J. Clin. Lab. Analysis 16: 47-51). This study evaluated the use of PCR combined with capillary electrophoresis for detection of pathogenic<br />
bacteria in bovine semen. Doses of semen free of pathogens were infected with decreasing concentrations of bacteria obtained by serial dilutions<br />
in base 10, to obtain samples containing from 1 to 10-7 bacterias/mL from the initial concentration of Lepstospira interrogans serovar pomona<br />
(108 bact/mL), Brucella abortus (2.8 x 108 bact/mL), Campylobacter fetus (1.5 x 105 bact/mL) and Haemophilus somnus (1.5 x 105 bact/mL).<br />
The samples were subjected to DNA extraction by the method of phenol/chloroform, and the extracted DNA was amplified by multiplex<br />
fluorescent PCR-using species-specific primers for each bacterium (each pair labeled with fluorescent substance or HEX FAN) for amplification<br />
of DNA fragments of 193 bp (B. abortus), 330 bp (L. pomona), 400 bp (H. somnus) and 415 bp (C. fetus). After multiplex PCR reaction,<br />
visualization of the fragments by electrophoresis on 8% polyacrylamide gel was stained with Silver Nitrate, and by capillary electrophoresis with<br />
automated analysis of DNA fragments. Could be detected simultaneously, in a single reaction, fragments of 193, 330, 400 and 415 base pairs<br />
of B. abortus, L. pomona, H. somnus and C. fetus, respectively. Using analysis in gel and capillary electrophoresis was detected in a dilution of<br />
10-2 and 10-3 times the initial concentration of each bacterium, respectively. Thus, capillary electrophoresis can be an alternative to PCR detection<br />
of pathogenic bacteria in semen and seems to be an effective and rapid method of pathogens detection, when compared to traditional electrophoretic<br />
system, which might be an excellent tool to quality health control of centers in artificial insemination and embryo transfer.<br />
Keywords: cattle semen, multiplex-pcr, pathogenic bacteria.<br />
A026 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
DIFFERENT PROTOC<br />
OCOLS OLS OF THAWING RAM SEMEN CRYOPRESER<br />
OPRESERVED IN EXTENDER CONT<br />
ONTAINING LOW-DENSIT<br />
W-DENSITY<br />
LIPOPROTEIN<br />
Luís Cláudio Oliveira Moura 1 , Maira Corona Da Silva 2 , Ana Cláudia Dumont Oliveira 3 , Mariana Machado Neves 4 , Paola Pereira Das<br />
Neves Snoeck 5 & Marc Roger Jean Marie Henry 6<br />
1,2,5<br />
UESC, ILHÉUS, BA, BRASIL. 3,6 UFMG, BELO HORIZONTE, MG, BRAZIL. 4 UFV, VIÇOSA, MG, BRAZIL.<br />
Artificial insemination is an important tool in genetic improvement of livestock species, although there are limitations on the use of<br />
cryopreserved ram semen, due to its low fertility compared to fresh semen. Several factors can affect the membrane integrity and fertilizing ability<br />
of sheep spermatozoa, including the velocity and temperature of thawing. The objective of the following experiment was to evaluate the influence<br />
of thawing time and temperature on the viability of cryopreserved spermatozoa in extender with low-density lipoprotein (LDL). Three Santa Inês<br />
rams were used as donors of semen. Five ejaculates per animal were collected. After fresh semen analysis, the ejaculates were fractionated and<br />
diluted in a control extender (E1-15% egg yolk) and extenders with different concentrations of LDL (E2-10% and E3-20%), in order to obtain<br />
400x106 sperm/mL. Samples were cooled from room temperature to 5°C using an average cooling rate of –0.25°C/min in TK4000 ® machine<br />
and then maintained in equilibrium (2 h at 5°C), after that, frozen at 4cm in nitrogen vapor and stored in cryogenic cylinder. Thawed samples (38°/<br />
30s and 50°C/15s) were subjected to computer analysis (CASA) for evaluation of motility immediately after thawing and after three hours of<br />
incubation at 38°C. The membrane integrity was evaluated by the hypoosmotic test (HOST) and by fluorescent probes. No significant<br />
differences (P > 0.05) between extenders in evaluations of motility immediately after thawing at 38°C were observed. The motility of E1 (74,0%)<br />
(38°C) was better (P < 0.05) than E1, E2 and E3 (49.2%; 47.2%; 45.7%, respectively) (50°C). The treatments did not differ in evaluations of<br />
motility at the end of the heat resistance test. E2 (38.1%) and E3 (39.9%) thawed at 50°C had lower (P < 0.05) percentage of cells with functional<br />
integrity than sperm diluted in E1 (57.2%) and E3 (52.6%) thawed at 38°C. E1 (47.8%) thawed at 50°C preserved better the structural integrity<br />
of sperm membranes (P < 0.05) than other treatments thawed at 38°C. It is possible to affirm that thawing at 50°C was more efficient than 38°C<br />
to preserve the structural integrity of sperm membranes when extender formulated with egg yolk was used. Also, it was demonstrated that the<br />
protocol of thawing at 38°C proved to be the most appropriate to preserve motility and functional integrity of plasma membrane in samples<br />
diluted in extender containing 20% of LDL.<br />
Keywords: santa inês, spermatozoa, extender.<br />
N<br />
s349
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A027 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EFFECT OF PORCINE SOMATOTR<br />
TROPIN (PST) ) ON TESTICULAR MORPHOLOGIC<br />
OGICAL AND FUNCTIONAL CHARACTERISTICS<br />
CTERISTICS<br />
FROM PREPUBERTAL BOARS<br />
Douglas Perazzoli, , Dante Ferrari Frigotto, Clairton Marcolongo Pereira, Viviane Rohrig Rabassa, Ivan Bianchi, Eduardo Schmitt & Marcio Nunes Corrêa<br />
UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS, RS, BRAZIL.<br />
The influence of exogenous growth hormone (GH) on testicular development has been characterized in several species; however,<br />
it is not well understood in boars. The aim of this work was to determine the effect of exogenous GH on testicular morphological and functional<br />
characteristics from prepubertal boars. In this study, 12 boars with 22 to 53 days of age were used. The animals were divided in two groups: GH<br />
Group (n = 6), which received 90 µg/Kg of porcine somatotropin (pST, Reporcin ® , Zamira Life Sciences Pty Ltd., Melbourne, Australia) every<br />
3 days; and Control Group (n=6), that received placebo injections (sodium chloride 0.9%) at the same interval. The boars were weighed every<br />
3 days to adjust pST dose. Orchietomy was performed when the boars reached 53 days of age. The testes were weighed to determine the<br />
gonadosomatic index, which is the relation between body weight and testes weight (%). Moreover, the central portion of the testicular<br />
parenchyma was sampled and stored in 10%formoline for immunohistochemical analysis. The monoclonal antibody Ki67 was used to identify<br />
germinative cells in proliferation and Vimentin was used to identify Sertoli cells. Cells were counted using ImageJ (ver.1.44, National Institutes<br />
of Health, Bethesda, USA). Statistical analysis was perfomed by analisys of variance with Tukey adjustment using SAS (SAS Institute Inc,<br />
USA). The number of Sertoli cells was higher in Control than GH Group (GH: 209.5 ± 12.3; Control: 372.0 ± 41.6; P = 0.0004). The same<br />
difference was found for the seminiferous tubules (GH: 28.7 ± 1.5; Control: 35.1 ± 1.3; P = 0.002). However, the diameter of the seminiferous<br />
tubules (GH: 202.9 ± 1.8; Control: 199.1 ± 1.8; P = 0.15), number of proliferating germinative cells (GH: 30.6 ± 3.2; Control: 32.3 ± 1.7; P =<br />
0.65) and gonadosomatic index (GH: 0.07 ± 0.01; Control: 0.08 ± 0.01; P = 0.38) were not different between groups. In conclusion, the use of<br />
pST in prepubertal boars reduced the number of Sertoli cells and seminiferous tubules, without affecting, however, the diameter of seminiferous<br />
tubules, number of germinative cells and gonadosomatic index.<br />
Keywords: exogenous growth hormone, testes, prepubertal boars.<br />
A028 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EFFECT OF DIFFERENT CONCENTR<br />
ONCENTRATIONS TIONS OF REDUCED GLUT<br />
UTATHIONE THIONE (GSH) FOR CRYOPRESER<br />
OPRESERVATION OF<br />
CANINE SPERM<br />
Camila Infantosi Vannucchi , Cristina Fátima Lucio, Liege Garcia Silva, Fernanda Machado Regazzi, Daniel Souza Ramos Angrimani,<br />
Marcílio Nichi & Valquiria Hyppolito Barnabe<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL, UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.<br />
Sperm cells are particularly susceptible to oxidative stress as their plasma membrane contains large amounts of polyunsaturated fatty<br />
acids and their cytoplasm has a low concentration of protective enzymes. The process of sperm cryopreservation induces the formation of free<br />
radicals, thereby diminishing sperm performance. Supplementation of the extender for cryopreservation with antioxidants may improve sperm<br />
quality after thawing. This study aimed to compare different concentrations of reduced glutathione (0, 10 and 20 mM GSH) in the extender for<br />
cryopreservation of canine semen. Eleven ejaculates were collected from 6 dogs and the samples were divided equivalently in three groups:<br />
GLU10 (10 mM glutathione), GLU20 (20 mM glutathione) and Control Group (CG - without antioxidant). The fresh and pos-thaw semen<br />
samples were evaluated for motility and vigor, computerized motility analysis (CASA), percentage of live sperm through eosin/nigrosin stain,<br />
oxidative stress by TBARS concentration, mitochondrial activity by the DAB staining, mitochondrial membrane potential by JC1 probe and<br />
membrane integrity by FITC/PI probes using flow cytometry. The variables were compared among groups by ANOVA and LSD at P < 0.05.<br />
The subjective motility suffered a significant reduction in the post-thaw treated groups and the vigor decreased only in the GLU20 group. The<br />
computerized assessment of motility detected a significant decrease in total and progressive motility in GLU20 group, with increased number of<br />
static sperm in relation to CG. There was a decrease in mitochondrial activity assessed by DAB of the GLU20 group. We observed a protective<br />
effect of the antioxidant supplementation, as the probes FITC / PI detected a lower percentage of acrosomal damage in the GLU20 and GLU10<br />
groups. No differences were observed for the assessment of JC1 and TBARS among the proposed groups. In conclusion, the addition of<br />
reduced glutathione in the semen extender presented a protective effect for the acrosome of the canine sperm. However, due to decrease in sperm<br />
motility, different concentrations of this antioxidant must be tested in order to detect the beneficial effects, without any pro-oxidative action.<br />
[FAPESP 09/52760-3].<br />
Keywords: antioxidants, sperm, canine.<br />
s350
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A029 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EFFECTS OF SELENIUM AND CHROMIUM ON SEMINAL QUALIT<br />
ALITY OF BUFF<br />
UFFAL<br />
ALOES SUPPLEMENTED WITH BYPRODUCT<br />
ODUCTS OF<br />
AMAZONIAN AGROINDUSTRY<br />
Alessandra Ximenes Santos 1 , Alexandre Rossetto Garcia 2 , Cristian Faturi 3 , Benjamim De Souza Nahúm 4 , José Brito Lourenço Junior 5 , Sâmia Rubielle Silva De<br />
Castro 6 , Geanne Rocha Silva 7 & Arnaldo Algaranhar Gonçalves 8<br />
1,7,8<br />
UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2,4 EMBRAPA AMAZÔNIA ORIENTAL, BELÉM, PA, BRAZIL. 3 UNIVERSIDADE FEDERAL RURAL DA AMAZÔNIA, BELÉM, PA,<br />
BRAZIL. 5 UNIVERSIDADE DO ESTADO DO PARÁ, BELÉM, PA, BRAZIL. 6 FACULDADES INTEGRADAS DO TAPAJÓS, SANTARÉM, PB, BRAZIL.<br />
The use of organic industrial byproducts available on Amazon, such as coconut meal (CM) and palm kernel cake (PKC), may<br />
constitute an bioeconomic alternative to replace conventional concentrates for ruminants, leading to environmental and productive gains.<br />
Therefore, the aim was to evaluate the semen quality of buffalo supplemented daily with experimental diets based on PKC or CM, with different<br />
levels of selenium (Se) and chromium (Cr) intake. Fifteen buffaloes (3.2±1.8 years, 578.6±101.9 kg) raised on pasture (Panicum maximum) at<br />
the Embrapa Eastern Amazon were daily supplemented with isoproteic concentrates (1% BW) during 252 days. Animals had similar initial<br />
semen quality and were divided into three groups: Control (5 bulls; conventional ration with 62% corn grain), T1 (5 bulls; ration with 69.3%<br />
CM) and T2 (5 bulls; ration with 69.3% of PKC). Bulls had semen collected weekly, starting 112 days after first day of supplementation and two<br />
complete spermatogenic cycles, according to Sharma and Gupta (1980, Animal Reproduction Science, 3, 217-224). The ejaculates (n = 173)<br />
were evaluated for sperm motility, integrity of plasma membrane and sperm morphology. The level of Se and Cr in the feedstuffs was determined<br />
according to EPA-Method 7742/6010 (2009, U.S. Environmental Protection Agency, 26p). Data were subjected to ANOVA, with comparison<br />
by t test and the association between variables was analyzed by Pearson correlation (P < 0.05). The average of daily intake of Se (mg/day) for<br />
Control, T1 and T2 was 1.37±0.35 B , 1.15±0.26 C and 2.80±0.66 A (P < 0.05), respectively. Daily consumption of Cr (mg/day) in Control, T1 and<br />
T2 was 7.17±1.87 B , 4.30±0.97 C and 20.75±4.94 A (P < 0.05), respectively. Sperm motility was significantly higher (P < 0.05) in T2 (71.7±15.1% A )<br />
compared to Control (59.3±20.5% B ) and T1 (56.7±24 8% B ). Higher level of plasma membrane integrity (P < 0.05) was also observed in T2<br />
(82.1±12.2% A ) compared to T1 (72.0±22.6% B ) and Control (69.2±19.4% B ). There was no significant difference in the sperm total defects<br />
(Control: 44.2±18.5%, T1: 41.3±16.1% and T2: 44.3±19.2%). Correlation between consumption of Se and Cr with sperm motility (r=0.36 and<br />
r=0.34, P < 0.0001) and with plasma membrane integrity (r=0.33 and r=0.32, P < 0.0001) were significant and of medium intensity. Thus it can<br />
be concluded that selenium and chromium presented positive effects on preservation of plasma membrane integrity and on sperm motility, but<br />
had no interference on cellular morphology.<br />
[Acknowledgments. Project “Network for Innovation in Animal Reproduction” (01.07.01.002)].<br />
Keywords: semen, micronutrients, bubalus bubalis.<br />
A030 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EFFICIENCY OF THE CAP<br />
APACIT<br />
CITATING TING MEDIA ON MOTILIT<br />
TILITY MAINTENANCE AND INDUCTION OF EQUINE<br />
SPERMATOZ<br />
OZOA HYPERACTIV<br />
CTIVATION<br />
TION<br />
Daniela Franco da Silva, Rubens Paes de Arruda, Thayna Pantoja Gardes, Rafaela Nogueira Rodrigues Cardoso, Juliana<br />
Nascimento, Henrique Fulaneti Carvalho, Kleber Menegon Lemes & André Furugen Cesar de Andrade<br />
N<br />
USP, PIRASSUNUNGA, SP, BRAZIL.<br />
Hyperactivation is characterized by a non-progressive sperm trajectory and an overt lateral displacement of the head, which is<br />
essential for the occurrence of penetration of the zona pellucida (Suarez, 2008; Human Reproduction Update, 14 (6), 647-57). The aim of this<br />
study was to test by CASA, the capacitating media F (Andrade et al., 2008; Animal Reproduction Science, 107, 304-5) and the capacitating media<br />
M (MCPARTLIN et al., 2008; Theriogenology, 69, 639-50) in equine spermatozoa, in order to determine which was most efficient in promoting<br />
hyperactivation after 5 h of incubation. Three ejaculates from three stallions was collected and cryopreserved (n = 9). The semen was thawed,<br />
selected in Percoll ® and tested in the two media. The motion characteristics analyzed by CASA were total motility (MT%) and progressive<br />
motility (MP%) after 0, 30, 60, 120 and 300 min of incubation at 38°C and 5% CO2. In addition, there were adjustments of the tool Edit / Sort<br />
to assess the percentage of hyperactivated cells. The equine sperm was considered as hyperactivated when VCL> 180 µm and ALH > 12 µm<br />
(Rathi et al., 2001; Biology of Reproduction 65, 462-470). The data were analyzed using ANOVA and Tukey test and are presented as mean and<br />
standard deviation. The mean value of MT in medium F was 64.23±19.22%; 58.9±21.74%; 49.38±13.21%; 47.23±15.44% and 28.44±10.22%<br />
in time 0, 30, 60, 120 300 respectively. The medium M mean value was 62.43±21.19%; 52.94±22.90%; 49.8±18.8%; 43.46±17.94% e<br />
22.51±11.41%. The MP values of medium F in time 0, 30, 60, 120 e 300 min were 50.17±15.81%; 45.27±17.78%; 39.20±9.93%; 37.91±13.41%;<br />
24.37±9.92%, respectively. The MP mean values of the M medium was 48.87±18.30; 43.18±19.35%; 41.52±16.26%; 36.20±15.62%;<br />
19.61±9.11%. There was no interaction between treatment and incubation time regarding to sperm hyperactivity variable and the results was<br />
higher in medium F 8.48±4.38%; 7.66±4.92%; 7.68±6.73; 6.86±4.65% e 4.84±4.52% when it was compared with the mean values of medium<br />
M 6.85±1.41%; 6.00±2.83%; 5.11±2.21%; 4.39±3.19% e 2.35±2.40%. There were no differences between the two tested medium. Although<br />
both medium was considered efficient in maintaining sperm motion characteristics after 300 min of incubation, the F medium was better in<br />
inducing hyperactivation of sperm cells. [We thank Fundação de Amparo à pesquisa do Estado de São Paulo – FAPESP (Process: 09/54906-<br />
5, 09/50474-3, 10/01912-5, 10/01916-0). We thank Botu-Pharma that kindly donated the cryoprotectant for this experiment].<br />
Keywords: capacity, hyperactivation, spermatozoa.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A031 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
IMPROVEMENT OF SPERM AND SEMINAL QUALITY IN NELORE VS. CANCHIM BULLS AFTER INCLUSION IN A FEEDLOT<br />
SYSTEM<br />
Monique Mendes Guardieiro 1 , José de Oliveira Carvalho Neto 1 , Fernanda Lavínia Moura Silva 2 ; Mariana Curci Martins Da Silveira 2 , Tito Nunes Rodrigues 1 ,<br />
Marta Borsato 1 , Pedro Leopoldo Jerônimo Monteiro Jr. 1 , Ricardo Silva Surjus 1 , Alexandre Barbieri Prata 1 , Renato Shinkai Gentil 1 , Flávio Augusto Portela<br />
Santos 1 & Roberto Sartori 1<br />
1<br />
ESALQ/USP, PIRACICABA, SP, BRAZIL. 2 UFRPE, PIRACICABA, SP, BRAZIL.<br />
The objective was to compare the testicle biometry and semen and sperm characteristics between Canchim (3/8 Bos indicus x 5/<br />
8 Bos taurus) and Nelore (Bos indicus) young bulls, during the transition from pasture to a feedlot system. Canchim (n = 15) and Nelore (n<br />
= 13) bulls were 20.7±1.4 and 18.1±0.9 months old at the beginning of the experiment, respectively. All bulls that were managed at a<br />
Brachiaria brizantha pasture system were moved to a feedlot system and fed the same nutritional regime (30.7% hay, 60.9% ground corn,<br />
6.0% soybean meal, 0.9% urea and 1.5% limestone and mineral salt in dry matter). Data were analyzed as repeated measures by the MIXED<br />
procedure of SAS (2009), using the initial body weight mean (IBW) of Canchim and Nelore bulls of 423.5±23.9 and 336.6±19.4 kg,<br />
respectively, as covariate. Scrotal circumference (SC) measurement and semen collection by electroejaculation were performed in three periods<br />
(P1, P2 and P3) with an interval of 15 days between P1 and P2 and 60 days between P2 and P3. Despite Canchim and Nelore bulls had<br />
different IBW, the average daily weight gains were similar (1.86 vs. 1.56 kg, respectively; P > 0.10) and the breeds had the same profile of<br />
sperm characteristics (vigorous movement, gross-motility, motility, concentration and % major or minor defects) during the three periods.<br />
Among sperm abnormalities, Canchim bulls had less proximal cytoplasmic droplets (PD, 5.0±3.0%) than Nelore bulls (22.3±3.3%; P < 0.01)<br />
in P1, suggesting that Canchim bulls were more sexually mature at the beginning of the evaluations. The % of major defects reduced 38.7%<br />
from P1 to P2 and 50.6% from P1 to P3, on average, for both breeds. Similarly, the % of PD was significantly reduced from P1 to P2 and from<br />
P2 to P3 (P < 0.01). Sperm concentration and motility increased from P1 to P2 and P3 (P = 0.08) and vigorous movement and gross-motility<br />
were improved from P2 to P3 (P = 0.03). The SC increased continuously over time for both breeds and, as expected, Nelore had smaller SC<br />
in all periods (P1: 29.4±0.7 vs. 33.6± 0.7; P2: 31.3±0.7 vs. 34.7±0.7; and P3: 33.4±0.7 vs. 37.7±0.7 cm, respectively; P < 0.01). We concluded<br />
that, despite Nelore were younger and lighter than Canchim bulls, the feedlot regime that was employed has allowed a similar development<br />
of sperm and semen characteristics between breeds, and potentially have hastened sexual maturity, especially in Nelore bulls. [Acknowledgements:<br />
Financial supports from CNPq, FAPESP, ALLTECH and EMBRAPA (Innovation Network on Animal Reproduction - 01.07.01.002)].<br />
Keywords: semen, pasture, feedlot.<br />
A032 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
INFLUENCE OF SEXING ON BOVINE SPERM NANOROUGHNESS OUGHNESS MEASURED BY ATOMIC FORCE MICROSC<br />
OSCOPY<br />
OPY<br />
Suelen Ribeiro Moita, , José de Oliveira Carvalho Neto, Margot Alves Nunes Dode & Luciano Paulino da Silva<br />
EMBRAPA, BRASILIA, DF, BRAZIL.<br />
The technique of sperm sexing applied to cattle increases economic advantages in systems in which production is favored by the<br />
offspring of a particular sex. Currently, flow cytometry is the most used method for sperm sexing. The present study aimed to analyze and<br />
characterize by atomic force microscopy (AFM) the surface of sexed sperm cells by flow cytometry, on a nanometric scale, since at this moment<br />
there is no study involving this level of characterization. Semen samples were obtained from three Nelore bulls and divided into four experimental<br />
groups: non-sexed (NS), X-sexed (SX), Y-sexed (SY) and pool of equal fractions of SX and SY (SXSY). To acquire the images of sperm cells,<br />
the atomic force microscopy SPM 9600 (Shimadzu, Japan) was operated in contact mode. The plane-fit correction of images and segmentation<br />
to isolate desired cells were performed, analyzing a total of 53 cells from each group/bull. Three distinct regions of the head of sperm cells were<br />
delimited for evaluation: A region, next to the acrosome; B region, equatorial region; and C region, post-acrosomal region. ANOVA statistics was<br />
performed and Tukey/Kramer test with a 5% (P < 0.05) of significance was used. The average value, median, maximum, minimum, mean<br />
roughness (Ra), root mean square (Rms), skewness, and kurtosis were the measured parameters. The results were compared among the A, B,<br />
and C regions, and among experimental groups. The average value, maximum, minimum, median, Rms and kurtosis parameters had no statistical<br />
differences among the studied regions of the same experimental group. However, for Ra parameter, the A region showed higher roughness (NS:<br />
51.9 ± 6.2 nm; SX: 55.9 ± 5.8 nm; SY: 60.2 ± 5.3 nm; e SXSY: 58.7 ± 5.7 nm) than the B region (NS: 14.2 ± 0.4 nm; SX: 16.8 ± 0.9 nm; SY:<br />
14.3 ± 1.6 nm; e SXSY: 15.7 ± 1.4 nm) and C region (NS: 43.9 ± 2.9 nm; SX: 44.1 ± 2.7 nm; SY: 41.1 ± 2.4 nm; e SXSY: 42.7 ± 1.8 nm).<br />
It was not possible identifying differences among experimental groups by ANOVA. It was concluded that atomic force microscopy is an<br />
analytical method that allowed the characterization of sperm cells nanoroughness, identifying different topographic aspects along the head.<br />
Keywords: atomic force microscopy, nanoroughness, sperm sexing.<br />
s352
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A033 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
THE INFLUENCE OF CULTURE MEDIUM ON OVIDUCT CELLS SPERM VIABILITY IN CATTLE<br />
TLE<br />
Patrícia da Silva Valleriote, Helga Fernandes Gomes, Carla Sobrinho Paes de Carvalho, Fausto Paes de Carvalho & Angelo José Burla Dias<br />
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
The aim of this study was to evaluate the influence of the secretions from oviduct cells on physical and functional plasma<br />
membrane of bovine spermatozoa. The oviducts were obtained from slaughterhouses and dissected in the laboratory. The oviduct epithelial<br />
cells were grown in culture bottles containing 5,0 mL of culture medium and maintained at 38,5°C; 5% CO 2<br />
for five days. The medium was<br />
centrifuged and the supernatant was concentrated 10 times in a protein concentrator (Amicon, Millipore). The total protein concentration was<br />
determined by the method of Bradford (1976). The straws of semen were thawed and submited to a Percoll gradient 45/90%. The sperm were<br />
resuspended in TALP-sp supplemented with BSA, at final concentration of 50x10 6 /ml and divided into three groups: T Ct (TALP-sp); T Hep<br />
(TALP-sp + 10 µg/mL heparin); T Ov (TALP-sp + 3,6 µg/mL of culture medium of oviduct cells). The semen was evaluated at 0h and after 3h<br />
incubation in relation to total motility (TM), progressive motility (PM), vigor (V) and hyposmotic test (TH). The samples were incubated<br />
in an incubator with an atmosphere of 5% CO 2<br />
; 38,5°C, for 3 h. In 0h the treatments T Ct , T Hep and T Ov presented similar results for the MT<br />
(85,0±11,2%; 80,0±10,4% and 86,4±4,75%, respectively). The results of the MP were 65,7 ± 23,1% (T Ct ); 58,6±19,3% (T Hep ) and<br />
69,3±13,7% (T Ov ), respectively. In the three treatments V was equal to 4,0. After 3h incubation, the MT values (mean ± SD) of T Ct , T Hep and<br />
T Ov were reduced in relation to 0h (65,4±13,0% ab ; 52,9±18,9% a e 75,0±5,0% b respectively). After three hours of incubation, we observed the<br />
following values of MP: T Ct (40,9 ± 21,6%); T Hep (36,4 ± 23,9%) and T Ov (48,6 ± 22,5%). The V values were: T Ct (3,1 ± 0,9%); T Hep (3,3 ±<br />
0,8%) and T Ov (3,4 ± 0,8%). The percentage of viable cells in TH 0h was 71,9±15,9% (T Ct ), 70,9±7,9% (T Hep ) and 66,7±20,8% (T Ov ). After<br />
3h incubation, we observed the following results: 64,3±8,9% (T Ct ), 65,1± 14,7% (T Hep ) and 64,4±16,3% (T Ov ). The secretions of bovine<br />
oviduct cells promoted increased values of MT sperm after 3h of incubation, indicating that these secretions may prolong the sperm viability.<br />
[Acknowledgment: FAPERJ].<br />
Keywords: spermatozoa, cell culture, seminal parameters.<br />
A034 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
INFLUENCE OF PERIOD OF THE YEAR IN SEMINAL QUALIT<br />
ALITY OF CAPTIVE COLL<br />
OLLARED PECARIES (PECARI<br />
TAJA<br />
AJACU) RAISED IN<br />
THE STATE TE OF PAR<br />
ARA<br />
Priscila Reis Kahwage 1 , Alexandre Rossetto Garcia 2 , Diva Anelie De Araujo Guimarães 3 , Otávio Mitio Ohashi 4 , Natalia Inagaki Albuquerque 5 , Mario Mansour<br />
Pinheiro Bartha 6 & Jakeline Dos Santos Pessoa 7<br />
1,3,4,6<br />
UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2,5 EMBRAPA AMAZÔNIA ORIENTAL, BELÉM, PA, BRAZIL. 7 UNIVERSIDADE FEDERAL RURAL DA AMAZÔNIA, BELÉM, PA,<br />
BRAZIL.<br />
The continuous assessment of collared peccaries (Pecari tajacu) semen characteristics might be useful to the development of semen<br />
collection procedures and gamete conservation during most favorable periods of the year. On the other hand, the environment in the Amazon<br />
region influences the reproductive characteristics and the behavior of the majority of domestic and wildlife species. Therefore, the aim of this<br />
research was evaluating physical and morphological characteristics of collared peccaries semen, considering two different periods of the year and<br />
its interference on semen quality. Eight adult males (52 ± 14.9 months and 19.8 ± 2.1 kg) from Embrapa Eastern Amazon (Belém, PA) herd were<br />
used as semen donors. Semen collection were carried out each fifteen days by electroejaculation, according Garcia et al. (2009, Revista Brasileira<br />
de Reprodução Animal, Supl.1, 462). Seminal samples (n=65) were evaluated for volume (mL), sperm concentration (million sptz/mL), pH (0-<br />
14), progressive motility (%), vigour (0-5), viability (%), major, minor and total sperm defects (%). The experiment occurred uring 17 months<br />
under tropical humid climatic type. Months from December to May were considered as “Intense Rainy Period” (IRP) while months from June<br />
to November were considered as “Soft Rainy Period” (SRP), according to Pachêco and Bastos (2007, Série Documentos - Embrapa Eastern<br />
Amazon, 300, 18). The effect of period over the seminal characteristics was evaluated using Student t test (P < 0.05). There was significant<br />
difference in the ejaculated volume (0.75 ± 0.5a mL in IRP X 0.3 ± 0.6b mL in SRP, P < 0.05). However, this feature depends on the semen<br />
collection method adopted and is also related to individual responses to electrical stimuli. Seminal features observed during IRP and SRP were,<br />
respectively: concentration of 109.8 ± 95.7 million sptz/mL and 167.2 ± 185 million sptz/mL, pH of 7.8 ± 0.6 and 7.8 ± 0.7, motility of 55.1 ±<br />
28, 9% and 53.5 ± 28.5%; vigour of 2.3 ± 0.9 and 2.0 ± 0.7, and viability of 56.1 ± 28.8% and 56.6 ± 28.8% (P > 0.05). Morphologically, there<br />
was 23.7 ± 14.0% and 21.7 ± 12.7% of major defects, 9.6 ± 7.7% and 8.6 ± 5.7% of minor defect and 33.3 ± 13.5% and 30.3 ± 13.7% of total<br />
sperm defects (P > 0.05). Differences between collared peccaries were observed (P < 0.05) for volume (0.3 ± 0.2 mL to 1.9 ± 0.8 mL in IRP,<br />
0.2 ± 0.1 mL to 1.0 ± 0 , 7 mL, in SRP), motility (18.3 ± 27.5% to 85.0 ± 4.0% in IRP and 18.2 ± 19.0% to 80.0 ± 6.1% IN SRP) , viability<br />
(19.6 ± 29.8% to 84.6 ± 3.2% in IRP and 21.1 ± 22.4% to 81.4 ± 7.5% in the SRP) and concentration (55.0 ± 48.0 to 167.19 ± 111.9 milion<br />
sptz/mL, in IRP and 40.53 ± 19.6 to 228.0 ± 150.0 million sptz/ Ml, in SRP). However, studied periods did not influenced on individual variation<br />
(P > 0.05).The absence of difference in the seminal features between periods reinforces the adaptability of collared peccaries to environmental<br />
variations of tropical humid climate. Thus, it was concluded that the period of the year in evaluated region did not interfere on physical and<br />
morphological seminal characteristics of Pecari tajacu. This fact enables the continuous research about biotechnologies of reproduction in this<br />
specie and leads to future establishment of ex situ germplasm collections.<br />
Keywords: andrology, spermatozoa, wildlife.<br />
N<br />
s353
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A035 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
INFLUENCE OF STOR<br />
ORAGE CONT<br />
ONTAINER (CYLINDRICAL AND FLATS) ON SEMEN EQUINE CRYOPRESER<br />
OPRESERVED<br />
Bruno Fagundes 1 , Juli Angélica Narváez 2 , Maurício Fraga Van Tilburg 3 , Marcus Antonio Barreto 2 , Wenitha Martins 2 & Jose Frederico Silva 2<br />
1<br />
UNIG, ITAPERUNA, RJ, BRAZIL. 2 UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL. 3 UFC, FORTALEZA, CE, BRAZIL.<br />
Cryopreservation is a process that reduces sperm viability of 50% (1). Focusing on the damage from freezing and thawing, some<br />
authors used different containers, for example: pellet (2,3), 0.25 and 0.5 mL straws (2,3), 5.0 mL maxi-straws or 5.0 mL aluminum tube (4)<br />
and plastic flat-pack ® (5,6). This study was to evaluate the influence of the shape of the storage container (cylindrical and flat “Flatbag”) on<br />
sperm motility and kinematics of Quarter Horse stallions after thawing (T0) and 30 min later (T30), kept in bath at 37°C. We compared straws<br />
(PAL) of 0.5 mL, maxi-straws (MAC) of 4 mL, flatbag (FB) of 0.5 mL and flatbag 4 mL, made with Ziploc ® , the following dimensions: 3 cm<br />
x 4 cm and 3 cm x 10 cm , respectively. The semen of the stallions were analyzed in CASA Hamilton Thorn Research ® 10.8 Keros, processed<br />
and diluted in the extender to freeze proposed by VIDAMENT et al. (2002). All samples were cooled to 5°C/20min subsequently placed at<br />
4 cm above liquid nitrogen for 10 min and then immersed in this. The samples were thawed at 37°C/30s. The variables of MT, MP, VCL, VAP<br />
and VS.L were evaluated at CASA. For data analysis we used the Tukey test, considering 5% level of significance compared with the<br />
containers as a function of time. At T0, MT was better in FB 0.5 mL compared with PAL 0.5 mL and MAX 4 mL. While the kinematics was<br />
observed that the PAL 0.5mL had better VCL in T30 when compared with other containers. It can be concluded that the types and volumes<br />
of containers used had an influence on the tested variables and that is likely to modify the curves of freezing and thawing according to the<br />
volume, if they can get better results in flat containers. [Reference: (1)Watson PF. 2000. Animal Reprod, 60:481; (2)Thomas PGAet al. 1993.<br />
Theriog, 40:1199; (3)Nöthling JO & Shuttleworth R. 2005. Theriog, 63:1469; (4)Ivanova-Kicheva et al. 1997. Theriog, 48:1343; (5)Eriksson<br />
& Rodriguez-Martinez. 2000. Reprod Science, 63:205; (6) Ekwall H el al. 2007. Theriog, 67:1472; (7)Vidament M.et al., Theriog. 58; 249-<br />
251. 2002].<br />
Keywords: semen, equine, flatbag.<br />
A036 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
IMPROVING THE QUALITY OF RNA FROM BOVINE SEMEN FOR USE IN PCR Q-REAL TIME<br />
Vivian<br />
Taís Fer<br />
ernandes Cipr<br />
ipriano<br />
iano, Anderson Mior<br />
ioranza,<br />
Adr<br />
driana Renzi,<br />
Est<br />
ster Silv<br />
ilveir<br />
eira Ramos<br />
& Raysildo Barb<br />
arbosa Lôbo<br />
FMRP - USP, RIBEIRAO PRETO, SP, BRAZIL.<br />
In an animal breeding program, several reproductive characteristics are analyzed (motility, sperm concentration, scrotal circumference)<br />
as the sperm quality is crucial to the fertilization. In order to assist the selection of bulls with greater reproductive capacity, molecular methods<br />
have been employed. In this context, the analysis of gene expression by RT-PCR can correlate the expression of some genes with important traits<br />
for the animal breeding. However, to obtain reliable results, the technique needs to be associated with a good RNA extracted, from which the<br />
cDNA is synthesized and subjected to analysis. In case of analysis expression in semen, Lalancette et al (Biology of reproduction. 2008, 78: 618-<br />
635) demonstrated the effectiveness of the reagent Trizol RNA extraction in this type of sample. The objective of this study was to standardize<br />
the RNA extraction from bovine semen for use in PCR qReal Time. Samples remained in liquid nitrogen (-196ºC) until their use. Two extractions<br />
were performed using the kit SV Total RNA Isolation System (Promega Corporation, São Paulo, Brazil) and 25 extractions by Trizol ® method<br />
(Invitrogen, São Paulo, Brazil). The concentration of RNAs was determinated by 260 nm absorbance preparations (1OD 260<br />
= 40mg/µL) and<br />
their quality assessed by the OD (optical density) 260/280 nm, which should be close to 2.0. In order to improve concentration and purity of<br />
RNA obtained by extraction with Trizol, it was changed: number of extracted cells (2.5 x 10 6 to 1.4 x 10 6 ), number (one to three) and duration<br />
of washing of the samples (5 to 15 min), temperature of Trizol used (0°C or 65°C), increase in the number of precipitations in isopropanol (one<br />
to three), change in temperature of extraction (0°C or 25°C) and final heating of the sample (65°C). As results, the RNA obtained by the SV Total<br />
RNA Isolation System earned an average of 22νg/µL and low quality (OD 260/280 = 0.23). The RNA extracted by modified Trizol method<br />
showed higher concentrations (120 mg/µL) and quality (OD 260/280 = 0.98) which was, however, still unsatisfactory. Over the changes made,<br />
it was observed that washing the sample and changes in precipitation decreased quality of RNA obtained. On the other hand, we obtained better<br />
quality (OD 260/280 = 1.7) and highest concentration of RNA (130 mg/µL) when the amount of semen to be extracted was decreased and the<br />
Trizol warmed.<br />
Keywords: animal breeding, bovine sperm, pcr qreal time.<br />
s354
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A037 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
EXPERIMENTAL MODEL TO PRESERVE<br />
WILD CANINE SPERM THROUGH<br />
THE FREEZE-DRYING PROCESS<br />
Luis Carlos Oña Magalhães, Cely Marini Melo e Oña, Mateus José Sudano, Letícia Ferrari Crocomo, Daniela Martins Paschoal, Fernanda da Cruz Landim<br />
Alvarenga & Maria Denise Lopes<br />
UNESP - BOTUCATU, BOTUCATU, SP, BRAZIL.<br />
The freeze-drying process might be an alternative to preserve the wild canine spermatozoa. Such technique has already been<br />
successfully used with the bovine, equine, murine and feline species. The objective of the present study was to assess both HTF and SOF<br />
efficiency in protecting the sperm nuclei of freeze-dried dog sperm. Three male dogs were used. Sperm was collected via digital manipulation.<br />
Sperm-rich fraction from ejaculates was divided into two aliquots, and then added with either HTF or SOF having a final concentration of 2x103<br />
sperm/mL. Samples were put into 2.0 mL Cryogenic Vials having a maximum volume of 0.5 mL per vial. Vials were taken to a refrigerator of<br />
stable temperature (Minitub do Brazil, Porto Alegre/ RS) and kept at 5ºC for 60 min. Soon after that, samples were kept in N2 vapor for 20 min,<br />
and finally plunged into N2. Vials were then taken to the freeze-drying machine in order to obtain a stable product and remove the liquid fraction.<br />
With a view to analyzing DNA integrity, it had been used the Acridine Orange test, and to analyze the membrane integrity it had been used the<br />
association of JC-1, FITC-PSA, and propidium iodide. Both SOF (77.3%) and HTF (84%) proved to protect the sperm cell DNA, and they<br />
might thus be used as extenders for the freeze-drying process (P < 0,05). Nevertheless, if we group up cell membrane damages, acrosome<br />
damages, and mitochondrial membrane potential, both extenders showed extensive percentage of sperm cell damage (SOF = 73,1±0,82%; HTF<br />
= 72,1±0,88 %) in the epifluorescence microscopy. Samples that used HTF as medium for both freeze-drying procedure and reconstitution<br />
required the use of Vortex followed by the use of Pasteur pipettes owing to the fact that sperm cells were strongly bonded. Despite the fact the<br />
both HTF and SOF are chemically defined extenders, and protect sperm DNA during the freeze-drying process, sperm cells that used the SOF<br />
medium were easier to isolate by a simple vial shake. [This research was supported by CAPES].<br />
Keywords: wild canine, freeze-drying, sperm.<br />
A038 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
SPERMATIC PROFILE OF EJACUL<br />
CULATED AND EPIDIDYMAL SEMEN FROM DOGS EPIDIDYMIDES AFTER COOLING<br />
Daniel Souza Ramos Angrimani, , Cristina Fátima Lucio, Gisele Almeida Lima Veiga, Andressa Dalmazzo , Marcílio Nichi & Camila<br />
Infantosi Vannucchi<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL - UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.<br />
The recovery of sperm directly from the epididymis allows for genetic material gaining after castration or post mortem, aside from<br />
being considered an alternative to the reproduction of high value animals and to permite studies on sperm maturation. However, the seminal<br />
characteristics of these samples have not been precisely determined in dogs, in addition to the lack of standardization of the epididymal<br />
processing. Therefore, the aim of this study was to compare the profile of the canine ejaculated sperm with samples from distinct epididymal<br />
segments after cooling the epydidimis. We used five dogs of different breeds aged 1 to 6 years. From the same animal, the semen sample was<br />
obtained by digital manipulation of the penis and, after 1 week, from the epididymal semen after orchiectomy. The epididymides were stored at<br />
5°C for 19 h and semen was subsequently collected through small incisions (< 1mm) of the head, body and tail, aspirated separately by automatic<br />
pipette and maintained in 100 µl TALP extender. The semen samples were evaluated for motility, vigor and Computer Assisted Sperm Analysis<br />
(CASA). The eosin/nigrosin stain was employed to evaluate the permeability of the sperm plasma membrane. Data were compared by ANOVA<br />
and Tukey test (P = 0.05). Ejaculated semen showed the highest motility at subjective analysis, followed by the tail, body and head samples, with<br />
significant difference among them. Considering sperm vigor, there was no difference between the ejaculate and the tail epididymal semen, this<br />
latest not different from the body. Semen from the head presented the smaller sperm vigor. Samples from the head and body of the epididymis<br />
showed higher membrane permeability in relation to the ejaculate. In the evaluation by CASA, an higher motility and percentage of fast<br />
spermatoza was observed in the ejaculated and tail semen, in comparison to the body and the head epidydimis. Progressive motility was<br />
significantly higher in the ejaculate and decreased progressively from the tail, body and head of the epidydimis. Samples of body and head<br />
showed a higher percentage of static sperm, differing significantly from the ejaculated and tail semen. In conclusion, the semen derived from the<br />
tail of the epididymis showed similar characteristics as the ejaculate, for motility, vigor and membrane permeability. The changes observed in the<br />
samples of the head and body of the epididymis may be due to sperm immaturity, hence not suitable for the use in sperm biotechnologies.<br />
Moreover, the cooling of the epididymis did not change the characteristics of the semen samples obtained from the epididymal tail.<br />
Keywords: epididymal semen; spermatic profile ; dogs<br />
N<br />
s355
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A039 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
LYOPHILIZED SEMINAL PLASMA IMPROVES<br />
THE MOTILIT<br />
TILITY OF FROZEN RAM SEMEN AND INCREASES THE CLEAVAGE IN<br />
HETEROLOGOUS IVF<br />
Renata Casali 1 ,Fabiano Carminatti Zago 2 , Michelle Federle 1 Jessica Nora Drum 1 , Mariana Sponchiado 1 , Norton Klein 1 , Maurício Seminotti Zanetti 1 , Saul<br />
, Gaudêncio Neto 1 , Alceu Mezzalira 1 , Ubirajara Maciel Da Costa 1 & Arnaldo Diniz Vieira 3<br />
1<br />
UDESC / CAV, LAGES, SC, BRAZIL. 2 EPAGRI, LAGES, SC, BRAZIL. 3 UFPEL, PELOTAS, RS, BRAZIL.<br />
The addition of heterologous seminal plasma (HSP) to the freezing extender of ram semen improved post-thaw viability<br />
parameters, and might be an alternative to improve intra-cervical insemination and in vitro fertilization (IVF) with frozen ram semen.<br />
However, data regarding such conditions are still very scarce, especially for lyophilized plasma. The aim of this study was to evaluate the<br />
effect of addition of lyophilized bovine seminal plasma (LBSP) and equine (LESP) to the freezing extender of ram semen. The evaluations<br />
performed were: progressive motility (PM) after thaw (AT) and PM after percoll selection (PS), acrosome integrity (AI) after PS and cleavage<br />
rates after IVF with heterologous bovine oocytes. Seminal plasma was obtained from five bulls and five stallions collected with artificial<br />
vagina. The ejaculates were centrifuged (5000 rpm) and lyophilized with Christ Alpha 1-4 Freeze Dryer. The ram semen was collected with<br />
artificial vagina from four Texel rams, pooled and diluted in 1 +1 in glycerolated Tris-egg yolk without seminal plasma (Tris control, TC) or<br />
added of 600µg/mL of LBSP or 600 µg/mL of LESP. The semen was then cooled at 0.3°C / min. until to 5ºC, stabilized for one hour, loaded<br />
in 0.25 mL straws in isothermal conditions and frozen at -80°C in LN2 vapor before store in cryogenic container at -196°C. The sperm<br />
viability was determined based on the PM-AT and PM-PS. The sperm were used for heterologous IVF (Garcia-Alvarez, 2009, Theriogenology,<br />
71: 643-650), and to evaluate the acrosome reaction by FITC method (Sukardi Ani Sci Rep 1997, 46: 89-96). Data were analyzed by Student<br />
T test with significance level of 5%. There was no difference between the average rates of post thaw progressive motility PM-AT of PSLB<br />
(27.5%) and PSLE (37.5%) groups, but both were higher than TC group (10%), improving the viability of thawed semen by the addition of<br />
heterologous seminal plasma. The rates of PM-PS were similar in all groups (TC 40.0%, 56.7% LBSP, LESP 70.0%). There was no difference<br />
in the IAPP evaluation of different groups (CT 58.4%, 67.3% LBSP, LESP 63.3%). There was a significant increase in cleavage rate of LBSP<br />
(45.7%) and LESP (43.7%) groups when compared to TC group (32.6%). The increased cleavage rate observed in heterologous IVF<br />
demonstrates that the frozen semen added of LEAP or LBSP has a greater oocyte penetration ability, which suggests that they may be more<br />
efficient for sheep artificial insemination.<br />
Keywords: cryopreservation, lyophilization, heterologous seminal plasma.<br />
A040 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY<br />
USE OF THE POWDERED COC<br />
OCONUT<br />
ONUT WATER AS AN ALTERNA<br />
TERNATIVE TIVE EXTENDER FOR THE CRYOPRESER<br />
OPRESERVATION OF COLL<br />
OLLARED<br />
PECCARIES (<br />
(TAYASSU<br />
ASSU TAJA<br />
AJACU<br />
CU) ) SEMEN<br />
Mariana Araújo Silva, , Lívia Batista Campos, José Artur Brilhante Bezerra, Gabriela Liberalino Lima, Gislayne Christianne Xavier Peixoto, Andréia Maria Da<br />
Silva & Alexandre Rodrigues Silva<br />
UFERSA, MOSSORÓ, RN, BRAZIL.<br />
Collared peccaries (Tayassu tajacu) are amongst the most hunted species due to the appreciation for its meat and international<br />
interest for his leather. This fact has contributed to a drastic decrease in its population in their natural habitats. In this sense, the development of<br />
protocols for the storage of semen would allow the formation of germplasm banks for its use in captive breeding programs. Once the literature<br />
is scarce, and only the Tris (Castelo et al., Cryobiology, 2010) extender has been used for this purpose, the present study aims to evaluate the<br />
efficiency of a powdered coconut water-based extender (ACP-116c ® ) as an alternative for the cryopreservation of collared peccary semen.<br />
Twelve adult males bred in captivity at the Centre of Multiplication of Wild Animals of UFERSA were used. They were mechanically restrained<br />
with the aid of a hand-net, and further anesthetized with propofol (5 mg/kg, IV). The electroejaculation was conducted, and the semen samples<br />
were evaluated for microscopic characteristics. Each sample was divided into two portions, the first was diluted in Tris-fructose and the second<br />
in ACP-116c ® , both plus 10% egg yolk. Samples were equilibrated for 240 min at 5°C, and further added of 3% glycerol, reaching a 100 x 106<br />
sperm/mL concentration. Samples were packed in 0.25 mL plastic straws and stored in liquid nitrogen. After one week, semen was thawed in<br />
a water-bath at 37ºC/30s and reevaluated. Data for sperm motility were Arcsin transformed and submitted to analysis of variance followed by<br />
Student’s t-test (P < 0.05). Sperm vigor was evaluated by the Mann-Whitney non-parametric test (P < 0.05). Fresh semen presented a<br />
concentration of 765 ± 313.8 x 106 sperm/mL, in a volume of 2.8 ± 0.7 mL, with 86.7 ± 2.6% motile sperm with vigor 4.4 ± 0.2. After thawing,<br />
a reduction was verified for all the parameters assessed in the use of both diluents (P < 0.05), which differed among themselves (P > 0.05).<br />
Values observed for sperm motility was 30.4 ± 5.7% and 40.8 ± 6.9%, and for vigor was 2.4 ± 0.2 and 2.9 ± 0.2 for Tris and ACP-116c ® ,<br />
respectively (P > 0.05). The results evidence an equivalence of diluents on the preservation of sperm quality. The use of ACP-116c ® is<br />
recommended as an alternative extender for the collared peccary semen cryopreservation.<br />
Keywords: powdered coconut water, cryopreservation, Tayassu tajacu.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A041 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
ADDITION OF INSULIN TO THE IN VITRO CULTURE MEDIUM PROMO<br />
OMOTES SURVIV<br />
VIVAL AND DEVEL<br />
VELOPMENT OF FOLLICLES<br />
PREANTRAL AL GOATS<br />
Roberta Nogueira Chaves, , Anelise Maria Costa Vasconcelos Alves, Luciana Rocha Faustino, Claudio Afonso Pinho Lopes, Kenio Patricio Lima Oliveira & José<br />
Ricardo de Figueiredo<br />
LABORATÓRIO DE MANIPULAÇÃO DE OÓCITOS E FOLÍCULOS PRÉ-ANTRAIS (LAMOFOPA), UECE, FORTALEZA, CE, BRAZIL.<br />
Insulin is usually added to the ovarian tissue culture as a factor that stimulates cell survival and mitosis, but no studies to<br />
determine the best concentration to be used. Thus, the aim of this study was to elucidate the effects of the addition of different insulin<br />
concentrations to the culture medium on the survival and development of goat preantral follicles after 7 days of culture. Ovaries (n=14) from<br />
adult, non-pregnant, mixed-breed goats (1-3 years of age) were collected from a local slaughterhouse. In the laboratory, the cortex from each<br />
ovarian pair was sliced and the fragments were fixed (fresh control) or cultured for 1 or 7 days in the absence or presence of insulin (0, 5, 10<br />
ng/mL and 5 or 10 µg/mL). Each treatment was repeated seven times. Non-cultured and cultured tissues were processed for classical histology,<br />
transmission electron microscopy and viability test using fluorescent probes (calcein-AM and ethidium homodimer-1). The data were<br />
subjected to analysis of variance and compared using the Dunnett’s, SNK and ÷2 tests (P < 0.05). After 7 days of culture in medium<br />
supplemented with 10 ng/mL insulin, 82% of the follicles present are morphologically normal. This percentage was significantly higher than<br />
those observed in control medium (71%) or supplemented with 5 (65%) and 10 µg/mL (66%), but did not differ from follicles cultured with<br />
5 ng/mL (74%) of insulin. The ultrastructural analysis and the viability test confirmed the integrity of follicles cultured for 7 days in medium<br />
containing 10 ng/mL of insulin. At the end of culture, all treatments significantly increased the percentage of developing follicles (0 ng/mL:<br />
52%, 5 ng/mL: 62%, 10 ng/mL: 76%, 5 µg/mL: 79%; 10 µg/mL: 60%) compared to day 0 (37%). The culture of ovarian tissue in the presence<br />
of 10 ng/mL or 5 µg/mL increased the percentage of developing follicles when compared to other cultivated treatments (P
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A043 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
APOPTOSIS OSIS IN FETAL BOVINE AND BUFF<br />
UFFAL<br />
ALOES OVARIES BY TUNEL METHOD<br />
Thiago Velasco Guimarães Silva , Simone do Socorro Damasceno Santos, Bruno Baraúna da Silva, Bruna Letícia Gentil Bittencourt, Rafael Vilar Sampaio,<br />
Marcela da Silva Cordeiro, Nathália Nogueira da Costa, Priscila Di Paula Bessa Santana, Veluma Priscila Duarte Corrêa, Moysés dos Santos Miranda &<br />
Otávio Mitio Ohashi<br />
UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL.<br />
Apoptosis was assessed in human fetal and sheep ovaries (Pol et al., 1998, Anticancer. Res 18: 3457-3461; Abir et al., 2002,<br />
Fertility and Sterility, 78: 259-264, Fulton et al., 2005, J Clin Endocrinol Metab., 90: 4664-70 and Aladaer et al., 2008, Reproduction Science,<br />
103: 179-186) and according to Levy et al., 2005, Gynécologie obstétrique & Fertilité, 33: 645-652, reaches about 80% of germ cells present in<br />
the human ovary. Although buffaloes are of significant importance in livestock, there are few studies related to the in situ and in vitro aspects of<br />
its folliculogenesis (Gupta et al., 2001, Veterinary Record 148: 543-544; Gupta et al., 2002, Theriogenology 57: 1839 -1854; Sharma et al.,<br />
2009, Livestock Science, 123: 300-305; Santos et al., 2006 Anim Reprod Sci 95, 1-2, 1-15 and Santos et al., <strong>2011</strong>, Reprod Dom Anim 46, e17-<br />
e22). The aim of this study was to evaluate the occurrence of apoptosis by TUNEL method in fetal ovaries of cattle and buffaloes. We collected<br />
ovaries from 9 bovine fetuses (2 of 4 months, 3 of 5 months, 2 of 7 months, 2 of 8 months) and 9 buffalo fetuses (3 of 4 months, 2 of 5 months,<br />
2 of 7 months and 2 of 8 months) estimated according to Abdel-Raouf & El-Naggar, 1968, (see UARJ. Vet Sci 5, p.37-43). After histological<br />
processing, sections were subjected to TUNEL assay (TACS-XL - Basic - TA 100, R&D Systems, Inc. USA), stained with HE and analyzed<br />
by optical microscopy, according to Feranil et al., 2005 (Reproduction in Domestic Animals 40, 111-116). We observed the overall appearance<br />
of the ovarian stroma and primordial, primary and secondary follicles, also evaluating cell number of each follicle, using ANOVA, with P < 0,05.<br />
The analysis showed that apoptosis occurs in cattle and buffaloes fetuses, with a similar pattern at different ages, observing a decrease in the<br />
number of apoptotic cells in 8 months buffalo fetus. In cattle it was observed that secondary follicles have a higher number of apoptotic cells (1,5<br />
± 1,8) than primordial and primary follicles (0,5 ± 1,0 Vs.. 0,5 ± 0,8), however, in buffaloes, the average of apoptotic follicular cells was similar<br />
in the three follicular classes (0,5 ± 0,9, 0,4 ± 0,7 and 0,5 ± 0,9). We also observed that the bovine secondary follicle has an average of apoptotic<br />
cells greater than the buffalo, which must take into account the greater number in bovine secondary follicle cells. Therefore, we conclude that<br />
apoptosis in ovarian follicles from bovine fetuses and buffaloes is similar in ages between 4 and 8 months.<br />
Keywords: follicular apoptosis, cattle, buffalo.<br />
A044 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EVAL<br />
ALUATION OF A FSH PROTOC<br />
OCOL OL FOR TRANSV<br />
ANSVAGINAL FOLLICULAR ASPIRATION<br />
Tobias Canan Canan Sov ernigo<br />
nigo, Éric<br />
ico da Silv<br />
ilva San<br />
ant os, R enat o Zanin, Paulo Rob<br />
ober<br />
ert o Adona,<br />
Samuel Guemer<br />
uemera,<br />
Paulo Júnior de<br />
Campos Filho, Raquel Cristina Gonçalves, Aline Tramontini Zanluch, Agostinho Ludovico, Maria Augusta Berlingieri, Luiz<br />
Fernando Coelho da Cunha Filho & Denis Marques Rossi<br />
UNOPAR, UNIVERSIDADE NORTE DO PARANÁ, ARAPONGAS, PR, BRAZIL.<br />
Studies have demonstrated that exogenous FSH may increase the number of viable oocytes per cycle (Sirard, 1985, Biol Reprod,<br />
33:487-494). Ovum pick up allows oocyte recovery once or twice a week without apparent reproductive tract trauma (Bols, 1995, Theriogenology,<br />
43:677-687). The aim of this experiment was to evaluate oocyte recovery every three days after FSH administration in three bovine breeds<br />
divided in three groups. In group FSH, animals received FSH on Day zero (D0) to synchronize follicular growth (beginning of treatment). On<br />
D1 animals received a single i.m. injection 200 mg of FSH. On D3 they were subjected to a follicular aspiration (first aspiration and beginning<br />
of D0). On D4 animals received the second FSH injection. On D6 a follicular aspiration was performed (second aspiration and beginning of D0).<br />
On D7 (D1) they had the third FSH injection. On D9 the third and last follicular aspiration was performed. All animals were aspirated 4 times:<br />
one to synchronize follicular growth (beginning) and 3 after FSH injections, in order to evaluate oocyte recovery. In control group, on D0,<br />
animals were submitted to follicular aspiration in order to synchronize follicular growth (beginning of control). The animals in this group were<br />
aspirated every 7 days. Four aspirations were performed; one to synchronize follicular growth, and 3 to evaluate oocyte recovery. A crossover<br />
design was performed, in which all animals were subjected to both treatments. Data were analyzed by Tukey test. FSH aspiration resulted in a<br />
decrease in 18.4% of total number of oocytes per aspiration. Nevertheless, there was no influence of breed or interaction between breed and FSH<br />
on this parameter. Although, numerically, the average of viable oocytes per aspiration was 12.7% lower in cows subjected to FSH. Considering<br />
percentage of viable oocytes, there was a breed influence. Girolando cows (n = 128; mean of 43.4%) had a lower average compared to Brangus<br />
(n = 82; mean of 60.3%) and Nelore (n = 70; mean of 55.9%). Brangus cows had a oocyte viability of 66.0% (n = 40) and 54.9%, with and<br />
without FSH (P < 0.05), respectively. Experiments in Holstein heifers treated with FSH have shown that over 75.0% of immature oocytes<br />
become blastocysts in vitro, compared with an average of 40.0% in non-treated animals (Blondin, 2002, Biol Reprod, 66:38-43). In another<br />
study, Andrade (2001, Rev Bras Reprod Anim, 25:412-13) reported that FSH treatment in animals subjected to follicular aspiration increased the<br />
quality of oocytes, indicating 60 h after treatment as the best interval for aspiration. Girolando cows presented a lower percentage of viable<br />
oocytes than Brangus and Nelore. The treatment with FSH decreased total oocyte count per aspiration, without affecting viability. Nonetheless,<br />
in Brangus cows, FSH allows a significant increase (20.3%) in the proportion of viable oocytes.<br />
Keywords: follicular aspiration, fsh, bovine.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A045 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
COMP<br />
OMPARISON BETWEEN LUTEAL PHASE PERIOD AND THE NUMBER OF FOLLICULAR<br />
WAVES IN BOS TAUR<br />
URUS<br />
US,<br />
BOS INDICUS AND BUB<br />
UBAL<br />
ALUS BUB<br />
UBALIS<br />
HEIFERS MAINTAINED AINED ON THE SAME NUTRITIONAL AND<br />
ENVIRONMENT<br />
ONMENTAL STATUS<br />
TUS<br />
Julia Maria Baldrighi, Manoel Francisco Sá Filho, José Antonio Visintin , Pietro Sampaio Baruselli & Mayra Elena Assumpção<br />
UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.<br />
Bovine luteal concentration after ovulation maintains the estrous cycle until luteolysis, when fertilization does not occur. During this<br />
cycle, corpus luteum (CL) development shows three phases: growth, static and regression. Thus, the luteal period involves the first two phases,<br />
when the CL is active. Considering these facts, an hypothesis is that the greater the luteal phase, the higher the number of follicular waves in one<br />
estrous cycle. The main objective of this study was to compare the luteal phase period with the number of follicular waves in one estrous cycle<br />
from Bos taurus (Holstein; n = 14), Bos indicus (Gir; n = 5) and Bubalus bubalis (Murrah; n = 15) heifers under the same nutritional and<br />
environmental conditions to avoid variations due to time and management influences. All 34 heifers were housed in pasture at the Department<br />
of Animal Reproduction, College of Veterinary Medicine, University of Sao Paulo, at Pirassununga Campus and they were synchronized with<br />
two doses of prostaglandin F2α (PGF; D-cloprostenol, 150 µg IM) fourteen days apart. From D0 (D0 = day of first ovulation), ultrasound scan<br />
of the ovaries was performed daily to follow their follicular dynamics (follicles = 3mm) and to record number of follicular waves, CL measures<br />
during inter-ovulatory interval (IOI) (duration of luteal phase) and the duration of the IOI in one estrous cycle. No significant difference was<br />
found between the IOI (22.2±0.65; 22.1±0.95; 24.0±0.65 days) for the Holstein, Gir and Buffalo, respectively. On the other hand, the number<br />
of follicular waves in one estrous cycle (2.8±0.15a, 3.4±0.24b, 2.8±0.13a waves; P < 0.05) and the duration of luteal phase (13.4±2.4a,<br />
16.0±0.0b, 15.4±0.74b days; P < 0.003) were statistically different for the three breed, Holstein, Gir and Murrah, respectively. Although there<br />
were no differences found the IOI among the three breeds, the zebu heifers showed higher number of follicular waves (60% with 3 waves and<br />
40% with 4 waves) and greater luteal phase than the other two breeds. After evaluating the three breeds at the same nutritional and environmental<br />
status it is possible to conclude that the CL activity may be involved in the number of follicular waves during one estrous cycle in Bos taurus,<br />
Bos indicus and Bubalus bubalis heifers.<br />
Keywords: ultrasound, corpus luteum, estral cycl.<br />
A046 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
BEHAVIOR OF A NEW POLYMORPHISM ON GDF-9 GENE (FECGSI) IN EWES OF SANTA A INÊS BREED IN FRONT OF<br />
SUPEROVUL<br />
OVULATION PROTOC<br />
OCOLS<br />
OLS<br />
Bianca Damiani Marques Silva 1 , Thiago Antonio de Souza Nascimento Silva 2 & Jairo Pereira Neves 3<br />
1<br />
EMBRAPA, ASA NORTE, DF, BRAZIL. 2,3 UNB, ASA NORTE, DF, BRAZIL.<br />
Many polymorphisms were described in GDF-9 and BMP-15 genes, but only one showed high ovulation and prolificacy in<br />
homozygosis without sterility. The aim of this study was evaluated the superovulatory answer and embryo yield on FecGSI polymorphism ewes<br />
in two superovulation protocols. Material and Methods Superovulation e embryo recovery was made in 18 Santa Inês ewes (6 E/E (mutated<br />
homozygote), 6 +/E (heterozygote) e 6 +/+ (wild homozygote)) to compare ovarian response among genotype. The ewes were randomly<br />
distributed in two protocols of superovulation: traditional protocol and Day 0 protocol. Each treatment was repeated twice in a cross-over model<br />
intercalated of two months each. Was used fresh semen for AI of one male with proved fertility. Five days after AI the embryos were recovered<br />
cirurgically, by laparotomy. The statistical analyses were factorial 3x2 with variance analyses by SAEG 9.1. Results There was no difference<br />
among evaluated parameters (P > 0.05) between Day 0 protocol (total CL 9.8 ± 5.3; total structures 4.5 ± 4.6; viable structures 1.6 ± 2.0) and<br />
traditional protocol (total CL 10.0 ± 6.0; total structures 3.5 ± 4.3; viable structures 1.7 ± 2.4), probably because of precocious acquisition of LH<br />
receptors changing the follicular dominance process. Corpora lutea in the ovaries (E/E 9.0±6.3; +/E 10.1 ±5.3; +/+ 10.5±5.3), total structure<br />
recovered (E/E 4.9±5.0; +/E 3.1±3.1; +/+ 4.1±5.2), and viable embryos (E/E 1.9±2.1; +/E 2.2±2.6; +/+ 0.9±1.7), there was no difference (P ><br />
0.05) among groups of genotype animals. Was observed higher number of animals answering the superovulation treatment when used Day 0<br />
protocol (10/18) in comparison with traditional protocol (6/18). The ewes that answered to superovulation protocol, regardless the protocol, by<br />
genotype, there were no difference E/E 6/12; +/E 5/12 e +/+ 5/12. Was not observed alteration in ovaries, in follicular growth and neither in viable<br />
embryo production when compare among genotypes. Conclusions Both protocols were efficient in superovulation and embryo production, but<br />
there were no difference among evaluated genotypes in ovulation rate, possibly for any alteration in FSH receptor, influenced by the new<br />
genotype or by the forward fase of FSH application.<br />
Keywords: ovulation rate, embryo, polymorphism.<br />
N<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A047 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
CANINE PREANTRAL AL FOLLICLES CULTURE AT DIFFERENT CONCENTR<br />
ONCENTRATIONS TIONS OF INSULIN IN PRESENCE OF FOLLICLE-<br />
STIMULATING TING HORMONE (FSH)<br />
Michelle Karen Brasil 1 , Gerlane Modesto Da Silva 1 , Ana Beatriz Graça Duarte 1 , Valdevane Rocha Araújo 1 , Ana Kelen Felipe Lima 2 ; Ticiana Franco Pereira Da<br />
Silva 1 , Franciele Osmarini Lunardi 1 & José Ricardo De Figueiredo 1<br />
1<br />
UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE FEDERAL DO TOCANTINS, ARAGUAÍNA, TO, BRAZIL.<br />
The composition of medium for in vitro culture is extremely important for achieving success in growing preantral follicles (PF).<br />
In this microenvironment follicles needs to be supported to survival and develop (Serafim et al., 2010). In vitro studies have shown that<br />
supplementation of culture medium with insulin has favored the growth, survival and also show low rates of follicular degeneration in cattle.<br />
However, in dogs the effect is unknown. Thus, this study aimed to evaluate the growth of canine PF at different concentrations of insulin added<br />
to the medium. Secondary PF were isolated by microdissection and in vitro cultured. The medium used was α-MEM supplemented by FSHrec<br />
(100, 500 and 1000 ng/mL on days 0, 6 and 12 of culture). The treatments were control (basic medium) and insulin at different concentrations<br />
(5 or 10 ng/mL and 10 mg/mL). The 18-days cell culture was performed in an incubator pre-calibrated at 39°C and 5% CO2 in air. The medium<br />
was changed every two days. The parameters evaluated were antrum formation and follicular diameter. The data were analyzed by ANOVA<br />
followed by Student t test and Kruskal-Wallis. For antrum formation evaluation we used the chi-square test (P < 0.05).The group insulin 10 ug/<br />
mL (411.05 ± 87.24 mM) was significantly higher when compared to other treatments (289.22 ± 56.60 mM) 5ng/mL insulin (347.38 ± 79.43<br />
mm) and 10ng/mL (336.10 ± 69.78 µm). About antrum formation, the insulin group 10 mg / mL (61.13%) was significantly higher than the<br />
control group (15.87%), 5 ng/mL (34.43%) and 10 ng/mL (50.79%). Thus it is concluded that the addition 10 mg/mL of insulin enhances the<br />
diameter and the antrum formation in canine PF after 18 days in culture.<br />
Keywords: canine, pre antral follicle, insulin.<br />
A048 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
FOLLICULAR DYNAMICS IN NELORE DONORS SUPERSTIMULATED<br />
TED WITH ECG ADMINISTERED IN A SINGLE<br />
DOSE OR FRACTIONED<br />
Lindsay Unno Gimenes<br />
1 , Gr aber Fidel Cássio Gr aber<br />
ert 2 , Mur<br />
urilo Gr aber<br />
ert t Lour<br />
ourenço<br />
2 , Juliano<br />
Tojal<br />
2 , Andréa M. San<br />
antilli Chanquetti<br />
2 &<br />
Karina Médici Madureira 2<br />
1<br />
UNIFIAN/ UNIABC, LEME/ SANTO ANDRÉ, SP, BRAZIL. 2 UNIFIAN, LEME, SP, BRAZIL.<br />
In the present study, follicular dynamics of Nelore donors superstimulated with 1500 IU of eCG administered in a single dose or<br />
fractioned was evaluated. For this purpose, 5 heifers and 4 cows with BCS of 2.9±0.1 and weighing 351.0±15.3 kg were used. On a random<br />
day of the estrous cycle (D0), all animals were treated with an intravaginal progesterone device previously used during 8 days (Primer ® ,<br />
Tecnopec) associated to 2 mg of estradiol benzoate (Gonadiol ® , Intervet Schering-Plough) and 0,5 mg of cloprostenol (PGF2α; Sincrocio ® ,<br />
Ouro Fino Saúde Animal). Four days later (D4), the animals were assigned in two groups to receive the following treatments: 1500IU of eCG<br />
(Folligon ® , Intervet Schering-Plough; G1x) or 1000 IU of eCG (G2x). On D6, all animals received another dose of PGF2α, and those of G2x<br />
were also treated with 500 IU of eCG. Intravaginal devices were removed after 36 h (D7), and 12 h later (D8) 0.01 mg of buserelin (Sincroforte ® ,<br />
Ouro Fino Saúde Animal) was administered. Another replicate was performed 14 days after administration of ovulation inducer in a cross-over<br />
design (n=9/group). Throughout the experimental period, all animals were evaluated by transrectal ultrasonography (Pie Medical Scanner 200)<br />
every 24 h, and ovulations were confirmed on D15. At this time, all animals received another PGF2α dose. Data were analyzed using PROC<br />
GLM of SAS, and the effects of treatment, replicate and treatment x replicate were included in the statistical model. Differences between groups<br />
were evaluated by Duncan’s test. No differences between G1x and G2x were found, respectively, for number of follicles on D4 (14.0±1.6 vs..<br />
17.0±2.2, P = 0.26) and number of follicles on D8 (20.7±1.6 vs.. 18.2±2.6, P = 0.47). However, an interaction treatment x replicate was found<br />
for the following variables: diameter of largest follicle on D8 (G1x replicate 1: 12.7±0.4 vs.. G1x replicate 2: 10.9±0.7 vs.. G2x replicate 1:<br />
9.5±0.6 vs.. G2x replicate 2: 12.4±0.6, P < 0.01), number of CL on D15 (G1x replicate 1: 12.4±1.0 vs.. G1x replicate 2: 4.3±0.5 vs.. G2x<br />
replicate 1: 7.5±2.1 vs.. G2x replicate 2: 9.8±2.7, P = 0.02) and ovulation rate (G1x replicate 1: 59.6±7.9% vs.. G1x replicate 2: 23.0±4.2% vs..<br />
G2x replicate 1: 42.2±11.2% vs.. G2x replicate 2: 53.3±11.9%, P = 0.03). In conclusion, the number of follicles was not affected by fractionation<br />
of eCG, although variables related to ovulation rate may have been affected by the diameter of follicles on D8. Further research including embryo<br />
collection outcomes need to be performed in order to verify the feasibility of the proposed protocol. [Acknowledgements: Unifian Leme - PIC<br />
Proc 110401and Prof. Pietro Baruselli].<br />
Keywords: superovulation, ultrasonography, bovine.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A049 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
THE EFFECT OF ECG IN MULTIPLE OVULATION PROTOC<br />
OCOLS OLS FOR HIGH PRODUCTION HOLSTEIN COWS EMBRYO DONORS<br />
Carlos Alberto Rodrigues 1 , Lais Mendes Vieira 2 , Andressa Lavezzo Ranieri 3 , Pericles Ricardo Lacerda e Silva 4 , Henderson Ayres 5 & Pietro Sampaio Baruselli 6<br />
1,4<br />
SAMVET, SAO CARLOS, SP, BRAZIL. 2,6 FMVZ USP, SÃO PAULO, SP, BRAZIL. 3 FCAV UNESP, JABOTICABAL, SP, BRAZIL. 5 INTERVET SCHERING-PLOUGH, BAURU, BULGÁRIA.<br />
The aim of this study was to evaluate the multiple ovulation response (number of corpora lutea – CL and number of viable<br />
embryos) in high production Holstein cows treated with eCG in the end of the protocol for multiple ovulation (MO) with FSH. The study<br />
was conducted in two periods (30 to 40 days interval). A total of 23 donors were used, whereas all donors passed through the two treatments,<br />
ending up with 46 MO. The cows were allocated in two experimental groups, according to the administration or not of eCG. Cows from group<br />
FSH (n = 23) received a norgestomet ear implant (Crestar ® , Intervet, Brazil) in the morning (AM) of a random day of the estrous cycle (D0)<br />
+ 2 mg estradiol benzoate (Gonadiol ® , Intervet, Brazil) + 50 mg progesterone (Index, Brazil) IM. From D4 on, each animal received 500 IU<br />
of FSH (Pluset ® , Hertape Calier, Brazil), diluted in 20 mL and divided in 8 applications of decreasing doses (100 IU, 100 IU, 75 IU, 75 IU,<br />
50 IU, 50 IU, 25 IU and 25 IU) 12h apart. On D6, 0.150 mg of sodic cloprostenol (Preloban ® , Intervet, Brazil) was administered AM and PM.<br />
On D7 PM, the implant was removed and on D8 PM 200 µg Gonadorelin (Fertagyl ® , Intervet, Brazil) IM, was administered. The artificial<br />
inseminations were done in fixed time on D9 AM and PM; embryo collection was performed on D15. Cows treated with eCG (n = 23)<br />
received similar treatment, except for the FSH (Pluset ® , Hertape Calier, Brazil) dilution, 18mL divided in 6 application for decreasing doses:<br />
111 IU, 111 IU, 83 IU, 83 IU, 56 IU and 56 IU. Also, a 500 IU of eCG (Folligon ® , Intervet, Brazil) was administrated on D7 AM. The number<br />
of formed CL was registered previously to each embryo collection. The binomial and continuous variables were analized by PROC<br />
GLIMMIX of SAS. Treatment with eCG had no effect on number of viable embryos (2.5 ± 0.7 vs. 3.5 ± 1.1; P = 0.10) per donor (average<br />
± SE); treatments without and with eCG, respectively. Although the eCG treatment effected the number of CL (6.4 ± 0.9 vs. 8.5 ± 1.1; P =<br />
0.01) and unfertilized oocytes rate - unfertilized oocytes per recovered structures – (34.8 ± 7.9 vs. 40.2 ± 9.0; P < 0.01) per donor (average<br />
± SEM); treatments without and with eCG, respectively. In conclusion, the addition of 500 IU of eCG in the end of the MO protocols with<br />
FSH in high production Holstein cows had effect on the protocol response, but did not enhance the number of viable embryos. [Acknowledgments:<br />
Fazenda Santa Rita (Agrindus) and Intervet Schering-Plough].<br />
Keywords: bovine, moet, ecg.<br />
A050 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EFFECT OF ACTIVIN-A ON THE DEVEL<br />
VELOPMENT OF BOVINE SECONDAR<br />
ONDARY FOLLICLES IN VITRO CULTURED<br />
José Renato de Sousa Passos, , Anderson Weiny Barbalho Silva, Gisvani Lopes de Vasconcelos, Maria Juliane Passos, José Jackson do Nascimento Costa,<br />
Rodr<br />
drigo Otávio Dec<br />
ecar<br />
aria de Salles Rossi,<br />
Francisc<br />
ancisco Taia Gomes Bezer<br />
erra,<br />
Joiane Araujo da Por<br />
orciuncula<br />
& José Rob<br />
ober<br />
erto Viana Silv<br />
ilva<br />
UNIVERSIDADE FEDERAL DO CEARA, SOBRAL, CE, BRAZIL.<br />
Recent studies have evaluated the effect of various hormones and growth factors during the in vitro culture of secondary follicles<br />
and, currently, in vitro oocyte maturation and embryo production was achieved in goats (Magalhães et al., Reprod. Domest. Anim., 46:134-140,<br />
2010). Despite the success of these studies, in vitro maturation and fertilization of oocytes from preantral follicles isolated and in vitro cultured<br />
has low efficiency, with a low rate of embryo production. In this context, the present study was conducted to verify the effect of Activin-A<br />
associated or not with FSH in the in vitro development of bovine preantral follicles. For this, secondary follicles (~0.2 mm) were mechanically<br />
isolated from ovaries (n = 20) and cultured in incubator with 5% CO2 in air at 39°C for 6 days in α-MEM (pH 7.2-7.4) supplemented with BSA<br />
(3.0 mg/mL), ITS (insulin 10 µg/mL, transferrin 5.5 µg/mL and selenium 5 ng/mL), glutamine (2 mM), hypoxanthine (2 mM), ascorbic acid (50<br />
µg/mL) under mineral oil. The treatment consisted of Activin-A (100 ng/mL), FSH (100 ng/mL) or the combination of FSH and Activin-A in<br />
the same concentrations. Data on follicular diameter were analyzed by Kruskal-Wallis test (P < 0.05). Throughout the culture period, no<br />
morphological signs of degeneration were observed. In all treatments, after 6 days of culture, a significant increase in follicular diameter (MEM:<br />
198.77±6.67 (P = 0.2828); Activin-A: 225.46±8.67 (P = 0.0052); Activin-A + FSH: 211.81±8.24 (P = 0.0203); FSH: 236.75±11.20 (P =<br />
0.0099), was observed when compared to day 0 (MEM: 182.64 ± 6.98; Activin-A: 187.17±11.85; Activin-A + FSH: 174.98±9.85; FSH:<br />
180.56±9.27). In addition, at the end of culture, Activin-A significantly increased follicular diameter (225.46±8.67) when compared with day 2<br />
(197.10±12.10). After 6 days, when comparisons among treatments were performed Activin-A + FSH (211.81±8.24) and FSH (236.75±11.20),<br />
significantly increased follicular diameter compared to MEM alone (198.77±6.67) (P = 0.0052). In conclusion, this study demonstrated that<br />
Activin-A and FSH promotes the growth of bovine secondary follicles in vitro.<br />
Keywords: follicle, activin-a, culture.<br />
N<br />
s361
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A051 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EFFECT OF LOW LEVEL LASER IRRADIA<br />
ADIATION ON MITOCHONDRIAL MEMBRANE POTENTIAL AND CYCLE CLE OF BOVINE<br />
CUMULLUS CELLS<br />
Carlos Alexandre Soares 1 , Kelly Annes 1 , Thiago Revers Dreyer 1 , Herculano Silva Martinho 2 , Samir Saldanha Nicolau 2 , Fernanda Maria Sevciuc 2 , Mayra Elena<br />
Assumpção 2 & Marcella Pecora Milazzotto 1<br />
1<br />
UNIVERSIDADE FEDERAL DO ABC, SANTO ANDRÉ, SP, BRAZIL. 2 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.<br />
Bovine in vitro embryo production aims to improve livestock desirable characteristics. However, the technique efficiency must<br />
be improved. An alternative to reach this goal may be the laser therapy. It works by using low level laser irradiation (LLLI) in biological<br />
samples to generate biomodulation; however its effect on female gametes is unknown. The objective of the study was to evaluate whether<br />
treatment with LLLI is able to promote changes in bovine cumullus cells in vitro cultured. Bovine ovaries were obtained from a local<br />
slaughterhouse. Follicles (2-8mm) were aspirated and the COCs were classified according to morphology. COCs (grades I and II) were<br />
irradiated with LLL (γ633nm) by using a dose of 1J/cm 2 . After that, they were IVM (TCM-199 media, 10% bovine fetal serum and hormones)<br />
in incubator at 38.5°C, 5% CO 2<br />
and high humidity. The control group received no irradiation. After 30 min (t30) or 24 h (t24) from the<br />
beginning of IVM, cumullus cells were mechanically removed from oocytes . After removal, cells were washed in PBS calcium and magnesium<br />
free and divided into two groups. The first group was incubated with 35uL of JC-1 (14.3ug/mL) for mitochondrial membrane potential<br />
evaluation. The second group was fixed in 70% ethanol and subsequently incubated with 50ug/mL of propidium iodide for the cell cycle<br />
evaluation. Data were collected by a flow cytometer Guava Easy Cyte Minib and graphs were evaluated by using Flojo software (v8. 7).<br />
Statistical analysis was performed using SAS system for Windows. Samples were classified as high or low mitochondrial membrane potential<br />
and G0/G1, S and G2/M of the cell cycle. There was no difference in mitochondrial membrane potential and cell cycle between between the<br />
control group and t30 (P = 0.633 and P = 0.86, respectively). However, after 24 h of IVM an increase of cells with high mitochondrial<br />
membrane potential was observed for the treated group when compared to control (P = 0.066). Regarding the cycle, there was an increase in<br />
the number of cells in both group S and G2/M. Moreover, there was a decrease in the number of cells in G0/G1 (P < 0.01). Based on these<br />
results, we concluded that the LLLI (γ633nm) with dose of 1J/cm 2 was able to induce changes in the metabolism of bovine cumullus cells<br />
cultured in vitro, which was responsible for an increased mitochondrial membrane potential and an increase of cells progressing in the cell cycle<br />
stages.<br />
Keywords: bovine, cumullus cells, low level laser.<br />
A052 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EFFECTS OF OVARIAN ORTOTOPIC OPIC AUT<br />
UTOTR<br />
TRANSPL<br />
ANSPLANT<br />
ANTATION TION IN REPRODUCTIVE BEHAVIOR IN GOATS (CAPR<br />
APRA A HIRCUS)<br />
Nayar<br />
ara a Almeida do Car<br />
armo<br />
mo, Samuel Amor<br />
morim Mar<br />
arcondes<br />
ondes, Fer<br />
ernanda Araujo dos San<br />
antos<br />
os, Gabr<br />
abriela Hémylin Fer<br />
erreir<br />
eira Mour<br />
oura,<br />
Talyta Lins Nunes<br />
unes, Valér<br />
aléria<br />
Ver<br />
eras de<br />
Paula,<br />
Raimundo Alv<br />
lves Bar<br />
arrêt<br />
rêto Junior<br />
unior, Michelly Fer<br />
ernandes de Mac<br />
acedo<br />
& Mar<br />
arcelo Barb<br />
arbosa Bezer<br />
erra<br />
UNIVERSIDADE FEDERAL RURAL DO SEMI-ÁRIDO, MOSSORO, RN, BRAZIL.<br />
Ovarian autotransplantation has been in use for wide goals related to reproduction in animals and humans. Once goats constitute<br />
experimental models for ovarian transplantation, this investigation aimed to evaluate the reproductive behavior of goats submitted to ortotopic<br />
ovarian autotransplantation. Thus, three crossbred goats were submitted to bilateral oophorectomy followed by autotransplantation of ovarian<br />
cortical fragments into infundibulum, ipsilateral to the donor ovaries. Fragments measuring approximately 3 x 1 x 1 mm were selected according<br />
to number and quality of preantral follicles and small antral follicles observed with the aid of an stereomicroscope (40X); after sedation, three<br />
fragments were transplanted into the infundibulum and sutured with nylon monofilament 6-0 with needle (black nylon, monofilament 6-0,<br />
Brasuture, São Sebastião da Grama, SP, Brazil). At an interval between 60 and 130 days post-transplantation, the reproductive behavior of goats<br />
was evaluated by the presence of a buck to observe the presumptive occurrence of estrus signs and possibility for mating. The results<br />
demonstrated that the first signs indicated similar behavior of proestrus that on average occurred at f 62 ± 3 days post-transplantation,<br />
accompanied by Flehmen reaction followed by courting, as well as mating attempts by the buck. This behavior was repeated at irregular intervals<br />
of 10 ± 6 days, ranging from 4 to 23 days, without mating acceptance by the goats. However, at 122 and 125 days post-transplantation, mating<br />
was observed in two goats, with one of them demonstrating persistent tail twitching and marked responsiveness to the presence of the buck prior<br />
to mating. This experiment demonstrated that ovarian tissues transplanted into the infundibulum underwent resumption of ovarian activity, but<br />
it is still reasonable to verify what variables may influences the estrus signs. More results are in progress.<br />
Keywords: autotransplantation, goat, fertility.<br />
s362
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A053 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EFFECT OF ECG ON OVULATION AND CONCEPTION RATES IN RED SINDHI COWS (<br />
(BOS<br />
TAUR<br />
URUS<br />
US INDICUS) TREATED<br />
TED WITH<br />
TWO SYNCHRONIZA<br />
ONIZATION OF OVULATION PROTOC<br />
OCOLS<br />
OLS<br />
Raquel Rodr<br />
drigues Costa Mello<br />
ello, Joaquim Esquer<br />
squerdo Fer<br />
erreir<br />
eira,<br />
Ana Paula<br />
Toledo Barb<br />
arbosa Silv<br />
ilva,<br />
Leandr<br />
eandro Mendes Masc<br />
ascar<br />
arenhas<br />
enhas, Ber<br />
ernar<br />
nardo Janella Fer<br />
erreir<br />
eira Silv<br />
ilva,<br />
Beatriz Oliveira Cardoso, Helcimar Barbosa Palhano & Marco Roberto Bourg Mello<br />
UFRRJ, SEROPEDICA, RJ, BRAZIL.<br />
The Red Sindhi zebu breed has excellent adaptability to adverse weather conditions and good management and ability to milk<br />
production. Although much research will be developed with these animals, studies of the reproductive traits are still scarce. Therefore, the<br />
objective of this study was to evaluate the effect of eCG on ovulation and conception rates of Red Sindhi cows during a treatment for<br />
synchronization of ovulation. Sixteen Red Sindhi cows with body condition score = 3.5 (scale of 1 to 5) were divided into two protocols for<br />
synchronization of ovulation. In protocol I, animals (n = 8) received on day 0 an intravaginal P4 device and an application of 2mg of estradiol<br />
benzoate (EB). On day 8, the device was removed and 10mg of Dinoprost (PGF2α) were applied, and on day 9, 1 mg of EB was applied, and<br />
the animals were fixed-time inseminated 24 h later; in protocol II, animals (n = 8) were submitted to the same treatment, and on day 8, 400 IU<br />
of eCG were applied. From day 0, all animals were evaluated daily by trans-rectal ultrasound device in order to monitor the follicular growth. The<br />
results were analyzed by ÷2 test, with significance level of 5%. It was observed that there was no statistical difference (P > 0.05) between<br />
protocols I and II concerning to ovulation rate, with 75% (6/8) and 100% (8/8), respectively. Regarding to conception rate, it was observed that<br />
there was statistical difference (P > 0.05) between protocols I and II (0% and 50%, respectively). The final conception rate in this study was low<br />
(25%), and this fact may be related to the high age of cows (average of 10 years old) and the long period in which they were no pregnant since<br />
they were being used as embryo donors. However, it can not conclude that treatment with eCG was efficient on the ovulation rate and conception<br />
rate in cows of Red Sindhi breed, once those are preliminary results, more research is needed on the reproductive physiology with a greater<br />
number of animals of this breed.<br />
Keywords: follicular growth, zebu, ftai.<br />
A054 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
TIMING EFFECT OF INSEMINATION USING SEX-SORTED SPERM IN EMBRYO PRODUCTION<br />
WITH NELORE (<br />
(BOS<br />
INDICUS) ) SUPEROVUL<br />
OVULATED DONORS<br />
Júlia Gle<br />
ley ci Soar<br />
oares<br />
1 , Nelcio Ant onio Tonizza Car<br />
arv alho 2 , Pietr<br />
ietro Sampaio Bar<br />
aruselli<br />
3 , Claudine<br />
laudiney Melo Mar<br />
artins<br />
4 , Mar<br />
arcílio Nichi 5 ,<br />
Manoel Francisco Sá Filho 6 , José Ricardo Garla Maio 7 , Ana Lúcia Abreu Silva 8 & Alessandra Corallo Nicacio 9<br />
1,3,5,6<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL, FMVZ-VRA-USP, SAO PAULO, SP, BRAZIL. 2 UNIDADE DE PESQUISA E DESENVOLVIMENTO DE REGISTRO/PÓLO REGIONAL DO<br />
D.S.A. DO VALE DO RIBEIRA/APTA, REGISTRO, SP, BRAZIL. 4 FERTILIZA CONSULTORIA ANIMAL, POÇOS DE CALDAS, MG, BRAZIL. 7 OURO FINO SAÚDE ANIMAL, RIBEIRÃO PRETO, SP,<br />
BRAZIL. 8 MESTRADO EM CIÊNCIA ANIMAL, UNIVERSIDADE ESTADUAL DO MARANHÃO (CCA/UEMA), SÃO LUÍS, MA, BRAZIL. 9 UNIVERSIDADE FEDERAL DO PAMPA, SÃO<br />
GABRIEL, RS, BRAZIL.<br />
The effect of the moment of TAI with sex-sorted sperm in embryo production of Nellore (Bos indicus) superovulated donors was<br />
evaluated. To this aim, 16 cycling donors were used, aged between 4 and 10 years, and postpartum around 40 days. The animals were sorted<br />
evenly into four groups, according to the moment of TAI (12 and 24 vs.. 18 and 30h after administration of pLH) and type of sperm used<br />
(conventional vs.. sex-sorted). The groups were sorted as follows: TAI with conventional sperm 12 and 24h (CSG12/24) and, 18 and 30h<br />
(CSG18/30); TAI with sex-sorted sperm 12 and 24h (SSG12/24) and, 18 and 30h (SSG18/30). The design was completely randomized, in a<br />
2x2 factorial scheme, with all the animals in their respective categories, submitted to all treatments (randomized cross-over) in a total of four<br />
replicates, with an interval of 42 days between replicates and 16 repetitions per treatment. Donors were synchronized with intravaginal<br />
progesterone device (P4) for 7 days. On the day of device insertion (D0) 2.0mg i.m. of Estradiol Benzoate were administered. From D4 to D7,<br />
all donors were treated with 8 decreasing doses of FSH (133mg i.m.; 12/12h). On D6, all animals received two PGF2α injections (0.53mg i.m.)<br />
after each FSH injection. At the last FSH dose (D7) the P4 device was removed and, donors received the pLH injection 12h later (25mg i.m.;<br />
D8). The embryonic structures were collected seven days after LHp injection (D15) and, embryos were quantified and classified according to<br />
morphology and quality (IETS, 1998). The variables were analyzed by Proc GLM procedure of SAS ® . In groups GSC12/24, GSC18/30,<br />
GSS12/24 and GSS18/30 were recovered, respectively: total of embryonic structures (2.9±1.1, 3.4±0.8, 2.6±1.2, 4.1±1.3); unfertilized oocytes<br />
(0.0±0.0, 0.1±0.1, 1.7±1.2, 0.4±0.2); transferable embryos (2.6±1.0, 2.7±0.7, 0.5±0.3, 2.2±1.0); freezable (2.4±1.0, 2.7±0.7, 0.5±0.3, 2.0±1.0)<br />
and degenerate (0.3±0.1, 0.7±0.4, 0.4±0.3, 1.5±0.8). There were no differences between groups (P > 0.05). As there was no interaction between<br />
treatments, it was possible to evaluate the main effects (type of sperm type and moment of insemination). As verified between groups, no<br />
difference was found for any of the analyzed variables between the main effects (P > 0.05). The results did not confirm the hypothesis that a delay<br />
in six hours on the FTAI with sex-sorted sperm in superovulation protocols results in increased production of viable embryos obtained from<br />
Nelore donors.<br />
Keywords: nelore, sex-sorted sperm, fixed-time superovulation.<br />
N<br />
s363
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A055 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EFFICIENCY OF DIFFERENT TREATMENT<br />
MENTS OF OVARIAN CYST<br />
STS S IN DAIRY COWS<br />
Tochimar<br />
chimara a Apar<br />
parecida Miy<br />
iyauchi<br />
1 , Car<br />
arlos Antônio de Car<br />
arvalho alho Fer<br />
ernandes<br />
1 , Mar<br />
arina Resende Pimen<br />
imenta Por<br />
ortinar<br />
tinari 1 , Mar<br />
arilu Mar<br />
artins Gioso<br />
1 , Bruno Fer<br />
ernandes<br />
Ludgero Alves 2 & Ana Cristina Silva de Figueiredo 1<br />
1<br />
UNIFENAS, ALFENAS, MG, BRAZIL. 2 BIOTRAN, ALFENAS, MG, BRAZIL.<br />
The incidence of the ovarian follicular cyst is close related to reproductive failure in dairy cattle. Effective treatment of this disease<br />
promotes the regression of this structure, the development of the corpus luteum and hastens the return of the normal ovarian cyclic activity. The<br />
benedict effect of the association between prostaglandin analogous and GnRH on treatment of the luteinized cystic structures has been reported.<br />
The study was designed to evaluate the Fertirelin acetate associated or not with cloprostenol on ovarian cyst treatments in dairy cattle. Hundredthirty-six<br />
Holstein cows with follicular cyst provided from four different were included for this purpose. Follicular cysts were detected by linear<br />
array 7.0 MHz probe (Esaote-Falco) by transrectal examination of the ovarian. Circular anechoic structures larger than 20 mm of the diameter<br />
without detecting luteal tissue were considered as follicular cysts. The animals were randomized in one of the five groups according to the drug<br />
injection: 2 mL of saline (Group 1; n = 16); 0.1mg of the Fertirelin acetate - Fertigen ® Schering Plough-Brazil (Group 2; n = 31); 0.1mg of the<br />
Fertirelin acetate and 0.530 mg of the Sodium Cloprostenol 10 days later - Ciosin ® Schering Plough-Brazil (Group 3; n = 28); 0.1mg of the<br />
Fertirelin acetate and 0.530 mg of the Sodium Cloprostenol at the same time (Group 4; n = 29); and 0.1mg of the Fertirelin acetate and two doses<br />
of the Sodium Cloprostenol, the first at the same time of the Fertirelin and the second 10 days later (Group 5; n = 32). All treated cows were<br />
reexamined by transrectal ultrasonography between 20 and 30 days after treatment. The therapy was considered efficient when the cystic<br />
structure was not present and the luteal tissue was detected at the second ultrasound examination. The percentage of recover after treatment was<br />
18.8c, 54.8b, 53.5b, 79.3a, 81.2%a for groups 1 to 5, respectively. The interval from treatment to first artificial insemination (AI) was<br />
61.2±17.9a, 44.5±16.4b, 30.9±12.6c, 26.2±14.2c, 18.3±10.2d days for groups 1 to 5, respectively. There was no effect of the farm and the<br />
number of AI per conception was similar among treatments. The interpretation of these results is that the association between Fertirelin and<br />
cloprostenol in two different protocols was efficient to promote luteolysis and to hasten the return to the reproductive activity after treatment.<br />
[Supported by Fapemig].<br />
Keywords: bovine, ovarian cysts, reproductive efficience.<br />
A056 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
STABILIT<br />
ABILITY OF REFERENCE GENES AND LEVELS OF MRNA FOR THE IGF SYSTEM STEM IN BOVINE OVARIAN<br />
FOLLICLES GROWN IN VITRO AND IN VIVO<br />
Gisvani Lopes de Vasconcelos, Anderson Weiny Barbalho Silva, José Jackson do Nascimento Costa, Maria Juliane Passos, Rodrigo<br />
Otávio Decaria de Salles Rossi, Emanuela de Lima Rebouças & José Roberto Viana Silva<br />
UNIVERSIDADE FEDERAL DO CEARÁ - UFC, SOBRAL, CE, BRAZIL.<br />
It is important to know the profile of gene expression, as well as suitable reference genes for normalization of data from real-time<br />
PCR (qRT-PCR) as internal controls. However, the expression of mRNA for the IGF system in secondary and early antral follicles in the bovine<br />
is not yet known. The aim of this study was to investigate the stability of seven reference genes, and the relative expression of IGF-I and II, the<br />
receptors of IGF-I and II (IGFR-I and-II) and IGF proteins (IGFBPs 1- 6) in bovine follicles before and after culture in vitro. In order to evaluate<br />
the stability of reference genes and the expression levels of IGF system, follicles with ~ 0.2 (n = 30), ~ 0.5 (n = 30) and ~ 1.0 mm (n = 30) were<br />
micro-dissected and then subjected to total RNA extraction and cDNA synthesis. GeNorm software was used to evaluate the stability of<br />
Glyceraldehyde-3 phosphatedehydrogenase (GAPDH), β-tubulin, β-actin, Fosfogliceroquinase, 18S rRNA, Ubiquitin(UBQ) and ribosomal<br />
protein-19 (RPL-19). To compare the levels of mRNA of IGF system before and after in vitro culture, secondary follicles of ~ 0.2 mm (n = 24)<br />
were isolated and cultured for 12 days to reach ~ 0.5 mm. These follicles were subjected to total RNA extraction and cDNA synthesis. The deltadelta<br />
CT method was used to normalize the data of mRNA expression. The nonparametric Kruskal-Wallis test was used to analyze mRNA levels<br />
for components of the IGF system (P < 0.05). The results demonstrated that GAPDH and UBQ were the most stable reference genes in preantral<br />
and antral bovine follicles. The levels of mRNA for IGFR-I and IGFR-II in follicles of 0.5 mm were significantly higher than in follicles of 0.2<br />
and 1.0 mm. A reduction in the levels of IGFBP-1 was observed during growth of the follicles. The levels of IGFBP-2 in follicles of 0.5 mm<br />
were significantly higher than in follicles of 1.0 mm. In contrast, 1.0 mm follicles showed higher levels of IGFBP-3 and IGFBP-4 than follicles<br />
of 0.2 and 0.5 mm. After in vitro culture, we observed significant reductions in levels of IGF-I, IGFR-I, IGFBP-3, -5 and -6 compared with noncultured<br />
follicles with ~ 0.2 mm. Comparing the 0.5 mm follicles grown in vitro with in vivo, the expression of IGFBP-1, -2, -3 e -6 were<br />
significantly lower in follicles cultured. Thus, we conclude that the UBQ and GAPDH are the most stable reference genes in bovine follicles with<br />
0.2, 0.5 and 1.0 mm, while the IGF-I and II, their receptors (IGFR-I and IGFR-II) and IGFBPs 1-6 show variable expression in follicular<br />
categories analyzed.<br />
Keywords: igf, bovine, mrna.<br />
s364
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A057 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EXPRESSION OF MRNA ENCODING FGFR1B AND FGFR2B IN BOVINE FETAL OVARIES DURING GESTATION<br />
TION<br />
Rubia Bueno Da Silva 1 , Ester Siqueira Caixeta 2 , Christopher Price 3 & José Buratini Junior 4<br />
1,2,4<br />
UNESP, BOTUCATU, SP, BRAZIL. 3 UNIVERSITÉ DE MONTREAL, MONTREAL, CANADÁ.<br />
Recent findings suggest the involvement of fibroblast growth factor 10 (FGF-10) in regulation of preantral follicle development.<br />
During gestational ovarian development, expression of RNAm FGF-10 is progressively upregulated with a significant increase at 120 days of<br />
gestation, coincident with the increase in primary follicles number (Castilho, data not published). FGF-10 signaling requires activation of FGFs<br />
receptors 1B or 2B (FGFR1B, FGFR2B). In the present study, we assessed mRNA expression patterns of FGFR1B and FGFR2B in bovine<br />
fetal ovaries during gestation. Bovine fetal ovaries were obtained in a local slaughterhouse, fetal age was estimated by the crown-rump length and<br />
samples were grouped according with days of gestation as follows: 60 (n = 7), 75 (n = 7), 90 (n = 7), 120 (n = 7), 150 (n = 7) and 210 (n = 7).<br />
Expression of mRNA encoding FGFR1B and FGFR2B was determined by semi-quantitative real time RT-PCR using bovine specific primers,<br />
and cyclophilin A as the endogenous control. Reverse transcription was performed with SuperScriptIII (Invitrogen, São Paulo, Brazil) and<br />
PCR with Power SYBR green master mix (Applied Biosystems, Foster City, USA) in an ABI Prism ® 7500. Relative expression values were<br />
determined by the Pfaffl equation, analyzed by ANOVA and groups were compared by orthogonal contrast test (JMP software). Expression of<br />
mRNA encoding FGFR1B and FGFR2B was detected throughout gestation. mRNA abundance of FGFR1B increased from 90 to 120 days of<br />
gestation, when we previously observed an increase in FGF-10 expression and in the number of primary follicles. In contrast, FGFR2B mRNA<br />
levels decreased from 60 to 90 days of gestation, around the time primordial follicle activation begins. In conclusion, both receptors of FGF-10<br />
were shown to be developmentally regulated during gestation. The temporal association between gene expression patterns and fetal follicular<br />
dynamics suggests that FGFR1B and FGFR2B signalling may be involved in the control of follicle activation.<br />
Keywords: fgfr1b, fgfr2b, bovine.<br />
A058 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
LEUKEMIA INHIBITOR<br />
ORY FACT<br />
CTOR STIMULATES<br />
TES THE IN VITRO DEVEL<br />
VELOPMENT OF SHEEP PREANTRAL AL FOLLICLES<br />
AND THE PRODUCTION OF EMBRYOS<br />
Valesca Barreto Luz 1 , Ticiana Franco Pereira da Silva 1 , Valdevane Rocha Araújo 1 , Ana Beatriz Graça Duarte 1 , Juliana Jales de<br />
Hollanda Celestino 2 , Deborah de Melo Magalhães Padilha 1 , Roberta Nogueira Chaves 1 , Ivina Rocha Brito 1 , Anderson Pinto<br />
Almeida 1 , Cristiano Feltrin 3 , Marcelo Bertolini 3 , Gerlane Modesto da Silva 1 & José Ricardo de Figueiredo 1<br />
1<br />
UECE, FORTALEZA, CE, BRAZIL. 2 UNILAB, REDENÇÃO, CE, BRAZIL. 3 UNIFOR, FORTALEZA, CE, BRAZIL.<br />
The aim of this study was to investigate the effects of leukemia inhibitory factor (LIF), with or without FSH on the development of<br />
sheep preantral follicles (PAF) and the influence embryo production. Ovaries of sheeps were dissected, and follicles (> 200 µm) were selected<br />
and cultured individually in drops of 100 µL of base medium, that consisted of α-MEM supplemented with 3 mg/mL of BSA, ITS (insulin 10<br />
µg/mL, transferrin 5.5 µg/mL and selenium 5 ng/mL), 2 mM of glutamine, 2 mM of hypoxantine, 50 µg/mL of ascorbic acid and LIF (50 ng/<br />
mL) in the presence or absence of sequential rFSH (day 0-6: 100 ng/mL, days 6-12: 500 ng/mL and days 12-18: 1000 ng/mL). In vitro culture<br />
(IVC) was performed at 39°C and 5% CO2 for 18 days, and all medium was replenished every six days. At the end of the IVC period, all healthy<br />
follicles were carefully opened and oocytes (>110 µm) with a homogeneous cytoplasm and surrounded by at least one compact layer of cumulus<br />
cells were selected for in vitro maturation (IVM). The selected cumulus-oocyte complexes were washed three times in maturation medium:<br />
TCM199B (TCM199 + sodium bicarbonate) supplemented with BSA (1 mg/mL), pyruvate (1 mM/mL), rFSH (0.5 µg/mL), LH (5 µg/mL),<br />
17β-estradiol (1 ug/mL), EGF (10 ng/mL), IGF-1 (50 ng/mL) and cysteamine (100 µM/mL). After washing, the oocytes were transferred to 50<br />
µL drops of maturation medium under mineral oil and then incubated for 40 h at 39ºC in 5% CO2. Mature oocytes from treatment with the highest<br />
rate of MII were activated by exposure to 5 µM ionomycin in TCM-HEPES supplemented with 0.4% BSA for 5 min followed by 3.5 h<br />
incubation in 2 mM 6-DMAP in SOFaa + 0.4% BSA at 39°C. Then, activated embryos were cultured in SOFaa + 10% SFB for 5 days.<br />
Cleavage and embryo rates were determined on days 2 and 5, respectively (Day 0: embryo activation). The results were compared by Chi-square<br />
test and were expressed as percentages. There were no significant differences between treatments regarding oocytes for IVM (= 110 µm) and<br />
meiosis resumption. In the group of follicles cultured with LIF in the absence of FSH showed a rate of MII of 29.63%, however in the presence<br />
of FSH this rate was of 16.66%. After parthenogenesis, which was only performed in treatment without FSH, were obtained 7 embryos (8 cels)<br />
from 12 mature oocytes. We concluded that the LIF associated or not to FSH stimulates the in vitro development of sheep PAF. Furthermore,<br />
embryos can be obtained from PAF cultured in the presence of LIF.<br />
Keywords: leukemia inhibitory factor, follicule, embryo.<br />
N<br />
s365
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A059 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EFFECTS OF FSH AND GROWTH DIFFERENTIATION TION FACT<br />
CTOR -9 (GDF-9) ON IN VITRO GROWTH OF BOVINE SECONDAR<br />
ONDARY<br />
FOLLICLES<br />
Regislane Pin<br />
into Rib<br />
ibeir<br />
eiro, Gisv<br />
isvani Lop<br />
opes de Vasc<br />
asconc<br />
oncelos<br />
elos, Anderson<br />
Wein<br />
einy Barbalho Silv<br />
ilva,<br />
Mar<br />
aria Juliane Passos<br />
assos, Francisc<br />
ancisco Taia Gomes Bezer<br />
erra,<br />
Katianne<br />
Freitas dos Santos, Emanuela de Lima Rebouças, Rodrigo Otávio Decaria de Salles Rossi, Ellen da Cunha de Vasconcelos & José Roberto Viana Silva<br />
UNIVERSIDADE FEDERAL DO CEARÁ, SOBRAL, CE, BRAZIL.<br />
Recently, isolation and culture of caprine secondary follicles of ~200µm has been carried out in order to produce oocytes capable<br />
of maturation (Magalhães et al., Reprod. Domest. Anim., 46:134-140, 2010). However, the effect of GDF-9 during the culture of bovine<br />
secondary follicles has not been evaluated yet. This study aimed to determine the effects of GDF-9 in the presence or absence of FSH on survival,<br />
growth and antrum formation in bovine preantral follicles cultured for 12 days. Secondary follicles were isolated and cultured, for 12 days,<br />
individually in α-MEM (control) or in α-MEM supplemented with GDF-9 (200 ng/mL), FSH (100 ng/mL from day 0 to 6, 500 ng/mL from<br />
day 6 to day 12) or GDF-9 associated with FSH. Each treatment was repeated four times, using around 24 follicles. The follicular survival was<br />
calculated according to the morphology of oocyte and granulosa cells. During culture, follicular diameter and antral cavity formation were<br />
evaluated. Data of follicular growth were compared by Kruskal-Walls test, while the percentage of viable follicles or follicles that formed an<br />
antrum were compared by chi-square test (P < 0.05). Based on the analysis, the treatments did not affect the percentage of surviving follicles after<br />
culture for 12 days. Compared to D0, during the culture of follicles with MEM alone or together with GDF-9, GDF-9+FSH or FSH there was<br />
a significant increase in follicular diameter on day 12, 8, 6 and 6, respectively. After 8 days of culture, GDF-9 (219.32 ± 15.21), GDF-9 + FSH<br />
(233.53 ± 10.57) and FSH (251.69 ± 15.49) significantly increased follicular diameter when compared to MEM (184.68 ± 9.74). Similar results<br />
were observed on day 10 (MEM: 193.78 ± 8.90; GDF-9: 232.25 ± 10.48; GDF-9 + FSH: 257.76 ± 17.18; FSH: 288.70 ± 16.09) and 12<br />
(MEM: 204.73 ± 8.93; GDF-9: 245.50 ± 13.38; GDF-9 + FSH: 266.02 ± 17.70; FSH: 307.80 ± 19 13). On day 12, FSH (70.83%) and GDF-<br />
9 (65%) significantly increased the rate of antrum formation when compared with control (31.81%) and GDF-9+FSH (43.82%). In conclusion,<br />
this study showed that FSH and GDF-9 promotes the growth and antrum formation of bovine secondary follicles in vitro.<br />
Keywords: gdf-9, follicles, culture.<br />
A060 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
INFLUENCE OF PROTOC<br />
OCOLS OLS FOR SUPEROVUL<br />
OVULATION ON INDUCED STRESS OF BROWN BROCKET DEER<br />
(MAZAMA GOUAZOUBIRA)<br />
Eveline Santos Zanetti<br />
& José Mauricio Barbanti Duarte<br />
FCAV - UNESP, JABOTICABAL, SP, BRAZIL.<br />
The knowledge of assisted reproduction techniques for wild animals should be useful for future in situ and ex situ conservation<br />
programs. However, stress is a limiting factor in assisted reproduction in wild animals maintained in captivity. The aims of the present study<br />
were: (1) to evaluate the influence of two different protocols designed to cause superovulation on induced stress of brown brocket deer, and (2)<br />
to evaluate the ovulation rate and the presence of functional corpora lutea after treatment by these two different protocols. For this, six hinds were<br />
assorted into 2 groups (n = 3): the first received CIDRÒ for 8 days, followed by 0.25mg i.m. injection of OB on D-8, 700IU of eCG on D-4<br />
and 265µg of PGF2α on D0 (Treatment 1) and the second received CIDRÒ for 7.5 days followed by 0.25mg i.m. injection of OB on D-7.5,<br />
130mg of FSH divided into eight equal doses [beginning on D-3 and ending D-0.5] and 265µg of PGF2α on D0 (Treatment 2). The treatments<br />
were crossed over with 44-48 day intervals after CIDRÒ removal (all the animals went through all 2 Treatments). Analysis was conducted using<br />
MINITAB ® Release 14 Software (P < 0.05). The induced stress (M1 – two days before the beginning of the treatments; M2 – all the days during<br />
the treatments; M3 – the last four days of the treatments and M4 – five days after the end of the treatments) and the treatment efficacy was<br />
evaluated by observation of CL and unruptured follicles via laparoscopy 7 days after the first copulation and analyze of fecal progestin<br />
concentration. There was no significant difference for fecal cortisol concentration (ng/g feces) in four different moments between the treatments;<br />
however M1 was significantly different from M3 within each Treatment ((T1): M1 = 98.52±8.99 vs.. M3 = 170.34±22.98 and (T2): M1 =<br />
91.40±10.93 vs. M3 = 190.54±31.82). The mean ovulation rate was higher (P = 0.05) in Treatment 1 (6.0±1.7) than in Treatment 2 (2.0±0.3),<br />
but the total ovarian stimulation (13.2±2.6 and 10.4±1.1, in 1 and 2, respectively) was not significantly different between treatments. Moreover,<br />
the mean fecal progestin concentration at the luteal phase in Treatment 1 (4293.69±769.47ng/g feces) was significantly different from Treatment<br />
2 (1571.26±240.28 ng/g feces). Thus, regardless of the treatment, the influence of the induced stress should be considered in the superovulation<br />
protocols and, in this case, the eCG seems to promote a better gonadotrophic stimulation to overcome its effects, since Treatment 1 induced better<br />
superovulation response in the species Mazama gouazoubira,with greatest mean ovulation rates and functional corpora lutea formation.<br />
Keywords: superovulation, stress, neotropical deer.<br />
s366
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A061 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
STEADY-ST<br />
Y-STATE TE LEVELS OF VASO<br />
ASOACTIVE INTESTINAL PEPTIDE MRNA IN GOAT OVARIES AND ITS EFFECT ON THE IN VITRO<br />
DEVEL<br />
VELOPMENT OF ISOLATED PREANTRAL AL FOLLICLES<br />
Jamily Bezerra Bruno 1 Valdevane Rocha Araújo 1 , Juliana Jales de Hollanda Celestino 2 , Márcia Viviane Alves Saraiva 1 , Rebeca Magalhães Pedrosa Rocha 1 ,<br />
,<br />
Ivina Rocha Brit<br />
ito 1 , Ana Bea<br />
eatr<br />
triz Graça Duar<br />
uarte 1 , Cleidson Mano<br />
anoel Gomes Silv<br />
ilva 1 , Isador<br />
sadora a Machado<br />
Teix<br />
eixeir<br />
eira Lima 1 , Mar<br />
aria Helena<br />
Tavar<br />
ares Matos<br />
3 & José Ric<br />
icar<br />
ardo<br />
de Figueiredo 1<br />
1<br />
UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE DA INTEGRAÇÃO INTERNACIONAL DA LUSOFONIA AFRO-BRASILEIRA, REDENÇÃO, CE, BRAZIL.<br />
3<br />
UNIVERSIDADE FEDERAL DO VALE DO SÃO FRANCISCO, PETROLINA, PE, BRAZIL.<br />
The aims of this study were to evaluate the levels of vasoactive intestinal peptide (VIP) mRNA in caprine ovaries, the effects of VIP<br />
and/or follicle-stimulating hormone (FSH) on follicular development and mRNA levels of VIP, and FSH receptor (FSHR) after in vitro culture<br />
of advanced secondary follicles for 6 days. All samples were maintained in á-minimum essential medium (α-MEM+) or in α-MEM+<br />
supplemented with FSH (100 ng/mL) and/or VIP (10 ng/mL). For expression analysis, caprine primordial, primary and secondary follicles<br />
were obtained. Cumulus-oocyte complexes (COCs), and clusters of granulosa/theca cells of small (3mm) antral goat<br />
follicles were also recovered. VIP and FSHR mRNA levels were quantified by real-time reverse transcription through the polymerase chain<br />
reaction (qRT-PCR). Glyceraldehyde-2-phosphate dehydrogenase (GAPDH) and β-actin were used as endogenous controls for fresh ovaries<br />
and for the culture follicles were used β-actin and ubiquitin (UBQ). The delta-delta-CT method was used to transform CT values into<br />
normalized relative steady-state mRNA levels. The survival, antrum formation, and follicular diameter were evaluated every 2 day. In each<br />
parameter analyzed was employed the most appropriate statistical test according to the behavior of data. (P < 0.05). qRT-PCR demonstrated<br />
that levels of VIP mRNA in secondary follicles were significantly higher than in primordial follicle. In addition, no significant difference was<br />
observed between COCs collected from small and large antral follicles. Granulosa/theca cells from large antral follicles had higher levels of VIP<br />
mRNA than small antral follicles. The addition of VIP and/or FSH to the culture medium had no effect on follicular development. However,<br />
the presence these substances significantly reduced VIP mRNA levels, but did not alter FSHR mRNA levels. Conclusions included that the<br />
VIP mRNA was detected in all goat follicular categories and cellular types, VIP and/or FSH did not affect the development of secondary<br />
follicles, and the expression of VIP mRNA levels after in vitro culture was reduced.<br />
Keywords: vip, fsh, expression.<br />
A062 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
STANDARD OF EMERGENCY FOLLICULAR<br />
WAVES IN LONG PROTOC<br />
OCOLS OLS (WITH OR WITHOUT CIDR REPLACEMENT<br />
CEMENT) ) AND<br />
SEASONALITY EFFECT IN SANTA A INES EWES<br />
Mar<br />
aria Emilia Franc<br />
anco Oliv<br />
liveir<br />
eira 1 , Car<br />
arla Cristina D’ama<br />
’amato 1 , Henderson Ayr<br />
yres<br />
2 , Luís Guilher<br />
uilherme Oliv<br />
liveir<br />
eira 1 , Pedr<br />
edro Paulo Maia<br />
Teix<br />
eixeir<br />
eira 1 , Jef<br />
eferson erson Fer<br />
erreir<br />
eira Fonsec<br />
onseca 3 &<br />
Wilter Ricardo Russiano Vicente 1<br />
1<br />
UNESP-FCAV, JABOTICABAL, SP, BRAZIL. 2 USP-FMVZ, SÃO PAULO, SP, BRAZIL. 3 EMBRAPA-CNPC, SOBRAL, CE, BRAZIL.<br />
N<br />
Superovulatory protocols in sheep show high variability of responses and, usually, not based on the follicular development. The<br />
present study evaluated the follicular dynamic in basis superovulation protocols in order to determine the pattern of follicular wave emergency.<br />
Season effect on variables was also studied. Seventy adult ewes were submitted to one of two synchronization protocols in three seasons<br />
(Factorial 2x3; Non-breeding: G-1CIDR, n = 12 and G-2CIDR, n=11; Transition: G-1CIDR, n = 12 and G-2CIDR, n=12; Breeding: G-<br />
1CIDR, n = 11 and G-2CIDR, n = 12). On D0 (randomized day of estrus cycle), estrus were synchronized with a P4 device (CIDR; Pfizer-<br />
Netherlands) for 14 days. However, in G-2CIDR, the CIDR was replaced by a new one on D7. At D0 and 14, 2.5 mg of dinoprost (Lutalyse,<br />
Pfizer-Netherlands), i.m., were administered in all ewes. Ovaries ultrasonographic exam (Aquila - Pie Medical, Netherlands) was performed<br />
daily by transrectal during treatments. Follicular wave was identified as a group of follicles (2-3 mm) that grew, being that one or more reached<br />
the minimum diameter of 4.5 mm. Emergence follicular wave day was considered when the largest follicle, still with 2-3 mm, was first identified.<br />
Data were analyzed by GLIMMIX using the SAS. A total of 31.43% of ewes had corpus luteum at the beginning of protocols. Experimental<br />
ewes presented two to five follicular waves emergencies during treatments (2 waves: 4.29%; 3 waves: 34.29%; 4 waves: 52.86% and; 5 waves:<br />
8.57% of ewes). There were no effect of treatment on emergency waves days (Wave 1: 2.05±0.42 vs.. 2.02±0.37; Wave 2: 5.69±0.42 vs..<br />
5.65±0.37; Wave 3: 9.77±0.42 vs.. 10.09±0.37; Wave 4: 11.85±0.39 vs.. 12.12±0.35 and; Wave 5: 12.5±0.40 vs.. 12.16±0.78 days for G-<br />
1CIDR and G-2CIDR respectively; P > 0.05). Similarly, there were no effect of seasons on these variables (Non-breeding: 2.01±0.46,<br />
5.11±0.47, 9.33±0.45, 12.27±0.40 and 13.50±0.40; Transition: 2.12±0.51, 5.95±0.52, 10.32±0.51, 11.61±0.50 and 11.16±0.78 and; Breeding:<br />
1.99±0.42, 5.95±0.42, 10.14±0.42 and 12.08±0.43 days for waves 1, 2, 3, 4 and 5, respectively, P > 0.05). In Breeding season no ewes showed<br />
five waves. Although these protocols are not developed to directly induce the follicular wave emergency, there was a pattern of emergency during<br />
both treatments, regardless of season. Furthermore, these informations will be important for the definition of new protocols (i.e. superovulation<br />
or synchronization treatments); which effectively based on ovarian activity and avoid deleterious effects.<br />
Keywords: follicular dynamic, superovulation, ovine.<br />
s367
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A063 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
BEHAVIOR<br />
VIORAL PAR<br />
ARAMETERS AMETERS OF DAIRY GOATS SUBJECTED TO SYNCHRONIZA<br />
ONIZATION<br />
WITH PGF2α<br />
Andressa Ferreira da Silva 1 , Luciana Vieira Esteves 1 , Felipe Zandonadi 1 , Renata do Carmo Cruz 2 & Jeferson Ferreira Fonseca 3<br />
1<br />
UFF, NITEROI, RJ, BRAZIL. 2 AUTONOMA, VIÇOSA, MG, BRAZIL. 3 EMBRAPA CAPRINOS E OVINOS, SOBRAL, CE, BRAZIL.<br />
Prostaglandin f2α has being used in the reproduction of small ruminants since it was identified as a luteolytic factor in the estrous<br />
cycle in sheep (Mc Cracken et al., 1984, Anim Reprod Sci 7, 31-55). Recently, the use of PGF2α has become more important as a result of<br />
restrictions on use of P4 in animal production in the United States and Europe (Menchaca e Rubianes, 2004, Reprod Fert Dev,16, 403-413).The<br />
aim of this study was to evaluate the effectiveness of estrus synchronization on the behavioral parameters of dairy goats. For synchronization of<br />
estrus, females (n = 29) during the breeding season (March and April) received two doses of d-cloprostenol (0,03 mg - Veteglan ® , Laboratories<br />
Calier, Barcelona, Espanha) intervals of 10 days. From the application of PGF2α the sexual behavior was evaluated every 12 h, with the use of<br />
males and the acceptance by the male mounts the confirmation of the signal beginning of estrus. The percentage of animals in estrus after the first<br />
dose of PGF2α (D0) and second (D10) after 36 h was 68.2% (15/22) and 91.3% (21/23) respectively, while that during the 48 h was 31.8%<br />
(7 / 22) and 8.7% (2 / 23). With regard to the percentage of estrus according to the time of event (diurnal and nocturnal) on D0, observed that<br />
diurnal and nocturnal estrus night had equal rates of 50.0% (11/22). The end of estrus was characterized by 63.6% (14/22) occurring during the<br />
daytime and 36.4% (8/22) at the end of estrus at night. In D10 at the beginning of diurnal estrus obtained the highest percentage (91.3% - 21/23),<br />
whereas at the beginning of estrus at night the lowest (8.7% - 2/23). The end of diurnal estrus, showed a rate of 34.8%, with most ending of estrus<br />
at night (65.2% - 15/23).The interval of application of PGF2α (D0) until the beginning of estrus (h) was 75.8 ± 53.9, while the duration of estrus<br />
35.4 ± 15.9. Since the interval of PGF2α (D10) at the beginning of estrus (h) was 47.7 ± 10.1 and duration of estrus was 26.8 ± 15.0. Concluded<br />
that the estrus can be efficiently synchronized in lactating pluripara goats by administrating two doses of PGF2α spaced at intervals of 10<br />
days.The data related to sexual behavior are important when you want to use IATF programs.<br />
Keywords: pgf2α, goat.<br />
A064 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
EXPRESSION PROFILE OF CANDIDA<br />
ANDIDATE GENES FOR THE ACQUISITION OF COMPETENCE DURING OOCYTE<br />
GROWTH IN BOVINE<br />
Isabela Rebouças Bessa , Rosana Camargo Nishimura, Fernanda Paulini , Maurício Machaim Franco & Margot Alves Nunes Dode<br />
EMBRAPA CENARGEN, BRASÍLIA, DF, BRAZIL.<br />
A variety of studies have shown that oocyte competence is determined by the amount and composition of maternal transcripts stored<br />
during oogenesis and folliculogenesis. However, little is known about the mechanisms involved and the molecular characteristics of the oocytes<br />
during the acquisition of competence. The aim of this study was characterize the expression pattern of genes possibly involved in competence:<br />
DNA methyltransferase 1 (DNMT1); DNA methyltransferase 3B (DNMT3B); histone acetyltransferase (HAT); histone deacetylase (HDAC)<br />
and histone methyltransferase supressor of variegation 3 homolog 1 (SUV39H1) during oocyte growth. First, we determined the diameter of<br />
oocytes obtained from different preantral follicles categories. Preantral follicles were isolated, morphologically classified according to their<br />
categories in primordial, primary, small secondary and final secondary follicles. Then, the oocytes were individually isolated and measured. After<br />
the determination of the oocyte size to be collected in each category of follicles, preantral and antral follicles were isolated from ovaries of Nelore<br />
cows by mechanical dissociation using a tissue chopper. To isolate the oocytes, follicles were exposed to collagenase type II solution (0.5 mg/<br />
mL) and then submitted to successive pipetting. The oocytes were measured with Motic Images Plus 2.0 program and classified according to<br />
their diameter into: 1) oocytes from primordial follicle: diameter < 20 µm; 2) oocytes from primary follicles: 25 to 35 µm; 3) oocytes from small<br />
secondary follicles: 40 to 60 µm; 4) oocytes from final secondary follicles: 65 to 85 µm; 5) oocytes from early antral follicles: 100 to 120 µm; and<br />
6) oocytes from final antral follicles: > 128 µm. Total RNA was extracted from 4 pools of 25 oocytes, subjected to reverse transcription reaction<br />
and genes quantified by qPCR. Target gene expression was normalized using CYC A. Data were analyzed by Kruskal-Wallis test (P < 0.05).<br />
The results show that the transcripts of SUV39H1 did not differ significantly between groups from preantral to antral follicles. For DNMT1,<br />
DNMT3B and HAT, the expression was lower in oocytes from preantral follicles when compared to antral follicles. From all the studied genes,<br />
only HDAC differed (P < 0.05) between antral follicles, showing less expression in competent oocytes (>128µm) when compared to initial<br />
antral follicles (100 a 120 µm). These results suggest that the stock of transcripts of the majority of the studied genes are accumulated before the<br />
oocyte reach their fully growth. The HDAC was the only gene in which expression could be related to oocyte competence. [Financial Support:<br />
Macroprograma 1 Embrapa].<br />
Keywords: oocyte bovine, preantral, gene expression.<br />
s368
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A065 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
ANTRAL AL AND PREANTRAL AL FOLLICULAR POPULATION IN NELORE OVARIES<br />
WITH HIGH AND LOW ANTRAL AL FOLLICULAR<br />
COUNTING:<br />
PRELIMINARY RESULTS<br />
Gusta<br />
ustavo Mar<br />
artins Gomes dos San<br />
antos<br />
os, Katia Cristina Silv<br />
ilva-S<br />
a-San<br />
antos<br />
os, Car<br />
arine Letícia Schneider, Letícia Schmidt Silot<br />
iloto, Fabio Mor<br />
orotti,<br />
otti, Thiago Nogueir<br />
gueira<br />
Marcantonio, Luciana Simões Rafagnin Marinho & Marcelo Marcondes Seneda<br />
UEL, LONDRINA, PR, BRAZIL.<br />
Aiming to compare and correlate antral and preantral follicle populations, Nelore cows (n = 20, Bos taurus indicus,72-96-monthold)<br />
were subjected to follicular aspiration randomly during their estrus cycle (D0) to withdraw all follicles =3mm and to induce the growth of<br />
a new follicular wave. On D4, D19, D34, D49 and D64 ovaries were examined by ultrasonography (Áquila PRO, Pie medical, Maastricht, The<br />
Netherlands) using a 7.5-convex array transducer and antral follicles =3mm were counted. After five evaluations cows were assigned to two<br />
groups: high antral follicular counting (AFC, mean =30 follicles, n = 6) and low AFC (=15 follicles, n = 5), and ovaries were collected at abattoir.<br />
Only one ovary per female was analyzed. After being processed for histological evaluation, the ovarian halves were serially sectioned at 7µm.<br />
One at every 120 sections was mounted and stained with periodic acid Schiff (PAS) and hematoxylin. Follicles were classified as primordial,<br />
primary and secondary and the number of preantral follicles was estimated using a correction factor (Gougeon e Chainy, 1987. J Reprod Fertil,<br />
81:433-442). There was an attempted to correlate the mean number of antral follicles with the mean number of preantral ones. There was a<br />
repeatability in the numbers of antral follicles during follicular waves per individual, in accordance to a previous study with Bos taurus taurus<br />
(Burns et al., 2005, Biol. Reprod., 73:54-62). The mean number of antral follicles for the high and low AFC groups was 34 ± 10 (mean ± SD)<br />
vs.. 10 ± 4 follicles. A large variation in numbers of preantral follicles was observed among individuals as reported previously (Silva-Santos et<br />
al., <strong>2011</strong>, Theriogenology, in press). Numbers of follicles varied from 10,719 to 152,999 for the high AFC and 1,754 to 62,348 for the low AFC<br />
group. The mean number of preantral follicles for animals from the high AFC group was 68,923 ± 58,705 (mean ± SD) and 28,324 ± 24,525<br />
follicles for the low AFC ones. However, two females with high AFC presented lower numbers of preantral follicles (10,719 and 16,585) than<br />
that of low AFC, and one female from low AFC group presented similar population of follicles (62,348) to the AFC group. Preliminary results<br />
suggest that there was a correlation among numbers of antral and preantral follicles and that females with high AFC present larger mean number<br />
of preantral follicles compared to that of low AFC ones.<br />
Keywords: estimate, follicles, Bos taurus indicus.<br />
A066 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
FOLLICULAR PROPOR<br />
OPORTION AND POPULATION OF RATS SUBMITTED<br />
TED TO DIFFERENT PROCEDURES OF OVARIAN<br />
AUT<br />
UTOL<br />
OLOGOUS<br />
OGOUS TRANSPL<br />
ANSPLANT<br />
ANTATION<br />
TION<br />
Michelly Fernandes De Macedo 1 , Marcelo Barbosa Bezerra 2 , Nayara Almeida Do Carmo 3 , Felipe Farias Pereira Da Câmara Barros 4 &<br />
Wilter Ricardo Russiano Vicente 5<br />
1,4,5<br />
UNESP, JABOTICABAL, SP, BRAZIL. 2,3 UFERSA, MOSSORÓ, RN, BRAZIL.<br />
N<br />
The ovarian autologous transplantation has attracted an increasing scientific interest due its contribution in basic or applied<br />
reproductive studies in animals and humans. This study was conducted to compare the population and proportion of follicles from rats after 180<br />
days of transplantation. Thus, ovarian cortical tissues from 24 Wistar rats were transplanted to the kidney subcapsular site (group A),<br />
subcutaneous tissue (group B) and bursa ovarica (group C), after bilateral ovariectomy. Three ovarian cortical fragments (1mm 3 ) were obtained<br />
after fragmentation of the left ovary and placed in each of the receptor sites. The contralateral ovaries served as controls. All the tissues,<br />
transplanted and control, were evaluated for light microscopy and stereological methods (GOUGEON, A.; J. Reprod. Fertil.; v.81; p.433-442)<br />
to determination of follicular population and determining the proportion in different categories of morphologically normal follicles (PEDERSEN,<br />
T.; J. Reprod. Fertil.; v.17; p.555-557). The proportion of follicles in the different categories was compared by chi-square test (P < 0.05). All<br />
transplanted tissues were successfully development. Observed the largest follicular population (1,765) in the transplanted fragments to the<br />
kidney subcapsular site. The transplanted group to the subcutaneous tissue demonstrated an intermediate follicular population (1,274), while the<br />
lowest number of follicles was observed in animals that received transplants in the ovarian bursa (276). In the tissues used as controls accounted<br />
for a population ranging from 28,823 to 39,347 morphologically normal follicles per ovary evaluated. The proportion of preantral follicles was<br />
94.97 to 97.35% in control ovaries and transplantation tissues has varied from 80.42 to 87.64%. In contrast, antral follicles were in a proportion<br />
from 2.64 to 5.04% in the control ovaries and from 12.36 to 19.57% in the transplanted fragments. These results demonstrated the follicular<br />
development and growth in the transplanted tissues when compared to that observed in intact ovaries. Analyzing separately the follicular<br />
categories, we noted that in group A there was a high proportion of primordial follicles activated (transition) after 180 days, indicating that this<br />
site provides a greater survival of tissues post-transplantation. In conclusion, all the receptor sites recommended were effective in maintenance<br />
follicular development and growth, and in the group of animals transplanted to the kidney subcapsular site was demonstrated the highest<br />
follicular population and percentage of health follicles with appropriate conditions for development.<br />
Keywords: transplantation, ovary, follicle.<br />
s369
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A067 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
FIRST SUCCESSFUL CESSFUL ESTROUS SYNCHRONIZA<br />
ONIZATION USING A PROST<br />
OSTAGL<br />
GLANDIN ANALOGUE IN COLL<br />
OLLARED PECCARIES<br />
(TAYASSU<br />
ASSU TAJA<br />
AJACU)<br />
Alexandre Rodrigues Silva 1 , Keila Moreira Maia 2 , Mariana Araújo Silva 3 , Gislayne Christianne Xavier Peixoto 4 m José Artur Brilhante Bezerra 5 m Andréia<br />
Maria Da Silva 6 & Moacir Franco Oliveira 7<br />
1,2,3,4,5,6<br />
LCGA - UFERSA, MOSSORO, RN, BRAZIL. 7 CEMAS - UFERSA, MOSSORO, RN, BRAZIL.<br />
The collared peccaries (Tayassu tajacu) are small mammals present in the whole Brazilian territory. Recently, they were classified<br />
as vulnerable to extinction, mainly due to the predatory hunting and deforestation. Its sustainable breeding under captivity could be an<br />
alternative for its conservation, as well as for production of meat and pelts that are good accepted in the international market. The development<br />
of methodologies for the control of estrous cycle could assist in the implementation of biotechniques such as artificial insemination and<br />
embryo transfer. Therefore, it was aimed to develop a program for the estrous synchronization in captive female collared peccaries. The<br />
estrous cycle of five females, aging 18 ± 0.4 months and weighting 20.6 ± 1.1 kg, bred under captivity in Centre of Multiplication of Wild<br />
Animals of UFERSA, were monitored through estrogen dosage using ELISA and evaluation of external genitalia during 15 days. Then, a<br />
prostaglandin F-2á analogue, cloprostenol (Veteglan ® , Hertape Calier, Belo Horizonte, Brazil) was administered at a 60 µg/Kg dosage, IM, and<br />
repeated after nine days. After first application, the monitoring of the cycle continues for 20 days, every two days, for the verification of<br />
estrous. The data were analyzed by descriptive analysis (Statview 5.0, Cary, USA). By monitoring the natural cycle, it was observed that<br />
three females were in follicular phase when the protocol begun. At 8 ± 1.7 days following the first prostaglandin administration, an estrogen<br />
peak was verified in three females that presented 27.7 ± 11.1 pg/mL, but only two of that presented external sings of estrous (66.7%). It was<br />
noted that such females who responded to the first application, only one was demonstrated in follicular phase during the administration of<br />
drugs. As a consequence of the second application, all animals achieved an estrogenic peak at 9.2 ± 1.1 days, showing levels of 28.1 ± 10.4 pg/<br />
mL of estrogen. Four of the five females (80%) showed external signs of estrus, such as vulvar opening, hyperemic vaginal mucosa and the<br />
presence of vaginal mucus. The response to the treatment was considered late when compared to domestic species, and it is suggested that<br />
higher prostaglandin doses could reduce the interval for estrous manifestation. It should be emphasized that this is the first description of a<br />
program for the estrous cycle control in collared peccaries, and the efficiency of the prostaglandin analogue use for the estrous synchronization<br />
of this species was demonstrated.<br />
Keywords: estrous cycle, peccary, synchronization<br />
A068 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
CELLUL<br />
ULAR PROLIFER<br />
OLIFERATION OF POLIOVULAR FOLLICLES IN ADULT NELORE OVARIES<br />
Katia Cristina Silva-Santos 1 , Gustavo Martins Gomes Dos Santos 2 , Reginaldo Luís Oliveira 3 , Letícia Schmidt Siloto 4 , Ana Paula Frederico Rogrigues Loureiro<br />
Bracarense 5 & Marcelo Marcondes Seneda 6<br />
1,2,3,4,5,6<br />
UEL, LONDRINA, PR, BRAZIL.<br />
Polyovular follicles identification has already been reported in many species. Thereafter, little information is available in the<br />
literature about these follicles in the ovaries from Nelore adult females (Silva-Santos et al., <strong>2011</strong>, Theriogenology, in press) and their function<br />
remains unclear. This way, aiming to evaluate cellular proliferation of polyovular follicles in the ovaries from Bos taurus indicus animals,<br />
ovaries were collected at abattoirs from adult Nelore females (24-30-months-old, n = 21), cut longitudinally, fixed in Bouin’s solution and<br />
processed for histology and imunohistochemical. The ovaries halves were dehydrated in alcohol, cleared in xilol, embbebed in paraffin and<br />
serially sectioned at 5µm. ovarian tissue was processed for histology and imunohistochemical. After being identified by histology, sections<br />
were stained with antibodies against proliferating cell nuclear antigen (PCNA, DAKO, Carpinteria, CA, USA), as described previously<br />
(Oktay et al., 1995, Biol. Reprod. 53:295-30). Polyovular follicles were classified according to the shape and number of granulosa cells. From<br />
ovaries analyzed by histology, four (19%) presented polyovular follicles containing two oocytes nuclei at primordial and primary stage. All<br />
polyovular follicles (n = 10) were PCNA positive, which indicates activity and proliferation of these structures. The majority of granulosa<br />
cells and oocyte nuclei from these follicles were PCNA positive, except two polyovular follicles in the same ovary, which one oocyte nuclei<br />
was PCNA positive and the other one was not, although both had PCNA-positive granulosa cells. It was concluded that polyovular follicles<br />
from adult Nelore females are proliferating and that these structures might be related with folliculogenesis.<br />
Keywords: polyovular follicles, proliferation, Bos taurus indicus.<br />
s370
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A069 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
SUPEROVUL<br />
OVULATOR<br />
ORY RESPONSE AND PREGANCY RATES OF HOLSTEIN COWS IN VIVO EMBRYO PRODUCTION<br />
WITHIN<br />
DIFFERENT CATAGORIES DURING SUMMER AND WINTER<br />
Lais Mendes Vieira 1 , Carlos Alberto Rodrigues 2 , Pericles Ricardo Lacerda E Silva 3 , José Nelio S. Sales 4 , Manoel Francisco Sá Filho 5 , Andressa Lavezzo Ranieri 6<br />
& Pietro Sampaio Baruselli 7.<br />
1,4,5,7<br />
FMVZ USP, SÃO PAULO, SP, BRAZIL. 2,3 CLÍNICA VETERINÁRIA SAMVET DE SÃO CARLOS, SÃO CARLOS, SP, BRAZIL. 6 FCAV UNESP, JABOTICABAL, SP, BRAZIL.<br />
The aim of this study was to evaluate the multiple ovulation response (MOr) and the pregnancy rates of in vivo production<br />
embryos from donors [Heifers (H), lactation cows (LC) and dry cows (DC)] within different seasons [Summer/Autumn (S/A) and winter/<br />
spring (W/S). The retrospective study was carried out in a commercial farm (Fazenda Santa Rita, Descalvado/SP, Brazil). The data set<br />
comprised 1,948 MO (H: 818; LC: 587; and DC: 543) of Holstein cows and was performed from 2007 to 2010. The animals were submitted<br />
to a superovulatory protocol with FTAI (P36) 12 and 24 h after GnRH treatment (ovulatory stimulus). The number of formed CL was<br />
registered previously to each embryo collection. Statistical analize was performed by PROC GLIMMIX of SAS. The data was presented<br />
with percentage or average ± SE (X ? Y ? Z within season and a ? b within category). Interaction between category and season was observed<br />
in MOr during Summer/Autumn (H-S/A: 81.1%Y; LC- S/A: 88.4%XY; DC- S/A: 95.2%X; P = 0.02), number of CL (H-W/S: 8.9 ± 0.28aZ;<br />
LC- W/S: 11,4 ± 0,44aX; DC- W/S: 10.0 ± 0.44Y; H-S/A: 6.8 ± 0.38bY; LC- S/A: 9.8 ± 0.58bX; DC-V: 10.3 ± 0.44X; P < 0.001), recovered<br />
structures (H–W/S: 6.5 ± 0.26bZ; LC– W/S: 9.7 ± 0.47aX; DC–W/S – 7.4 ± 0.44Y; H–S/A: 4.9 ± 0.31bZ; LC–S/A: 8.5 ± 0.59bX; DC– S/A:<br />
7.7 ± 0.44Y; P < 0.001), recovery rate (H–W/S: 58.5%Y; LC– W/S: 69.9%X; DC– W/S: 58.6%Y; H–S/A: 54.0%Z; LC–S/A: 68.1%X; DC–<br />
W/S: 63.8%Y; P < 0.001), viable embryos rate (H–W/S: 67.8%aX; LC– W/S: 44.9%aY; DC– W/S: 40.9%aZ; H–S/A: 55.7%bX; LC– S/A:<br />
38.1%bY; DC– S/A: 30.8%bZ; P < 0.001) and pregnancy rate from transferred fresh embryos (H-W/S: 36.9%Y; LC- W/S: 39.3%X; DC- W/<br />
S: 39.8%X; H-S/A: 34.9%Y; LC- S/A: 36.8%Y; DC- S/A: 42.2%X; P < 0.001). Viable embryos number was affected by category (H: 4.49 ±<br />
0.18X; LC: 4.42 ± 0.25X; e DC: 3.1 ± 0.20Y; P < 0.001) and season (W/S: 4.57 ± 0.16 vs.. S/A: 3.11 ± 0.17; P < 0.001). In conclusion, heifers<br />
present lower MO response between the categories. The viable embryo production was reduced during summer/autumn, even more,<br />
considering categories, production was higher among heifers and lactation cows. In addition, viable embryos rate was inferior in all categories<br />
during summer/autumn. Moreover, dry cows had higher pregnancy rate per embryo transfer during the summer, and this rate was similar to<br />
lactation cows during the winter/spring. [Acknowledgments: Agrindus S.A. and Intervet Schering-Plough].<br />
Keywords: bovine, moet, season.<br />
A070 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
SUPEROVUL<br />
OVULATION OF NELORE HEIFERS USING SINGLE INJECTION OF FOLICLE STIMULANT HORMONE CARRIED INTO<br />
POLIMERIC MATRIX<br />
Edgar Maurício Mogollon-Waltero 1 , Hector Javier Narvaez 2 , Ricardo Da Costa 3 , Célia Raquel Quirino 4 & Reginaldo Silva Fontes 5<br />
N<br />
1,2,4,5<br />
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL. 3 IZ, NOVA ODESSA, SP, BRAZIL.<br />
Among the reproductive biotechnologies used in the livestock industry, is increasingly common to require the use of multiple<br />
ovulation or superovulation. However, it is not possible to reduce the negative effect generated by the handling of animals because of the short<br />
half-life of the hormone FSH ranging from 5 to 12 h approximately. The conventional process of superovulation in cattle need eight intramuscular<br />
injections of FSH which increases handling and stress degree of animals. The aim of this work were to reduce the number of injections for one<br />
and the amount of hormone FSH to 50% by incorporating it into a biodegradable and biocompatible polymeric matrix. In this experiment were<br />
used 30 Nelore heifers were randomly divided into three groups, the control group received 100 mg of FSH in three injections each 36 h, while<br />
the other groups received only one injection of 100 or 50 mg of hormone loaded in the polymer matrix. The superovulatory response was<br />
determined according to the number and size of growing follicles, day 0, 4, 6, 8 and 14 of the protocol and the number of corpora lutea (CL) two<br />
days before collection by ultrasound examination furthermore the quantity and development state of the collected structures in the uterine washed.<br />
There was effect, in both doses and number of applications hormone FSH on the number of collected structures (P > 0.05), the result being<br />
higher in the group with a mean 4.0 ± 7.42 embryos per collection Vs. . 1.7 ± 0.5 and 4.03 ± 1.58 in control and group 2 respectively, the number<br />
of corpora lutea was 8.10 ± 1.52, 5.56 ± 3.88 and 2.40 ± 1 , 71 for control and groups 1 and 2 respectively and the number of follicles (> 6 mm)<br />
on day 8 (LH injection) was 14.70 ± 3.53, 17.33 ± 5.57 and 9.30 ± 4 , 08 for control and groups 1 and 2 respectively. We performed the analysis<br />
of variance (Proc GLM, SAS, 1999) variables: mean number of follicles >6 mm in day 8, mean number of CL in day 14 and number of collected<br />
embryos. When the data did not show normal distribution, they were transformed by logarithm base 10. The treatment means were compared<br />
by “t” test. It is concluded that FSH loaded in polimeric matrix produce efficient super ovulation in Nelore heifers with only one subcutaneous<br />
injection.<br />
Keywords: superovulation, polimeric matrix, fsh.<br />
s371
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A071 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
ENERGY SUPPLEMENTATION TION IN SANTA A INÊS SHEEP<br />
Sabr<br />
abrina Silv<br />
ilva Ven<br />
entur<br />
turi 1 , Isab<br />
sabel Roussouliér<br />
oussouliéres Soar<br />
oares<br />
2 , Andr<br />
ndressa Fer<br />
erreir<br />
eira Da Silv<br />
ilva 3 , Elizab<br />
lizabeth Cruz Car<br />
ardoso<br />
4 , André Rios Rodr<br />
drigues<br />
5 , Rodolpho Almeida<br />
Tor<br />
orres<br />
Filho 6 , Jeferson Ferreira Fonseca 7 & Felipe Zandonadi 8<br />
1,2,3,4,5,6,8<br />
UNIVERSIDADE FEDERAL FLUMINENSE, NITEROI, RJ, BRAZIL. 7 EMBRAPA CAPRINOS E OVINOS, CORONEL PACHECO, MG, BRAZIL.<br />
Follicular growth is mainly controlled by gonadotropins and locally produced growth factors. However, many environmental<br />
factors such as nutrition can influence follicular development, oocyte quality, the rate of ovulation and consequently, fertility. Caloric supplementation<br />
causes an increase in circulating concentrations of glucose and insulin, leading to a rise in the number of follicles as well as an increase in follicular<br />
diameter and ovulation rate (SOMCHIT et al., 2007, Theriogenology 68, 1037-1046, VIÑOLES et al.,2009, Animal Reproduction Science 113,<br />
82-92). This study aims to evaluate the effects of increasing energy by 20% through dietary supplementation as an induction procedure and<br />
synchronization of estrus in ewes on ovulation rate, follicle diameter and circulating concentrations of glucose and insulin. Forty-three Santa Inês<br />
sheep were chosen and separated into two experimental groups where the Gcontrol group received a maintenance diet and the GM+20%<br />
received a maintenance diet with a supplementation providing a 20% increase of energy during the induction period and synchronization of<br />
estrus. The GM+20% group underwent a 6-day procedure where an intravaginal sponge was inserted containing 60 mg of medroxiprogesterone<br />
acetate (Progespon ® , Shering Plough Saúde Animal, São Paulo, Brazil). There was administration of 37.5 µg of d-cloprostenol (Veteglan ® , Lab.<br />
Hertape-Calier Saúde Animal S/A, MG, Brazil) and 200 IU of eCG (equine chorionic gonadotropin - Novormon 5000 ® , Syntex Ind. Biochemistry,<br />
Buenos Aires, Argentina) 24 h before sponge removal. The monitoring of ovarian follicular dynamics was carried out to determine the timing<br />
and number of ovulations, and the diameter of preovulatory follicles. Ultrasound examinations were conducted to evaluate follicular diameter and<br />
ovulation rate. Bloods was collected to determine plasma insulin and glucose concentrations and were made with the animals before diet was<br />
offered. Statistical analysis was performed through Duncan´s Test. In relation to plasma insulin concentration, it was observed that the GM+20%<br />
presented with high concentrations (1,66 ± 1,51 vs. 3,04 ± 3,66 (ng/mL) – P > 0,05). Plasma glucose concentrations did not change (70,20 ±<br />
7,72 vs. 69,70 ± 25,46 – (mg/dL) P > 0,05). The results showed no differences in follicular diameter (6,00±0,20 vs. 5,90±0,60- P > 0,05) or in<br />
the rate of ovulation (1,00 vs. 1,13 - P > 0,05) between the groups. Therefore the supplementation addressed in this study led to an increase in<br />
insulin, however was not sufficient to affect reproductive parameters and plasma glucose concentration.<br />
Keywords: sheep, supplementation, santa inês.<br />
A072 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
VARIA<br />
ARIATION ON VASCUL<br />
ASCULARIZA<br />
ARIZATION OF FOLLICLES INDUCED TO OVULATE<br />
WITH HCG AND GNRH EVAL<br />
ALUATED<br />
BY DOPPLER ULTR<br />
TRASONOGR<br />
ASONOGRAPHY APHY - PRELIMINARY RESULTS<br />
Renata Cristina Uliani 1 , Luciano Andrade Silva 2 & Marco Antonio Alvarenga 3<br />
1,3<br />
FMVZ UNESP, BOTUCATU, SP, BRAZIL. 2 FZEA USP, PIRASSUNUNGA, SP, BRAZIL.<br />
Only few studies are avaliable in the literature with use of Doppler in equine reproduction which has been used for study of<br />
vascularization of preovulatory follicle, follicular and oocyte quality check, vascularization of corpus luteum and uterus, establishment of<br />
pregnancy and evaluation of pregnancy. Recent studies with Doppler have shown changes in blood flow and vascularization of the follicular<br />
wall at the time close to ovulation. For this reason Doppler transrectal ultrasonography has been used in studies of ovarian and follicle<br />
hemodynamics in large animals. Ovulation is an outstanding event in the study of reproduction, which makes its induction a biotechnology of<br />
reproduction that deserves special attention. The products commercially available for ovulation induction are hCG and deslorelin, a GnRH<br />
analogue. HCG is a protein hormone with LH action. Its composition is similar to equine LH but with a half life much longer because contains<br />
much sialic acid. The deslorelin administered intramuscularly induces the release of endogenous LH by the mare. Objectives: Is absent in the<br />
literature studies concerned in compare the follicular vascularization after administration of hCG and GnRH, thus, the objective of this work was<br />
to check the variation on vascularization of the follicle after the ovulation induction with each one of the inductors. Material and methods: Two<br />
estrous cycles of five mares (
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A073 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
VIABILITY OF CANINE PREANTRAL AL FOLLICLES CULTURED<br />
IN VITRO AFTER CONSER<br />
ONSERVATION IN AN OOCYTE<br />
TRANSPOR<br />
ANSPORTER<br />
Rodrigo José Sousa Gonçalves 1 , Vanessa Raquel Pinto Barros 2 , Anderson Pinto Almeida 3 , Ana Beatriz Graça Duarte 4 , Valdevane Rocha Araújo 5 , Cleidson<br />
Mano<br />
anoel Gomes Silv<br />
ilva 6 , Gio<br />
iovanna Quin<br />
uintino Rodr<br />
drigues<br />
7 , Ger<br />
erlane Modest<br />
desto Da Silv<br />
ilva 8 , José Ric<br />
icar<br />
ardo De Figueir<br />
igueiredo<br />
edo 9 & Mar<br />
aria Helena<br />
Tavar<br />
ares Matos<br />
10<br />
1,2,10<br />
UNIVERSIDADE FEDERAL DO VALE DO SÃO FRANCISCO, PETROLINA, PE, BRAZIL. 3,4,5,6,7,8,9 UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.<br />
Maintenance of follicular quality after collection and transportation of the ovaries to the laboratories of reproductive techniques<br />
is a limiting factor for the success of in vitro culture of preantral follicles (PAF) in the canine species. In this way, the aim of this study was<br />
to assess the viability of canine PAF cultured in vitro after storage for 12 or 24 h in an oocyte transporter ® - Compact, Wta-Brazil (TO). After<br />
collection of canine ovaries,180 secondary preantral follicles with a diameter higher than 200 mm were mechanically isolated, and destined to<br />
conservation into the oocyte transporter at 39°C in α-Minimum Essential Medium supplemented with BSA (3 mg/mL), glutamine (2 mM),<br />
hypoxanthine (2 mM), ITS (insulin 10 µg/mL, transferrin 5.5 µg/mL, and selenium 5 ng/mL), ascorbic acid (50 ag/ml) and recombinant FSH<br />
(100 ng/ml). The PAF were further destined to in vitro culture using the same conservation medium. Six treatments were used: PAF assessed<br />
by fluorescence microscopy (FM) after isolation (T1), PAF cultured after isolation (T2 - cultured control), PAF destined to (FM) after 12<br />
(T3) or 24 h (T4) of conservation, PAF destined for the culture after 12 (T5) or 24 h (T6) of conservation. PAF were individually cultured in<br />
100 mL drops of medium, at 39°C and 5% CO2 for 7 days. Every 2 days, 60 ìl of the culture medium was changed by a fresh medium. The<br />
follicular viability was analyzed using the fluorescent markers calcein-AM (4µM) and ethidium homodimer-1 (2µM) for viable and nonviable<br />
PAF, respectively. At the end of the isolation, preservation in the TO and the culture for 7 days, PAF of all the treatments (100%) analyzed<br />
by FM were stained in green by calcein-AM demonstrating that all them are viable the that explains the absence of statistical analysis of this<br />
experiment. In conclusion, canine preantral follicle preserved for 12 and 24 h in an oocyte transporter maintain the viability in vitro after<br />
culture for 7 days.<br />
Keywords: canine, preantral follicle, preservation.<br />
A074 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION<br />
ASCORBIC ACID IMPROVES<br />
THE SURVIV<br />
VIVAL AND IN VITRO GROWTH OF ISOLATED CAPRINE PREANTRAL AL FOLLICLES<br />
Gerlane Modesto da Silva 1 , Carlos Henrique Lobo 2 , Valdevane Rocha Araújo 1 , Ana Beatriz Graça Duarte 1 , Arlindo Alencar Moura 2 & José Ricardo de<br />
Figueiredo 1<br />
1<br />
UECE, FORTALEZA, CE, BRAZIL. 2 UFC, FORTALEZA, CE, BRAZIL.<br />
Ascorbic acid (AA) may act to regulate the matrix metalloproteinases and their inhibitors associated with the turnover of collagen<br />
on the basement membrane during follicle growth (Murray et al., 2001; Journals of Reproduction and Fertility 121, 89-96). The present study<br />
aims to investigate the influence of AA on the survival, growth, antral formation, extruded oocytes and mRNA expression of the matrix<br />
metalloproteinases-9 (MMP9) and their tissue inhibitor-2 (TIMP2) on caprine preantral follicles (FOPA). Isolated FOPA were individually<br />
cultured without (MEM+) or with AA at 50 µg/mL (AA50) or 100 µg/mL (AA100) during 18 days. To culture at least 32 follicles were used<br />
per treatment. Three pools of 10 follicles from non cultured (fresh control), MEM+ alone, AA50 and AA100 were collected to quantified the<br />
genes MMP9 and TIMP2 by real-time polymerase chain reaction (qPCR). The percentage of survival, antrum formation, extrusion rate and<br />
maturation were compared by x2 test (StatView for Windows). The diameter and growth rates were compared by Kruskal-Wallis test. For qPCR<br />
differences between the control and treatments was assessed with the T test of SAS 9.0. At the end of culture, AA50 significantly increased the<br />
percentage of follicular survival (59.38% vs. MEM+: 29.03%; AA100: 44.83%; P < 0.05) and antral formation (65.63% vs. MEM+: 34.78%;<br />
AA100: 52.00%; P < 0.05) compared with MEM+ alone. After 6 days of culture, AA50 (319.14±89.70) increased follicular diameter (mm),<br />
compared with the other treatments (MEM+: 229.59±78.13; AA100: 269.80±68.50; P < 0.05). At day 12, both concentrations of AA (AA50:<br />
452.38±136.81; AA100: 451.98±165.45) increased follicular diameter when compared with control medium (242.11±131.69; P < 0.05).<br />
Moreover, mean increase in follicular diameter daily (µm/day) was higher in the presence of both concentrations of AA (AA50: 18.15±10.57<br />
and AA100: 20.94±10.43) than in MEM+ alone (11.38±9.46; P < 0.05). Lower percentages of extruded oocytes were observed in medium<br />
containing AA50 (18.75%) compared with control medium (56.52%); P < 0.05. With respect to the maturation, all fully grown oocytes showed<br />
an intact germinal vesicle. The qPCR assays showed that AA50 show mRNA expression for MMP9 was higher 2.4 times when compared to<br />
MEM+ alone (P < 0.05). In addition, mRNA expression for TIMP2 gene was higher 2.08 times in AA100 than in MEM+ (P < 0.05). In<br />
conclusion, AA at 50 µg/ml increase the caprine FOPA viability and development after in vitro culture and influences the enzyme MMP9<br />
involved with basement membrane remodeling.<br />
Keywords: mmp9, timp2, ascorbic acid.<br />
N<br />
s373
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A075 FTAI, FTET AND AI<br />
FOLLICULAR AND REPRODUCTIVE TRAITS IN SHORT-TERM PROGESTERONE AND PGF2ALPHA BASED ESTROUS-<br />
SYNCHRONIZA<br />
ONIZATION PROTOC<br />
OCOL OL IN SHEEP<br />
Rodrigo V Allende, , Fernando R Saravia, Evelyn N Lara, Andrea F Leiva, T Díaz & José Franscisco Cox<br />
UNIVERSIDAD DE CONCEPCIÓN, CHILLÁN, CHILE.<br />
The conventional protocols to estrous synchronization based on 12-day progestagen treatments and eCG at intravaginal device<br />
removal has no particular advantage based on follicular dynamics in sheep (Bartlewski et al., <strong>2011</strong>). The aims of this study were to describe size<br />
and static phase of preovulatory follicles, interval to ovulation and fertility post AI of ewes subjected to a short-term progesterone treatment. At<br />
mid breeding season, 20 Suffolk ewes under controlled feeding receive an intravaginal progesterone-release devise (CIDR G; 0.3 g progesterone,<br />
Pfizer) for 7 days combined to cloprostenol (0.125 mg im Estrumate, Intervet) at CIDR removal (n = 10 control group) or the same protocol plus<br />
eCG (400 IU Novormon, Syntex) at CIDR removal (n = 10). Antral follicular dynamics was followed by daily transrectal ultrasonography (10-<br />
MHz probe; Honda 2010Vet, Japan). Follicles > 2,5 mm and CLs were measured daily and recorded by sketches on ovarian charts. To determine<br />
ovulation time, ewes were scanned every 8 h since 12 h after estrus detection until ovulation, and this moment was the middle period between<br />
the last identification of preovulatory follicles and their disappearance. The preovulatory follicular diameter was measured on ovulated<br />
follicles and the persistency of the static phase was calculated as the interval between they reached 4.0 mm in diameter until ovulation. To<br />
assess fertility after treatment, 256 ewes (2.5-3.0 BCS, 1-5 scale) were synchronized by the progesterone treatment plus eCG and were<br />
inseminated at estrus with fresh semen. Follicular size and persistency and interval to estrus and ovulation were compared by the Student’s<br />
t-test. The results showed that ovulatory follicles from ewes treated with eCG have similar features than those from control group<br />
(6.3mm±0,21 vs.. 6.5mm±0,22), similar behavior at persistency (4.47±0.51 vs.. 3.92±0.29 days) and so for ovulatory rate (1.63 vs.. 1.63).<br />
However, the intervals between CIDR retrieval and estrus and ovulation were shorter for eCG group than for control (32.8±3.23 vs..<br />
45.8±2.42 h and 61.9±2.48 vs.. 82.2±4.97 h, P < 0.05). In addition, the corpus luteum diameter size at day 10 was also different (11.7±0.4<br />
vs.. 10.9±0.28 mm for eCG and control respectively; P < 0.05). The preliminary results show that 83.7% ewes were inseminated with a<br />
conception rate of 53.2% (checked by ultrasound at 30 day after AI). The cumulative results indicate that a short-term progesterone protocol<br />
combined to PGF2a and eCG allows estrous synchronization with normal fertility after AI. [Reference: Bertlewski PM, Baby TE, Giffin GL.<br />
<strong>2011</strong>, Anim Reprod Sci, 124:259-268. Funded by Consorcio Ovino (Project FIA FIC-CS-C-2006-1-P-001-22)].<br />
Keywords: estrus synchronization, folliculogenesis, sheep.<br />
A076 FTAI, FTET AND AI<br />
EFFECT OF CIDR ® REUTILIZATION TION IN A PROGESTER<br />
OGESTERONE-PGF2ALPHA ONE-PGF2ALPHA BASED ESTROUS-SYNCHR<br />
OUS-SYNCHRONIZA<br />
ONIZATION PROTOC<br />
OCOL OL ON<br />
ESTROUS PRESENTATION TION AND OVULATOR<br />
ORY PERFORMANCE IN SHEEP<br />
Fernando R Saravia 1 , Rodrigo V Allende 2 , Evelyn N Lara 3 , Andrea F Leiva 4 , T Diaz 5 , P Rojas 6 , M Recabarren 7 & José Franscisco Cox 8<br />
1,2,3,4,5,6,7,8<br />
UNIVERDSIDAD DE CONCEPCIÓN, VICENTE MENDEZ 595, CHILE.<br />
The use of short-term progesterone (P4) treatments allows the intensive use of P4 vaginal inserts. The study was aimed to<br />
describe the P4 profiles and follicular size and ovulation after the use of new and 7-day used inserts in short-term P4 protocols. In Experiment<br />
1, 12 Suffolk ewes under controlled feeding and during anestrus season, were randomly grouped and treated by a new P4-release device (CIDR<br />
G ® , Pfizer) for 7 days (n = 6) or a used one (n = 6). Blood was collected to measure plasma P4 by RIA (Coat a Count, DPC, USA). In<br />
Experiment 2, 38 Suffolk ewes (2 replicates) were synchronized during the breeding season by a 7-day treatment with new (n = 22) or used<br />
CIDR G ® (n = 16) plus cloprostenol (0,125 mg Estrumate ® , Intervet) at CIDR ® retrieval. Half of the ewes in each group received 400 IU of<br />
eCG (Novormon ® , Syntex). Estrous detection was carried out thrice a day by teaser rams. The diameters of ovulatory follicles were measured<br />
by daily ultrasonography (10-MHz linear array probe; Honda 2100Vet). Since 8 h after estrus, follicles =3.5 mm in diameter were measured<br />
each 8 h and sketched on ovarian charts to identify ovulation. Ten day later, ovaries were scanned to identify corpora lutea to confirm<br />
ovulation points. The moment of ovulation was defined as the middle point between the last identification of preovulatory follicles and their<br />
disappearance. Data were calculated and comparisons were done P4 by the Student’ t test and by Factorial ANOVA. Results in Exp 1 show<br />
that P4 profiles differed in ewes treated with new and used CIDR (0.2±0.1 vs.. 0.3±0.07; 5.3±1.48 vs.. 2.1±0.64; 4.7±1.3 vs.. 1.9±0.24;<br />
3.6±0.77 vs.. 1.9±0.25; 2.5±0.26 vs.. 1.5± 0.25; 2.2±0,58 vs.. 1.2±0.16 ng/mL at days 0,1,2,3,5 and 7 for CIDRs new and reutilized<br />
respectively; P < 0.05). Results in Exp 2, show that ewes treated with new and used CIDR have similar reproductive response (P < 0.10),<br />
therefore data were grouped and used to assess the effect of eCG in the 7-day P4 protocol. eCG has no effect on ovulatory follicle diameter<br />
nor on ovulatory rate or number of follicles ovulated (6.2±0.14 vs.. 6.2±0.2 mm; 100% (22/22) vs.. 100% 16/16); 1.69 (37/22) vs.. 1.69 (27/<br />
16) for eCG and untreated controls respectively; P > 0.10). However eCG increases the diameter of CL and reduces the intervals to estrus and<br />
ovulation (11.3±0.3 vs.. 10.7±0.2 mm; 32.0±1.78 vs.. 45.5±1.71 h; 61.2±1.49 vs.. 80.7±2.81 h for eCG and control groups respectively; P <<br />
0.05). The reduced interval to ovulation and the increase in CL diameters suggests that eCG increases the estradiol production of ovulatory<br />
follicles in short-term P4 protocols. [Funded by Consorcio Ovino (FIA FIC-CS-C-2006-1-P-001-22)].<br />
Keywords: estrous synchronization, ovulation, sheep.<br />
s374
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A077 FTAI, FTET AND AI<br />
EFFECT OF ESTRADIOL BENZOATE AT CIDR INSERTION ON OVARIAN<br />
TRAIT<br />
AITS AFTER SHORT-TERM PROGESTER<br />
OGESTERONE ONE AND<br />
PGF2ALPHA BASED ESTROUS-SYNCHR<br />
OUS-SYNCHRONIZA<br />
ONIZATION PROTOC<br />
OCOL OL IN SHEEP<br />
José Franscisco Cox 1 , Evelyn N Lara 2 , Andrea F Leiva 3 , H Muñoz 4 , Rodrigo V Allende 5 & Fernando R Saravia 6<br />
1,2,3,4,5,6<br />
UNIVERSIDAD DE CONCEPCIÓN, CHILLÁN, CHILE.<br />
Estradiol benzoate (EB) has been used in cattle to control follicular emergence and to potentiate progesterone to accelerate<br />
dominant follicle turnover. The study was aimed to study the effect of EB at CIDR insertion on ovulatory follicular traits and interval to<br />
ovulation during the mid breeding season in sheep in ewes subjected to short-term progesterone protocol for estrous synchronization. Fifty<br />
four ewes under controlled feeding were synchronized by the insertion of intravaginal progesterone-release device (CIDR G, 0.3 g progesterone,<br />
Pfizer) for 6 days combined to cloprostenol (0.125 mg im Estrumate, Intervet) and eCG (400 IU Novormon, Syntex) at CIDR removal (n =<br />
22) or as before plus 0,5 mg EB at CIDR insertion (n = 32). Antral follicular dynamics was followed daily since CIDR retrieval and each 8 h<br />
since 12 h after estrus detection by transrectal ultrasonography using a 10-MHz linear array probe connected to a B-mode scanner (Honda<br />
HS 2010Vet, Japan). Follicles = 2,5 mm and CLs that were recorded by sketches on ovarian charts. The preovulatory follicular diameter was<br />
measured on ovulated follicles that were later confirmed at CL measure 10 days after estrus. To determine ovulation time, ewes were scanned<br />
until ovulation, and the moment of ovulation was defined as the middle point between the last identification of preovulatory follicles and their<br />
disappearance. Therefore the interval between both CIDR retrieval and estrus detection to ovulation were the periods in hours between these<br />
points. Follicular and CL traits and the interval to estrus and ovulation between treatments were compared by the Student’s t-test and a P <<br />
0.05 was considered significant. The results show that both the size of ovulatory follicles and that of CLs at day 10 after estrus were similar<br />
(6.0±0.09 vs.. 6.1±0.13 mm; 11.1 vs.. 10.5 mm in diameter for treated and untreated groups respectively; P > 0.10). However, EB increases<br />
the intervals from the end of treatment to estrus and ovulation (40.3±1.84 vs.. 34.1±1.97 h; 70.1±1.91 vs.. 64.1±1.96 h for treated and<br />
untreated controls respectively; P < 0.05) and reduces the ovulatory response compared to untreated ewes (82.1% vs. 95.7%; 1.26 vs.. 1.82;<br />
for ovulation rate and number of follicles ovulated for treated and untreated ewes respectively; P < 0.05). Cumulative information suggest that<br />
estradiol benzoate added at CIDR insertion has an inhibitory effect on FSH profiles that delays the emergence of antral follicles and reduces<br />
the ovulatory rate and the number of follicles ovulated in treated ewes without other observable changes in follicular development and in CL<br />
development after ovulation. [Funded by Consorcio Ovino (FIA FIC-CS-C-2006-1-P-001-22)].<br />
Keywords: estrous synchronization, estradiol, sheep.<br />
A078 FTAI, FTET AND AI<br />
TAI INCREASES THE REPRODUCTIVE PERFORMANCE OF LACT<br />
CTATING TING PRIMIPAR<br />
AROUS ACY<br />
CYCLIC CLIC NELORE COWS<br />
Rodrigo Ribeiro Cunha 1 , Marilu Martins Gioso 2 , Regis José De Carvalho 3 , Carlos Antônio De Carvalho Fernandes 4 , Miller Pereira Palhão 5 , Marina Resende<br />
Pimenta Portinari 6 & Thais Camargo Rossi 7<br />
1,2,3,4,5,6,7<br />
UNIFENAS, ALFENAS, MG, BRAZIL.<br />
N<br />
The objective of this experiment was to evaluate the efficiency of a Fixed Time Artificial Insemination (TAI) protocol in an<br />
attempt to improve the reproductive performance of primiparous lactating acyclic Nelore cows. The experiment was conducted for a period<br />
of breeding season (BS) of 180 days. Were used 350 Nelore cows with body condition score (BCS) greater than 2.5 (average 2.65 ± 0.01) and<br />
days postpartum average of 67.35 ± 1.0 days. The animals were divided into four groups: G1 (n = 61) and G3 (n = 116) - cows with ovarian<br />
follicles smaller than 8mm in diameter and absence of CL. G2 (n = 57) and G4 (n = 116) - cows with follicles greater than 8mm and absence<br />
of CL. G1 and G2 remained with the bulls in the management of natural mating (NM) throughout the experimental period, from day 0 of BS.<br />
The G3 and G4 were treated (Day 0) with an intravaginal device containing 1.0 g of P4 for 8 days and 2mg (2mL) of estradiol benzoate (EB)<br />
IM. The withdrawal of P4 (Day 8) were applied IM 400 IU (2mL) of eCG and 75µg (2mL) of cloprostenol and 24 h later (Day 9),<br />
administered 1mg (1mL) IM BE. The TAI was performed between 51-54 h after withdrawal of P4 (Day 10) using semen from a single bull.<br />
After five days of the TAI, cows in groups G3 and G4 were submitted with bulls in the management of NM. All animals were evaluated by<br />
ultrasound for cyclicity rates (presence of CL) and pregnancy diagnosis at 35 and 180 days after onset of BS. For the analysis of the presence<br />
of CL and pregnancy rates at 35 and 180 days, between groups, were performed a chi-square. For the variable: period of service was used<br />
ANOVA and Duncan. At 35 days of BS, the G3 (42.24% A) and G4 (51.70%) presented higher rates of pregnancy (P < 0.05) than G1 (0%<br />
B) and G2 (35% B). A greater number of ovulations was also found (P < 0.05) in G3 and G4 when compared to animals maintained only during<br />
NM (6.56% C, 28.07% B 50.86% A, 62, 93%, for G1, G2, G3 and G4, respectively). Pregnancy rates at 180 days were: 22.95% C (G1),<br />
45.61% B (G2), 40.51% B (G3) and 65.51% A (G4). For percentage of animals with CL at 180 days of MS, the G4 (71.55% A) obtained better<br />
results than Groups 1, 2 and 3 (42.62% B 56.14% B 48.27% B, respectively). Additionally, groups of TAI + eCG had shorter periods of<br />
service (days, P < 0.05): G1 (134.00 ± 11.70 A), G2 (119.65 ± 8.27 A), G3 (79, 91 ± 5.38 B) and G4 (81.40 ± 4.59 B). The work suggests<br />
that the TAI was effective to improve the reproductive efficiency of primiparous lactating acyclic Nelore cows in compared to females<br />
subjected only NM. In addition, primiparous cows subjected to TAI at the beginning of BS had the reduced postpartum anoestrus period and<br />
increased pregnancy rates than cows subjected only to NM.<br />
Keywords: ecg, body score condition, zebu breed.<br />
s375
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A079 FTAI, FTET AND AI<br />
OCCURRENCE CURRENCE OF ESTRUS AFTER SYNCHRONIZA<br />
ONIZATION IMPROVE<br />
THE USE OF SEX SEMEN IN TIMED ARTIFICIAL INSEMIANTION<br />
PROTOCOL<br />
Pietro Sampaio Baruselli 1 , Eric K. Abe 2 , Rodrigo Vasconcellos Sala 3 , Marcílio Nichi 4 , Evanil Pires Campos Filho 5 & Manoel Francisco Sá Filho 6<br />
1,3,4,6<br />
UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 2 AGROPECUÁRIA ABE, ANANÁS, TO, BRAZIL. 5 SEXING TECHNOLOGIES, SERTÃOZINHO, SP, BRAZIL.<br />
Previous studies have demonstrated that lactating Nelore cows displaying estrus after synchronization of ovulation protocol<br />
have higher risk of pregnancy than those cows that did not display estrus. The objective of present study was evaluate if the occurrence of<br />
estrus following the synchronization can improve the use of sex sorted (SX) sperm in the timed artificial insemination (TAI) protocols. A total<br />
of 491 multiparous Nelore (Bos indicus) cows were treated with P4 device (Sincrogest ® , Ouro Fino Animal Health) and 2mg of estradiol<br />
benzoate i.m. (BE, Sincrodiol ® , Ouro Fino Animal Health). Eight days later, the P4 devices were removed and were administrated i.m. PGF2á<br />
(SINCROCIO ® , Ouro Fino Animal Health) and 300 IU of equine chorionic gonadotropin (eCG; Folligon ® , Intervet Shering-Plough). The<br />
ovulation was induced using 1 mg EB i.m. 24 h after the P4 device removal. Estrus was determined by using an adhesive detection of estrous<br />
aid (Estrotect, IVP, Spring Valley). At P4 device removal, cows received the detection of estrus aid, placed between the hips and the tail head.<br />
Estrus was determined at TAI by the activation of each device. Immediately before the TAI cows were reassigned to receive SX (2×106 sperm<br />
cells/ insemination dose) or CON (20x106 sperm cells/insemination dose) semen according the occurrence of estrus. Two Nelore bulls had<br />
previously proved satisfactory pregnancy outcomes in TAI programs were used in all inseminations. The ejaculates from each bull were<br />
equality distributed to obtain similar amount of non-sex sorted and sex sorted sperm doses. The overall occurrence of estrus was 74.7% (367/<br />
491). There was no interaction (P = 0.87) between type semen and the occurrence of estrus on conception rate [No Estrus - CON = 43.6%,<br />
27/62; Estrus - CON = 58.5%, 107/183; No Estrus - SX = 33.9% and 21/62; Estrus - SX = 50.0%; 92/184]. The use of SX semen (45.9%, 113/<br />
246) resulted lower conception rate (P = 0.05) than the use of CON semen (54.7%, 134/245). In addition, cows displayed estrus had higher<br />
(P = 0.003) conception rates (54.2%, 199/367) than those cows that did not display estrus (38.7%, 48/124).Therefore, the occurrence of<br />
estrus after synchronization protocol improves the efficiency of TAI programs in lactating Nelore cows, regardless of the type of semen. It<br />
is suggested that this information can be used to improve the use of sexed semen in TAI programs. [Acknowledgements: Sexing Technologies;<br />
Ouro Fino Saúde Animal; Agropecuária Abe].<br />
Keywords: Bos indicus, ftai, reproductive efficiency.<br />
A080 FTAI, FTET AND AI<br />
THE USE OF BIOABOR<br />
ABORTOGEN<br />
OGEN ® AND BIOLEPTOGEN<br />
OGEN ® VACCINES CINES MAY IMPROVE REPRODUCTIVE EFFICIENCY OF BEEF COWS<br />
Marcos Sivieri 1 , Roberta Machado Ferreira 2 , Danilo Paik Borges 3 , Thiago Silva Antonio 3 , Reuel Luiz Gonçalves 3 & Pietro Sampaio Baruselli 2<br />
1<br />
VETERINÁRIO AUTÔNOMO, SACRAMENTO, MG, BRAZIL. 2 FMVZ-USP, SÃO PAULO, SP, BRAZIL. 3 BIOGÊNESIS-BAGÓ, CURITIBA, PR, BRAZIL.<br />
The aim of the present study was to evaluate the effect of the vaccines Bioabortogen ® and Bioleptogen ® on pregnancy rate (30<br />
and 60 days after insemination) and pregnancy loss (between 30 and 60 days of pregnancy) of beef cows fixed time artificial inseminated<br />
(FTAI). The study was conducted during the reproductive season in three commercial farms in Sacramento and Presidente Olegário cities,<br />
state of Minas Gerais, Brazil. On random days of the estrus cycle, post partum Nelore (Bos indicus) cows (n = 268) received a progesteronereleasing<br />
intravaginal device associated to 2 mg of estradiol benzoate. Also, blood samples were taken from the coccigea vein of a subset of<br />
cows (n = 18) of all farms. They were used to do a serological investigation* to infectious bovine rhinotracheitis virus (IBRV), bovine viral<br />
diarrhea virus (BVDV) and Leptospirosis. After eight days the device was removed and 150 µg of D-cloprostenol, 300 IU of eCG and 1 mg<br />
of estradiol cypionate IM were administered. Following two more days, the cows were homogenously allocated to one of two experimental<br />
groups (vaccinated cows and nonvaccinated control cows) and inseminated. Vaccinated cows received 5 mL of Bioabortogen ® + 5 mL of<br />
Bioleptogen ® SC, and control cows were administered two doses of 5 mL of saline solution as placebo. Vaccine booster was given 30 days after<br />
the first dose (30 days following FTAI). Ten days after FTAI, cows were exposed to clean-up bulls. Pregnancy rate and pregnancy loss were<br />
analysed with PROC GLIMMIX from SAS. The serological profile revealed high infection rate for the three pathogens: 100% of positive<br />
cows for BVD, 62% for IBR and 54% for Leptospirosis. No interations among farms were found. The 30 days pregnancy rate was greater<br />
in vaccinated cows [42.7% (58/136) vs. 53.8% (71/132); P = 0.05]. The 60 days pregnancy rate [41.2% (56/136) vs. 51.5% (68/132); P =<br />
0.09] and the rate of pregnancy following FTAI plus the first service of clean-up bull [62.5% (85/136) vs. 72.7% (96/132); P = 0.06] tended<br />
to be greater in vaccinated cows compared to nonvaccinated control cows. However, pregnancy loss was unaffected by vaccination [3.5% (2/<br />
42) vs. 4.2% (3/42); P =0.93]. The results are suggestive that it is important to adopt control procedures and prevention strategies against IBR,<br />
BVD and Leptospirosis, including the performance of vaccination programs at farms where these pathogens have high percentage of<br />
occurrence. [Acknowledgments: Farms Manancial, São Judas e Bela Vista].<br />
Keywords: bovine, vaccine, reproductive deseases.<br />
s376
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A081 FTAI, FTET AND AI<br />
EVAL<br />
ALUATION OF ARTIFICIAL INSEMINATION IN GIROL<br />
OLANDO HEIFERS WITH SEXED SEMEN<br />
Marcio Ferreira Mendanha, , Silvia Ferrari & Daniela Stockmann Silva<br />
UNIVERSIDADE ANHEMBI-MORUMBI, SÃO PAULO, SP, BRAZIL.<br />
The objective of this experiment was to compare three protocols of estrus synchronization and artificial insemination with sexed<br />
semen and also evaluate the use of artificial insemination in the uterine horn. Virgin Girolando heifers (n = 45) selected based on their body score<br />
(2,5 – 3,5) weight (2/3 of adult weight) and age (18 – 24 months) were enrolled in this study. The cyclicity of heifers was confirmed through<br />
trans-rectal ultrasound exams. The animals were divided in three groups of 15. The cows in AIEstr treatment ( AIEst- artificial insemination on<br />
estrus) received 2 mL of Cloprostenol on day one and on day 11. Observation of estrus occurred three times a day and the artificial insemination<br />
was done 12 h after observation of estrus. The AITF(artificial insemination on fixed time) protocol was provided by Ourofino company. Cows<br />
enrolled in the AITF treatment received an intravaginal device with 1g of progesterone and 2 mL of benzoate of estradiol on day zero. After 8 days<br />
the devices were withdrawn and 400 UI of eCG and 0,5mL of ciprionato of estradiol were administered on the animals. On day 10, 2,5 mL of<br />
GnRH were administered and the cows were inseminated. Cows enrolled in the AITFUS treatment received injections of 2 mL of clorprostenol<br />
until the follicles reached 14mm, at which time cows received 2,5 mL of GnRH, followed 4 h later by artificial insemination deep in the<br />
ipsilateral horn to the ovary with the largest follicle. Similar pregnancy rates were observed at 45d after AI (AIEst =55%, AITF=40%, and<br />
AITFUS= 55%). Cows enrolled in the AIEstr treatment showed more variations within the estrus. In conclusion, the pregnancy rates were<br />
similar between treatments and acceptable pregnancy rates were obtained in all of them. The technique that uses GnRH hormone as ovulation<br />
inductor with artificial insemination in the uterine horn might be used obtaining pregnancy rates similar to the other tested protocols.<br />
Keywords: sexed semen, girolando heifers, ai.<br />
A082 FTAI, FTET AND AI<br />
EVAL<br />
ALUATION OF DIFFERENT DOSES OF ECG IN THE TAI CONCEPTION RATE IN CYCLING CLING NELORE HEIFERS SYNCHRONIZED<br />
WITH INTRAVAGINAL PROGESTER<br />
OGESTERONE ONE DEVICE PREVIOUSL<br />
VIOUSLY USED FOR 9 DAYS.<br />
Everton Rodolfo Carvalho 1 , Adnan Darin Pereira Rodrigues 2 , Thiago Martins 2 , Hugo Borges Graff 1 , Rogério Fonseca Guimarães Peres 1 & Amanda Prudêncio<br />
Lemes 3<br />
1<br />
AGROPECUÁRIA FAZENDA BRAZIL, BARRA DO GARÇAS, MT, BRAZIL. 2 UNESP, BOTUCATU, SP, BRAZIL. 3 ESALQ-USP, PIRACICABA, SP, BRAZIL.<br />
The TAI in beef cows has equivalents results to conventional AI, however, in nelore heifers the results with the TAI are lower<br />
when compared to conventional AI (Dias, C. C. et al., 2009, Theriogenology, 72:378-385). The use of eCG increase the diameter of the N<br />
dominant follicle, ovulation rate, P4 concentration after TAI and conception rate in heifers synchronized with intravaginal progesterone<br />
implant (CIDR ® , Pfizer Animal Health, Brazil) (Peres et al., 2009, Theriogenology, 72:681-689) and norgestormet ear implant (Crestar ® ,<br />
Intervet Schering Plough) (Sá Filho et al., 2010, Anim Reprod Sci, 118, 182-187). The aim of this study was determine the effect of the dose<br />
of eCG in the conception rate in Nelore heifers synchronized with CIDR of second use. Heifers (n = 626) with average body condition score<br />
(BCS) of 2.92, were used. The heifers was considered cycling when observed presence of CL in one of two ultrasound (Mindray -DP<br />
2200VET) evaluation on D-7 and D0 of the TAI protocol. All heifers receive the first day (D0) intravaginal progesterone implant (CIDR ® ,<br />
Pfzier Animal Health, Brazil) and 2 mg estradiol benzoate (BE), after seven days (D7) 12,5 mg dinoprost trometamine (PGF2α, Lutalyse ® ,<br />
Pfizer Saúde Animal, Brazil). On day nine (D9) 0.5 mg estradiol cypionate (ECP ® , Pfzier Animal Health, Brazil) and implant withdrawal, and<br />
TAI was performed 48hs after (D11). On D9, the heifers were grouped randomly to receive 100 IU (n = 160), 200 IU (n = 158), 300 IU (n<br />
= 152) or 400 IU (n = 156) of eCG. The datas were analized per logistic regression using PROC LOGISTIC in SAS ® . in SAS. There was no<br />
effect of eCG (P = 0,9479) in the conception rate when utilized 100(38,75%), 200(36,08%), 300(36,84%) and 400 (37,18%) IU. This results<br />
shown that even using higher doses of eCG, this has no effect on conception rate in pubertal Nellore heifers synchronized with CIDR<br />
previously used for 9 days.<br />
Keywords: tai, ecg, heifers.<br />
s377
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A083 FTAI, FTET AND AI<br />
EVAL<br />
ALUATION OF THE EFFECTS OF HEAT STRESS ON REPRODUCTIVE PERFORMANCE OF NELORE HEIFERS DURING THE<br />
BREEDING SEASON<br />
Milt<br />
ilton Matur<br />
turana Filho<br />
1 , Namíbia Apar<br />
parecida<br />
Teix<br />
eixeir<br />
eira 2 , Saar<br />
aara a Car<br />
arolina Scolar<br />
olari 3 , Aline Kehrle<br />
4 , Patrícia Helena Paiv<br />
aiva a Migue<br />
iguez 5 , Thiago San<br />
antin<br />
6 & Ed Hoffman<br />
Madureira 7<br />
1,2,3,4,5,6,7<br />
USP, PIRASSUNUNGA, SP, BRAZIL. 2,3 USP, PIRASSUNUNGA, SP, BRAZIL.<br />
Many studies involving the effects of heat stress have been developed in dairy cattle, but a few has been discussed this issue in<br />
slaughtering cattle. The aim of this study was to evaluate the effects of heat stress on reproductive performance of Bos indicus beef heifers during<br />
the breeding season. The experiment was conducted from November 2009 to January 2010. We had been using 102 heifers of Nellore, cyclic,<br />
with 24 months. To TAI was used semen of one bull of the Nelore breed. The animals were maintained on Brachiaria brizantha, with water and<br />
mineralization ad libitum. At the beginning of the protocol were evaluated parameters of body condition score (BCS, also evaluated at TAI and<br />
pregnancy diagnosis (PD). Rectal temperature (RT) and temperature and humidity index (THI) were evaluated at TAI. The THI is calculated from<br />
the model defined by Thom (1959) were: THI (0.8 x T ° C + (RH (%) / 100) x (T-14, 4) + 46.4) where: T = temperature °C, RH = relative<br />
humidity. The climate data used were obtained from the Meteorological Station of USP. The following hormonal protocol was used: D0 =<br />
implant insertion vaginal P4 + application of 2 mg EB, D8 = implant removal vaginal P4 + application of 300 IU of eCG, according to the body<br />
score + 25 mg D-Cloprostenol; application 1mg D9 = BE; D10 = TAI. The heifers were retrospectively separated into groups according to the<br />
THI at the time of TAI, which is equivalent to: Class 1 (69-74), Class 2 (75-79) and Class 3 (80-84), the latter being characterized by conditions<br />
more extreme temperature and humidity. The pregnancy rate (PR) was evaluated by ultrasonography (Aloka 500 with 5.0 MHz linear probe) on<br />
days 30, 60 and 90 days after TAI, and separated according to the THI and RT classes. The data were subjected to frequency analysis using<br />
PROC FREQ and logistic regression analysis by PROC LOGISTIC, using the Statistical Analysis System (SAS, 9.2) by adopting a significance<br />
level of 5%. The rate of pregnant at 30 days of the heifers which were in the condition for a THI 1 was 57.8% (n = 19), for heifers 2 of THI was<br />
57.7% (n = 38) and THI was 3 28.9% (n = 45). So, there was a reduction (P < 0.01) the pregnancy rate for heifers in the third class of THI. Thus,<br />
the fertility of heifers may be affected by the effects of heat stress.<br />
Keywords: heat stress, temperature and humidity index, nelore heifers.<br />
A084 FTAI, FTET AND AI<br />
THE CHARACTERISTICS OF THE FOLICULAR DYNAMICS OVER THE DIAMETER OF THE CORPUS LUTEUM IN<br />
FIXED TIME EMBRYO TRANSFER RECIPIENTS<br />
A n tônio De Lisboa Rib<br />
ibeir<br />
eiro Filho<br />
1 , Br uno Henr<br />
enrique De Ar aújo Andr<br />
ndrade<br />
2 , Tess Picq Coutinho<br />
3 , Leandr<br />
eandro Mour<br />
oureir<br />
eira De Castr<br />
astro<br />
Feitosa 4 , Marcus Vinícius Galvão Loiola 5 , Alexandra Soares Rodrigues 6 , Priscila Assis Ferraz 7 , Marcos Chalhoub 8 , Endrigo Adonis<br />
B r aga De Ar aujo 9 , Sidnei Nunes Oliv<br />
liveir<br />
eira 10 , Mar<br />
arc os Rib<br />
ibeir<br />
eiro 11 & Yeda Fumie<br />
Wa tanabe 12<br />
1,2,3,4,5,6,7,8,9,10<br />
ESCOLA DE MEDICINA VETERINÁRIA - UFBA, SALVADOR, BA, BRAZIL. 3,11 UNIME, LAURO DE FEITAS, BA, BRAZIL. 12 VITROGEN PESQUISA E DESENVOLVIMENTO EM<br />
BIOTECNOLOGIAS DE REPRODUÇÃO LTDA, CRAVINHOS, SP, BRAZIL.<br />
A positive correlation between the preovulatory follicle and the diameter of the corpus luteum (CL) in cows submitted to fixed time<br />
artificial insemination (FTAI) protocols has been verified (Andrade et al., <strong>2011</strong>, Congresso Brasileiro de Reprodução Animal 19, 116). Our<br />
objectives were to evaluate the dominant follicle growth characteristics with the diameter of the CL in embryo recipients heifers submitted to<br />
ovulation synchronization protocols. For this purpose 12 Bos taurus taurus x Bos taurus indicus heifers were used. Heifers were treated with<br />
the following protocol: the animals received an intravaginal progesterone device (P4 - CIDR ® , Pfizer, São Paulo, Brazil), 2mg of Estradiol<br />
Benzoate i.m. (EB - Gonadiol ® , Shering-Plough, São Paulo, Brazil) and a part of animals were treated with 250µg of Cloprostenol i.m.<br />
(Sincrocio ® , Ouro-Fino, São Paulo, Brazil) on the day named D0. On day 8 (D8) the P4 was removed followed by the application of 300IU of<br />
eCG (Novormon ® , Shering-Plough, São Paulo, Brazil), 250µg Cloprostenol i.m. The animals had ovulation induced with 1 mg of EB or 1 mg<br />
of Estradiol Cypionate i.m. (ECP ® , Pfizer, São Paulo, Brazil) on day 8 or 1 mg of EB i.m. on Day 9. On D10 the examinations started by<br />
transrectal ultrasound at 6 hour intervals, for the following 70 h in order to follow up on the preovulatory dynamics. The CL diameter was<br />
evaluated by the same technique seven days after the first ultrasound evaluation (D17). The PROC CORR resource from SAS ® software was<br />
used for statistical analysis of the correlation between the results (P < 0.05). The average diameter of the follicles 48h after P4 removal (FOL-<br />
D10), of the preovulatory follicle (FOLPRE) and of the CL were 10.71±3.44, 12.53±2.95 e 17.55±1.86mm, respectively. The diameter of the<br />
FOL-D10 presented strong negative correlation with the growth rate (R=0.70, P = 0.02) and strong positive correlation with the preovulatory<br />
diameter (R=0.84, P = 0.0007). Although, the diameter of the preovulatory follicle did not present correlation to the CL diameter. These results<br />
show that the smaller the diameter of the FOL-D10, the larger its growth rate. Nevertheless, the diameter of the FOLPRE was not correlated with<br />
the diameter of the CL, making it necessary that more studies are developed to elucidate these and other questions exposed in this study.<br />
Keywords: cl, ftet, recipients.<br />
s378
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A085 FTAI, FTET AND AI<br />
FOLLICULAR FEATURES IN MURRAH,<br />
MEDITERRANEAN AND CROSSBRED BUFF<br />
UFFAL<br />
ALO HEIFERS SUBMITTED<br />
TED TO EXOGENOUS<br />
CONTR<br />
ONTROL OL OF OVULATION<br />
Alexandre Rossetto Garcia 1 , Geanne Rocha Silva 2 , Benjamim De Souza Nahúm 3 , Jakeline Dos Santos Pessoa 4 , Arnaldo Algaranhar Gonçalves 5 & Alessandra<br />
Ximenes Santos 6<br />
1,3<br />
EMBRAPA AMAZÔNIA ORIENTAL, BELEM, PA, BRAZIL. 2,5,6 UFPA/EMBRAPA/UFRA, BELÉM, PA, BRAZIL. 4 UNIVERSIDADE FEDERAL RURAL DA AMAZÔNIA, BELÉM, PA, BRAZIL.<br />
Ultrasound studies about follicular development in buffaloes are important because they can enhance ovarian physiological<br />
knowledge, and enable increasing the efficiency of estrus synchronization protocols and fixed-time artificial insemination (FTAI). This study<br />
aimed to evaluate the effect of racial characteristics on ovarian dynamics and conception rates of buffalo heifers submitted to Ovs.ynch protocol.<br />
Twenty one cycling females presenting corpus luteum and/or development of follicular structures on ultrasonographic scanning were used.<br />
Females were divided in Murrah Group (n = 7; 2.52±0.43 years and 359.7±76.1 kg), Mediterranean Group (n = 9; 2,32±0,33 years and<br />
328.6±65.5 kg) and Crossbred Group (n = 5; 2.24±0.35 years and 348.6±60.9 kg) and they were synchronized using Ovsynch (100 µg IM<br />
Lecirelin on D0 and D9/ 0.530 mg IM cloprostenol on D7). Ovarian structures were scanned and classified by ultrasonography at D0, D7 and<br />
D9, according to their diameter (small:
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A087 FTAI, FTET AND AI<br />
COMP<br />
OMPARISON OF THE CONCEPTION RATES OF NELORE LACT<br />
CTATING TING COWS SUBMITTED<br />
TED TO FTAI AND TREATED<br />
TED WITH<br />
PORCINE PITUITAR<br />
ARY EXTR<br />
TRACT AND EQUINE CHORIONIC GONADOTR<br />
TROPIN<br />
Suellen Migue<br />
iguez Gonzále<br />
onzález 1 , Jeff<br />
efferson<br />
erson Tadeu Camp<br />
ampos<br />
2 , Wanessa Blaschi<br />
3 , Thales Ric<br />
icar<br />
ardo Rigo Bar<br />
arreir<br />
eiros<br />
4 & Mar<br />
arcelo Mar<br />
arcondes Seneda<br />
5<br />
1<br />
UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ - CAMPUS LUIZ MENEGUEL, BANDEIRANTES, PR, BRAZIL. 2,5 UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA, PR,<br />
BRAZIL. 3,4 UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL.<br />
The objective of this trial was to evaluate the effect of different gonadotrophin treatment in lactating Nelore cows submitted to FTAI.<br />
Nelore cows (n=457) were used with 30 to 45 days postpartum and body condition score ranging from 3.0 to 3.5 (range = 1-5 ECC) in the region<br />
of Iby Yau, Paraguay. The animals were divided intro three groups treatments basead on body condition and postpartum period. In the first group<br />
(G-C, n = 128), the animals received an intravaginal device containing 1.9 g progesterone (CIDR ® , Pfizer, Brazil) and an application of 2 mg<br />
of estradiol benzoate (EB), (Estrogin ® , Farmavet, Brazil), intramuscularly (IM). On the eighth day the devices were removed 150µg of<br />
cloprostenol (Veteglan ® , Hertape-Calier, Brazil)was injected intramuscularly and 1 mg of estradiol cypionate (ECP, Pfizer ® , Brazil). In the<br />
second group (G-EHS, n = 154), cows were treated similarly to G-C, were administered 50 UI of porcine pituitary extract (Pluset ® , Hertape-<br />
Calier, Spain) intramuscularly, simultaneous CIDR ® removal. In the third group (G-eCG, n = 154), cows were treated similarly to the G-C, and<br />
administered 300 IU of equine chorionic gonadotropin (Novormon ® , Schering-Intervet, Brazil), intramuscularly, simultaneous CIDR removal.<br />
All animals were inseminated after 48 to 52 h of CIDR removal. Pregnancy diagnosis was conducted at 30 days after artificial insemination by<br />
transrectal ultrasonography (Aloka SSD 500, 5 mHz). The results were analyzed by chi-square test (P < 0.05). The conception rates for the GC,<br />
G-G-EHS and eCG were respectively (22.6%, 29/128) (51.9%, 80/154) (50.8%; 89/172) (P > 0.05). From the observed results, it is concluded<br />
that equine chorionic gonadotropin showed conception rates comparable to those obtained using porcine pituitary extract, becoming an option<br />
in the composition of FTAI protocols in Nelore cows in postpartum period.<br />
Keywords: fsh, ftai, nelore.<br />
A088 FTAI, FTET AND AI<br />
COMP<br />
OMPARISON OF RATES OF CONCEPTION AND PREGNANCY LOSSES IN DIFFERENT YEARS IN NELORE COWS<br />
UNDER FTAI<br />
VACCINA<br />
CINATED OR NOT AGAINST BVD<br />
VDV V AND BOHV-1<br />
Thales Ricardo Rigo Barreiros 1 , Armando Alfonso De Oliveira Friedel 2 , Robson Augusto Fagundes Stellato 3 , Suellen Miguez<br />
González 4 , Wanessa Blaschi 5 & Luiz Henrique Aguilera Turrussi 6<br />
1,2,4,5,6<br />
UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL. 3 LABORATÓRIOS PFIZER, SAÚDE ANIMAL, BANDEIRANTES, PR, BRAZIL. 6 UNIVERSIDADE<br />
ESTADUAL DO NORTE DO PARANÁ, LONDRINA, PR, BRAZIL.<br />
The aim of this study was a retrospective analysis of cows vaccinated and unvaccinated against bovine herpes virus type 1 (BoHV-<br />
1) and bovine viral diarrhea virus by comparing conception rates and pregnancy loss in cows receiving FTAI. The study was conducted in two<br />
farms located in Santa Amelia and Congoinhas -PR in-breeding seasons 2008/2009, 2009/2010 and 2010/<strong>2011</strong>. We used 1070 lactating Nelore<br />
cows with 30 to 60 days postpartum and body condition score between 2,5 and 3,0. The animals received an intramuscular (IM) 2 mg of estradiol<br />
benzoate (Estrogin ® , Farmavet, Brazil) and an intravaginal device containing 1,9 g progesterone (CIDR ® , Pfizer, Brazil). Seven days later he was<br />
administered 12,5 mg of IM dinaprost (Lutalyse ® , Pfizer, Brazil). The devices were removed 48 h later, simultaneously application of 400 UI<br />
of eCG (Novormon ® , Schering-Intervet, Brazil) and 1 mg of estradiol cypionate (ECP ® , Pfizer, Brazil). The FTAI was performed 48 to 52 h after<br />
withdrawal of CIDR ® , with semen from two bulls in a manner deemed in each property. Pregnancy diagnosis was performed by transrectal<br />
ultrasonography 30 and 60 days of gestation and pregnancy loss were defined by differences in conception rates. Cows in the breeding season<br />
2008/2009 (G-2008, n = 267) were analyzed only the pregnancy loss. In the 2009/2010 breeding season, 328 cows were treated so as to body<br />
condition score and postpartum period in two groups or not vaccinated against BVDV and BoHV-1 (Cattle Master ® , Pfizer, Brazil), (G-V, n =<br />
155, G-NV = 173, respectively). In the 2010/<strong>2011</strong> breeding season, 475 cows were vaccinated against BVDV and BoHV-1. The results were<br />
compared by chi-square test (P < 0,05). The conception rates for 2008/2009 breeding season were 53,1% (142/267) and 50,5% (135/267),<br />
respectively at 30 and 60 days of gestation, resulting in 4,9% (7 / 142) of pregnancy loss. The conception rates for 2009/2010 breeding season<br />
were higher (P < 0,05) at 30 and 60 days of gestation for the G-V 49%, (76/155), 46,4% (72/155) compared to G -NV 32,9%, (57/173), 30,6%<br />
(53/173). The pregnancy loss were similar (P > 0,05) for the G-V 5,2%, (4 / 76) and G-NV 7%, (4 / 57). The conception rates for 2010/<strong>2011</strong><br />
breeding season were 60,6% (288/475) and 59,3% (282/475), respectively 30 and 60 days gestation, resulting in 2,0% (6 / 288) of pregnancy<br />
loss. From the results it is concluded that vaccination increased the conception rate in cows subjected to FTAI, but there was no reduction in<br />
pregnancy loss.<br />
Keywords: pregnancy losses, ftai, vaccine.<br />
s380
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A089 FTAI, FTET AND AI<br />
CONCEPTION RATES IN POSTPAR<br />
ARTUM NELORE COWS TREATED<br />
TED WITH ECG BEFORE AND/OR AFTER FIXED-TIME ARTIFICIAL<br />
INSEMINATION<br />
Amanda Prudêncio Lemes 1 , Adnan Darin Pereira Rodrigues 2 , Rogério Fonseca Guimarães Peres 3 , Hugo Borges Graff 4 , Everton Rodolfo Carvalho 5 , Alexandre<br />
Henryli Souza 6 & Roberto Sartori 7<br />
1,7<br />
UNIVERSIDADE DE SÃO PAULO, ESCOLA SUPERIOR DE AGRICULTURA, PIRACICABA, SP, BRAZIL. 2 UNIVERSIDADE ESTADUAL PAULISTA, BOTUCATU, SP, BRAZIL.<br />
3,4,5<br />
AGROPECUÁRIA FAZENDA BRAZIL, BARRA DO GARÇAS, MT, BRAZIL. 6 DEPARTMENT OF DAIRY SCIENCE, UNIVERSITY OF WISCONSIN-MADISON, MADISON, ESTADOS UNIDOS.<br />
The objective of this study was to evaluate synchronization and conception rates in postpartum Nelore cows treated with eCG before<br />
and/or after TAI. Multiparous cows with body condition score (BCS) of 3.03±0.03 (scale from 1 to 5) and kept at pasture were used. Cyclicity<br />
status was assessed based on the presence of corpus luteum detected by ultrasound evaluation in a sub sample of these animals (n = 100). An<br />
incidence of 90% of anestrus was detected at the beginning of treatments at 34.4±0.3 days postpartum. In order to synchronize ovulation for TAI,<br />
the following protocol was used: D0 - intravaginal progesterone device (DIB ® , Syntex S.A., Argentina), 2 mg estradiol benzoate (Gonadiol ® ,<br />
i.m., Syntex S.A.) and 12.5 mg dinoprost tromethamine (Lutalyse ® , i.m., Pfizer Animal Health); D8 - 12.5 mg dinoprost tromethamine, 0.8 mg<br />
estradiol cypionate (ECP ® , i.m., Pfizer Animal Health) and withdrawal of DIB; D10 - AI using semen from three bulls equally distributed<br />
among treatments. Cows were grouped randomly to receive 300 IU eCG (Novormon 5000 ® , Syntex S.A.) on D8 of the protocol (n = 227),<br />
on D24 of the protocol (14 days after AI, n = 229), on D8 and D24 (n = 224), or not to receive eCG (Control, n = 224) in a 2x2 factorial design.<br />
The DIB was used three times and the reuses were made in a balanced way among groups. Ultrasound evaluations were performed on days<br />
7, 30 and 60 after AI. Data were analyzed using PROC GLIMMIX of SAS ® , with the experimental unit “cow” treated as a random variable.<br />
The means are presented as least squares ± SE. When cows were treated with eCG 14 days after AI, there was no improvement in conception<br />
at 30 (41.6±3.7% vs. 42.7±3.6%, P = 0.75) or 60 days (39.8±3.7% vs. 40.2±3.6%, P = 0.93). However, although the use of eCG on D8 of<br />
the protocol did not significantly improve conception rate at 30 days (44.3±3.7% vs. 40.1±3.6%, P = 0.11), this treatment resulted in a greater<br />
synchronization rate (90.9±1.4% vs. 80.5±1.9%, P < 0.0001) and conception rate at 60 days after AI (42.9±3.7% vs. 37.2±3.6%, P = 0.045).<br />
An effect of ECC in the synchronization rate and conception at 30 and 60 days was also observed (P < 0.05). Cows with lower BCS (
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A091 FTAI, FTET AND AI<br />
EFFECT OF NEW AND REUSED VAGINAL INSERTS S ON PLASMA PROGESTER<br />
OGESTERONE ONE PROFILE AND FOLLICULAR SIZE AT THE END<br />
OF PROGESTER<br />
OGESTERONE ONE AND PROST<br />
OSTAGL<br />
GLANDIN F2ALPHA ESTROUS SYNCHRONIZA<br />
ONIZATION<br />
TREATMENT IN BEEF COWS<br />
Evelyn N Lara 1 , Andrea F Leiva 2 , Carolina Baeza 3 , Alfredo Rodríguez 4 , Pedro Rojas 5 , Rodrigo V Allende 6 , Fernando R Saravia 7 & José Francisco Cox 8<br />
1,2,3,4,5,6,7,8<br />
UNIVERSIDAD DE CONCEPCIÓN, CONCEPCIÓN, CHILE.<br />
The aims of this study were to assess plasma progesterone (P4) profiles in beef cows with new and used CIDR and DIB and to<br />
assess whether those inserts can affect follicular size at the end of 7-day treatments in cycling cows. The study used 210 cows, = 2.5 points BCS,<br />
with CL detected by ultrasonography (7.5 MHz probe, Aloka 500D, Japan), > 60 days post partum and under controlled feeding. In Exp. 1, 24<br />
cows received 0.5 mg Cloprostenol (Estrumate ® , Schering-Plough) and 48 h later, a new CIDR (1.38 g P4, CIDR B ® , Pfizer; n = 5) or DIB (1,0<br />
g P4, DIB ® , Syntex; n = 5) and an 8-day used CIDR (n = 7) or DIB (n = 7). Blood samples were collected daily into heparinized tubes to collect<br />
plasma that were stored at -20ºC until assayed. P4 was measured by solid phase RIA (Coat a count ® , DPC). In Exp. 2, 98 cows were grouped<br />
at random and received a new (n = 57) or a reused CIDR (n = 41) for 7 days and ovarian scanning was used to measure follicular diameters as<br />
before. In Exp. 3, 88 cows were grouped randomly and received a new CIDR without (w/o, n = 38) or plus 2 mg estradiol benzoate (BE,<br />
Syntex; n = 24) and a 7-day used CIDR w/o (n = 13) or plus 2 mg BE at insertion (n = 13). CIDRs were maintained by 7 d when ovaries were<br />
scanned to measure the largest follicles. Results were analyzed by 2x2 factorial ANOVA and by the Student’s t test. In Exp 1, cows with new<br />
inserts showed similar P4 profiles up to day 7 (P > 0.10) that differed from those carrying used inserts in the amount of P4 released in the first<br />
3 days and in day 7 (P < 0.05); when P4 profiles were compared between cows with used inserts they showed similar results (P > 0.10),<br />
except for day 7, where cows with CIDR showed higher concentrations (1.9±0.2 vs.. 1.1±0.2 ng/ml; P < 0.05). In Exp 2, new CIDR reduced<br />
follicular diameter at day 7 compared to used CIDR (11.5±0,45 vs..12.9±0.47 mm for new and used respectively; P < 0,02); in Exp 3, 2 mg<br />
BE at insertion did not affect follicular diameter in cows with new inserts (11.1±0.4 vs.. 10.9±0.4 mm for CIDR plus and wo BE, P = 0.40),<br />
but tended to reduce those with used CIDR (12.4±0.4 and 11.4±0.6 for groups plus or wo BE respectively; P = 0.89). Results suggest that<br />
P4 released by vaginal inserts affects follicular diameter at the end of treatment, but the treatment length can by standardize by the addition<br />
of 2 mg BE at insertion. In addition, only new DIB inserts are reliable to be used in estrous synchronization protocols for AIFT.<br />
Keywords: iatf, estrous synchronization, follicular diameter.<br />
A092 FTAI, FTET AND AI<br />
DOSE EFFECT OF ESTRADIOL BENZOATE ASSOCIATED<br />
WITH PROGESTER<br />
OGESTERONE ONE ON THE SYNCHRONIZA<br />
ONIZATION OF FOLLICULAR<br />
WAVE EMERGENCE IN BOS INDICUS AND BOS TAUR<br />
URUS<br />
US COWS<br />
Michele Ricieri Bastos 1 , Ricardo Silva Surjus 2 , Alexandre Barbieri Prata 3 , Murillo Alves Porto Meschiatti 4 , Marta Borsato 5 , Gerson Barreto Mourão 6 , Alexandre<br />
Mendonça Pedroso 7 , Alexandre Vaz Pires 8 & Roberto Sartori 9<br />
1<br />
FMVZ, UNESP, BOTUCATU, SP, BRAZIL. 2,3,4,5,6,8,9 ESALQ, USP, PIRACICABA, SP, BRAZIL; 7 EMBRAPA PECUÁRIA SUDESTE, SÃO CARLOS, SP, BRAZIL.<br />
The effect of dose of estradiol benzoate (EB: 1, 2 and 4 mg) associated with progesterone (P4) on the synchronization of follicular<br />
wave emergence was evaluated in nonlactating Nelore (n = 13) and Holstein (n = 11) cows receiving a maintenance diet (NRC, 2000). The body<br />
condition score and body weight were kept at 3.5±0.1 and 3.0±0.2 (scale of 1 to 5) and 535±14 and 600±23 kg for Nelore and Holstein cows,<br />
respectively. Two doses of PGF2α (530 µg, im, Sincrocio, Ourofino, Brazil) were given 11 days apart, and simultaneously with the second<br />
PGF2α, cows were treated with an intravaginal P4 device, which remained for 10 days (Sincrogest, Ourofino), and EB (im, Sincrodiol,<br />
Ourofino). There were three replicates in a Latin Square design. Ovarian dynamics was monitored daily by means of ultrasonography using a<br />
7.5 MHz linear transducer for 10 days after EB treatment. Only cows with follicular wave emergence synchronized by the protocol were<br />
included in the statistical analysis, ie, when the emergency occurred between 1 and 6 days after treatment with EB+P4. The results were analyzed<br />
using PROC GLIMMIX of SAS and are presented as least squares means ± SE. The follicular wave emergence was not synchronized in 2.5%<br />
(1/39) of the Nelore cows, and this one was treated with 1 mg of EB. In Holsteins, 15.1% (5/33) had no wave emergence synchronized, and three<br />
cows received 1 mg and the other two, 2 or 4 mg of EB. The follicular wave emergence occurred 3.0±0.3, 3.3±0.1 and 3.8±0.2 days, after<br />
treatment with 1, 2 and 4 mg of EB, respectively (P = 0.03), independent of breed, although plasma concentrations of estradiol 24 h after treatment<br />
with EB was higher (P < 0.05) in Nelore cows (1 mg: 46.5±3.6 vs.. 26.0±2.4; 2 mg: 88.4±5.2 vs.. 40.6±5.0 and 4 mg: 152.6±11.5 vs.. 68.9±4.2<br />
pg/mL). The diameter of the greatest follicle 9 days after treatment was 12.5±0.7, 11.3±0.7 and 10.4±0.7 mm in cows that received 1, 2 and 4<br />
mg of EB (P = 0.09). At follicular wave emergence, the number of 2 to 5 mm follicles present in the ovaries was higher in Nelore (25.0±4.4) than<br />
in Holstein cows (13.2±2.7; P = 0.03). Furthermore, follicle deviation occurred, on average, 3.0±0.2 days after wave emergence, independent<br />
of breed, when the diameter of the greatest follicle reached 7.3±0.4 and 9.0±0.5 mm in Nelore and Holstein cows, respectively. We concluded<br />
that the timing of follicle wave emergence after treatment with EB+P4 was EB dose-dependent for both, Nelore and Holstein cows. Moreover,<br />
the breed effects observed in this study corroborate previous results of our research group (BASTOS et al., Acta Sci.Vet., 38, Supl 2: s776,<br />
2010). [Acknowledgments: FAPESP, CNPq and Ourofino Agronegócio].<br />
Keywords: synchronization, follicular wave, cattle.<br />
s382
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A093 FTAI, FTET AND AI<br />
EFFECT OF ESTROUS DETECTION ON PREGNANCY RATE IN MULTIP<br />
TIPAR<br />
AROUS NELORE (<br />
(BOS INDICUS) ) COWS SUBMITED TO A<br />
TIMED AI IN THE BRAZILIAN SOUTH PANT<br />
ANTANAL<br />
ANAL<br />
Juliana Correa Borges 1 , Márcio Ribeiro Silva 2 , Isabela Cristina Canavari 3 , Raissa Reverete Raineri 4 , José Francisco Massoneto 5 & Daniel Barros Marinho 6<br />
1,3,4<br />
UNESP - JABOTICABAL, JABOTICABAL, SP, BRAZIL. 2 MELHORE ANIMAL, JABOTICABAL, SP, BRAZIL. 5,6 AGROPECUÁRIA EMA PANTANAL, CORUMBA, MS, BRAZIL.<br />
It is known that estrous behavior is related to estrogen concentration, which correlates with LH surge and ovulation. Although<br />
TAI does not require estrous detection, if is reasonable to speculate if cows showing estrous around TAI have greater pregnancy rate. The<br />
objective of this experiment was to test the effect of expression of estrus immediately prior to or at TAI and body condition score (BCS) status<br />
on pregnancy rate. The study was conducted in three farms, located in the sub-region Paiaguás, Corumbá-MS. 5491 Nelore cows (40 to 60<br />
days postpartum) classified by BCS status (BCS1;thin) and (BCS2; moderate) were used. At unknown stages of the estrous cycle (D0), all<br />
cows received 2 mg of estradiol benzoate (RIC-BE ® , Syntex, Argentina) and a progesterone-releasing intravaginal device (PRIMER ® ,<br />
Tecnopec, Brazil). On Day 8, at the time of progesterone device removal, cows received 150µg of d-Cloprostenol (Prolise ® , ARSA,<br />
Argentina), 300U of eCG (5000 Folligon ® , Intervet, Holland) and 1mg of estradiol benzoate. On day 9 estrous was observation in the<br />
afternoon. The TAI was performed on day 10 about 44 and 48 h after progesterone device removal. Pregnancy diagnosis was performed by<br />
ultrasonography (VET ® DP 2200, Mindray, China) 30-45 days after FTAI. The body condition scores were grouped into two classes, BSC1<br />
(BCS = 2.75, n=2264) and BSC2 (BCS> 2.75, n = 3227). Data were analyzed using the GLIMMIX procedure of SAS. Model included<br />
estrous detection (yes or no) and body condition score class (thin or good). The BCS averaged 2.71±0.09 and 3.21±0.26 for groups BSC1 and<br />
BSC2, respectively. Pregnancy rates of 48.4% (1084/2240) and 40.2% (1307/3251) differed (P < 0.01) between estrous manifestation and<br />
conventional FTAI, respectively. Pregnancy rates between the estrous detection group [49.5% (711/1437); 46.5% (373/803)] and conventional<br />
TAI group [42.1% (753/1790); 38.0% (554/1461)] differed (P < 0.01) between BCS2 and BCS1, in order. Estrous detection rates differed (P<br />
< 0.01) between BCS2 [44.5% (1437/3227)] and BCS1 [35.5% (803/2264)]. Estrous detection was 40.8% (2240/5491) and differed among<br />
farms (P < 0.05). In conclusion, estrous detection at TAI was greater in BSC2 group and increased pregnancy rate in multiparous Nelore (Bos<br />
indicus) cows submitted to a TAI program in the Brazilian south Pantanal. [Acknowledgments: EMA Pantanal].<br />
Keywords: boby condition, estrous detectation, pregnancy rate.<br />
A094 FTAI, FTET AND AI<br />
EFFECT OF HCG AND/OR CIDR IN LACT<br />
CTATING TING DAIRY CATTLE TLE ON CIRCUL<br />
CULATING PROGESTER<br />
OGESTERONE ONE AND CONCEPTION RATES<br />
Anibal Ballar<br />
allarotti Nascimen<br />
ascimento, Ale<br />
lexandr<br />
xandre Henr<br />
enrily Souza,<br />
Mar<br />
ary Her<br />
erlih<br />
lihy, Abdulk<br />
dulkadir Keskin,<br />
Fer<br />
ernando Panser<br />
ansera a Dalla Costa,<br />
Glaucio Lop<br />
opes Jr, Jer<br />
erry Guen<br />
uenther &<br />
Milo Charles Wiltbank<br />
UNIVERSITY OF WISCONSIN, MADISON, ESTADOS UNIDOS.<br />
Adequate circulating P4 after AI is important for pregnancy success. Lactating dairy cattle have lower P4 and this problem may<br />
be compounded by ovulation of smaller follicles using timed AI protocols, such as Double-Ovs.ynch (~14mm ovulatory follicle). In trial 1,<br />
our objective was to determine the supplementation strategy, after Double-Ovs.ynch, which resulted in P4 that approached the concentrations<br />
in heifers. Lactating Holstein cows (n = 72) were synchronized with Double-Ovs.ynch (Ovs.ynch-7d-Ovs.ynch-timed AI). After AI (Day<br />
5) cows were assigned randomly to receive No treatment (Control), CIDR, 3,300 IU hCG, or CIDR+hCG. A group of heifers after normal<br />
estrus was followed as controls (heifers; n = 10). Ultrasound and blood samples were used to determine ovulation after hCG and circulating<br />
P4. In trial 2, our objective was to determine the conception rates in lactating dairy cows after supplementation with P4 to concentration that<br />
mimic heifer levels. Lactating Holstein cows (n = 794) were also synchronized with Double-Ovs.ynch. On Day 5 after AI cows were<br />
randomly assigned to one of four groups: No treatment (Control), CIDR, 2,000 IU hCG, or CIDR+hCG. For the first trial, circulating P4<br />
profiles for each treatment were compared to heifers using repeated measures (Proc Mixed, SAS; Treat effect=T; TreatXtime interaction=TXt).<br />
Heifers had greater P4 than cows at all times after Day 5 (heifers: 2,84 ng/mL; cows: 1,17 ng/mL on d 5; and heifers: 9.30 ng/mL; cows: 4.80<br />
ng/mL on d 15) (T and TXt P
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A095 FTAI, FTET AND AI<br />
EFFECT OF THE OVULATOR<br />
ORY FOLLICLE DIAMETER ON THE PREGNANCY RATE OF LACT<br />
CTATING TING COWS FIXED TIME<br />
INSEMINATED<br />
WITH LOW PROGESTER<br />
OGESTERONE ONE PROTOC<br />
OCOLS<br />
OLS<br />
Luiz Francisco Machado Pfeifer 1 , Sofia Del Carmen Bonilla De Souza Leal 2 , Augusto Schneider 2 , Eduardo Schmitt 2 & Marcio<br />
Nunes Corrêa 2<br />
1<br />
EMBRAPA, PORTO VELHO, RO, BRAZIL. 2 UFPEL, PELOTAS, RS, BRAZIL.<br />
Previous studies indicated that ovulatory follicle diameter at insemination influenced pregnancy rates and embryonic/fetal<br />
mortality. The objective of this study was to evaluate the influence of the diameter of ovulatory follicle (OF) on the reproductive performance<br />
of lactating beef cows subjected to low progesterone fixed-time artificial insemination (FTAI) protocols. Ninety three lactating beef cows (60<br />
– 80 days postpartum) at random stages of estrous cycle were given a luteolytic dose of prostaglandin F2α (500 µg cloprostenol; PGF) twice,<br />
11 d apart. Ten days after the second PGF treatment, cows were given 1.5 mg of estradiol benzoate im and a progesterone-releasing<br />
intravaginal device (Cue-Mate) with a single pod containing 0.78 g progesterone (Day 0). Cows received another luteolytic dose of PGF on<br />
Day 0. On Day 8, the Cue-Mate was removed. Fifty-four hours to 56 h later, cattle received 12.5 mg of porcine LH (pLH) i.m. and were<br />
concurrently artificially inseminated. Ultrassonographic exams of the ovaries were performed on Day 10 and 17 to evaluate the diameter of<br />
OF and CL, respectively. The dominant follicles were classified according to its diameter, in one of the five categories: 10 – 12 (n = 8); 13 –<br />
15 (n = 26); 16 – 18 (n = 22); 19 – 21 (n = 22) and > 22 mm (n = 15). The pregnancy rate was 25, 64, 45, 32 and 27 %, for dominant follicles<br />
of 10 – 12, 13 – 15, 16 – 18, 19 – 21 and > 22 mm, respectively. Cows which presented OF > 19 mm resulted in larger CL than cows that had<br />
OF < 15 mm in diameter (P < 0.001). However, cows with OF between 13 – 15 mm had higher pregnancy rate than others categories of OF<br />
(P < 0.05). In conclusion, although large ovulatory follicles results in large CL and consequently high progesterone production, the optimal<br />
size of ovulatory follicles (13 – 15 mm) may result in positive benefits on pregnancy rate for cows subjected to FTAI with low progesterone<br />
concentration protocols.<br />
Keywords: dominant follicle, fertility, progesterone.<br />
A096 FTAI, FTET AND AI<br />
EFFECT OF SYNCHRONIZA<br />
ONIZATION FOLLICULAR<br />
WAVE METHOD ON BOVINE SUPEROVUL<br />
OVULATOR<br />
ORY RESPONSE<br />
Joaquim Esquer<br />
squerdo Fer<br />
erreir<br />
eira 1 , Raquel Rodr<br />
drigues Costa Mello<br />
1 , Ana Paula<br />
Toledo Barb<br />
arbosa Silv<br />
ilva 1 , Leandr<br />
eandro Mendes Masc<br />
ascar<br />
arenhas<br />
1 , Ber<br />
ernar<br />
nardo Janella Fer<br />
erreir<br />
eira<br />
Silva 1 , Beatriz Oliveira Cardoso 1 , Pedro Afonso Moreira Alves 2 , Helcimar Barbosa Palhano 1 & Marco Roberto Bourg Mello 1<br />
1<br />
UFRRJ, SEROPÉDICA, RJ, BRAZIL. 2 PESAGRO, SEROPÉDICA, RJ, BRAZIL.<br />
Although the bovine embryo transfer technique has been widely employed around the world, the variability of donors’ response<br />
to the superovulatory treatment still is a great limitation. Therefore, the aim of this study was to evaluate the effect of synchronization method<br />
of the follicular wave on superovulatory response of the bovine embryo donors. Thus, nine Girolando cows were randomly distributed in<br />
three treatments according to synchronization method of the follicular wave before the superovulation: 1) synchronization with GnRH; 2)<br />
synchronization with progestin implant and Estradiol Benzoate admistration; 3) without synchronization (estrous base observation – control<br />
group). The embryo donors were submitted to follicular wave synchronization treatment, and superstimulated with FSH given in eight<br />
decreasing doses, administered twice daily, intramuscular, every 12 h. At the same time of fifth FSH injection, a prostaglandin dose was<br />
administrated. The progestin implant was removed at the moment of last FSH application. All donors received a GnRH dose to induce and<br />
synchronizate the ovulation. The fixed time artificial insemination of donors was performed with conventional semen twice. The first<br />
insemination was performed 12 h after GnRH injection and the second one 12 h after the fist insemination. The superovulatory response was<br />
assessed based on the number of corpora lutea present in both ovaries on the day of embryo flushing as well as the number and the quality<br />
of recovered structures. The data collected were submitted to variant analysis (ANOVA), to the Tukey test at 5% probability. The average<br />
of corpora lutea observed was 4.5; 5.7 and 9.3 for groups 1, 2 and 3, respectly. The average of recovered structures per donors and the<br />
percentage of viable embryos was 02 and 100%; 03 and 65%; 07 and 95% for groups 1, 2 and 3, respectly. No statistic differences were<br />
observed. Since that data of this study are still preliminary, we could not conclude if the synchronization follicular wave method does interfere<br />
with superovulatory response of Girolando embryo donors.<br />
Keywords: superovulation, girolando, follicular wave<br />
s384
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A097 FTAI, FTET AND AI<br />
EFFECT OF TREATMENT<br />
WITH GNRH ANALOGUE AT THE MOMENT OF INSEMINATION ON PREGNANCY RATE IN<br />
PRIMIPAR<br />
AROUS NELORE (<br />
(BOS BOS INDICUS) ) COWS SUBMITED TO A TIMED AI IN THE BRAZILIAN SOUTH PANT<br />
ANTANAL<br />
ANAL<br />
Márcio Ribeiro Silva 1 , José Francisco Massoneto 2 , Daniel Barros Marinho 2 , Isabela Cristina Canavari 3 , Raissa Reverete Raineri 3 & Juliana Correa Borges 3<br />
1<br />
MELHORE ANIMAL, JABOTICABAL, SP, BRAZIL. 2 AGROPECUÁRIA EMA PANTANAL, CORUMBA, MS, BRAZIL. 3 UNESP/FCAV, JABOTICABAL, SP, BRAZIL.<br />
Primiparous cows have lower reproductive efficiency in FTAI programs, especially in environments with limited quality of<br />
forage, as the Pantanal. The objective this experiment was to evaluate the effect of GnRH analogue administration (lecirelin) at the moment of<br />
insemination on pregnancy rate of primiparous. The study was conducted at Piratininga farm, located in the sub-region Paiaguás, Corumbá-MS.<br />
We used 327 Nelore heifers (40 to 60 days postpartum) with 3 (PRI3, n = 132) and 4 years old (PRI4, n = 195), average of 38.5 and 49.7 months<br />
respectively. At unknown stages of the estrous cycle (D0), all cows received 2 mg of estradiol benzoate (RIC-BE ® , Syntex, Argentina) and one<br />
progesterone-releasing intravaginal device. On Day 8, at the time of progesterone device removal, cows received 150µg of d-Cloprostenol<br />
(Prolise ® , ARSA, Argentina), 300U of eCG (5000 Folligon ® , Intervet, Holland) and 1mg of estradiol benzoate. On day 10 was performed<br />
artificial insemination (AI), where 183 cows (GnRH group) received 25µg of lecirelin IM (Gestran Plus ® , ARSA, Argentina). The FTAI was<br />
performed by same technician. Pregnancy diagnosis was performed by ultrasonography (VET ® DP 2200, Mindray, China) 30 days after<br />
FTAI. The body condition scores (BCS, 1-5) were grouped into classes, BCS1 or Thin (BCS = 2.75, n = 199) and BCS2 or Good (BCS> 2.75,<br />
n = 128). Binomial data were analyzed using the GLIMMIX procedure of SAS. Model included pregnancy rate (dependent variable) and<br />
GnRH treatment, age, body condition score and interactions, as independent variables. Error probability less than 5% were considered<br />
significant. There was no effect of administration of GnRH, as well as, age, body condition, and interactions (P > 0.05). The BCS averaged 2.85<br />
± 0.25 and 2.87 ± 0.24 for groups PRI3 and PRI4, respectively. The treatments with progesterone lasted 188.85 ± 3.14 and 188.99 ± 2.95 h,<br />
and the intervals between device removal and AI were 47.97 ± 1.94 and 48.51 ± 2.05 h to PRI3 and PRI4, in order. There were no interactions<br />
among treatment and the explanatory variables. Pregnancy rates of 43.8% (63/144) and 54.1% (99/183) did not differ (P > 0.05) between<br />
control and GnRH. Pregnancy rates between the control group [46.6% (57/109) 41.9% (36/86)] and GnRH [56.8% (42/74), 52.3% (57/109)]<br />
did not differ (P > 0.05) between classes PRI3 and PRI4, in order. For body condition, pregnancy rates of 44.0% (37/84) and 53.0% (61/115)<br />
for BCS1 and 43.3% (26/60) and 55.9% (38 / 68) for BCS2, did not differ (P > 0.05) between control and GnRH, respectively. In conclusion,<br />
although GnRH group have showed superiority percentage, the lecirelin administration on TAI did not increase primiparous pregnancy rate<br />
in Nelore cows at 3 and 4 years in the Brazilian South Pantanal. [Acknowledgments: Agener União Saúde Animal].<br />
Keywords: body condition score, pregnancy rate, reprodutive efficiency.<br />
A098 FTAI, FTET AND AI<br />
EFFICIENCY OF DIFFERENT STIMULI GONADOTR<br />
TROPIN IN RESYNCHRONIZA<br />
ONIZATION NELLORE LACT<br />
CTATING TING COWS SUBMITTED<br />
TED TO<br />
FTAI<br />
Jeff<br />
efferson<br />
erson Tadeu Camp<br />
ampos<br />
1 , Suellen Migue<br />
iguez Gonzále<br />
onzález 2 , Wanessa Blaschi<br />
2 , Thales Ric<br />
icar<br />
ardo Rigo Bar<br />
arreir<br />
eiros<br />
2 & Mar<br />
arcelo Mar<br />
arcondes Seneda<br />
1<br />
1<br />
UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA, PR, BRAZIL. 2 UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL.<br />
N<br />
The aim of this study was to evaluate the efficiency of different gonadotropic stimuli resynchronization in Nelore cows. Were<br />
used 814 cows with BCS between 3.0 to 3.5 in the region of Iby Yau, Paraguay. The cows received an intravaginal device containing 1 g of P4<br />
(DIB, Syntex, Argentina) and application of 2 mg of estradiol benzoate (EB) (estradiol benzoate, Syntex, Argentina) IM. Eight days later, the<br />
devices were removed while the application of 1 mg estradiol cypionate (ECP, Pfizer ® , Brazil), 300 UI of eCG (Novormon, Syntex,<br />
Argentina) IM and 125 mg of cloprostenol (cyclase, Syntex, Argentina) IM. All animals were inseminated 48 to 54 h after removal of the DIB.<br />
The cows were divided into five groups. The control group (GC, n = 186) was subjected to heat detection from 18 to 23 days after FTAI. In<br />
the second group (G-R23, n = 159), cows were subjected to 23 days after the resynchronization FTAI with the same protocol mentioned<br />
above, without the application of eCG and the diagnosis of pregnancy by ultrasound (Aloka SSD500, 5 MHz, Japan) on the day of DIB<br />
removal. The last three groups received similar treatment, with the addition of 200UI of eCG (G-R23/200, n = 142), 300 UI of eCG (G-R23/<br />
300, n = 150) simultaneous removal of the DIB or performance the temporary removal of calves from the removal of the DIB and FTAI (G-<br />
R23/RTB, n = 177). All cows were subjected to natural breeding after the second insemination, the proportion of one bull to 25 cows.<br />
Pregnancy diagnosis was performed after 31 days and 30 days after removal of bulls. The results were analyzed by ANOVA (P < 0.05). The<br />
conception rates at FTAI were similar (P > 0.05) [GC=47.8%(89/186); G-R23=54%(86/159); G-R23/200=50.7% (72/142), G-R23/<br />
300=50.6%(76/150) and G-R23/RTB=55.3%(98/177)]. In the resynchronization G-R23/300 G-R23/RTB and had higher conception rates (P<br />
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A99 FTAI, FTET AND AI<br />
EFICIENCY OF TIMED ARTIFICIAL INSEMINATION PROTOC<br />
OCOL OL FOR HOLSTEIN COWS REARED UNDER SEMIARID CONDITION<br />
Sebastião Inocêncio Guido 1 , Antonio Santana Dos Santos Filho 2 , Lucas Carvalho Pereira 3 , Joaquim Corrêa De Oliveira Andrade 4 , Fabiani Coutinho Lordão<br />
Guido 5 & Claudio Coutinho Bartolomeu 6<br />
1,2,3,4<br />
INSTITUTO AGRÔNOMICO DE PERNAMBUCO - IPA, RECIFE, PE, BRAZIL. 3,4,5 VETERINÁRIO AUTONÔMO, MACEIÓ, AL, BRAZIL. 4,5,6 VETERINÁRIO AUTONÔMO, RECIFE, PE, BRAZIL.<br />
6<br />
UFRPE, RECIFE, PE, BRAZIL.<br />
This study was conducted at the Experimental Station of São Bento do Una- (ESSBU/IPA ) Pernambuco, Brazil, (Latitude 08 31'<br />
35’’ and Longitude 036 27’ 34.8'’) with the aim to evaluate the efficiency of protocol for TAI in dairy cows reared in semi-arid conditions during<br />
the dry season (relative humidity of 53.5% and mean average environment temperature of 25.3°C). There were used lactating cows with over<br />
100 days of lactation with an average milk production of 7,200 kg/cow/lactation in 305 days and age ranging from 36 to 108 months. The herd<br />
was kept in semi-intensive system, receiving a diet composed of cactus pear (Opuntia ficus indica, Mill), sorghum silage (Sorghum bicolor (L.)<br />
Moench) and concentrated with 18% crude protein (CP), and a mineral supplement and water ad libitum. All cows in the voluntary waiting<br />
period (VWP) were submitted to gynecological examination by rectal palpation, being distributed in two treatments T1 and T2: T1 (n = 30) was<br />
composed of cows with palpable CL which received an application of 0.530mg of cloprostenol, and were artificial inseminated 12 h after being<br />
observed in estrus. At T2 (n = 28) cows with no palpable CL were submitted to TAI. The protocol consisted of an intravaginal progesterone<br />
device inserted at D0 along with 2mg of estradiol benzoate (EB), at D7 application of 0.530mg cloprostenol and 300 IU of eCG, at D8 device<br />
removal and at D9 application of 1 mg EB. Timed artificial Insemination was performed 54h after device removal at D10. Data were subjected<br />
to analysis of variance and means were compared by Tukey test at 5% by the statistical package SPSS 16 for windows. The cows on T2<br />
underwent ultrasound evaluation of the ovulatory response during estrus at the time of EB application. All cows in both groups T1 and T2 were<br />
artificial inseminated with conventional semen. Pregnancy diagnosis was performed by rectal palpation aided by ultrasound in the 45th day after<br />
AI. The conception rate (CR) was 26.66% (8/30) in T1 and 25.00% (7/28) in T2, (P > 0.05). Regarding to the occurrence of estrus in cows from<br />
T1, it ranged from 48 to 72 h after application of PGF2a. In T2 follicular diameter measured at D9 ranged from 10.9 to 25.4mm. In the conditions<br />
of this experiment, the protocols used showed low CR. However the treatment used in the T1 has a better cost-benefit.<br />
Keywords: tai, bovine, holstein.<br />
A100 FTAI, FTET AND AI<br />
LUTEOL<br />
UTEOLY TIC EFECTIVENESS OF PROST<br />
OSTAGL<br />
GLANDINA<br />
TOR<br />
ORTUGA<br />
® IN BOVINE CAT TLE<br />
M arc os Sampaio Bar<br />
aruselli<br />
1 , Rob<br />
ober<br />
erta Machado Fer<br />
err eira 2 , Mano<br />
anoel Fr ancisco Sá Filho<br />
3 , Luis Fer<br />
ernando Mon<br />
ont eiro Tamassia<br />
4 , Camila<br />
Alves Nascimento 5 , Renan Oliveira Fernades 6 & Pietro Sampaio Baruselli 7<br />
1,4,5,6<br />
TORTUGA CIA ZOOTECNICA AGRARIA, SÃO PAULO, SP, BRAZIL. 2,3,4,5,6,7 FMVZ-USP - DEPARTAMENTO DE REPRODUÇÃO ANIMAL, SÃO PAULO, SP, BRAZIL.<br />
The aim of the present study was to evaluate the effect of the administration of 0.150 mg of Prostaglandina (PGF) Tortuga ® , D (+)<br />
Cloprostenol, on (1) the morphological regression of the corpus luteum (CL) and (2) the reduction of serum progesterone (P4) concentration on<br />
days 8 and 12 of the estrous cycle in Nelore (Bos indicus) heifer. Pubertal heifers were homogenously allocated in one of Five experimental<br />
groups to receive none PGF (Control; n = 11), PGF Tortuga ® on the 8th (n = 12) or 12th (n = 14) day of the estrous cycle, or Ciosin ® on 8th (n<br />
= 14) or 12th (n = 12) day of the estrous cycle. At random stages of the estrous cycle, all heifers were treated with a norgestomet ear implant<br />
(Crestar ® , Intervet, Brazil) and 2 mg of estradiol benzoate (Gonadiol ® , Intervet, Brazil). After eight days, the implant was removed and 1 mg of<br />
estradiol cypionate (E.C.P. ® , Pfizer, Brazil) plus 0.530 mg of sodic cloprostenol (Ciosin ® , Intervet, Brazil) were administered. Morphological<br />
and functional regression of the CL was evaluated by ultrassonography and P4 concentration, respectively. Daily ultrassonographic exams were<br />
done for four days following PGF administration. Blood samples were collect right before PGF administration (0h) and then 6, 12, 24, 48, 72<br />
and 96 h after treatment. P4 concentration was determined by radioimmunoassay (Coat-A-Count ® , Siemens, USA). Luteolysis rate (%; heifers<br />
reaching serum P4 concentration below 1ng/ml after treatment / all heifers treated) was analyzed using test FREQ of SAS for Windows.<br />
Progesterone concentration was analyzed using PROC GLIMMIX from SAS. No interaction was found among treatment and day of treatment<br />
(8th or 12th day of the estrus cycle), thus data was grouped. Luteolysis rate (%) was greater (P < 0.01) when heifers were treated with P4<br />
Tortuga ® and Ciosin ® than Control heifers (88.5% vs. 80.8% vs. 0.0%, respectively). Morphological regression of the CL was evidenced in both,<br />
heifers treated with PGF Tortuga ® (4,283.0 ± 1,849.3 mm3 before treatment and 915.2 ± 880.2 after 96h) and treated with Ciosin ® (4,074.0 ±<br />
1,746.7 mm 3 and 715.5 ± 588.6 mm3, respectively). Similarly, serum P4 concentration was reduced (P < 0.05) after treatment with both, PGF<br />
Tortuga ® and Ciosin ® , respectively (0h: 3.4 ± 1.7 vs. 4.3 ± 1.9 ng/mL; 96 h: 0.18 ± 0.20 vs. 0.25 ± 0.31 ng/mL). There was no difference (P ><br />
0.05) on the CL regression and the reduction of P4 concentration between heifers treated with PGF Tortuga ® and Ciosin ® . In conclusion, the<br />
administration of 0,150 mg of PGF Tortuga ® efficiently promotes luteolysis in heifers, leading to morphological and functional regression of the<br />
CL.<br />
Keywords: bovines, protaglandin, luteolysis.<br />
s386
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A101 FTAI, FTET AND AI<br />
ESTABLISHMENT OF A SYNCHRONIZA<br />
ONIZATION OF OVULATION PROTOC<br />
OCOL OL FOR FIXED TIME EMBRYO TRANSFER APLLIED ON<br />
DAIRY CATTLE TLE GENETIC IMPROVEMENT PROGR<br />
OGRAM OF ACRE STATE<br />
TE<br />
Rodolpho Satrapa 1 , Jefferson Viana Alves Diniz 2 , Francisco Lopes Dantas 2 , Rosano Ramos Marcelino 2 , Neilton Lima Vasconcelos 2 , Marcos Nereu Luckner 2 ,<br />
José Marques Carneiro Junior 3 & Rafael Augusto Satrapa 4<br />
1<br />
UFAC, RIO BRANCO, AC, BRAZIL. 2 SECRETARIA DE ESTADO DE AGROPECUARIA, RIO BRANCO, AC, BRAZIL. 3 EMBRAPA, RIO BRANCO, AC, BRAZIL. 4 UNESP, SAO PAULO, SP, BRAZIL.<br />
In Acre State, the low technological level of the milk-producing farms, mostly with crossbred zebu-crossed and low genetic<br />
pattern, with average production of around 3 to 5 kg / day during the local rainy season has prompted the Acre Agriculture State Department<br />
to implement a genetic improvement program for dairy cattle based on the technique of embryo transfer (ET), an important tool that facilitates<br />
the rapid multiplication of animals of high genetic potential. The program also aims social gains, since an increase in productivity of livestock,<br />
may be reflected in improved economic conditions of farmers. For this purpose, the Improvement and Dissemination of Animal Genetics<br />
Station (EMDGA) was installed and equipped with a laboratory for embryo production by superovulation or IVF procedures from Gyr<br />
donors and sexed semen from Holstein bulls. With this purpose, a synchronization of ovulation (Ovs.ynch) protocol of recipients for FTET<br />
was established, which, logistically and economically, best suited with the distances to be traveled to the properties, many of them<br />
inaccessible due to the characteristics of the region. In 30 properties we evaluated 525 crossbred cows with varying degrees of blood (Bos<br />
taurus x Bos indicus), between 3 and 6 years of age and calved between 60 and 90 days. Before the start of treatment 197 cows (197/525 =<br />
37.52%) were rejected because of their poor body condition score (BCS) ( / = 18mm by U.S. (Aloka SSD 500,<br />
Aloka, Japan) each of 235 cows (235/328 = 71.65%) received one fresh embryo (morula or blastocyst stage, grade 1 or 2); 93 cows (93/235<br />
= 28.35%) were rejected by BCS
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A103 FTAI, FTET AND AI<br />
INFLUENCY OF THE UTERINE LAVAGE<br />
TO ENDOMETRIAL CYTOL<br />
OLOGY OGY ON THE BOVINE FERTILIT<br />
TILITY<br />
Helder Esteves Thomé, Rubens Paes De Arruda, Bruna Marcele Martins De Oliveira, Milton Maturana Filho, Guilherme Cain De Oliveira, Shirley Andrea<br />
Rodrigues Florez, Carina De Fátima Guimaraes & Eneiva Carla Carvalho Celeghini<br />
DEPARTAMENTO DE REPRODUCAO ANIMAL - FMVZ - USP, SAO PAULO, SP, BRAZIL.<br />
Uterine defense mechanisms against environmental microorganisms include anatomical and functional barriers, as non-specific<br />
immune responses (polymorphonuclear inflammatory cells) and specific (humoral antibodies). This rapid and intense influx of inflammatory<br />
cells at the site of semen deposition, after the artificial insemination (AI), is described as “post-breeding inflammatory response” and can be<br />
intensified with the deposition of a higher number of dead sperm during AI and delay the uterine clearance, featuring a subclinical endometritis,<br />
which may develop into clinical endometritis. The diagnosis of subclinical endometritis have been described by several techniques, being<br />
characterized by the proportion of polymorphonuclear cells in a cytological sample taken from the uterus of clinically healthy cows, through<br />
uterine biopsy, cytobrush or cotton swab and uterine lavage. However, these methods are invasive and can influence the fertilization rate of these<br />
animals. Given this, it was aimed to evaluate the conception rate in cows submitted to uterine lavage four hours after the AI. Have been used<br />
127 Nellore cows, lactating, undergoing a protocol of timed artificial insemination (TAI), which consisted of inserting a subcutaneous implant<br />
containing 3 mg of norgestomet (Crestar ® ) plus the IM application of 2 mg of estradiol benzoate (Estrogin ® ), eight days after the progestin<br />
implant was removed, administrating 1 mg of estradiol benzoate, 0.5 mg of sodium cloprostenol (Sincrocio ® ), and 300 IU of equine chorionic<br />
gonadotropin (Novormon ® ), performing a TAI 36 h later. The uterine lavage was performed in 35 cows, four hours after AI by the intrauterine<br />
infusion of 40 mL of sodium chloride solution 0.9%, keeping it for 10 seconds and then the liquid was removed. Pregnancy diagnosis was<br />
performed 35 days after TAI. The qui-square analysis was used to determine the frequency of the pregnancy rate by PROC FREQ of SAS,<br />
version 9.2 (SAS, 2010). From the animals that went through uterine lavage, was obtained a pregnancy rate of 54.28% (19/35) against the rate<br />
of 55.43% (51/92) of the animals that had not manipulated its uterine environment, being observed no difference (P > 0.05) in pregnancy rate<br />
between the group which was submitted to uterine lavage and that which was not. In this way, it can be concluded that the uterine lavage<br />
performed four hours after AI for uterine cytological evaluation does not influence fertility in cows. [Acknowledgment: FAPESP, process<br />
number 09/50365-0].<br />
Keywords: pathology, cow, endometritis.<br />
A104 FTAI, FTET AND AI<br />
ARTIFICIAL INSEMINATION IN PHEASANTS S LODGED IN INDIVIDUAL CAGES<br />
Osvaldo Almeida Resende 1 , Jaci De Almeida 1 & Edward Pompeu Ponte 2<br />
1,2<br />
UBM - CENTRO UNIVERSITARIO DE BARRA MANSA, BARRA MANSA, RJ, BRAZIL. 3 SITIO DAS AVES, MAGÉ, RJ, BRAZIL.<br />
The exploration of ornamental birds has been being practiced for the birds and feathers commercialization, in small farms in the<br />
Brazil. However the handling of natural matings (NM) of these birds, in individual or collective pens has not been presenting compatible<br />
performance with the commercial needs. Thus a venerated pheasants’ reproductive project (40 males and 200 females) using the method of<br />
artificial insemination (AI) was developed in a particular poultry farm, located in the Rio de Janeiro’s State, objectifying make possible the<br />
commercial exploration of male tails feathers. The pheasants were housed at the 16 age weeks, in individual galvanized wire cages, measuring<br />
100x80x82cm (male) and 25x44x42cm (female), kept in triple arrays, in covered shed, side long wire screened, measuring 30.0x5.0x2.3m The<br />
semen collection technique was accomplished by means of abdominal and loin-sacrum massages (Resende et al., 1983, Rev. Bras. Reprod.<br />
Anim., 7, 2: 23), twice a week with the males supported on a on a table. The semen was gathered in 0.25mL straw, coupled in insulin syringe<br />
plastic. To avoid spermatic losses was put in the straw a drop of lactate ringer solution and afterwards to collect diluted in the same extender, in<br />
proportion considering the volume and the concentration of the ejaculate. The artificial inseminations were made in oviduct twice a week, after<br />
14:00hs, in the dose of 0.02mL of the diluted semen, using straw of 0.25mL coupled to the plastic insulin syringe, with the female supported on<br />
top of the cage. The eggs were kept by 7 days, in cold chamber (12-15 o C). The incubations were done in small incubators electric, with electronic<br />
adjustment of humidity, temperature and turns of eggs, according to the technical recommendations for the specie. The evaluated reproductive<br />
data were the averages of the seminal characteristics, egg production and fertility embracing the relative activities to periods of 41 to 48 age weeks.<br />
The general average results obtained in the evaluated periods were volume of 0.05-0.3mL, motility of 70-90%, vigor of 2-5 (0-5) to the seminal<br />
characteristics (selected males), eggs production of 86.8±4.7% and fertility of 94.1±3.2%. Results these best (P < 0,05) to the average values of<br />
egg production (61.7±23.8%) and fertility (72.2±16.1%) obtained in the natural mating control groups of the venerated pheasants, managed<br />
concomitantly, in 40 collective pens (1male and 2-3 females). The lodging system of pheasants in individual cages of wire and the reproductive<br />
method did not affect the bird’s health (breast and foot lesions were not observed). The project using AI showed to be technically viable for<br />
pheasants’ reproductive activities lodged in individual cages.<br />
Keywords: artificial insemination, pheasants, cages.<br />
s388
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A105 FTAI, FTET AND AI<br />
EARLY EMBRYO MORTALIT<br />
ALITY IN BEEF COWS AFTER FIXED TIME ARTIFICIAL INSEMINATION (FTAI) AND HORMONAL<br />
TREATMENT<br />
MENTS<br />
Rui Machado, , Mateus José Sudano, Marco Aurelio Bergamaschi, Guilherme De Paula Nogueira, Claudia Maria Bertan Membrive & Mário Binelli<br />
EMBRAPA PECUÁRIA SUDESTE, SAO CARLOS, SP, BRAZIL.<br />
Failure in the maternal recognition of pregnancy is implicated with early embryo mortality. This study evaluated strategies to<br />
optimize luteal function of cows after FTAI. Forty-five Nelore (Bos taurus indicus) cows received one progesterone (1.9g) intravaginal device<br />
(P4D) and 2mg estradiol benzoate. Seven days later received 0.25mg of PGF2α analogue. Two days after that the P4D was removed and 0.5<br />
mg of estradiol cipionate was given. FTAI (D0) took place 48 h after P4D removal. Females were distributed into: Tcont (n = 9) - nothing further;<br />
TPGF (n = 10) - 0.25mg of PGF2α analogue on D14, D16 and D18 (negative control group); TGnRH-hCG (n = 14) – 200mcg of gonadorrelin<br />
(GnRH) on D5 and 2500 IU of hCG on D12; and TeCG (n = 12) - 400 IU eCG at P4D withdrawal. All drugs but P4D were given IM. Cows<br />
were submitted to: plasma progesterone determination [P4] on D14, D16, D18, as well as ultra-sound examination of ovaries and transcervical<br />
embryo collection on D18. Concepti were classified according to integrity and length (whole = WH, fragmented = FR or severely fragmented<br />
= SF). Results were analyzed through ANOVA or the Chi-square test. Regardless of treatment, recovery rate was 75.5% (34/45) and retrieved<br />
concepti were 12 WH, 4 FR and 18 SF. Early embryonic losses were: 40% (2/5) for Tcont; 41.7% (5/12) for TGnRH-hCG ; 25% (2/8) for TeCG<br />
and 100% (9/9) for TPGF. The number and the size of CL were smaller (P < 0.05) for TPGF when compared to the other groups. TPGF cows<br />
produced only SF concepti and their [P4] were subluteal ( 0.05) between [P4]<br />
of Tcont and TeCG (both above 1ng/mL). The [P4] on D18 was subluteal (
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A107 FTAI, FTET AND AI<br />
OVARIAN PAR<br />
ARAMETERS AMETERS OF DAIRY GOATS UNDERGOING SYNCHRONIZA<br />
ONIZATION<br />
WITH PGF2Á ASSOCIATED OR NOT WITH THE<br />
USE OF HCG IN THE BEGINNING OF ESTRUS<br />
Luciana Vieira Esteves, , Felipe Zandonadi, Andressa Ferreira Da Silva, Sabrina Silva Venturi & Jeferson Ferreira Fonseca<br />
UFF, NITEROI, RJ, BRAZIL.<br />
The estrous synchronization refers to the concentration of the occurrence of estrus in restricted period (24 to 72 h) during the<br />
breeding season. The aim of present study was to evaluate the ovarian responses after estrous synchronization protocol using two doses of<br />
prostaglandin associated of use of hCG at onset of estrus in dairy goats. A total of 29 females were used (n = 10 Toggemburg, n = 10 Alpine and<br />
n = 9 Saanen breeds). The females were assigned into two experimental groups according the use of 250IU hCG at onset of estrus (T1= Control;<br />
1 mL of saline solution and T2=hCG). Both groups received two doses of 30µg d-cloprostenol via submucosa vulvar with 10 days apart.<br />
Ultrasonography exams were performed twice daily after the second dose of PGF2α. All females were inseminated 16 h after onset of estrus.<br />
All animals that did not display estrus (20.7%; 6/29) after the second dose of PGF2α did not ovulate. However, all females that displayed estrus<br />
ovulated. There is a higher incidence of ovulation during the night period 69.6% (16/23) than during the diurnal period 30.4% (7 / 23). The<br />
intervals from the second application of PGF2α to the occurrence of ovulation (h) and from onset of estrus to ovulation (h) did not differ<br />
between treatments groups (P > 0.05), 85.7 ± 10.4 h vs. 87.5 ± 12.8 h, 41.0 ± 13.2 h vs. 38.8 ± 11.9 h, respectively for T1 and T2. The<br />
diameter of the largest follicle and the second largest follicle (mm) and number of ovulations did not also different between treatments (P ><br />
0.05), 6.8 ± 0.5 vs. 7.7 ± 1.9, 6 , 2 ± 0.5 vs. 6.9 ± 1.7, 1.8 ± 0.6 vs.. 1.8 ± 0.6, respectively. The conception rates did not differ (P > 0.05) among<br />
females that did receiving (T2= 54.4%; 6/11) or not hCG (T1= 66.7%; 8/12). The results of the present study demonstrated that the estrus<br />
of the dairy goats can be efficiently synchronized through the administration of two doses of PGF2α 10 days apart. Also the administration<br />
of hCG at the time of onset of estrus did not affect the results.<br />
Keywords: synchronization, goat, estrus.<br />
A108 FTAI, FTET AND AI<br />
THE PROTOC<br />
OCOL OL FOR FTAI<br />
WITH ESTRADIOL CYPIONATE DOES NOT REQUIRE ADITTIONAL TIONAL GNRH AND ALLOWS<br />
INSEMINATION IN TWO PERIODS<br />
Felip<br />
elipe e Pitanga<br />
Tor<br />
orres<br />
1 , Rob<br />
ober<br />
erta Machado Fer<br />
erreir<br />
eira 2 , Flávio Aragon Lima 3 , Márcio Bar<br />
arciela<br />
Ver<br />
eras<br />
4 , Felip<br />
elipe e Rangel França Silv<br />
ilva 5 , Thiago Guz<br />
uzella Guida<br />
6 , Rodr<br />
drigo<br />
Vasconcellos Sala 7 & Pietro Sampaio Baruselli 8<br />
1,3,4,5,6<br />
POLICLINICA VETERINÁRIA PIONEIROS LTDA, CARAMBEÍ, PR, BRAZIL. 2,7,8 FMVZ-USP, SÃO PAULO, SP, BRAZIL. 5 UENP, BANDEIRANTES, PR, BRAZIL. 6 UNESP, BOTUCATU, SP,<br />
BRAZIL.<br />
The aim of the present study was to evaluate time to insemination and the necessity of administering GnRH in high-producing<br />
Holstein cows treated for fixed time artificial insemination (FTAI). The study was conducted in ten commercial dairy farm located around<br />
Carambeí city, state of Paraná, Brazil. A total of 424 Holstein cows received 2mg estradiol benzoate (Gonadiol ® , Intervet, Brazil) and one<br />
progesterone-releasing intravaginal device (CIDR ® , Pfizer, Brazil) on Day 0 (D0). On D8, device was removed and all animals received 25mg<br />
dinoprost (Lutalyse ® , Pfizer, Brazil) and 1 mg of estradiol cypionate (E.C.P. ® , Pfizer, Brazil). Then, cows were homogenously allocated to one<br />
of four experimental groups accordingly with time to insemination and the use of GnRH. (Fertagyl ® , Intervet, Brazil):1) AI 48h after device<br />
removal and without concurrent the administration of GnRH, 2) AI 48h with GnRH, 3) AI 56h without GnRH, 4) AI 56h with GnRH. Statistical<br />
analysis was done using PROC GLIMMIX from SAS. No interactions were observed among time to insemination and treatment with GnRH.<br />
Thus, data was grouped. Similar P/AI 30 [without GnRH = 37.3% (n = 212) vs. with GnRH = 41.5% (n = 212); P = 0.28] and 60 days after<br />
FTAI [without GnRH = 33.0% (n = 212) vs. with GnRH = 35.9% (n = 212); P = 0.61] was observed between cows treated or not with GnRH.<br />
Pregnancy loss between 30 and 60 days after FTAI was also similar among groups [without GnRH = 11.4% (n = 79) vs. with GnRH = 13.6%<br />
(n = 88); P = 0.16]. This protocol allowed the performance of AI in both periods 48 (AM) and 56 h (PM) following device removal, without<br />
compromising pregnancy rates 30 [48h = 40.3% (n = 216) vs. 56h = 38.5% (n = 208); P = 0.63] and 60 days [48h = 33.8 (n = 216) vs. 56h =<br />
35.1% (n = 208); P = 0.72] after FTAI. Pregnancy loss was also unaffected by time to AI [48h = 16.1 (n = 87) vs. 56h = 8.8% (n = 80); P =<br />
0.67]. The results are suggestive that the administration of additional GnRH at FTAI in not necessary when estradiol cypionate is used at device<br />
removal. Besides, the protocol used herein allows the performance of AI both in the morning (48h) and in the afternoon (56h) enabling the<br />
synchronization of large number of animals (same protocol with different time to AI and similar efficiency). [Acknowledgments: Chaparral,<br />
Erica, Frizia, Gruno, IMKJE, Ipê, São Cristóvão, Tainha, Três Lagoas, Pfizer Saúde Animal].<br />
Keywords: bovine, estradiol cypionate, gnrh.<br />
s390
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A109 FTAI, FTET AND AI<br />
SYNCHRONIZA<br />
ONIZATION AND RESYNCHRONIZA<br />
ONIZATION USING A NORGEST<br />
GESTOMET EAR IMPLANT IN NELORE HEIFERS TIMED<br />
ARTIFICIAL INSEMINATED<br />
Manoel Francisco Sá Filho 1 , Raphael A.L Oliveira 2 , Rodrigo Vasconcellos Sala 3 , Gustavo G. Macedo 4 , Renato W Girotto 5 , Luciano Penteado 6 & Pietro Sampaio<br />
Baruselli 7<br />
1,2,3,4,5,6,7<br />
UNIVERSIDADE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 5 RG GENÉTICA AVANÇADA, ÁGUA BOA, MT, BRAZIL. 6 FIRMASA-IATF, CAMPO GRANDE, MS, BRAZIL.<br />
Different synchronization (Exp. 1) and resynchronization (Exp. 2) protocols using norgestomet ear implant (NORG; Crestar ® )<br />
in heifers (Bos indicus) for timed artificial insemination (TAI) were tested. In Exp 1, 417 cyclic heifers were randomly assigned at the beginning<br />
of the synchronization protocol into two groups according to the number of uses of NORG (new or previously used for 9 days) associated with<br />
2mg of estradiol benzoate (EB, Gonadiol ® ). Eight days later, the NORG implants were removed and were administered 150 g of D-cloprostenol<br />
(PGF; Preloban ® ), 300U of eCG (Folligon ® ) and 0.5 mg of estradiol cypionate (ECP ® ). On the day of TAI, the females were newly reassigned<br />
to be inseminated at 48 or 54 h after NORG withdraw (Factorial 2 x 2). There was no effect of type of implant (P = 0.70) or time of TAI (P =<br />
0.26), however, there was an interaction (P = 0.02) between the type of implant and on the moment of TAI conception rate [New (48 h) =<br />
47.6%; 50/105ab; New (54 h) = 53.3%a; 57/107; Used (48 h) = 56.6%; 56/99a and Used (54 h) = 40.0%; 42/105b)]. In Exp 2, 475 heifers<br />
receiving a previous TAI 22 days before were randomly assigned into two experimental groups according to the inducer of follicular wave<br />
emergency (1mg of EB or 100µg GnRH) associated with the insertion of used NORG (Resynchronization protocol). Seven days later,<br />
pregnancy diagnosis was performed by ultrasonography exam. In pregnant heifers only the NORG implant was removed, while in non<br />
pregnant heifers received the additional treatment with PGF and 0.5 mg of ECP at NORG removal and TAI was performed 48 h afterward.<br />
The conception rate of the first TAI were similar (P = 0.97) between heifers treated with EB (41.9%, 101/241) or GnRH (41.4%, 97/234),<br />
however, females treated with EB (49.2%, 51/140) achieved higher (P = 0.04) conception rates after resynchronization protocol than those<br />
heifers treated with GnRH (37.2%, 51/137). Furthermore, the moment of TAI in heifers depends of the type of NORG (new or previously<br />
used), in which females synchronized with used NORG should be inseminated 48 h after NORG withdrawal. In addition, it is possible to<br />
resynchronize heifers 22 days after the first TAI using 1mg EB at the beginning of the resynchronization protocol using a NORG implant<br />
without compromising the pregnancy results. [Acknowledgements: Intervet Schering-Plough, Agropecuária Couto Magalhães S/A e Piveta<br />
Agropecuária].<br />
Keywords: ftai, resyncrhonization, crestar.<br />
A110 FTAI, FTET AND AI<br />
SYNCHRONIZA<br />
ONIZATION OF OVULATION IN WATER-B<br />
TER-BUFF<br />
UFFAL<br />
ALO HEIFERS WITH ESTRADIOL BENZOATE OR GONADORELIN<br />
Mar<br />
arcílio Nichi 1 , Nelcio Antonio<br />
Tonizza Car<br />
arvalho<br />
alho 2 , Júlia Gle<br />
leyci Soar<br />
oares<br />
1 , Diego Cavalc<br />
alcan<br />
ante Souza<br />
3 & Pietr<br />
ietro Sampaio Bar<br />
aruselli<br />
1<br />
N<br />
1<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL, VRA-FMVZ-USP, SÃO PAULO, SP, BRAZIL. 2 UNIDADE DE PESQUISA E DESENVOLVIMENTO DE REGISTRO–PÓLO REGIONAL DO D.S.A.<br />
DO VALE DO RIBEIRA/APTA, REGISTRO, SP, BRAZIL. 3 ESCRITÓRIO DE DESENVOLVIMENTO RURAL DE REGISTRO-CATI, REGISTRO, SP, BRAZIL.<br />
The aim of this study was to evaluate the efficiency of different ovulation inducers – Estradiol Benzoate and Gonadorelin - in a<br />
protocol of fixed-time artificial insemination (FTAI) in water-buffalo heifers during the non-breeding season (spring and summer). Nineteen<br />
Murrah buffalo heifers were allocated into two Groups (GEB, n = 10; GGo, n = 9), homogeneously distributed according to age, weight, body<br />
condition score and ovarian activity. At random stage of the estrous cycle (Day 0 = D0; pm), all heifers received a second use intravaginal<br />
progesterone device (DIB ® , Intervet/Schering-Plough, Brazil) plus 2.0mg of Estradiol Benzoate (i.m.; EB; Gonadiol ® , Intervet/Schering-<br />
Plough, Brazil). In D9 (pm), females received 150µg of an analogue of PGF2α (i.m.; d-cloprostenol sodic, Preloban ® , Intervet/Schering-<br />
Plough, Brazil) plus 400IU of eCG (i.m.; Novormon ® , Intervet/Schering-Plough, Brazil), followed by the device removal. In D10 (pm) the GEB<br />
ovulation was induced with 1.0mg of Estradiol Benzoate (i.m.; Gonadiol ® , Intervet/Schering-Plough, Brazil), while in GGo ovulation was<br />
induced twenty four hours later (D11 - pm) using 100µg of GnRH (i.m.; Gonadorelin, Fertagil ® , Intervet/Schering-Plough, Brazil). The<br />
ultrasonographic evaluation (Mindray DP2200Vet, China) was performed on D0 to assess ovarian activity, in D9 to measure the follicular<br />
diameter and from D11 to D14 (12/12 h per 60 h) to establish the moment of ovulation. Data were analyzed by PROC GLM of the SAS for<br />
Windows program and by Fisher´s exact test. The maximum diameter (Ø) of the dominant follicle (13 ± 0 vs.. 13 ± 0 mm), the Ø of ovulatory<br />
follicle (13 ± 1 vs.. 13 ± 0 mm), the interval between DIB removal and ovulation (75.0 ± 2.0 vs.. 80.0 ± 2.7 h) and ovulation rate [80.0% (8/10)<br />
vs.. 66.7% (6/9)] did not differ between GEB and GGo (P > 0.05). However, as expected, the interval between Estradiol Benzoate administration<br />
and ovulation was greater (51.0 ± 2.0 h) than the interval between Gonadorelin administration and ovulation (32.0 ± 2.7 h; P < 0.01). The results<br />
of the present study suggest that the treatment with progesterone associated to Estradiol Benzoate (D0), followed by the administration of eCG<br />
and PGF2α (D9), with the subsequent induction of ovulation using Estradiol Benzoate (D10) or Gonadorelin (D11) resulted in satisfactory<br />
follicular response and ovulation rate, with possible indication for the use in synchronization of ovulation and FTAI in water-buffalo heifers<br />
during the non-breeding season. However, further studies with a larger sample size are needed to confirm these results.<br />
Keywords: buffalo heifers, ovulation induction, ftai.<br />
s391
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A111 FTAI, FTET AND AI<br />
SYNCHRONIZA<br />
ONIZATION AND INDUCTION OF FERTILE ESTRUS OF PACA A (<br />
(CUNICUL<br />
CUNICULUS US PACA A L.)<br />
Vânia Maria França Ribeiro 1 , Rodolpho Satrapa 2 , Jefferson Viana Alves Diniz 3 , Rafael Augusto Satrapa 4 & Rodolfo Rumpf 5<br />
1,2<br />
UNIVERSIDADE FEDERAL DO ACRE (UFAC), RIO BRANCO, AC, BRAZIL. 3 SECRETARIA DE ESTADO DE AGROPECUÁRIA DEPARTAMENTO DE MELHORAMENTO E DIFUSÃO<br />
GENÉTICA DO ESTADO, RIO BRANCO, AC, BRAZIL. 4 DEPARTAMENTO DE FARMACOLOGIA, UNESP/BOTUCATU, SAO PAULO, SP, BRAZIL. 5 EMPRESA BRASILEIRA DE PESQUISA<br />
AGROPECUÁRIA/CENARGEN, BRASILIA, DF, BRAZIL.<br />
To verify the effect of progestin implants associated with two different dosis of gonadotropin on induction of estrus synchronization<br />
and fertile on paca, 18 females composed the G1, G2 and G3, with six animals each. In females of G1 and G2 were inserted 1.5 mg norgestomet<br />
(Crestar, Intervet Shering Plough), which were removed after eight days (D8), and 0.25 IU and 0.50 IU of eCG (Novormon 5000, Intervet<br />
Shering Plough), taught in G1 and G2 respectively. Twenty-four hours before the D8, females received 0.5 mL of prostaglandin (Ciosin, Intervet<br />
Shering Plough). G3 did not receive treatment and was paired with males on the same day that G1 and G2. G1 females presented estrus five,<br />
seven, 10, 11 (paca /day) and 14 (two pacas/day) days and the G2 three (two pacas/day), eight, 11 (three pacas/day) days after D8, respectively.<br />
G3 came in heat one, five, seven, eight, nine and 13 days after pairing with males. G1, G2 and G3 pregnancy rates were 100% (6 females), 66%<br />
(4 females) and 50% (3 females), respectively. G1 and G2 and G3 calving rates were 83.3% and 100%, respectively. With respect of litter per<br />
offspring, 100% of G1 and G3 produced one, while 50% of G2 produced two. The hormonal protocols used were effective in inducing and<br />
synchronizing fertile estrus and improved the reproductive efficiency of pacas. [Financial support: Acre State Department of Agriculture].<br />
Keywords: estrus synchronization, cuniculus paca l., progesterone.<br />
A112 FTAI, FTET AND AI<br />
SUPPLEMENTATION TION OF NELORE COWS WITH RUMEN-PR<br />
UMEN-PROTECTED FAT BEFORE AND/OR AFTER TAI<br />
Igor Miglioli Sokoloski 1 , Monique Mendes Guardieiro 2 , Alexandre Barbieri Prata 3 , Luis Henrique Dantas Carrijo 4 , Gerson Barreto<br />
Mourão 5 & Roberto Sartori 6<br />
1,2,3,5,6<br />
ESALQ, PIRACICABA, SP, BRAZIL. 4 INTEGRAL NUTRIÇÃO ANIMAL, GOIÂNIA, GO, BRAZIL.<br />
Previous studies have shown an increase in conception rate of Nellore cows supplemented with rumen-protected fat rich in linoleic<br />
acid, from the beginning of the ovulation synchronization protocol or immediately after TAI (Lopes et al. 2009, J Anim Sci 87:3935-3943). In<br />
contrast, supplementation with the same product previously to AI in Nellore embryo donors, have compromised in vitro embryo development<br />
after cryopreservation (Guardieiro et al. 2010, Reprod Fertil Dev 22:205-206). Furthermore, in vitro supplementation with linoleic acid inhibited<br />
oocyte development and maturation (Marei et al. 2010, Reproduction 139:979-988). Based on these contradictory results, the objective was to<br />
evaluate the effect of supplementation of cows with rumen-protected fat before and/or after TAI on conception rate. We hypothesized that fat<br />
supplementation before TAI compromises conception rates, whereas after TAI it increases. A total of 399 primiparous and multiparous Nellore<br />
cows (60-80 days postpartum) with a mean BCS of 4 (scale of 1 to 9) were kept on pasture and randomly divided into four treatments according<br />
to the daily supplementation: 100 g-Megalac-E ® (Arm & Hammer Church & Dwight Company, Brazil) per cow for 30 days before TAI (30B,<br />
n = 98); for 30 days after TAI (30A, n = 101); for 30 days before and 30 days after TAI (60d, n = 100) or without Megalac-E ® (0d; Megalac-<br />
E ® was replaced by kaolin, n = 100). Megalac-E ® was offered to cows as part of a supplement called Fertigral 200 ® (Integral Produbon Nutrição<br />
Animal, Brazil). Cows were inseminated using the following ovulation synchronization protocol: D0 - CIDR ® (progesterone intravaginal<br />
device, Pfizer Animal Health, Brazil) and 2.0 mg estradiol benzoate (Estrogin ® , i.m., Farmavet, Brazil), D6 - 12.5 mg dinoprost tromethamine<br />
(Lutalyse ® , i.m., Pfizer Animal Health), D8 - 0.5 mg estradiol cypionate (ECP ® , i.m., Pfizer Animal Health), 300 IU eCG (Novormon 5000 ® ,<br />
i.m., Intervet Schering-Plough, Brazil) and CIDR ® removal; D10 - AI. Pregnancy diagnosis was performed by ultrasonography 45 days after<br />
TAI. The results were analyzed by the GLIMMIX procedure of SAS ® and are presented as least squares means ± SE. There was no difference<br />
in conception rates at 45 days among groups (30B: 55.6 ± 6.7; 30A: 53.7 ± 6.6; 60d: 56.7 ± 6.6 and 0d: 49.9 ± 7.4%; P = 0.90), independently<br />
of animal category. Contrary to our hypothesis, supplementation with rumen-protected fat rich in polyunsaturated fatty acids both before and/or<br />
after TAI did not affect conception rate in postpartum Nellore cows. [Acknowledgments: To CNPq, FAPESP, Integral Produbon, Agropecuária<br />
Vale do Sonho, Arm & Hammer and EMBRAPA (Innovation Network on Animal Reproduction - 01.07.01.002)].<br />
Keywords: polyunsaturated fatty acids, reproduction, tai.<br />
s392
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A113 FTAI, FTET AND AI<br />
PREGNANCY RATES OF DIFFERENT PROTOC<br />
OCOLS OLS FOR FIXED TIME TRANSFER OF IN VITRO PRODUCED BOVINE EMBRYOS<br />
Tess Picq Coutinho<br />
1 , Yeda Fumie<br />
Watanab<br />
tanabe 2 , Leandr<br />
eandro Mour<br />
oureir<br />
eira De Castr<br />
astro Feit<br />
eitosa<br />
3 , Mar<br />
arcus<br />
Vinícius Galvão Loiola<br />
3 , Bruno Henr<br />
enrique De Araújo Andr<br />
ndrade<br />
3 ,<br />
Alexandra Soares Rodrigues 3 , Priscila Assis Ferraz 3 , Marcos Chalhoub 3 , Endrigo Adonis Braga De Araujo 3 , Sidnei Nunes Oliveira 3 , Marcos Ribeiro 1 & Antônio<br />
De Lisboa Ribeiro Filho 3<br />
1<br />
FACULDADE DE CIÊNCIAS AGRÁRIAS E DA SAÚDE - UNIME, LAURO DE FREITAS, BA, BRAZIL. 2 VITROGEN PESQUISA E DESENVOLVIMENTO EM BIOTECNOLOGIAS DE<br />
REPRODUÇÃO LTDA, CRAVINHOS, SP, BRAZIL. 3 ESCOLA DE MEDICINA VETERINÁRIA - UFBA, SALVADOR, BA, BRAZIL.<br />
The objective of this study was to evaluate the effect of three different estrus synchronization protocols over the pregnancy rates of<br />
in vitro produced bovine embryos. A total of 1.248 recipients Bos taurus taurus x Bos taurus indicus were randomly distributed in three<br />
experimental groups, according to the estrus synchronization protocol used: the females in group P1 (n = 421), in a random day of the estrous<br />
cycle named day zero (D0), received an intravaginal progesterone implant (CIDR ® , Pfizer, São Paulo, Brazil), 2mg of Estradiol Benzoate i.m.<br />
(EB, Gonadiol ® , Shering-Plough, São Paulo, Brazil), and 250µg Cloprostenol, i.m. (Sincrocio ® , Ouro-Fino, São Paulo, Brazil). On day eight<br />
(D8), the progesterone implants were removed followed by the administration of 300IU of eCG i.m. (Novormon ® , Shering-Plough, São Paulo,<br />
Brazil), 250µg of Cloprostenol i.m. and 1mg of EB i.m. The animals in group P2 (n = 389) received on D0 a progesterone implant associated<br />
with 2mg of EB i.m., on D8 the progesterone implants were removed, the animals were also treated with 400IU of eCG i.m. and 500µg of<br />
Cloprostenol i.m., and finally on D9 1mg of EB i.m. was administered. The recipients in group P3 (n = 438) received on D0 progesterone<br />
implants, 2mg of EB i.m. and 250µg Cloprostenol i.m. On D8 the progesterone implants were removed and subsequently 300IU of eCG i.m.,<br />
500µg of Cloprostenol i.m and 1mg of Estradiol Cipionate i.m. (EC, ECP ® , Pfizer, São Paulo, Brazil) were administered. The in vitro produced<br />
embryos were transferred on day 17 (D17) of each estrus synchronization protocol. The pregnancy diagnose was accomplished by transrectal<br />
ultrasound 30 days after fertilization and the results were processed and analyzed by the chi-square test (÷2) available in SAS statistical software,<br />
version 6.0 (P < 0.05). The pregnancy rates of groups P1, P2, P3 were, respectively, 27.08% (114/421); 24.68% (96/389) and 31.74% (139/<br />
438). On comparison of the pregnancy rates between groups P1 and P2 no difference was verified (P > 0.05), the same pattern was observed<br />
when comparing groups P1 and P3, on the other hand, group P3 showed superior pregnancy rates (P < 0.05) when compared to group P2. The<br />
results suggest that the three protocols used were efficient in estrus synchronization for recipients of in vitro produced bovine embryos,<br />
nevertheless, the protocol that used EC stood out amongst the others for providing pregnancy rates equivalent or superior, besides reducing the<br />
number of handlings to which the animals were submitted.<br />
Keywords: estradiol benzoate, cipionate, ftet.<br />
A114 FTAI, FTET AND AI<br />
PREGNANCY RATE AND EMBRYONIC/FET<br />
ONIC/FETAL LOSS AFTER ECG ADMINISTRATION TION GIVEN 5 OR 10 DAYS AFTER<br />
INSEMINATION IN CYCLING CLING EWES<br />
C. Gar<br />
arcía-P<br />
cía-Pin<br />
int os, P. C. Dos San<br />
ant os-Net<br />
eto & A. Menchac<br />
enchaca<br />
INSTITUTO REPRODUCCIÓN ANIMAL URUGUAY, MONTEVIDEO, URUGUAY.<br />
Regarding a recent report in which eCG given twice -at intravaginal device removal and 14 days after insemination- improved<br />
pregnancy rate in beef cattle (Nuñez et al., <strong>2011</strong>; Reprod. Fertil. Dev.;23:163.), we evaluated the use of eCG after insemination in sheep. The<br />
experiment was performed during breeding season (April, 33º S, Uruguay) on a total of 408 cycling multiparous ewes with a body condition<br />
score of 2.8±0.3 (scale 0–5). Ewes received a Short-term Protocol, which consists of 6 days treatment with intravaginal sponges containing 60<br />
mg of medroxiprogesterone acetate (Syntex, Argentina). One dose of PGF2α analogue (125ug cloprostenol, Ciclase DL, Syntex) and 300 IU<br />
of eCG (Novormon, Syntex) was given at the time of device removal. Fixed-time intrauterine insemination was performed by laparoscopy with<br />
fresh semen (100 millions of spermatozoa) between 48 to 56 h after sponge removal. Ewes were randomly assigned to three experimental groups<br />
to receive a second dose of 300 IU of eCG on Day 5 (n = 137) or Day 10 (n = 138) after insemination, or no eCG was administrated after<br />
insemination (n = 133). Non return to estrus was determined by estrus detection with vasectomized rams twice daily from Day 15 to 24 after<br />
insemination. Pregnancy rate was determined by transrectal ultrasonography (5 MHz, Well-D, China) 30 days after insemination. Birth rate was<br />
determined by inspection of parturition twice a day from 140 to 155 days after insemination. Embryonic loss was determined by number of<br />
pregnant ewes at 30 days after insemination on number of ewes that non return to estrus, and fetal loss by number of newborn at parturition on<br />
number of fetuses at 30 days after insemination. Neither non return to estrus rate, pregnancy rate, nor birth rate was affected by eCG<br />
administration, averaging 44.8% (183/408), 34.8% (142/408), and 32.1% (127/396), respectively (P = NS; logistic regression). Number of<br />
fetuses/pregnant ewes and number of fetuses/inseminated ewes, as well as number of newborns/ewes giving birth and number of newborns/<br />
inseminated ewes were not different between experimental groups (P = NS; Poisson regression). In addition, no effect of eCG was found in<br />
embryonic/fetal loss (P = NS; logistic regression). In conclusion, a second dose of 300 IU of eCG given 5 days or 10 days after insemination<br />
in cycling ewes has not significant influence on pregnancy rate, birth rate and embryonic/fetal loss.<br />
Keywords: embrionic/fetal loss, ecg, insemination sheep.<br />
N<br />
s393
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A115 FTAI, FTET AND AI<br />
CONCEPTION RATES IN BEEF CATTLE TLE GIVEN A NEW OR PREVIOUSL<br />
VIOUSLY USED PROGESTER<br />
OGESTERONE ONE DEVICE IN FIXED-TIME<br />
ARTIFICIAL INSEMINATION<br />
Aline Kehrle 1 , Manoel Pereira Neto 2 , Milton Maturana Filho 1 , Patrícia Helena Paiva Miguez 1 , Gabriel Carvalho Gomes 1 , Orlando De Oliveira Junior 3 , Thiago<br />
Silva Antonio 3 & Ed Hoffman Madureira 1<br />
1<br />
UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL. 2 FAZENDA GUARÁ, ALIANÇA DO TOCANTINS, TO, BRAZIL. 3 BIOGÉNESIS-BAGÓ, CURITIBA, PR, BRAZIL.<br />
Re-utilization of Progesterone (P4) inserts has been widely used, as the costs of a hormonal protocol can be reduced by device reutilization.<br />
An experiment was conducted aiming to compare FTAI conception rates (CR) in beef cattle given a new or previously used<br />
(Cronipres ® - Biogenesis-Bago, Argentina) P4 devices. In this experiment 763 lactating Nellore (Bos indicus) and crossbreed Simental x Nellore<br />
(Bos taurus x Bos indicus) cows with age ranged from 3-10 years, body condition score 4 to 7 (1-9 scale) were distributed in four FTAI groups.<br />
All animals were treated with 2 mg i.m. of Oestradiol Benzoate (Cronibest ® - Biogenesis-Bago, Argentina) and received the Cronipres ® P4<br />
(containing 1 g of P4) device (CRO1 - new devices, n = 194; CRO2 – devices used once, n = 190; CRO3 – devices used two times and added<br />
with one P4 ring (0.1 g P4), n=191 and CRO4 – devices used three times and added with two more P4 rings, n = 188). Together with device<br />
withdrawal (day 8) all animals received 300 IU i.m. of eCG (Novormon ® , Schering–Plough, USA) and 150 µg i.m. of D–cloprostenol,<br />
(Croniben ® , Biogenesis-Bagó, Argentina). On day 9, all cows were treated with 1 mg of Oestradiol Benzoate (Cronibest ® - Biogenesis-Bago,<br />
Argentina) and FTAI was performed 30h later. In each group, treatments were equally distributed. The data were submitted to analysis of<br />
variance (PROC GLIMMIX – Statistical Analysis Sistem - SAS ® ), using as explanatory variables the effect of treatment (CRO1, CRO2, CRO3<br />
e CRO4), body condition score (BCS5), ciclicity (cycling and non–cycling), FTAI groups (1 to 4) parity (5 mounths) and breed (Nellore and Simental X Nellore) and its interactions, eliminating the variables from the final model<br />
when P > 0.2. The final model included treatment (P = 0.18), breed (P = 0.11) and FTAI group (P = 0.14). CR did not differ among treatments<br />
(CRO1 – 55.1%a, CRO2 – 47.9%ab, CRO3 – 48.7%ab e CRO4 – 44.15%b) (P = 0.18). Overall CR was 49.1%. There was no statistical<br />
difference between treatments, however a larger number of animals should be tested.<br />
Keywords: previously used devices, ftai, progesterone.<br />
A116 FTAI, FTET AND AI<br />
USE OF DIB ® AND CRESTAR<br />
® TO SYNCHRONIZE OVULATION AND FTAI IN BUFF<br />
UFFAL<br />
ALO HEIFERS DURING THE<br />
NON-BREEDING SEASON<br />
Nelcio Antonio<br />
Tonizza Car<br />
arvalho<br />
alho 1 , Júlia Gle<br />
leyci Soar<br />
oares<br />
2 , Ever<br />
erton Luiz Reis<br />
3 & Pietr<br />
ietro Sampaio Bar<br />
aruselli<br />
2<br />
1<br />
APTA, REGISTRO, SP, BRAZIL. 2 VRA-FMVZ-USP, SÃO PAULO, SP, BRAZIL. 3 INTERVET/SHERING-PLOUGH, COTIA, SP, BRAZIL.<br />
The aim of this study was to evaluate the effect of intravaginal progesterone device (DIB ® ) and subcutaneous implant of progestogen<br />
(CRESTAR ® ) on follicular response and on conception rate in buffalo heifers synchronised to fixed time artificial insemination (FTAI) during<br />
the non-breeding season (spring and summer). For this, one hundred and third heifers (n = 130) were divided into two groups (DIB ® = GD, n<br />
= 65; CRESTAR ® = GC, n = 65) according to age, weight, body condition score and ovarian activity. One part of this animals (GD=13 and<br />
GC=17) were randomly selected to verify the follicular dynamics. At random stages of estrus cycle (Day 0 = D0; PM), buffaloes in GD received<br />
a DIB ® (1,0gr. of P4, Intervet/Schering-Plough, Brazil) and in GC a CRESTAR ® (3.0mg of Norgestomet, Intervet/Schering-Plough, Brazil). At<br />
the same moment (D0, PM), all animals received more 2.0mg of Estradiol Benzoate (i.m.; Gonadiol ® , Intervet/Schering-Plough, Brazil). On D9<br />
(PM), the devices and implants were removed and was administrated 150µg of PGF2a analogue (i.m.; d-cloprostenol sodic, Preloban ® , Intervet/<br />
Schering-Plough, Brazil) plus 400IU of eCG (i.m.; Folligon ® , Intervet/Schering-Plough, Brazil) in all buffaloes. After 48h (D11, PM), the<br />
ovulation was induced by 10µg of GnRH (i.m.; Buserilin Acetate, Conceptal ® , Intervet/Schering-Plough, Brazil). The AI was realised with<br />
applicator and sheath specific for sheep and goats (IMV, France) 16h after the last hormonal administration (D12, AM). The ultrasound<br />
(Mindray, DP2200Vet, China) examination was performed in all heifers on D0 to verify the ovarian status and on D42 – thirty days after the<br />
FTAI – to the pregnancy diagnosis. From D9 to D11 (24/24h) and from D11 to D14 (12/12h for 60h) the 30 heifers selected to verify the<br />
follicular dynamics was submitted to access the follicular diameter and the moment of ovulation by ultrasonography. Data were analyzed by<br />
ANOVA of the SAS for Windows program and by Chi-square test. There were no significant differences (P > 0.05) between groups GD and<br />
GC for the dominant follicle diameter (Æ) on D9 (1.0 ± 0.1 vs.. 1.0 ± 0.0 cm), the ovulatory follicle Æ (1.4 ± 0.0 vs.. 1.4 ± 0.0 cm), the interval<br />
GnRH administration/ovulation (29.0 ± 3.7 vs.. 30.0 ± 1.9 h), the ovulation rate [92.3% (12/13) vs.. 94.1% (16/17)] and to the conception rate<br />
[47.7% (34/65) vs.. 47,7% (34/65)]. Both hormonal protocols used at the present study provided satisfactory follicular response and conception<br />
rate in FTAI buffalo heifers during the non-breeding season.<br />
Keywords: ftai, buffalo heifers, non-breeding season.<br />
s394
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A117 FTAI, FTET AND AI<br />
REPRODUCTIVE ULTR<br />
TRASONOGR<br />
ASONOGRAPHY APHY FOR USE OF HIGH COST SEMEN IN FTAI PROGR<br />
OGRAMS<br />
José Ribamar De Souza<br />
Tor<br />
orres Júnior, Hans Muller<br />
Vaz Braga Aguiar & Paulo Henr<br />
enrique Cavalc<br />
alcan<br />
anti<br />
UNIVERSIDADE FEDERAL DO MARANHÃO, CHAPADINHA, MA, BRAZIL.<br />
The objective was to evaluate the impact of reproductive ultrasonography based on costs of semen in FTAI programs. Lactating<br />
Nelore cows (n = 796), were allocated in different handled treatments according to use or not of reproductive ultrasound examination at FTAI<br />
(US vs. noUS) (US; CHISON D600VET, USPBrazil Eletromedicina, Brazil) and inseminated of two manners: aleatory or dominant follicle<br />
diameter dependent (DF = or < 11mm). We hypothesized that the biggest DF have best chances of increases ovulation and conception rates. The<br />
DF=11mm animals were inseminated with high cost semen (T35US; $35.00/batch) and the DF
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A119 OPU-IVP AND ET<br />
ULTR<br />
TRASOUND-GUIDED FOLLICLE ASPIRATION DO NOT REDUCE BLOOD FLOW IN THE FOLLICULAR<br />
WALL - PRELIMINARY<br />
RESULTS<br />
Alberto Mansur Ghetti 1 , Felipe Zandonadi 2 , Luiz Gustavo Bruno Siqueira 3 , Luiz Sérgio Almeida Camargo 4 , Eduardo Kenji N. Arashiro 5 , Carlos Alberto Soares<br />
Paim 6 & João Henrique Moreira Viana 7<br />
1,2<br />
UFF, NITERÓI, RJ, BRAZIL. 3,4,7 EMBRAPA CNPGL, JUIZ DE FORA, MG, BRAZIL. 5 UFMG, BELO HORIZONTE, MG, BRAZIL. 6 CENTRO DE ENSINO SUPERIOR DE JUIZ DE FORA, JUIZ DE<br />
FORA, MG, BRAZIL.<br />
Removal of the follicular content by transvaginal ultrasound-guided aspiration (OPU) may not induce immediate atresia and loss of<br />
function, resulting in the occurrence of residual or persistent follicles (Viana et al. 2001; Dorea et al., <strong>2011</strong>). The presence of steroidogenic<br />
activity post-aspiration in these structures may compromise the efficiency of the synchronization of follicular growth by removing the dominant<br />
follicle. The mechanisms associated with maintaining or not of the activity of the remaining follicular wall cells, however, are not clear. The aim<br />
of this study was to evaluate the changes in blood flow caused by follicular aspiration in functional dominant follicles. Holstein-Zebu cows (n<br />
= 10), lactating, cyclic and with body condition score of 2.4 ± 0.36 were used. The follicular growth was synchronized (D0) by 1 mg of<br />
oestradiol benzoate (Sincrodiol, OuroFino Agronegócios, Cravinhos, SP), and insertion of a progesterone releasing device (Sincrogest,<br />
OuroFino). In D0 the cows received 0.5 mg of clorprostenol sodium (Sincrocio, OuroFino) to prevent any interference of any corpus luteum<br />
in the evaluation of vascular dynamics. The dominant follicle of the subsequent wave (mean diameter 12.9±0.8 mm) was aspirated using<br />
conventional OPU procedures. The blood flow (BF) was monitored by color Doppler immediately before and every 12 h after aspiration for<br />
three days, using an ultrasound device equipped with a 7.5 MHz linear transducer (MyLabVet 30, Esaote, Genoa, Italy). The BF of each follicle<br />
was measured by calculating the ratio of the maximum area of the Doppler signal and the area of the follicular wall in the center section of the<br />
follicle. All aspirated follicles showed persistence, characterized by initial formation of a clot in the cavity 12 h after follicular aspiration, followed<br />
by retraction and formation of fluid-filled cavity with anechoic appearance, similar to the antrum of non-aspirated follicles. In one animal (10%)<br />
a reduction (87.0%) in BF was observed subsequent to aspiration, in the other animals the BF showed a quadratic effect according time postaspiration<br />
(y= -1.16x2 +11.33x +16.26; R² = 0.76), with an increase up to 48h and a reduction thereafter (P < 0.01). The increase in BF after<br />
aspiration may be due to the inflammatory reaction associated with the procedure, and could indirectly contribute to the maintenance of the<br />
steroidogenic activity in the remaining cells of the follicular wall. The preliminary results suggest that the follicle aspiration affects the pattern of<br />
vascular wall, leading to a transient increase in blood flow. [Acknowledgment: Nutricell Nutrientes Celulares, CAPES, Fapemig and Embrapa<br />
project 01.07.01.002].<br />
Keywords: opu, blood flow, doppler.<br />
A120 OPU-IVP AND ET<br />
EVAL<br />
ALUATION OF OOCYTES RECOVERED FROM COWS UNDERGOING IMMUNOSUPPRESSIVE PROTOC<br />
OCOL<br />
OL WITH<br />
DEXAMETHASONE<br />
André Penido Oliveira, Paula Maria Pires Do Nascimento, , Juliana Marques Bicalho, Guilherme Gomes De Carvalho, Rejane Silva<br />
Diniz, Jenner Karlisson Pimenta Dos Reis, Rômulo C Leite & Magali D´Angelo<br />
UFMG, BELO HORIZONTE, MG, BRAZIL.<br />
Glucocorticoids have immunosuppressive and antiinflammatory activity, affect the release of GnRH (Nangalama and Moberg,<br />
1991, Journal of Endocrinol. 137, 87-94) and can affect programs of fixed-time AI, in vivo and in vitro embryo production. The objective of the<br />
study was to evaluate the production of COCs recovered by OPU after dexamethasone administration in cows. We used nine (n = 9) females,<br />
with average weight of 454 ± 55.7 kg and 3.5 ± 0.5 score of body condition, aged between four and six years old. The animals were divided<br />
evenly into two groups: treatment group (Gt, n = 6) and control group (Gc, n = 3). In the D0 Gt received 0.1 mg / kg of dexamethasone (iv),<br />
repeated every 24 h for 5 consecutive days. The Gc group received saline solution (0.1 mg / kg IV) like Gt. Both groups underwent OPU every<br />
four days (D0, D4, D8, D12, D16 and D20), in a total of six aspirations in the experimental period (20 days). COCs recovered were classified<br />
as viable or unviable, according to Viana et al. (2004, Anim. Reprod. Sci. 84: 1-12). The data were analysed by t test or correlation analysis.<br />
Difference was observed between the number of viable COCs and unviable during the experimental period. No differences were observed in the<br />
total number of COCs between days of treatment (P < 0,05). Low correlation (r = 0,07) between the number of viable COCs and treatment day,<br />
so the dexamethasone protocol did not affect the quality and quantity of recovered COCs.<br />
Keywords: oocyte, dexametason, bovine.<br />
s396
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A121 OPU-IVP AND ET<br />
EVAL<br />
ALUATION OF A MEDIUM BASED ON COC<br />
OCONUT<br />
ONUT WATER (COC<br />
OCOS OS NUCIFERA)<br />
TO IN VITRO EMBRYO PRODUCTION OF<br />
BOVINE<br />
Marcela Da Silva Cordeiro 1 , Priscilla Do Carmo De Azevedo Ramos 2 , Nathália Nogueira Da Costa 2 , Thiago Velasco Guimarães Silva 2 , Priscila Di Paula Bessa<br />
Santana 2 , Bruno Baraúna Da Silva 2 , Davi César Nascimento Dos Santos 2 , Andre Luiz Alves De Sá 2 , Stefanne Dhullia Braga Conceicão 2 , Carlos Leonardo De<br />
Aragão Araújo 2 , Simone Do Socorro Damasceno Santos 2 , Moysés Dos Santos Miranda 2 & Otávio Mitio Ohashi 2<br />
1<br />
INSTITUTO FEDERAL DE EDUCAÇÃO, CIÊNCIA E TECNOLOGIA DO PARÁ, ABAETETUBA, PA, BRAZIL. 2 UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL.<br />
Studies on the chemical composition of coconut water in natura reveal that some of its features are quite different from those<br />
presented by the TCM 199 (Ramos, et al., 2010, Acta Scientiae Veterinariae, 38: 764) which limits its use as a culture medium in IVP. Therefore<br />
the aim of this study was to evaluate a supplemented medium based on coconut water (S-BCWM) to be used in the process of IVM, IVF and<br />
IVC. The S-BCWM consisted of 20 mL of in natura coconut water (5 months of age) + 80 mL of ultrapure water + osmolytes (0.46g NaCl and<br />
0.018g MgSO4, values determined in a previous analysis of chemical composition of coconut water in natura). Amino acids, myo-inositol, trisodium<br />
citrate and sodium bicarbonate were added according to basic formulation of the SOF (Tervit et al., 1972 Journal Reproduction Fertility,<br />
30: 493-497). For IVM period the TCM 199 and S-BCWM media were supplemented with 0.5µg/mL FSH, 50µg/ml LH, 50µg/mL of<br />
gentamicin, 22µg/mL of pyruvate and 10% FCS. In the IVF the SOF medium (without glucose) and S-BCWM were add with 10µg/mL heparin,<br />
2µM penicillamine, 1µM hypotaurine, 0.25 µM epinephrine, 50µg/mL of gentamicin, 22µg/mL of pyruvate and 6 mg/mL BSA. In the<br />
process of the IVC the SOF (1 mM glucose) and S-BCWM were supplemented with 6 mg/mL BSA, 10 µg/mL insulin, 50µg/mL of gentamicin<br />
and 10% FCS. COCs from slaughterhouse were selected and randomly distributed among the following groups: G1 (IVM in TCM199, IVF<br />
in IVC and IVC in SOF), G2 (IVM in S-BCWM, IVF in SOF and IVC in SOF), G3 (IVM in TCM 199, IVF in S-BCWM and IVC in SOF)<br />
and G4 (IVM in TCM199, IVF in SOF and IVC in S-BCWM). Fertilization was performed 18 h after the onset of IVM with semen from a<br />
single bull (2x10 6 sperm/mL). After 30h of IVF, presumptive zygotes were transferred to culture media drops for co-culture with a cumulus<br />
cells monolayer. IVP process was performed in incubator with 5% CO 2<br />
, at 38.5°C with high humidity. Fertilization, cleavage and blastocyst<br />
rates were analyzed by ANOVA (significance level of 5%). It was observed that the maturation of oocytes resulted in S-BCWM Cleavage<br />
(82.50 ± 5.2%) and blastocysts (36.07 ± 8.8%) similar to those obtained by maturation in TCM 199 (82.61 ± 6.7% to 41.47 ± 7.9%, P ><br />
0.05). However, the use of S-BCWM during the IVF period blocked the fertilization process, once 100% of the oocytes were arrested in MII<br />
stage after 18h IVF. When S-BCWM was used for embryo cultivation there was no embryonic development. Therefore, the S-BCWM can<br />
be an option only as a maturation medium in the process of IVP of bovine. Acknowledgements: FAPESPA and UNOPAR.<br />
Keywords: coconut water, cocos nucifera, ivp.<br />
A122 OPU-IVP AND ET<br />
EVAL<br />
ALUATION OF A NEW METHOD FOR IDENTIFYING ENDOMETRITIS IN ANESTROUS MARES THROUGH<br />
ULTR<br />
TRASSONOGR<br />
ASSONOGRAPHY<br />
APHY<br />
Gabriel Maksoud Greco 1 , Leticia Mendes Pupio Maia 1 , Maria Manoela Castro Chaves 2 & Marco Antonio Alvarenga 42<br />
N<br />
1<br />
MÉDICO VETERINÁRIO AUTÔNOMO, CONSELHEIRO LAFAIETE, MG, BRAZIL. 2 UNIVERSIDADE ESTADUAL PAULISTA, BOTUCATU, SP, BRAZIL.<br />
At the beginning and end of the breeding season, when the percentage of recipient mares showing normal estrous cycles is low<br />
compared to donors, progesterone-treated acyclic recipients have been routinely used in embryo transfer programs. However, anestrous<br />
recipients are often selected for treatment through the evaluation of their body score condition and reproductive history, since their poor uterine<br />
tone and endometrial atrophy usually impair it´s evaluation through ultrassonography and rectal palpation. The objective of the present study was<br />
to test the hypothesis that a single estrogen injection could aid in the ultrassographical identification of endometritis in anestrous mares. Twentyfour<br />
anestrous mares that did not show air, endometrial edema or uterine fluid through ultrassography were submitted for endometrial cytology<br />
with a sterile uterine brush. A minimum of ten fields were evaluated per Giemsa-stained slide. Mares were classified as positive for cytology<br />
(moderate endometritis) when more than two neutrophils were microscopically identified per field under oil immersion (×100). Animals were<br />
subsequently injected with 5.0 mg of estradiol benzoate (Estrogin ® , Farmavet, São Paulo, Brazil) I.M., being evaluated through ultrassonography<br />
past 48 h to detect uterine fluid accumulation. The presence and depth of intra-uterine fluid was recorded. Results were statistically analyzed<br />
through the Chi-square test, significance levels set as P < 0.05. Of the twenty-four anestrous mares, eleven were positive and thirteen negative<br />
for cytology. All of them presented some grade of endometrial edema 48 h post-injection. Intra-uterine fluid was detected in all of the positive<br />
cytology mares (100%; 11/11), which was fairly echogenic in three (27.3%; 3/11) and had a depth of a least 20 mm in eight (72.7%; 8/11). On<br />
the other hand, five of the thirteen negative cytology mares (38.5%; 5/13) showed fluid accumulation and only a single mare (20.0%; 1/5) had<br />
more than 20 mm of uterine fluid. Positive cytology mares had higher chances of accumulating intra-uterine fluid, which can exceed 20 mm in<br />
depth (P < 0.05). In conclusion, mares showing uterine fluid 48 h post-treatment with 5.0 mg of estradiol benzoate should not be used as embryo<br />
recipients, for they have higher chances of suffering from endometritis. Moreover, authors suggest that this method should be routinely<br />
implemented during reproductive evaluations of anestrous recipient mares, especially prior to purchase.<br />
Keywords: mare, anestrous, endometritis.<br />
s397
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A123 OPU-IVP AND ET<br />
HEMODINAMIC EVAL<br />
ALUATION OF THE UTERUS US IN MARES USED AS RECIPIENT: PAR<br />
ARTIAL RESULTS<br />
Fernanda Saules Ignácio, , Jair Camargo Ferreira, Roberta Pahim De Melo, Natalia Claro De Abreu, Gustavo Henrique Marques Araújo & Cezinande Meira<br />
UNESP-BOTUCATU, BOTUCATU, SP, BRAZIL.<br />
An important limitant factor in embryo transfer programs is the number and quality of available recipient mares during the<br />
breeding season. Real time doppler ultrassonography has been recent used to study uterine hemodynamic. However, this is the fisrt report<br />
of Doppler ultrassonography evaluation in recipient mares. The aim of this work was to evaluate recipient mares using power Doppler. A total<br />
of seven recipient mares were ultrassonographicaly followed for ovulation induction (1600IU of hCG). Inovulation was done among D4 to<br />
D6 (D0 = ovulation). Vascular perfusion and spectral Doppler data from vessels of the mesometrium attachment were daily collected from<br />
horns separately (right and left) from D0 to D15. In the pregnant mares (5/7), embryo was located to compare horns with (+) and without (-<br />
) embryo from D9 to D15. Vascular perfusion of both horns were subjectively estimated by scoring (from 1 to 4) the extent of colored areas<br />
in the endometrium during real-time cross-sectioning of the middle segment of each horn during continuous scan of 1 min. For the spectral<br />
mode, one cardiac cycle was arbitrarily chosen from each of the three scans made. Analysis of variance followed by Tukey (P < 0.05) were<br />
used for comparison between right and left horns, pulsatility (PI) and resistance (RI) indexes. Analysis of variance followed by Kruskal-Wallis<br />
were used for comparing (+) and (-) horns. For evaluation of vascular perfusion during the days, Student-Newman-Keuls test was used. For<br />
vascular perfusion, RI and PI Statistical differences were not detected (P > 0.05) when right and left horns were compared, but difference (P<br />
< 0.05) along the days was detected. However, this difference was not detected by Tukey test for the RI. An increase in uterine vascular<br />
perfusion in recipient mares during the days of inovulation (PI at D5 = 0.96±0.10) and, in pregnant mares, a gradual increase kept until D15<br />
(PI = 0,75±0,75). Even though, more studies are still needed to confirm and to add more information to these findings. Uterine hemodynamic<br />
evaluation using the power Doppler and Spectral Doppler showed a great potential for a reliable evaluation of recipient mares and for the<br />
transferred embryos.<br />
Keywords: embryo transfer, recipient, doppler.<br />
A124 OPU-IVP AND ET<br />
COMP<br />
OMPARISON OF THE PREGNANCY RATE OF EMBRYOS COMING OF OVULATION IN MARES SINGLE OR DOUBLE RACE POLO<br />
ARGENTINO<br />
Talita Dias Dos Reis<br />
1 , Mar<br />
arcos Massao Higuti 1 , Jamile Toth De Paula<br />
1 , Suellen Migue<br />
iguez Gonzále<br />
onzález 1 , Eduar<br />
duardo do Ber<br />
ernar<br />
nardi<br />
2 & Thales Ric<br />
icar<br />
ardo Rigo Bar<br />
arreir<br />
eiros<br />
1<br />
1<br />
UNIVERSIDADE ESTADUAL DO NORTE DO PARANÁ, BANDEIRANTES, PR, BRAZIL. 2 CENTRAL DE REPRODUÇÃO EQUINA-MALALHUE, CASBAS, ARGENTINA.<br />
The aim of this study was to evaluate the pregnancy rate of embryos from double ovulation or simple. The study was conducted<br />
through analysis of results in Equine Reproduction Center-Malalhue Casbas in the city of the province of Buenos Aires, Argentina. Experimental<br />
period was the season of 2010 to February <strong>2011</strong>. We used 90 mares bred Polo Argentino as embryo donors, and 247 mares breed with embryo<br />
recipients. Donors were inseminated with semen from 14 stallions Polo Argentino. The donors were examined by transrectal ultrasonography<br />
(Chisone D600, 7.0 MHz, China) every 24 h and were inseminated with fresh semen and sperm concentration of 800x106, when the largest<br />
follicle had 35 mm in diameter and uterine edema grade 2 (scale of 0 to 3), repeating the procedure every 48 h until the time of ovulation. The<br />
collection of embryos were seven, eight or nine days after ovulation and the embryos were transferred into recipients that ovulated the same day<br />
or within five days after the donors. The results were analyzed by chi-square test (P < 0,05). 315 samples were performed, resulting in 69,2%<br />
(219/315) recoveries of embryos, which resulted in the recovery of two embryos in 9,5% (30/315) of procedures, and increased 13,7% (30 /<br />
219) of embryos recovered. The pregnancy rate was higher for embryos from double ovulation in relation to embryos from single ovulation<br />
(83,3%, 50/60 vs. 64,7%, 103/159). From the results presented it is concluded that embryos obtained by double ovulation can increase the<br />
pregnancy rate in embryo transfer programs.<br />
Keywords: embryo transfer, mares, ovulation.<br />
s398
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A125 OPU-IVP AND ET<br />
COMP<br />
OMPARISON BETWEEN<br />
THE NUMBER OF ASPIRADED ADED FOLLICLES BY VIDEOLAP<br />
APAR<br />
AROSC<br />
OSCOPY OPY AND THE OOCYTE RECOVER<br />
OVERY IN<br />
DORPER AND SANTA A INÊS EWES<br />
Alexandra Soares Rodrigues 1 , Janaína Silva Chacon 2 , Antônio De Lisboa Ribeiro Filho 1 , Bruno Henrique De Araújo Andrade 1 , Marcus Vinícius Galvão Loiol 1 ,<br />
Priscila Assis Ferraz 1 , Leandro Moureira De Castro Feitosa 1 , Endrigo Adonis Braga De Araujo 1 , Sidnei Nunes Oliveira 1 , Marcos Chalhoub 1 & Alexandre Faria<br />
Tab<br />
abet<br />
2<br />
1<br />
ESCOLA DE MEDICINA VETERINÁRIA - UFBA, SALVADOR, BA, BRAZIL. 2 AUTONOMO, SÃO PAULO, SP, BRAZIL.<br />
The follicular aspiration by videolaparoscopy has emerged as an important biotechnology for oocyte recovery in sheep, being<br />
less invasive, providing high efficiency and repeatability in this specie. However, several factors inherent in the ewes can interfere in the<br />
follicular development and oocyte recovery, amongst which the breed of the donors stands out. Therefore, the objective of this study was to<br />
compare the number of punctured follicles, oocytes recovered and oocyte recovery rate (recovered oocytes / follicles aspirated) amongst<br />
Dorper and Santa Inês ewes that underwent videolaporoscopic aspiration. The groups were composed of 29 Dorper and 15 Santa Inês<br />
animals, comprising a total of 44 oocyte donors, all on average three years old. All animals were synchronized and superstimulated according<br />
to Baldassare et al. (2004, Animal Reproduction Science 82-83, 255-266). The aspirations were performed by videolaparoscopy using three<br />
laparoscopic ports in the abdominal cavity. For this, we used a 16G catheter attached to an aspiration system that had a vacuum pump and<br />
a container for collection of oocytes. The follicles were aspirated and counted independently of their size or maturity. The follicular contents<br />
aspirated were filtered and washed in solution at 37°C, consisting of DPBS (Embriocare,Cultilab, Campinas, Brazil) added with heparin<br />
(Linquemine ® , Roche, Rio de Janeiro, Brazil) placed in petri dishes, observed with stereomicroscope with the purpose of identifying and<br />
counting the oocytes recovered. To compare the number of punctured follicles, oocytes recovered and the oocyte recovery rate the package<br />
SAS version 6.0 was used (Procedures MEANS and GLM with P < 0.05). The average number of follicles punctured in Dorper ewes was<br />
19.74±11.36 not differing (P = 0.06) from those obtained for Santa Inês 13.42±6.93. However, the number of oocytes recovered for Dorper<br />
ewes (11.89±7.17) was higher (P = 0.02) than Santa Inês (9.00±4.46). No differences were noticed between Dorper (61.88%) and Santa Inês<br />
(60.84%) ewes when the oocyte recovery rate was considered. The results in this study suggest that Dorper ewes provide a greater number<br />
of oocytes in relation to Santa Inês, although the number of follicles punctured and the oocyte recovery rate were similar between both breeds.<br />
Thus, further research is needed to determine whether differences found in this study are intrinsic characteristics of the breeds.<br />
Keywords: opu, videolaparoscopy, sheep.<br />
A126 OPU-IVP AND ET<br />
CORREL<br />
ORRELATION OF KINETICS OF DEVEL<br />
VELOPMENT AND SEX OF IN VITRO PRODUCED BOVINE EMBRYOS<br />
Antônio Roncada Pupulim 1 , Álida Reis Buzzo 2 , Josmar Mazucheli 3 , Flávio Vieira Meirelles 4 , Raquel Zanetti Puelker 5 & Isabele Picada Emanuelli 1<br />
N<br />
1,2<br />
CESUMAR, MARINGÁ, PR, BRAZIL. 3 UEM, MARINGÁ, PR, BRAZIL; 4 FZEA, PIRASSUNUNGA, SP, BRAZIL. 5 PROGEST, BOTUCATU, SP, BRAZIL.<br />
Approaches to improve the culture medium for in vitro production (IVP) of bovine embryo have been continuous due to the high<br />
commercial demand and part of it attempts the production of female cattle (Dairy cows and stud cattle). However, in some embryonic in vitro<br />
culture systems, the kinetic of development in male are faster than in female embryos 1,2. The aim of this work was to relate the kinetics of<br />
blastocysts expansion with the production rates of male and female embryos. Cumulus-oocyte Complex (n = 917; class I and II) of cows from<br />
slaughterhouse were matured with TCM-199 bicarbonate and 10 % FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen/thawed semen<br />
in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10 % FSB (5% O2, 38.5°C). Seven days<br />
after IVF, embryos were divided in two groups, according to their kinetic stage of development: BL (n = 175) – not expanded blastocysts; and<br />
BX (n=146) – hatched and expanded blastocysts. Hence, embryos were individually frozen in liquid nitrogen and stored in cryotubes. After<br />
thawing, Proteinase K (16 mg/mL) was added in each tube and incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and<br />
the content of each tube was divided in two samples (A and B) and subjected to PCR technique. Two pairs of primers for specific sequence of<br />
Y chromosome were employed to amplify the sequence of 210 pb and 250 pb for male bovine; and also a pair of primers for autosomal bovine<br />
sequence with a 280 pb fragment. Female embryos were the ones with a 280 pb product observed in sample A and none in sample B. The<br />
presence of two amplicons (280 and 210 pb) in sample A and one of 250 pb in sample B indicated that embryo was male. Chi-square test was<br />
used to evaluate the homogeneity. The analysis of the percentage of males and females between the experimental groups was performed by<br />
logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of male and female in group BL<br />
(49.71 and 50.29%; P > 0.05). In group Bx, the amount of males (65.75%) was statistically different from the females (34.25%), i.e. the chance<br />
of the embryo to be male was twice higher (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development<br />
between male and female embryos and the blastocoels expansion can point that out. In vitro culture media with FCS support the development of<br />
expanded male blastocysts. Further researches in culture medium modifications (FCS, the energy source, amino acids and others) are needed for<br />
trending the production of sex defined embryos. (1) Avery B, Mol Reprod Dev, v.32(3), p.265-70, 1992.(2) Xu KP, Mol Reprod Dev, v.31(4),<br />
p.249-50, 1992.<br />
Keywords: ivp, kinetics, sex embrionic.<br />
s399
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A127 OPU-IVP AND ET<br />
DIFFERENT VOL<br />
OLUMES AND CENTRIFUGATIONS<br />
TIONS TIME OF PERCOLL GRADIENT DO NOT AFFECT THE SPERM QUALIT<br />
ALITY AND<br />
EMBRYO DEVELOPMENT OF IN VITRO PRODUCED BOVINE EMBRYOS<br />
Liziane Lemos<br />
Vianna<br />
1 , Jor<br />
orgea Pradieé<br />
2 , Elisa Car<br />
aroline Da Silv<br />
ilva San<br />
antos<br />
3 , Ale<br />
lexander Oliv<br />
liveir<br />
eira Gonçalv<br />
onçalves<br />
4 , Ledi Anghinoni<br />
5 , Mar<br />
aria Gabr<br />
abriela<br />
Tavar<br />
ares<br />
Rheingantz 6 , Luiz Francisco Machado Pfeifer 7 , Margot Alves Nunes Dode 8 , Marcio Nunes Corrêa 9 & Ligia Margareth Cantarelli Pegoraro 10<br />
1,2,3,4,6,9<br />
UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS, RS, BRAZIL. 5,10 EMBRAPA CLIMA TEMPERADO, PELOTAS, RS, BRAZIL. 7 EMBRAPA RONDÔNIA, PORTO VELHO, PORTO<br />
VENLHO, MS, BRAZIL. 8 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL.<br />
The objective of this study was to compare the embryo development and sperm quality and sex ratio of in vitro produced bovine<br />
embryos. Cumulus-oocyte complexes (COCs) were aspirated, from ovaries collected from slaughterhouse, selected and matured for 22 to 24h.<br />
For sperm selection, the Percoll (Sigma, Aldrich, St. Louis, USA) was preparated in the convencional way - P Group (90 e 45% with total<br />
volume of 4mL) and minigradient way PM Group - (90 e 45% with total volume of 800 µL). For IVF, the semen was thawed and added to the<br />
gradients. The P Group was centrifuged for 20 min at 700 xg and the PM Group for 5 min at 700 xg. In both the second centrifugation was<br />
performed for 5 min at 700 xg. The CCOs were randomly divided between treatments (P Group n = 830; PM Group n = 897) inseminated and<br />
maintained for 18h in IVF medium. Briefly, they were transferred to culture medium (SOFaa) and incubated for 7 days, at 39 o C and 5% CO2.<br />
Cleavage and embryonic global rates (delevoped embryos/inseminated oocytes) were observed at D2 and D7 respectively (Day 0 = FIV). For<br />
this study, 11 routines of IVP were performed. Embryos of each groups (P Group n = 115; PM Group n = 102) were harvested at D8 of culture<br />
and prepared for sexing determination by PCR. To evaluate the integrity of the plasmatic membrane we used 6-carboxiphluorescein diacetate<br />
(FDA) with propidium iodide (PI) and to acrosome integrity fluorescein isocianate (FITC) conjugated with peanut agglutinin (PNA)<br />
(Molecular Probe ® , Eugene, Oregon, USA). Two evaluations were performed: before (Control Group) and after treatments (3 replicates). A<br />
chi-square analysis was performed to compare cleavage, embryonic global rates between groups and sex ratio to expected of 50%. The semen<br />
characteristics were analyzed by one-way ANOVA using the General Linear Models (GLM) procedure. Tukey test was used to detect<br />
differences between means. The P Group had higher cleavage rate than PM Group (70.4% P versus 65.8% PM, P < 0.05). However, no<br />
difference was observed between groups on the embryonic development (19.2% P versus 16.3% MP). Semen quality evaluations (membrane<br />
and acrosome integrity) did not differ between groups. The sex ratio observed was similar between treatments. The results showed no<br />
influence of volume and time of centrifugation in the use of Percoll Gradient, on the embryo production and sperm quality.<br />
Keywords: bovine; sex ratio; sperm selection.<br />
A128 OPU-IVP AND ET<br />
CUMULATIVE EFFECT OF BOVINE RECOMBINANT SOMATOTR<br />
TROPIN (RBST) ) ON THE IN VITRO PRODUCTION OF EMBRYOS<br />
FROM LACT<br />
CTATING TING GUZOL<br />
OLANDO FEMALES<br />
Felip<br />
elipe e Jesus Mor<br />
oraes Junior<br />
1,18 , Luiz Fer<br />
ernando Schütz 1 , Cristiano Feltr<br />
eltrin<br />
1 , Leonar<br />
eonardo<br />
Tondello Mar<br />
artins<br />
1 , Saul Gaudêncio Net<br />
eto 1 , Maurício Barb<br />
arbosa Salviano<br />
1 , Kaio<br />
Cesar Simiano<br />
Tavar<br />
ares<br />
1 , Juliana Lop<br />
opes Almeida<br />
1 , Vic<br />
ictor Hugo Vieir<br />
ieira Rodr<br />
drigues<br />
1 , Jamir Machado Junior<br />
1 , Cesár Car<br />
arneir<br />
neiro Linhares Fer<br />
ernades<br />
2 , Barbar<br />
arbara a Mar<br />
ara<br />
Bandeira Santos 2 , Carlos Enrique Mendez Calderón 3 , Luis Henrique Aguiar 1 , Eudes Vieira Castro 3 , Francisco Jose Magalhães Barbosa 4 , Luciana Relly<br />
Ber<br />
ertolini<br />
1 , Jose Adalmir<br />
Tor<br />
orres Sousa<br />
5 & Mar<br />
arcelo Ber<br />
ertolini<br />
1<br />
1<br />
LABORATÓRIO DE BIOLOGIA MOLECULAR E DO DESENVOLVIMENTO, UNIVERSIDADE DE FORTALEZA (UNIFOR), FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE ESTADUAL DO CEARÁ -<br />
UECE, FORTALEZA, CE, BRAZIL. 3 UNIVERSIDADE COOPERATIVA DA COLOMBIA – UCC, BUCARAMANGA, COLÔMBIA; 4 GRUPO EDSON QUEIROZ, FORTALEZA, CE, BRAZIL. 5 LAB. DE<br />
REPRODUÇÃO ANIMAL, UNIVERSIDADE FEDERAL DO PIAUÍ – UFPI, TERESINA, PI, BRAZIL.<br />
The aim of this study was to evaluate the cumulative effect of different doses of recombinant bovine somatotropin (rbST; Boostin ® ,<br />
Schering-Plough) on the number and quality of cumulus-oocyte complexes (COCs) obtained in vivo from lactating Guzolando cows, along with<br />
its effect on the in vitro production (IVP) of embryos. For that purpose, 14 half-blood Guzolando cycling multiparous females were selected at<br />
67.3 ± 16.5 days of lactation, body condition score of 3.3 ± 0.2, and 6.6 ± 0.4 years of age. Females were randomly allocated to three<br />
experimental groups (Control, 250 mg and 500 mg rbST) and subjected to six sessions of in vivo follicular aspiration (ovum pick up, OPU) at<br />
a 14 day interval. Six days prior to each OPU session, a 1.5 mL dose of saline solution (Control, n = 4), 250 mg rbST (n = 5) or 500 mg rbST<br />
(n=5) was injected in the base of the tail in each animal. Recovered COCs were morphologically Graded as I to IV (Leibfried & First 1979, J<br />
Anim Sci 48:76-86), and subjected to IVP. Rates of in vitro embryo development to the compact morula and blastocyst stages were evaluated on<br />
Day 6.5 of culture. The mean numbers of recovered COCs, viable COCs (Grades I, II, and III), and embryos produced by OPU-IPV per<br />
session/female were compared by the Student’s t test, whereas the proportion of recovered COCs, based on the morphological grade, and rate<br />
of embryo development on Day 6.5, were compared by the ÷ 2 test, for P < 0.05. When compared with the control group, the administration of<br />
250 mg and 500 mg rbST increased the mean number of recovered COCs (8.6 ± 1.6 vs.. 13.3 ± 1.5 and 11.3 ± 1.6) and viable COCs (5.8 ± 1.1<br />
vs.. 7.6 ± 0.8 and 6.5 ± 0.8), for a lower proportion of viable COCs (66.7% vs.. 56.8% and 57.8%), respectively. In addition, the 250 mg and<br />
500 mg rbST groups yielded a greater mean number of Grade III (5.3 ± 0.7 and 4.8 ± 0.6 vs.. 4.0 ± 0.7) and Grade IV COCs (6.0 ± 1.0 and 4.8<br />
± 0.9 vs.. 2.9 ± 0.6) when compared with the control group, respectively. Nevertheless, despite the increase in COCs numbers and decrease in<br />
COCs quality seen in the 250 mg and 500 mg treatment groups relative to the controls, no differences were observed in the rates of embryo<br />
development (80/227, 35.2%; 77 / 370, 36.0%; and 57/138, 41.3%) or the mean number of viable embryos per session/donor (2.7 ± 0.4; 2.6 ±<br />
0.4; and 2.4 ± 0.4) between groups, respectively. In summary, our preliminary results indicated that the administration of 250 mg and 500 mg<br />
rbST in Guzolando cows six days prior to in vivo oocyte retrieval was followed by an increase in the number of COCs, with a slight reduction<br />
in quality, which were not translated into differences in the in vitro embryo production efficiency in any group.<br />
Keywords: bovine, ccos, rbst.<br />
s400
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A129 OPU-IVP AND ET<br />
EFFECT OF HIGH ENERGY DIET ON METABOLIC,<br />
ENDOCRINE AND REPRODUCTIVE PAR<br />
ARAMETERS AMETERS ON BOS INDICUS AND BOS<br />
TAUR<br />
URUS<br />
US COWS<br />
José Nelio S. Sales<br />
1 ; Lilian Tam<br />
amy Iguma 2 ; Car<br />
arolina Cap<br />
apobiango R. Quin<br />
uintão<br />
3 ; Mar<br />
arco A.S. Gama<br />
4 ; Célio Freitas<br />
5 ; Michele Munk Per<br />
ereir<br />
eira 6 ; Luiz Sérgio Almeida<br />
Camargo 7 ; João Henrique Moreira Viana 8 ; Pietro Sampaio Baruselli 9<br />
1,9<br />
UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 2,3,4,5,6,7,8 EMBRAPA, JUIZ DE FORA, MG, BRAZIL. 5,10 EMBRAPA, VALENÇA, RJ, BRAZIL.<br />
The effect of different energy levels in diet [maintenance (M) and high energy (1.7M)] on metabolic, endocrine and reproductive<br />
parameters of non lactating Bos indicus (n = 14; Gir) and Bos taurus (n = 14; Holstein - HPB) cows submitted to ultrasound guided ovum pick<br />
up (OPU) followed by in vitro embryo production (PIV) was evaluated. The oocyte donors were housed in Tie Stall System and the diets were<br />
given twice daily (8:00 AM e 4:00 PM). During 21 days prior to the beginning of experiment, animals were fed with the maintenance diet for<br />
their adaptation. After this period, the experimental diets (M and 1.7M) were given and cows (Gir and HPB) were submitted to nine OPU<br />
procedures, 14 days apart each. The donors were synchronized before OPU (D0 - 2mg EB + norgestomet ear implant; D5 - OPU). There were<br />
no interaction between breeds and energy level in diet and effect of diet for the qualitative or quantitative variables. However, Bos indicus cows<br />
showed higher number of recovered structures and better oocyte quality when compared to Bos taurus donors. Similar to what was found in<br />
oocyte quality and number, the in vitro embryo production also did not differ between diets and it was observed that Bos indicus cows had higher<br />
production of embryos. However, we verified that the high energy reduced in vitro embryo production in Bos indicus cows after 60 days of high<br />
energy diet. Moreover, Gir cows that recevied high energy showed higher transcript abundance for HSP70.1, GLUT1, IGF1R e IGF2R genes.<br />
Cows fed with excess of energy in diet presented higher serum and follicular fluid concentrations of glucose and cholesterol. However, the ureic<br />
nitrogen concentration was higher in cows which received maintenance diet. When comparing the two genetic groups, it was observed that Bos<br />
indicus cows presented higher concentrations of glucose, cholesterol, NEFA and ureic nitrogen both in follicular fluid and in blood. Furthermore,<br />
cows that presented decreased blastocyst rate (high energy - Bos indicus) exhibited high follicular fluid concentrations of insulin. In conclusion,<br />
increasing energy in diet did not interfere in oocyte number and quality. However, the high energy reduced the in vitro embryo production in Bos<br />
indicus cows after 60 days of diet. Moreover, Bos indicus cows showed better oocyte quality, higher number of viable oocytes and increased in<br />
vitro embryo production than Bos taurus.<br />
Keywords: opu-fiv, energy, oocytes.<br />
A130 OPU-IVP AND ET<br />
EFFECT OF THE SPERM SELECTION WITH ISOLATE<br />
® OR PERCOLL<br />
® ON SPERM QUALIT<br />
ALITY AND IN VITRO BOVINE<br />
EMBRYO DEVEL<br />
VELOPMENT FOR FROZEN-THA<br />
OZEN-THAWED SEXED AND NON-SEXED SEMEN<br />
Paula Ro drigue<br />
iguez V illamil 1 , M aria Eugenia Gar<br />
arcia Gome<br />
omez 2 , Mar<br />
ariana Fer<br />
ernande<br />
nandez Tar<br />
aranc<br />
anco 2 , M ariana Caccia<br />
2 & Gabr<br />
abriel Bó 1<br />
1<br />
INSTITUTO DE REPRODUCCION ANIMAL DE CORDOBA, CORDOBA, ARGENTINA. 2 UNIVERSIDAD DE VILLA MARIA, VILLA DEL ROSARIO, ARGENTINA.<br />
N<br />
The aim of this study was to compare the effects of spermatozoa separation techniques Isolate ® (Irving-Scientific, California, USA)<br />
and Percoll ® (Nutricell, SP, Brazil), on sperm quality and in vitro embryo production using sexed and non-sexed semen. Oocytes (n = 5046) were<br />
obtained from slaughterhouse ovaries and fertilized with frozen/thawed sexed or unsexed semen of four Holstein bulls. The sperm quality<br />
(motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection by the two<br />
methods. Oocytes were maturated in TCM-199 supplemented with 0.4% of BSA for 24 h in controlled atmosphere and those matured were<br />
selected and randomly allocated into four different groups. Group 1: oocytes fertilized with sexed semen selected by Percoll ® ; Group 2: oocytes<br />
fertilized with sexed semen selected by Isolate ® ; Group 3: oocytes fertilized with non-sexed semen selected by Percoll ® ; Group 4: oocytes<br />
fertilized with non-sexed semen selected by Isolate ® . Fertilization was performed in Fert-talp medium during 18 h at same conditions as<br />
maturation. Consequently, presumptive zygotes were cultured for 7 days in SOF medium in a 39°C humidified incubator with 5% CO2, 5% O2<br />
and 90% N2. The results of cleavage and embryo production were evaluated on Day 2 and 7 after fertilization. All percentages were transformed<br />
by square root and compared by ANOVA. Regardless of sperm selection technique, sperm motility and percentage of normal sperm increased<br />
(P < 0.005) compared with the initial sperm parameters. For non-sexed semen, Percoll ® gradient increased the recovery rate of sperm (57.3 ±<br />
2.7) compared with Isolate ® (46.0 ± 1.8, P < 0.05). Furthermore, sperm selected by Isolate ® presented significant improvements compared with<br />
Percoll ® gradient, on membrane integrity of sexed (41.0 ± 0.6 vs. 38.8 ± 0.8) and non-sexed semen (60.8 ± 1.6 vs. 58.8 ± 0.5, P < 0.05). Finally,<br />
blastocyst development rates were greater (P < 0.05) for sexed (Group 2: 14.0 ± 1.0) or non-sexed semen (Group 4: 22.0 ± 1.1) selected by<br />
Isolate ® when compared to those produced with sexed (Group 1: 10.5 ± 1.5) or non-sexed semen (Group 3: 17.0 ± 2.1) selected with Percoll ® .<br />
In conclusion, Isolate ® may be a better alternative than Percoll ® as a sperm selection method for IVP systems using sexed or non-sexed bovine<br />
semen.<br />
Keywords: selection, sexed, non-sexed.<br />
s401
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A131 OPU-IVP AND ET<br />
THE EFFECT OF FOLLICLE WAVE SYNCHRONIZA<br />
ONIZATION AND ECG TREATMENT ON CUMULUS US OOCYTE COMPLEX RECOVER<br />
OVERY<br />
RATES AND QUALIT<br />
ALITY IN BRANGUS AND ANGUS DONORS<br />
Felipe Ledur Ongaratto 1 , Paula Rodriguez Villamil 1 , Andres Tribulo 1 & Gabriel A. Bo 2<br />
1<br />
INSTITUTO DE REPRODUCCION ANIMAL, CORDOBA, ARGENTINA. 2 IRAC - UNVM, ESTRANGEIRO, ARGENTINA.<br />
Two experiments were designed to evaluate the effect different treatments of follicle wave synchronization and superstimulation<br />
on the number and quality of COCs aspirated per OPU session. Experiment 1 was designed to test the hypothesis that synchronizing the<br />
emergence of follicular wave using estradiol benzoate (EB) and P4 would improve the number and quality of oocytes aspirated by OPU.<br />
Brangus (n = 10) and Angus (n = 10) cows were randomly allocated in two treatment groups and treated two times in a cross-over design. On<br />
Day 0, donors in Group 1 received 2.5 mg EB (Zoovet, Argentina) and 50 mg P4 (Syntex, Argentina) intramuscularly (im); whereas, donors<br />
in Group 2 (Control) did not receive any hormonal treatment. On Day 6, OPU was performed by ultrasound-guided follicular aspiration and<br />
COCs were classified based on cytoplasm appearance and cumulus cell numbers as described by Chaubal et al. 2006. Data was analyzed by<br />
ANOVA. The mean number of follicles aspirated on Day 6, total number of oocytes retrieved and the number of viable oocytes were higher<br />
(P < 0.05) in cows in Group 1(12.9 ± 1.1; 7.9 ± 1.3 and 5.2 ± 0.9) than in those in Group 2 (7.3 ± 0.7; 3.2 ± 0.5 and 2.1 ± 0.4). Experiment<br />
2 was designed to test the hypotheses that: 1) induction of luteolysis 3 days prior to aspiration (to avoid the presence of the CL at the time<br />
of OPU) and 2) treatment with eCG (to stimulate follicle development), would improve the number and quality of oocytes aspirated by OPU.<br />
Brangus (n = 20) cows were randomly allocated into 3 treatment groups and treated 3 times in a cross-over design. On Day 0, all donors<br />
received 2.5 mg EB and 50 mg P4 im (Lab. Rio de Janeiro, Argentina). On Day 4, donors in Group 1 received 150 µg D(+) cloprostenol (PGF,<br />
Ciclar, Zoovet, Argentina) and donors in Group 2 received PGF plus 800 IU eCG (Novormon, Syntex); whereas donors in Group 3 did not<br />
receive any treatment. OPU was performed on Day 7 and COCs were classified and data analyzed as in Experiment 1. The number of follicles<br />
aspirated on Day 7, total number of oocytes retrieved and the number of viable oocytes were higher (P < 0.05) in cows in Group 1 (11.4 ±<br />
1.2 e 7.9 ± 1.1) than in those in Groups 2 (7.5 ± 1.4 e 4.5 ± 1.0) and 3 (7.1 ± 0.8 e 4.7 ± 0.7). In Conclusion, synchronization of follicle wave<br />
emergence and the induction of the luteolysis prior to OPU, but not the superstimulation treatment with eCG positively affected the number<br />
and quality of COCs.<br />
Keywords: emergence wave synchronization, ecg, oocyte.<br />
A132 OPU-IVP AND ET<br />
EFFECT OF FSH INCORPOR<br />
ORPORATED IN A POLIMERIC MATRIX OF PLUR<br />
URONIC F-127 ® ABOUT THE QUALIT<br />
ALITY OF OOCYTES<br />
COLLECTED BY FOLLICULAR ASPIRATION IN ZEBU HEIFERS<br />
Hector Javier Narvaez, , Reginaldo Silva Fontes, Gina Marcela Mican, Célia Raquel Quirino<br />
UNIVERSIDADE ESTADUAL DO NORTE FLUMINENSE DARCY RIBEIRO, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
This study aimed to evaluate the effect of slow release of FSH when incorporated in a biodegradable polymer matrix of pluronic<br />
F-127 ® on the quality of oocytes recovered by transvaginal follicular aspiration (OPU) in Nelore x Brahman heifers. Eight zebu heifers were<br />
used (Bos indicus), as oocyte donors age between 20 to 24 months. In a random day of the estrous cycle heifers were pre-synchronized by<br />
the association of an intravaginal gadget containing 1g of progesterone (Sincrogest ® , Ouro Fino, Brazil ) plus 2mg of estradiol benzoate (RIC-<br />
BE ® , Tecnopec, Brazil). After 8 days, the intravaginal progesterone gadget was removed, we administered 150 µg D-Cloprostenol (Prolise ® ,<br />
Tecnopec, Brazil) and 24 h later all females received an application of 1mg estradiol benzoate. 8 days after pre-synchronization heifers received<br />
a 150µg dose of D-cloprostenol to eliminate the luteum corpus present and 36 h after animals were subjected to the aspiration of follicles >5mm<br />
diameter, in order to synchronize the emergence of a new follicular wave, and considered as D0. As this moment, females were assigned to two<br />
experimental groups. In the control group (G1) (n = 4), animals didn’t receive the polymer matrix of pluronic F-127 ® FSH. Group 2 (n = 4),<br />
animals that received the polymer matrix of pluronic F-127 ® containing 50mg de FSH-p (Folltropin-V ® , Bioniche, Canada) subcutaneously. The<br />
animals in both experimental groups were subjected to three replicas with an interval of 10 days, including the synchronization of the wave with<br />
puncture of follicles >5mm and the OPU performed 5 days after this synchronization procedure. For the preparation of the polymeric matrix was<br />
used pluronic F-127 ® 20% (Sigma -Aldrich chemical Co, USA) and 1% de PVA (Sigma-Aldrich Chemical Co, USA) diluted in PBS was later<br />
performed the addition of FSH, the polymer matrix as it was cooled to 4°C use. Analysis of variance was performed using the SAS statistical<br />
program. There was statistical difference (P < 0.05) for the average number of follicles seen in G2 (24.7±14.3) compared to G1 (15.1±7.5).The<br />
number oocytes retrieved, the G1 to G2 was higher (14±9.4 and 7.9±4.2, respectively). Regarding the quality oocytes, the G2 had more grade<br />
I structures (5.8±3.2) compared to G1 (1.2±0.7). And the total number of viable oocytes was significant for G2 compared to G1 (13.8±6.5 and<br />
7.4±2.1). It can be concluded that the application of FSH incorporated in a polymer matrix of pluronic F-127 ® was effective in improving the<br />
quality of oocytes picked.<br />
Keywords: opu, fsh, pluronic f-127 ®<br />
s402
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A133 OPU-IVP AND ET<br />
EFFECT OF CORPUS LUTEUM ON THE RECOVER<br />
OVERY RATE AND NUMBER OF OOCYTES IN CROSSBRED ZEBU HEIFERS<br />
Bruno Pena Carvalho, , Fernanda Queiroz Costa, Felipe Borges Rosa, Aline Matos Arrais, Roger Cardozo Maia & Angelo José Burla Dias<br />
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
The presence of the corpus luteum (CL) on the small ovaries of heifers hinders the manipulation during the ovum pick-up (OPU)<br />
procedure. Petyim et al. (2003, Theriogenology, 60, 175-188) reported that the presence of a CL on the ovary does not seem to affect the number<br />
of aspirated follicles and recovery rate during the luteal phase in Bos taurus heifers. However, studies that evaluated the influence of the CL on<br />
the recovery rate in zebu heifers are scarce. The aim of this study was to compare the mean number of oocytes per animal, number of oocytes per<br />
session and the oocyte recovery rate in zebu heifers with or without a CL on the ovaries. For this, data from 48 sessions of OPU in cyclic zebu<br />
crossbred heifers (Nellore x Brahman) were analyzed, with body condition score ranging between 3 and 4 (scale of 1 to 5) and mean age of 2<br />
years, maintained on pasture with daily supplementation of 4.5 kg of concentrate and mineral salt ad libitum. Sessions were performed by<br />
ultrasound-guided transvaginal OPU (DP-2200, Mindray, 6.5 MHz) with a minimum interval of seven days, in which the follicles > 3 mm were<br />
counted and aspirated and the CL, when present, were measured, thus obtaining, to each aspiration session two groups of animals: G1 - ovaries<br />
without the presence of CL and G2 - ovaries with the presence of CL. All animals were aspirated with and without CL and within each group<br />
were determined the mean number of oocytes per animal, number of oocytes per session and its recovery rate, which was calculated by the ratio<br />
between the number of follicles and the number of oocytes recovered, expressed as a percentage. The means of the variables were compared<br />
between groups G1 and G2 by t test (LSD) with significance level of 5%. The mean number of oocytes per animal, mean number of oocytes<br />
per session and the recovery rate, followed by standard deviations in G1 and G2 was respectively: 15.0 ± 7.9, 12.9 ± 10.5, 80% ± 0 , 24, and<br />
13.5 ± 6.4, 10.9 ± 6.9, 80% ± 0.31. There was no significant difference among the groups (P > 0.05). Data demonstrated that the presence of<br />
a CL did not affect the mean number of oocytes per animal and per session of OPU, as well as the oocyte recovery rate, evidencing that even<br />
in heifers, whose ovaries are smaller, the presence of a CL does not impair the outcome of an OPU. [Acknowledgment: FAPERJ].<br />
Keywords: opu, Bos indicus, recovery rate.<br />
A134 OPU-IVP AND ET<br />
EFFECT OF STAGE OF DEVEL<br />
VELOPMENT ON THE SEX RATIO OF PREGNANCIES ORIGINATED FROM<br />
IN VITRO PRODUCED<br />
EMBRYOS<br />
Lucas Maciel Gouvêa<br />
CONCEPÇÃO REPRODUÇÃO BOVINA, ARACATUBA, SP, BRAZIL.<br />
The objective of the present study was to verify whether the stage of development of in vitro produced bovine embryos<br />
influences the sex rate of Day 60 pregnancies. It is known that the proportion of male embryos that develop in vitro is skewed 2:1. Studies<br />
suggest that this difference is due to a differential expression of genes that are exclusive to the Y chromosome, mostly genes related to glucose<br />
metabolism, oxygen reactive species and cellular mitosis (Kochhar, H.P., et al., Theriogenology 55, 3-14, 2001). Three embryonic stages were<br />
used in the experiment: early blastocyst (n = 230), blastocyst (n = 347) and expanded blastocyst (n = 400) of Nelore breeds obtained in the<br />
years 2010 and <strong>2011</strong>, by the company Concepção Reprodução Bovina. Fetal sexing was done at day 60 of pregnancy with a Mindray DP<br />
2200 vet ultrasound. The percent of male fetus in each stage of development as well as the standard error was calculated by ANOVA of the<br />
GLM procedure of SAS (SAS for Windows, Version 9.0, Cary, NC). The difference was considered significant when P < 0.05. Data were also<br />
analyzed by orthogonal contrast and CATMOD, both GLM commands. Were included in the analysis the effects of bull, donor, farm and<br />
laboratory, without any interaction. All embryos were transferred and fetal sexing was done by the same veterinarian. The percent of male<br />
fetus at Day 60 pregnancy was 61.74±3.2; 55.04±2.6; 58.75±2.4 for early blastocyst, blastocyst and expanded blastocyst, respectively.<br />
There was no statistical difference between the stages of the development on the sex ratio (P = 0.26). Results indicate that the stage of<br />
development of the in vitro produced bovine embryo does not influence the sex ratio of day 60 pregnancies. At every stage there were more<br />
males than females, but no interaction between stage and sex.<br />
Keywords: in vitro production, embryonic stage, sex.<br />
N<br />
s403
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A135 OPU-IVP AND ET<br />
EFFECT OF THE METHOD FOR THE RETRIEVAL OF CUMULUS-OOCY<br />
US-OOCYTE COMPLEXES ON THE IN VITRO PRODUCTION<br />
EFFICIENCY OF SHEEP EMBRYOS<br />
Maurício Barb<br />
arbosa Salviano<br />
1 , Cristiano Feltr<br />
eltrin<br />
2 , Juliana Lop<br />
opes Almeida<br />
2 , Kaio Cesar Simiano<br />
Tavar<br />
ares<br />
2 , Felip<br />
elipe e Jesus Mor<br />
oraes Junior<br />
2 , Luiz Fer<br />
ernando Schütz 2 ,<br />
Saul Gaudêncio Net<br />
eto 2 , Leonar<br />
eonardo<br />
Tondello Mar<br />
artins<br />
2 , Vic<br />
ictor Hugo Vieir<br />
ieira Rodr<br />
drigues<br />
2 , Raquel Rib<br />
ibeir<br />
eiro Freir<br />
eire 2 , Tatiane Mar<br />
aria Mon<br />
onte Nobr<br />
obre 2 , José Baptista<br />
Sant´Ana Neto 2 , Priscila Ferreira Alencar Silva 2 , José Luis Rigo Rodrigues 1 , Luciana Relly Bertolini 2 & Marcelo Bertolini 2<br />
1<br />
LABORATÓRIO DE EMBRIOLOGIA E BIOTÉCNICAS DE REPRODUÇÃO DA FACULDADE DE VETERINÁRIA DA UFRGS, PORTO ALEGRE, RS, BRAZIL. 2 LABORATÓRIO DE BIOLOGIA<br />
MOLECULAR E DO DESENVOLVIMENTO DA UNIFOR, FORTALEZA, CE, BRAZIL.<br />
Obtaining high quality oocytes in small ruminants for the IVP of embryos is usually a challenge. Recovery methods may<br />
significantly affect the quality of cumulus-oocyte complexes (COCs), especially the cumulus cell vestment. The aim of this study was to compare<br />
the efficiency of follicular aspiration and ovary slicing for the retrieval of COCs for the IVP of sheep embryos. Sheep ovaries collected at a local<br />
slaughterhouse were transported to the laboratory in PBS (34.3±1.7 o C), being separated into two groups for the COC retrieval by follicular<br />
aspiration or ovary slicing. For the aspiration, 2 to 8 mm follicles were aspirated using a 21G needle coupled to a 5 mL syringe, with the follicular<br />
contents placed into 15 mL tubes. For the slicing, ovaries were placed in PBS supplemented with 1% FBS, 25 µg/mL heparin, and 50 µg/mL<br />
gentamicin, on a Petri dish. The ovarian cortex was randomly scarified using a scalpel blade, followed by tissue washing in PBS medium. The<br />
ovarian contents from both groups were examined under a stereomicroscope to search for COCs, which were morphologically graded as I to IV<br />
(Leibfried & First, 1979, J Anim Sci 48:76-86). All COCs were used for the IVP of embryos using commercial media (Nutricell ® , São Paulo).<br />
After 25 h of IVM, COCs were subjected to IVF using frozen ram semen segregated using Percoll ® gradient (a pool of three rams, with 1.5<br />
million sperm cells/mL). After 24 h co-incubation, zygotes were in vitro-cultured for six days at 38.5 o C, 5% CO 2<br />
and saturated humidity. Data<br />
were analyzed by the Student´s t test or by the ÷ 2 test (P < 0.05). After four replications, a total of 328 COCs were recovered from 32 ewes<br />
(10.2 COCs/female), with 131 (8.2 COCs/female) obtained by follicular aspiration and 197 (12.3 COCs/female) by ovary slicing. The COC<br />
recovery rate by slicing was 50.4% higher, with a greater proportion of Grades I and II COCs, than follicular aspiration (74/197, 37.6% vs..<br />
23/131, 17.6%, respectively). However, in spite of the similar rates of embryonic developmental between the slicing (53/197; 26.9%) and<br />
aspiration (35/131; 26.7%) procedures, the higher number of COCs obtained by ovary slicing was reflected into a comparative increase<br />
(51.4%) in the number of viable embryos on Day 6 by ovary slicing (3.3 embryos/female) than by follicular aspiration (2.2 embryos/female).<br />
In addition, no differences were observed regarding stage of development and embryo quality on Day 6 between groups. In summary, the<br />
retrieval efficiency of viable COC per female was higher after ovary slicing than follicular aspiration, which was translated into a higher ovine<br />
IVP embryo yield on a per female basis.<br />
Keywords: sheep, ivp of embryos, oocyte.<br />
A136 OPU-IVP AND ET<br />
EFFICIENCY OF OPU-IVP-ET OF FRESH AND VITRIFIED EMBRYOS IN BUFF<br />
UFFAL<br />
ALO<br />
Wilson Pardini Saliba 1 , Roberti M Drumond 1 , Henrique X Bayão 1 , Múcio Túlio Alvim 1 , Pietro Sampaio Baruselli 2 , Lindsay Unno Gimenes 2 , Rômulo C Leite 3 ,<br />
Eduardo Bastianetto 3 & Bianca Gasparrini 4<br />
1<br />
CENATTE EMBRIÕES LTDA, PEDRO LEOPOLDO, MG, BRAZIL. 2 FMVZ-USP, SAO PAULO, SP, BRAZIL. 3 UFMG, BELO HORIZONTE, MG, BRAZIL. 4 UNINA, NAPOLI, ITÁLIA.<br />
OPU and IVP are promising techniques in buffalo, inversely to recovery of in vivo embryos which is still unsatisfactory. Scientific<br />
studies show that buffaloes are able to respond to superovulatory protocols, however, embryo recovery is low (Baruselli et. al., 1999).<br />
Moreover, success in the transfer of fresh embryos (ET) has not reached pregnancy rates higher than 15% (Sa Filho et al., 2005). The present<br />
study aims to report OPU-IVP-ET outcomes of fresh and vitrified embryos in buffalo. For this purpose, 36 buffalo donors were submitted to<br />
11 OPU sessions (n = 201) between July and December 2010. 998 total oocytes (5.0 ± 0.5 / donor / session) and 584 viable oocytes (2.9 ± 0.3<br />
/ donor / session) were recovered. Viable oocytes (grades 1, 2 and 3) were subjected to IVM, IVF (D0) and IVC. On day 2 (D2) after IVF 54.5%<br />
of cleavage rate was obtained. Embryo production was evaluated on days 5 (D5), 6 (D6) and 7 (D7) after IVF. Embryo yield was 44.9% (grade<br />
1: 229 embryos, grade 2: 5 embryos and grade 3: 28 embryos). 115 fresh and 21 vitrified embryos were transferred into recipients previously<br />
synchronized with ETFT protocol, 49 of the 115 ET were done with 5 days-embryos (Group D5) to synchronized 6 days after ovulationrecipients,<br />
and 66 of the 115 ET were done with 6 days-embryos (Group D6) to 7 days after ovulation-recipients. On 30th day, 46.9% (23/49)<br />
of pregnancy of 5 days-embryos, and 40.9% (27/66) of 6 days-embryos were confirmed. On 60th day, 44.9% (22/49) of pregnancy of 5 daysembryos,<br />
and 39.4% (26/66) of 6 days-embryos were confirmed. Only two pregnancy losses (4.2%), one in each group, were found. No<br />
significant differences (P > 0.05) were found between groups D5 and D6 on pregnancy rates at 30 or 60 days, and on pregnancy loss between<br />
30 and 60 days (by chi-square test in SAS). From vitrified embryos, 33.3% (7/21) of pregnancy rate at 30 days, and 28.6% (6/21) after 60 days<br />
of gestation (14.3% pregnancy loss) was verified. No abortion (between diagnosis of pregnancy and birth to 60 days) and stillbirth (14 births<br />
so far) occurred until present moment. Results are unprecedented in science and opens possibilities for the commercial use of the techniques of<br />
OPU, IVP and ET of fresh and vitrified embryos in buffaloes. [Acknowledgements: FAPEMIG (APQ - 0591-5.04/08), FINEP, FIEMG].<br />
Keywords: ivp, et, buffalo.<br />
s404
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A137 OPU-IVP AND ET<br />
EFFICACY CY OF SHORT PROTOC<br />
OCOLS<br />
OLS WITH CIDR ® ON ESTRUS SYNCHRONIZA<br />
ONIZATION OF EMBRYOS RECIPIENTS EWES<br />
Fausto Stecca D´Angieri 1 , Julio Cesar Barbosa Da Silva 2 , Wagner Kodato Okabe 2 , Milton Maturana Filho 3 & Anneliese De Souza Traldi 3<br />
1<br />
PFIZER SAÚDE ANIMAL, SÃO PAULO, SP, BRAZIL. 2 EMBRYO SYS REPRODUÇÃO ANIMAL, OURO FINO, MG, BRAZIL. 3 DEPARTAMENTO DE REPRODUÇÃO ANIMAL - FMVZ ,USP,<br />
PIRASSUNUNGA, SP, BRAZIL.<br />
The aim of this study was to compare two estrus synchronization protocols, characterized by different priming times with Eazibreed<br />
device CIDR ® (CIDR, Pfizer, Brazil), and assess their efficacy and viability by analysing estrus and pregnancy rates. One hundred and<br />
forty-five hair sheep (SI) and Dorper crossbred (MD) recipients were divided into two experimental groups, of which 78 were synchronized<br />
with a 6-day protocol (CIDR 6) and 67 with a 9-day protocol (CIDR 9). At device withdrawal every animal received 400IU of eCG (Folligon,<br />
Intervet, Brazil) and 10mg of Dinoprost (Lutalyse, Pfizer, Brazil). Embryos at early blastocyst (EB), blastocyst (BL) and expanded blastocyst<br />
(XB) stage from Dorper, White Dorper and Santa Inês donor breeds were transferred randomly on D6 to 48 recipients of the CIDR 6 group and<br />
43 recipients of the CIDR 9 group of wich 62 hair sheep and 29 Dorper crossbred. Data was analyzed by logistic regression (PROC LOGISTIC<br />
from SAS software, 2010), with a significance level of 5%. Statistical model included effects of treatment on estrous behavior and pregnancy<br />
rate (CIDR 6 vs.. CIDR 9), breed of recipient (SI vs.. MD), and embryonic developmental stage (EB, BL and XB). There was no effect of the<br />
length of treatment on estrous behavior (CIDR 6: 91%; CIDR 9: 88%) and pregnancy rate (CIDR 6: 75%; CIDR 9: 63%), no difference between<br />
breed of recipient (69% identical for both breeds) and between EB, BL and XB (74%, 71% and 75%, respectively). In comparison to the classical<br />
protocols of 12 and 14 days, known to be effective for the ovine species, both tested protocols resulted in adequate response regarding estrus of<br />
the recipients, with an average of 90% (130/145), and pregnancy rate of 69% (63/91), also adequate for the species. This results indicate that short<br />
protocols are appropriate for synchronization of recipients during embryo transfer programs in sheep. In addition, the 6-day protocol may<br />
be an alternative for recipient synchronization due to its efficiency and practicality.<br />
Keywords: cidr; estrus synchronization; recipients<br />
A138 OPU-IVP AND ET<br />
FERTILIT<br />
TILITY OF RECIPIENTS TRANSFERRED<br />
WITH EMBRYOS IN OVIDUCT BY TWO DAYS OF SANTA A INÊS AND DORPER SHEEP IN VITRO PRODUCED<br />
Endrigo Adonis Braga De Araujo 1 , Sidnei Nunes Oliveira 1 , Andrea Cristina Basso 2 , Janaína Silva Chacon 3 , Marcos Chalhoub 1 , Antônio De Lisboa Ribeiro<br />
Filho 1 , Alexandra Soares Rodrigues 1 , Leandro Moureira De Castro Feitosa 1 , Marcus Vinícius Galvão Loiola 1 , Priscila Assis Ferraz 1 , Bruno Henrique De Araújo<br />
Andr<br />
ndrade<br />
1 & Ale<br />
lexandr<br />
xandre Far<br />
aria<br />
Tab<br />
abet<br />
3<br />
1<br />
ESCOLA DE MEDICINA VETERINÁRIA – UFBA, SALVADOR, BA, BRAZIL. 2 IN VITRO BRAZIL SA, MOGI MIRIM, SP, BRAZIL. 3 AUTÔNOMO, SÃO PAULO, SP, BRAZIL.<br />
The pregnancy rate in IVEP is influenced by several little-known aspects in sheep. Therefore, this study aimed to compare the<br />
fertility of recipients transferred in oviduct with in vitro produced embryos of two-days-old from Santa Ines and Dorper sheep. The estrous cycle<br />
of 260 recipients was synchronized with the use of 60 mg of a medroxyprogesterone acetate intravaginal sponge (Progespon ® , Shering-Plough<br />
Intervet, Sao Paulo, Brazil) during nine days. On day seven, the animals were subjected to 300IU eCG (Novormon ® , Shering-Plough Intervet,<br />
Sao Paulo, Brazil) and 350 µg of d-cloprostenol i.m (Prolise ® ,Tecnopec, São Paulo, Brazil). On day 11, the animals were randomly divided into<br />
two groups: 75 recipients were transferred with Santa Ines embryos (GI) and 185 recipients with Dorper embryos (GII) two days after their IVF.<br />
Initially, the cumulus-oocytes of Santa Ines and Dorper donors raised in the State of Bahia were obtained by follicular aspiration performed by<br />
videolaparoscopy. They were then placed in cryovials containing maturation medium on temperature, humidity and CO2 concentration appropriated<br />
and sent to a commercial laboratory in São Paulo. At this laboratory the stages of maturation, fertilization and in vitro culture were carried on. Two<br />
days after fertilization, the embryos produced were sent to the state of Bahia, in adequate medium and environment, where the embryo tranfers<br />
were proceeded in oviduct, ipsilateral to the ovary with the most recently ovulated corpus luteum. Pregnancy diagnosis was performed by<br />
transabdominal ultrasound, 45 days after IVF. To compare the pregnancy rates in both groups, the chi-square test available in the SAS statistical<br />
software, version 6.0 (P > 0.05), was used. The overall pregnancy rate was 42.69% (111/260). The sheep embryo tranfers with Santa Ines<br />
embryos (GI) had a pregnancy rate of 54.67% (41/75), which was significantly higher than the 37.84% rate (70/185) obtained from tranfers with<br />
Dorper embryos (GII). This study suggests a superiority in the fertility of recipients transferred with embryos from Santa Ines when compared<br />
to those from Dorper. In addition, it is presented here as a possibility the transportation of sheep oocytes and early embryos (two days after IVF)<br />
over long distances maintaining their viability.<br />
Keywords: ivep, videolaparoscopy, pregnancy.<br />
N<br />
s405
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A139 OPU-IVP AND ET<br />
APOPTOSIS IN FRESH AND VITRIFIED IN VITRO AND IN VIVO PRODUCED BOVINE EMBRYOS<br />
Mateus José Sudano 1 , Daniela Mar<br />
artins Paschoal<br />
1 , Tatiana Da Silv<br />
ilva Rasc<br />
ascado<br />
ado 1 , Mid<br />
idyan Dar<br />
aroz Guastali<br />
1 , Rosiar<br />
osiara a Rosár<br />
osária Dias Mazier<br />
aziero 1 , Letícia Fer<br />
errar<br />
ari<br />
Crocomo 1 , Luis Carlos Oña Magalhães 1 , Bianca Andriolo Monteiro 1 , Joao Ferreira De Lima Neto 1 , Alicio Martins Júnior 2 , Rui Machado 3 & Fernanda Da Cruz<br />
Landim Alvarenga 1<br />
1<br />
FMVZ-UNESP, BOTUCATU, SP, BRAZIL. 2 FMVA-UNESP, ARAÇATUBA, SP, BRAZIL. 3 EMBRAPA PECUÁRIA SUDESTE, SÃO CARLOS, SP, BRAZIL.<br />
In vitro produced (IVP) bovine embryos have a reduced criotolerance than those produced in vivo (VIVO). It is believed that<br />
some peculiarities observed in IVP embryos directly affect the quality of these embryos. The aim of this study was to evaluate the incidence<br />
of apoptosis in different bovine embryos source (in vitro and in vivo produced) and condition (fresh and vitrified), in a 2 x 2 factorial<br />
experimental design, respectively. For the IVP of embryos, oocytes were matured and fertilized in vitro (D0). Zygotes (Bos indicus) were<br />
cultured in SOFaa supplemented with 2.5% FCS and 0.5% BSA. The embryos were maintained for seven days at 38.5 ° C in 5% O2, 5% CO2<br />
and 90% N2. For the in vivo production, embryos were recovered (n = 30) from cows (Bos indicus) superstimulated. The blastocysts (D7)<br />
were vitrified (Campos-Chillon et al., 2006, Theriogenology, 65, 1200-14). After vitrification, embryos were warmed and cultured for 12 h on<br />
SOFaa + 10% FCS + 0,5% BSA. Apoptosis was evaluated before (n = 15) and after (n = 15) vitrification by TUNEL assay. For statistical<br />
analysis, data were analyzed by ANOVA followed Tukey’s test using PROC GLM of SAS (Inst. Inc., Cary, NC, USA). Sources of variation<br />
in the model including embryos source (in vitro and in vivo produced), the condition (fresh and vitrified) and first order interaction; all factors<br />
were considered fixed effects. Data were subjected to arcsine transformation, and are reported as untransformed means±standard error. The<br />
level of significance was 5%. Apoptosis in fresh blastocysts was lower than in vitrified in vitro (18.5 ± 2.0% vs. 42.9 ± 2.1%, P
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A141 OPU-IVP AND ET<br />
INFLUENCE OF THE SPERM PRE-INCUBATION ON CLEAVAGE RATES,<br />
BLAST<br />
ASTOCY<br />
OCYST PRODUCTION AND SEX DISTRIBUTION OF<br />
IN VITRO PRODUCED BOVINE EMBRYOS<br />
Alinne Gloria Curcio, Reginaldo Silva Fontes, Steveen Ribeiro Leal, Célia Raquel Quirino, Gina Marcela Mican, Carla Sobrinho Paes de Carvalho & Gonçalo<br />
Apolinario de Souza Filho<br />
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
Studies on bovine embryos produced in vitro have showed that the sex ratio may differ from 1:1. This works suggest that Y-<br />
chromosome-bearing spermatozoa have a selective advantage for early fertilization (Kochhar et al. Animal Reproduction Science 77 (2003) 33–<br />
49). On the other hand, X-chromosome-bearing show greater longevity and penetrate the majority of oocytes when they become ready for<br />
fertilization (Dominko and First, Theriogenology 47(1997), 1041–1050). The objective of this study was to determine the influence of bovine<br />
sperm pre-incubation in the IVP and the sex ratio of bovine embryos on days 8 and 9 of development. A group of 277 oocytes from<br />
slaughterhouse cows from Campos dos Goytacazes, RJ were matured for 22h in modified TCM199 and in vitro fertilizated with frozen-thawed<br />
semen bull. Prior the fertilization, the sperm were pre-incubated in Fec-Talp supplemented with heparin for 1 h (G-1) or 4 h (G-4) in a humidified<br />
atmosphere of 5% CO2 in air at 38ºC in IVF droplets adjusted to a final concentration of 1x106/mL. In the control group (GC) there wasn´t<br />
sperm pre-incubation. The IVC was done in TCM199 supplemented with FSB 10%. All the experiment was repeated for five times. All reagents<br />
used were obtained from Sigma (St. Louis, USA) Cleavage and blastocyst production rates were analyzed on the days 3 and 7-8 of development<br />
respectively, by ANOVA test (SAS, 2002). Blastocysts at different development stages on days 8 e 9 were sexed by amplification of bovine<br />
amelogenine gen (Leal S.R. (2007), Dissertation (UENF). There were no significant differences in cleavage rates between the groups G-1, G-<br />
4 and CG (59,40 ± 18,82; 43,60 ±17,76; 63,80 ± 10,03, respectively), but the sperm pre-incubation for 1 h increased the rate of blastocyst<br />
production (37,20 ±19,43) when compared with CG (28,20 ± 15,75) and G-4 (11,00 ± 5,83). More than 90% of embryos were successfully<br />
sexed. The sperm pre-incubation did not lead to a significant alteration (X2, P > 0,05) in the sex ratio (M:F) among experimental groups (GC:<br />
0,90; G-1: 1,15; G-4: 0,8). The sperm pre-incubation for one hour increased the IVP, but did not change the sex proportion of embryos collected<br />
on eight or nine day of development. In this way, further studies should be performed to determine how these factors act on developmental<br />
competence and in the sex distribution of in vitro produced bovine embryos.<br />
Keywords: in vitro production, oocyto, amilogenine.<br />
A142 OPU-IVP AND ET<br />
INFLUENCE OF SEMEN ON THE PREGNANCY RATE IN MARES UNDER EMBRYO TRANSFER IN THE NORTHEAST<br />
OF THE STATE TE OF PAR<br />
ARA<br />
S ebastião Tavar<br />
ares Rolim Filho<br />
,<br />
Fr anklin Oliv<br />
liveir<br />
eira Barb<br />
arbosa Filho<br />
ilho, Haroldo Fr ancisco Loba<br />
obat o Rib<br />
ibeir<br />
eiro, Ellen<br />
Yasmin Eguchi Mesquita,<br />
Keitiane Colares Sousa & Henry Daniel Manrique Ayala<br />
CURSO DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL, UNIVERSIDADE FEDERAL DO PARÁ (UFPA), BELEM, PA, BRAZIL.<br />
The aim of this study was to evaluate the pregnancy rate of mares recipients transferred with embryos from donor inseminated with<br />
semen of different types in the northeastern state of Para, assessing such variables as influenced by the type of semen on conception rate of<br />
donors. 120 mares were used, which were 43 donor embryo, pure breeds of Quarter Horse and Paint Horse (with age ranging from 3 to 18 years)<br />
weighing between 350 and 600 kg were selected for high genetic value and excellent characteristics for livestock or skills in equestrian events<br />
and 77 of embryo recipient mares, breed aged between 3 and 10 years, weighing between 350 and 500 kg being performed gynecological<br />
examinations and evaluation of reproductive history. 16 stallions were used, since they are the breeds Quarter Horse and Paint miles, with age<br />
ranging between 3 and 20 years, with different types of semen (frozen, fresh and chilled). The frozen semen was imported from other regions<br />
of the country and transported and stored in liquid nitrogen canister. Insemination with fresh semen were taken immediately after collection.<br />
Already cooled semen was diluted with the diluent “Max Semen” in a 2:1 ratio and transported in appropriate containers “Equitainer” to 5°C for<br />
24 h. Also cooled semen was used from the southeast and northeast. In this study we found a conception rate of 88.13%, 68.42%, 73.07%, using<br />
fresh semen, cooled and frozen, respectively. Significant difference in conception rate from the donor, using fresh and cooled semen (P < 0.05)<br />
did not differ in relation to frozen semen (P > 0.05). The conception rate in donor insemination with cooled semen was lower than the results<br />
obtained with fresh semen and frozen, assuming failure in quality shipping chilled semen, which can interfere with sperm quality.<br />
Keywords: mares, embryo transfer, semen.<br />
N<br />
s407
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A143 OPU-IVP AND ET<br />
IGF-I (INSULIN-LIKE GROWTH FACT<br />
CTOR I) INCREASES IN VITRO PRODUCTION,<br />
BUT DOES NOT PROTECT EMBRYOS FROM<br />
DELETERIOUS EFFECT OF HEAT STRESS IN NELORE (BOS INDICUS) AND HOLSTEIN (BOS TAUR<br />
URUS) US) BREEDS<br />
Anthony César de Souza Castilho 1 , Rafael Augusto Satrap 1 , Eduardo Montanari Razza 1 , Cintia Fernandes Silva 1 , Renato Simões 1 , Thais Nabhan 1 , Janahi<br />
Sousa Ticianelli 1 , Josmar Mazucheli 2 & Ciro Moraes Barros 1<br />
1<br />
UNESP, BOTUCATU, SP, BRAZIL. 2 UEM, MARINGÁ, PR, BRAZIL.<br />
To better understand the differences between taurine and zebu cattle about their resistance to heat stress (HS) we aimed to verify<br />
a) whether the addition of IGF-I (Insulin-like growth factor I) to the culture medium in the absence of fetal calf serum (FCS) would be able to<br />
maintain embryo production rates similar to those obtained in medium with FCS, b) whether the addition of IGF-I to the culture medium is more<br />
effective in decreasing the deleterious effects of HS in Holstein embryos (HOL) when compared to Nelore (NEL), and c) whether the deleterious<br />
effect of HS in embryonic development and in apoptosis rates is higher in HOL embryos when compared with the NEL. In experiment 1, NEL<br />
oocytes were matured, fertilized with NEL semen and, 10 h post insemination (10 hpi), embryos were randomly divided into four groups: FCS<br />
(FCS 5% + 0 ng/mL of IGF-I, n=165), IGF (FCS 0% + 100 ng/mL of IGF-I, n = 163); FCS + IGF (n=169) and control (0% FCS + 0 ng/mL<br />
of IGF- I, n = 168). We evaluated the rates of cleavage (Cli), morula (Mo), blastocyst (BL) and hatched blastocyst (HB). In experiments 2 and<br />
3, HOL and NEL oocytes were matured, fertilized with semen from HOL and NEL bulls, respectively, and cultured until the BL stage. After 10<br />
hpi embryos were randomly divided into four groups: control (cultured in absence of IGF at 39°C), HS (cultured in the absence of IGF and<br />
exposed to 41°C, for 9 h at 96 hpi, and then returning to 39°C), IGF (cultured in the presence of IGF at 39°C) and HS/IGF (IGF + HS). In<br />
experiment 2 were evaluated rates of Cli, Mo and BL and in experiment 3 we evaluated the apoptosis rate of embryos. Data were analyzed using<br />
SAS Proc GENMOD, and logistic regression and Binomial Negative-regression were performed. In experiment 1, the rate of BL was similar<br />
(P > 0.05) between groups FCS (40.6±3.4) and IGF (36.2±5.6). In experiment 2, the HS decreased BL rates, regardless of IGF treatment in<br />
Nelore (29% vs. 24%) and HOL (20% vs. 15%). The addition of IGF-I in the culture medium, regardless of HS increased (P < 0.05) the<br />
production rates of both BL in Nelore (23% vs. 29%) and HOL (15% vs. 21%). In experiment 3, HS, regardless of treatment with IGF, the rate<br />
of apoptosis was significantly increased in NEL (3.3±0.2 vs. 4.1±0.3) and especially in HOL (4.8±0,3 vs. 6.0±0.4). We conclude that: a) the<br />
addition of IGF-I to the culture medium improved embryo production in both breeds and can be used in replacement of FCS; b) for both breeds,<br />
the addition of IGF-I to the culture medium did not protect embryos from deleterious effect of HS; c) Holstein was not more sensitive the<br />
deleterious effects of HS than Nelore, in both breeds there was reduction in the blastocyst production and higher incidence of apoptosis after HS.<br />
Keywords: igf-I, heat stress, bovine.<br />
A144 OPU-IVP AND ET<br />
TREATED TED OOCYTES<br />
WITH RETINOIDS AND WITH RETINOIDS AND GROW TH FACT<br />
CTOR IMPROVE<br />
THE IN VITRO<br />
EMBRYOUS PRODUCTION AND INHIBIT THE APOPTOSIS?<br />
Ricardo de Macedo Chaves 1 , Juliana Costa Conceição 2 , Madson Atila Vidal Silva 3 , Edivaldo Rosas dos Santos Júnior 4 , Leopoldo<br />
Mayer Freitas Neto 5 , Fabíola Freitas de Paula-Lopes 6 , Paulo Bayard Dias Gonçalves 7 , Paulo Fernandes Lima 8 & Marcos Antonio<br />
Lemos de Oliveira 9<br />
1,3<br />
UEMA, SÃO LUIS, MA, BRAZIL. 2,4,6,9 UFRPE, RECIFE, PE, BRAZIL. 5 FACULDADE PIO DÉCIMO, ARACAJU, SE, BRAZIL. 7 UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP,<br />
BRAZIL. 8 UNIVERSIDADE FEDERAL DE SANTA MARIA, SANTA MARIA, RS, BRAZIL.<br />
The objective was to evaluate in vitro the effect of adding retinol (RT) and retinoic acid (RA) in half and maturation of oocytes of both<br />
retinoids and IGF-1 in the middle of developing goat embryos to foster blastocyst production in vitro and inhibit apoptosis measured by enzyme<br />
activity of group II caspases and DNA fragmentation by TUNEL assay. Ovaries were obtained from a slaughterhouse and transported to the<br />
laboratory in warm saline at 30°C. The oocytes (n = 4320) were collected from follicles measuring 2-6 mm in diameter by aspiration with a<br />
syringe needle attached. The oocytes, tested in 10 replicates, were initially washed in medium itself, then screened and distributed in droplets of<br />
TCM 199 supplemented with RT or with RA, before finally being incubated at 39°C in a humidified atmosphere containing 5% CO2 for 24 h.<br />
Thereafter, oocytes were processed through mDM and exposed to sperm for 18 h in the same atmospheric conditions mentioned. Subsequently,<br />
presumptive zygotes were placed in droplets of KSOM medium supplemented with RT (G1, 2, 3, 7 and 8), RA (G4, 5, 6, 9 and 10), or IGF-<br />
I (G2 and 5) alone or in association with each other (G1, 3, 4, 6, 7, 8, 9 and 10), beyond the monolayer of cells of the oviduct. Finally they were<br />
incubated at 39 ° C in humid atmosphere with 5% CO2 during ten days. At the end of the 10th day of co-culture blastocysts were prepared for<br />
localization of the changes characteristic of apoptosis, using the reagent PhiPhiLux-G1D2 for determination of enzyme activity caspases group<br />
II and solutions of 4% paraformaldehyde and Phosphate -Buffered Saline + 1 mg/mL of polyvinylpyrrolidone to determine the number of<br />
blastomeres positive for apoptosis by TUNEL reaction test (DNA fragmentation). For statistical analysis, variance analysis was performed by<br />
the method of least squares. The results showed that the groups of oocytes and embryos treated only with retinoids produced fewer blastocysts<br />
(P < 0.05) than in groups of oocytes and embryos treated with retinoids associated with IGF-I. As to the blastocyst positive for apoptosis, is<br />
assessed by the activity of enzymes caspazes or DNA fragmentation, there was no difference (P > 0.05) between groups. The results suggest<br />
that the combination of retinoids with IGF-I is beneficial and can be recommended to maximize production in vitro blastocyst goats, but does not<br />
inhibit apoptosis.<br />
Keywords: retinoids, apoptosis, blastocyst.<br />
s408
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A145 OPU-IVP AND ET<br />
GENE EXPRESSION PROFILE OF IGF SYSTEM STEM MEMBERS ON IN VITRO PRODUCED BOVINE BLAST<br />
ASTOCY<br />
OCYST<br />
STS:<br />
S: COMP<br />
OMPARISON BETWEEN NELORE (<br />
(BOS INDICUS) ) AND<br />
HOLSTEIN (<br />
(BOS<br />
TAUR<br />
URUS<br />
US)<br />
Rafael Augusto Satrapa 1 , Anthony César de Souza Castilho 2 , Renato Simões 3 , Eduardo Montanari Razza 4 , Cintia Fernandes Silva 5 , Thais Nabhan 6 , Marcelo<br />
Pegorer 7 , Raquel Zanetti Puelker 8 , Henrique Eduardo 9 , José Buratini Junior 10 & Ciro Moraes Barros 11<br />
1,2,3,4,5,6,8,11<br />
DEPARTAMENTO DE FARMACOLOGIA, INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU (IBB), BOTUCATU, SP, BRAZIL. 7 DEPARTAMENTO DE REPRODUÇÃO ANIMAL - FMVZ,<br />
UNIVERSIDADE ESTADUAL PAULISTA (UNESP), BOTUCATU, SP, BRAZIL. 9 VETERINÁRIO AUTÔNOMO, BOTUCATU, SP, BRAZIL. 10 DEPARTAMENTO DE FISIOLOGIA, INSTITUTO DE<br />
BIOCIÊNCIAS DE BOTUCATU (IBB), BOTUCATU, SP, BRAZIL.<br />
Evidences indicate that the IGF system is related to embryo quality. To better understand the differences on in vitro embryo<br />
development of Zebu and European cattle, we aim to determine the effect of heat stress on the pattern of mRNA expression of IGF-I and II,<br />
IGFR-I and II, IGFBP-2 and 4 and PAPP-A in blastocysts from Nelore (NEL) and Holstein (HOL) breeds. NEL and HOL oocytes were<br />
aspirated by OPU, matured in TCM199, fertilized with semen from NEL (n = 6) and HOL (n = 6) bulls, respectively, and cultured in SOF<br />
medium (synthetic oviduct fluid, without FCS) to the blastocyst stage. Ninety-six h post-insemination (96 hpi) embryos = 16 cells were<br />
randomly separated in two groups: control and Heat Stress (HS). Embryos from control group were continuously cultured at 39°C and embryos<br />
from HS group were exposed to 41°C for 9 h, and returned to 39°C afterwards. Embryos at 168 hpi were removed from culture medium and<br />
allocated into five pools with five embryos in each experimental group, which were subjected to RNA extraction and stocked at -80°C. The<br />
expression of target genes was performed by RT-PCR in real time with oligo-dT in reverse transcription and specific primers for the bovine<br />
species. Expression of cyclophilin-A was used as endogenous control. The means of mRNA levels of target genes were compared using T-test<br />
or Man-Whitney test whether or not data were normally distributed. The mean values of IGF-I expression were higher (P < 0.05) in blastocysts<br />
undergoing HS when compared to control group, in both NEL (22.0±11.0 vs. 0.2±0.01, respectively) and HOL (8.0±5.0 vs. 1.0±0.3), and this<br />
increase was more evident in Nelore. Moreover, relative values of gene expressions of IGFBP-2 and 4 were higher (P < 0.05) in HOL<br />
blastocysts pools undergone HS (110.0±80.0 and 6.0±1.0, respectively) when compared to NEL undergone HS (2.0±0.1 and 0.1±0.01) or not<br />
(0.2±0.01 and 0.1±0.01) and HOL control (4.0±0.1 and 0.1±0.02). The higher IGF bioavailability verified in NEL blastocysts under HS can<br />
contribute to a greater tolerance of this breed to the HS when compared to HOL. [We acknowledge FAPESP for the financial support].<br />
Keywords: igf, gene expression, bovine.<br />
A146 OPU-IVP AND ET<br />
POTENTIAL OF THE GYR (BOS TAUR<br />
URUS US INDICUS) AND HOLSTEIN (BOS TAUR<br />
URUS<br />
US TAUR<br />
URUS) US) DONORS FOR EMBRYO<br />
PRODUCTION<br />
J oão Henr<br />
enrique Mor<br />
oreir<br />
eira V iana 1 , Ta timara a Mar<br />
aria Miy<br />
iyauchi<br />
2 , To chimara a Apar<br />
parecida Miy<br />
iyauchi<br />
3 , Eduar<br />
duardo do Ramos de Oliv<br />
liveir<br />
eira 4 , Jose<br />
Antonio D. Garcia 5 , Marilu Martins Gioso 6 , Carlos Antônio de Carvalho Fernandes 7 & Miller Pereira Palhão 8<br />
1<br />
EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2,4 BIOTRAN, ALFENAS, MG, BRAZIL. 3,5,6,7,8 UNIFENAS, ALFENAS, MG, BRAZIL.<br />
N<br />
The ovarian follicle pick-up (OPU) and oocyte retrieval are critical steps for IVEP from living donors. Recently, variability in<br />
oocytes recovery has been studied in Gyr breed and it seems to be related to intrinsic potential of the donor (Oliveira et al., XXVI World Buiatrics<br />
Congress, 2010). The objective was to evaluate the potential for oocyte retrieval in two important breeds of dairy cows. Results from 245 OPU<br />
sections in Gyr (n = 132) and Holstein (n = 113) breeds were included for this purpose. Five days before OPU, the ovarian follicular wave was<br />
synchronized with auricular implant (norgestomet - Crestar ® - Intervet Schering- Plough Animal Health) and intramuscular injections of 2 mg<br />
of estradiol benzoate (2 mL of Gonadiol ® - Intervet Schering- Plough Animal Health) and 0,250 mg of D-cloprostenol (Veteglan ® - Hertape<br />
Calier Saúde Animal S.A). The OPU sections were separated in quartiles regarding the total oocytes recovered within each breed. Thereafter, the<br />
oocytes were classified according to the degree of quality and considered viable or not viable. Sex-sorted (X) semen was used for in vitro<br />
fertilization. Data from recovered oocytes (total and viable) and embryo produced in each quartile were submitted to one-way ANOVA and<br />
means between breeds were compared by “t” test (P < 0.05). The oocytes recovered (total and viable) were greater (P < 0.0003 and P < 0.0001)<br />
in OPU sections from Gyr compared to Holstein donors (34.3±9.9 vs. 25.2±7.5 oocytes and 27.8±9.5 vs. 18.4±7.0 viable, respectively, for the<br />
first quartile,). These differences were kept in OPU sections assigned in the 2nd e 3rd quartiles. However, the sections below the 3rd quartile did<br />
not change (P < 0.3 and P < 0.2) between Gyr and Holstein (5.5±2.8 vs. 4.8±2.0 oocytes and 4.0±2.2 vs. 3.5±2.0 viable, respectively). The OPU<br />
sessions with higher number of the recovered oocytes (1st quartile) confirmed the highest production of embryo, and did not differ (P < 0.6)<br />
between breeds (7.6±6.3 vs. 5.9±4.5 embryos, respectively, for Gyr vs. Holstein). However, for the other quartiles (2nd, 3rd and the remained<br />
OPU sections), the embryo production was greater in Gyr compared to Holstein donors (5.4±4.0 vs. 2.0±1.9, 3.1±2.0 vs. 1.6±1.5 and 1.6±1.3<br />
vs. 0.9±1.2 embryos, respectively). In the 1st quartile, the conversion rate of the viable oocytes to embryo was greater (P < 0.06) in Holstein<br />
(32.1%, 154/479) than in Gyr breed (27.3%, 243/890). Previous studies have shown the importance on selecting Gyr donors using the total<br />
oocytes recovered. The results suggest that the same principles could be useful to select Holstein donors. [Support: FAPEMIG Projeto CVZ<br />
AQP 01654/09, Embrapa Projeto Rede de Inovação em Reprodução Animal (01.07.01.002)].<br />
Keywords: dairy cattle, opu, ivep.<br />
s409
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A147 OPU-IVP AND ET<br />
PRESERVATION OF INTRAFOLLICUL<br />
AFOLLICULAR AR OOCYTES AT 4°C<br />
Roberto Solano Ferro 1 & Onel Solano Garcia 2<br />
1<br />
UEMA, SÃO LUIS, MA, BRAZIL. 2 AGRITEC, BACABAL, MA, BRAZIL.<br />
These works investigated the possibility of preserving bovine ovaries at 4°C and still have oocytes capable of maturation and<br />
fertilization. For that, 180 ovaries obtained at the slaughterhouse were transported to the laboratory in saline solution at 25 -30°C and divided in<br />
3 experimental groups of 60 ovaries each. Ovaries were washed three time in phosphate-buffered saline (PBS) supplemented with penicillin<br />
(100 UI /mL) and streptomycin (0.1 mg/mL) upon arrival to the laboratory. Oocytes were obtained for the first group immediately after washing.<br />
Ovaries for the second and third groups were directly plunged into 600 mL of previously cooled to 4°C PBS + antibiotics. In all cases oocyte<br />
selection for in IVM was based upon morphological appearance of cumulus oocytes complexes (CoCs ). Os Treatments were compared among<br />
each other using (ANOVA followed the test of tukey (P < 0.05). Puncture was performed after 12 or 24 h of preservation respectively. In For<br />
IVM, TCM-199 supplemented with 20% estrous cows serum and 10ug/mL de FSH was employed. Culture conditions were 39°C, 5% C0 2<br />
in<br />
air for 22-24 h. For evaluating 30 CoCs from each group were denuded by pipeting in trypsin /EDTA (0.1% /0.2%) and the presence of first polar<br />
body was taken as the evidence for maturation. Fertilization and in vitro culture were performed according to SEIDEL. Cleavage was evaluated<br />
72 h after the start of fertilization. The results showed no significant difference among groups P > 0.05 in maturation or cleavage no control group<br />
(90.0 and 56.0%) in relation to experimental groups 12h (83.0; 52,0) and 24h (86.0; 50.0%) respectibily despite a slight tendency to decrease<br />
embryonic cleavage with the increasing in preservation length. Although, we did not study either metabolic changes or cooling rates. These<br />
findings indicate the possibility of preserving intrafollicular oocytes for short periods of time (12 to 24 h) without decreasing their capability for<br />
maturation, fertilization and cleavage oocytes.<br />
Keywords: ovaries, oocytes, fiv.<br />
A148 OPU-IVP AND ET<br />
PRODUCTION OF OVINE OOCYTES FROM SLAUGHTERHOUSE OVARIES USING T WO COLLECTION PROCEDURES<br />
Paula Mariana Rafaelli 1 , Patricio Zenón Diaz Pumará 1 , Pedro Ortiz 1 & Juan Ignacio Ernesto 2<br />
1<br />
LABORATORIO DE BIOTECNOLOGÍA DE LA REPRODUCCIÓN, VETERINARIA, UNIVERSIDAD DEL SALVADOR, BUENOS AIRES, ARGENTINA. 2 INSTITUTO DE BIOLOGÍA Y MEDICINA<br />
EXPERIMENTAL CONICET, BUENOS AIRES, ARGENTINA.<br />
Ovaries from slaughterhouse in sheep are not as abundant as in cattle, on the other side these from young females generally come<br />
with low and variable functional activity. For this reason it can not expect the same amount of good quality oocytes per ovary were processed,<br />
many of them appear denuded. Our aim is development of IVF with the ultimate goal of generating transgenic sheep using the technique defined<br />
as SMGT (sperm mediated gene transfer) and we consider this source of oocytes as primordial. 203 ovaries were processed by follicular<br />
puncture 5 cc syringe and needle of 21 G (0.8 x 25 mm) filled with 1 cc of PBS plus FCS (fetal calf serum). Were punctured follicles of more<br />
than 1.0 mm in diameter. In a method called “1)” was only observed the contents collected in the syringe as is the practice in bovine ovaries. In<br />
a method called “2”) the ovary was manipulated on a 90 mm petri dish with a small amount of culture medium, in order to collect any losses on<br />
the punching. This means that after washing this media was observed along with that collected in the syringe. With the aim of verifying the<br />
concomitant production of oocytes, in both cases, were recorded the number of punctured follicles per ovary. For the method “1)” the amount<br />
of processed ovary was 103 and the total number of follicles punctured were 457, which gives 4.4 follicles per ovary. In the method “2”) 100<br />
ovaries were processed with 451 total punctures, it mean 4.5 follicles incised per ovary. The oocytes obtained were classified into three<br />
categories: a) covered more than one layer of granulosa cells; b) partially covered by granulosa cells; d) nude. It further registered the number of<br />
empty zonae (EZP) collected in each case. For the method “1)” We collected a total of 60 OO and 8 EZP; 12 oocytes were type a), type b) were<br />
35 ; and type d) 13. In the method “2)” 106 OO were collected and 4 EZP; 36 OO were type a); 31 type b); and 39 type c). The amount of follicular<br />
punctures was similar in both methods. However the number of oocytes obtained with the method “2)” were significantly higher than method<br />
“1)” (106/100 vs. 60/103). Chi-square statistical analysis resulting in highly significant differences between both samples (÷2 = 11,31, df = 1,<br />
P < 0,0008) It is also significant difference between oocytes kind a) in Method “2)” versus Method “1)” (36/100 vs. 12/103) and type c) (39/100<br />
vs. 13/103). Significative differences between both methods were found in therms of EZP collected, Method 1: 8; Method 2): 4). We conclude<br />
that many oocytes are lost during the follicular puncture in ovine model which does not happen in the usual methodology in cattle.<br />
Keywords: oocyte, empty zona pellucida, in vitro fertilization.<br />
s410
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A149 OPU-IVP AND ET<br />
IN VITRO EMBRYO PRODUCTION (IVEP) AFTER THE LAP<br />
APAR<br />
AROSC<br />
OSCOPIC OPIC OOCYTE RECOVER<br />
OVERY (LOR) IN CANINDÉ GOATS<br />
Ale<br />
lexsandr<br />
sandra a Fer<br />
ernandes Per<br />
ereir<br />
eira,<br />
Raylene Ramos Mour<br />
oura,<br />
Ribr<br />
ibrio io Ivan<br />
Tavar<br />
ares Per<br />
ereir<br />
eira Batista,<br />
Joanna Mar<br />
aria Gonçalv<br />
onçalves de Souza,<br />
Agostinho Soar<br />
oares de<br />
Alcân<br />
lcântar<br />
tara a Net<br />
eto, Car<br />
arlos Henr<br />
enrique Sousa de Melo<br />
elo, Iana Sales Camp<br />
ampelo<br />
elo, Maiar<br />
aiara a Pinheir<br />
inheiro Vieir<br />
ieira,<br />
Dárcio Ítalo Alv<br />
lves<br />
Teix<br />
eixeir<br />
eira,<br />
Luciana Magalhães Melo & Vic<br />
icen<br />
ente<br />
José de Figueirêdo Freitas<br />
UECE, FORTALEZA, CE, BRAZIL.<br />
Currently, a major concern of livestock is the biodiversity preservation. In Northeast Brazil, there are several naturalized goat<br />
breeds at risk of extinction, including the Canindé. Reproductive biotechnologies could participate in this process. From these, IVEP after<br />
LOR may accelerate the genetic material recovery. Nevertheless, few studies demonstrate the real efficiency of this system in goats. Therefore,<br />
the aim of this study was to evaluate the IVEP coupled with LOR as biotechnique to create an embryo bank for the preservation of Canindé<br />
goats. Thus, 20 adult and cyclic goats (five females per session) received intravaginal sponges with 60 mg medroxyprogesterone acetate<br />
(Progespon, Buenos Aires, Argentina) for 11 days associated with 70 µg cloprostenol (Prolise, Buenos Aires, Brazil) in the eighth day. Thirtysix<br />
hours before sponge removal, animals received 70 mg pFSH (Folltropin, Ontario, Canadá) and 200 IU eCG (Novormon, Buenos Aires,<br />
Argentina). The follicles, visualized by laparoscopy, were classified as small (< 3mm), medium (3 to 4 mm) and large (> 4mm) and aspirated<br />
just after the sponge removal using an aspiration system for small ruminants (Watanabe, Cravinhos, Brazil). Cumulus-oocyte complexes<br />
(COCs) were recovered and classified (grade I to IV) based in the presence of cumulus cells and cytoplasm homogeneity. Grade I and II<br />
structures were matured in modified TCM199, for 24 h at 38.5°C and 5% CO 2<br />
. After this period, COCs were fertilized with fresh<br />
spermatozoa (2x10 6 sperm/mL) in SOF-FIV medium supplemented with heparin for 16 h in the same maturation conditions. The presumptive<br />
zygotes were cultured in SOF-CIV medium, in the same fertilization conditions, for seven days. A total of 245 follicles were punctured and<br />
distributed in small (31.5%), medium (35.9%) and large (32.6%). The oocyte recovery rate was 74.3% (182/245) with an average of 9.1<br />
oocytes per goat. Regarding to oocyte quality, 13.2% (24/182), 68.1% (124/182), 5.5% (10/182) and 13.2% (24/182) were classified as grade<br />
I, II, III and IV, respectively. The average of COCs submitted to maturation (grade I and II) was 7.5 per goat. From the presumptive zygotes<br />
in vitro incubated, 58.3% (84/144) cleaved after 48 h of culture. The blastocyst rate was 52.1% (75/144) regarding the total number of<br />
structures in culture. The total percentage of blastocyst in relation to cleaved embryos was 89.3% (75/84). In conclusion, the IVEP-LOR<br />
system was efficient to produce Canindé goat blastocysts and may be used in the creation of an embryo bank in order to preserve the breed.<br />
Keywords: goat, Canindé, embryo.<br />
A150 OPU-IVP AND ET<br />
IN VITRO EMBRYO PRODUCTION USING FROZEN SEMEN PREVIOUSL<br />
VIOUSLY INFECTED WITH BOVINE HERPESVIRUS<br />
TYPE 5<br />
Diego Gouvêa Souza<br />
1 , Alicio Mar<br />
artins Júnior 1 , Rena<br />
enata Sanches Calegar<br />
alegari 2 , Jéssica Oliv<br />
liveir<br />
eira Caldeir<br />
aldeira 1 , Camila Silv<br />
ilva-F<br />
a-Frade<br />
ade 3 & Ter<br />
ereza Cristina Car<br />
ardoso<br />
3<br />
N<br />
1<br />
DCCRA-FMVA, UNESP, ARAÇATUBA, SP, BRAZIL. 2 DRARV- FMVZ, UNESP, BOTUCATU, SP, BRAZIL. 3 DAPSA-FMVA, UNESP, ARAÇATUBA, SP, BRAZIL.<br />
Bovine herpesvirus type 5 (BoHV-5) is recognized as the ethiological agent of bovine encephalitis and its natural transmission has<br />
been recently described via contaminated semen. Despite it has been detected in bull semen, no reports were found in the literature with regard<br />
to its influence on in vitro embryo production. The aim of this study was to verify the susceptibility of in vitro matured bovine oocytes fertilized<br />
with frozen semen, which was experimentally infected with BoHV-5 before freezing process. The endpoints assessed were the subsequent<br />
embryonic development and the possible contamination of zygotes via infected spermatozoa. The ejaculates of three Nelore bulls, with negative<br />
PCR for BoHV-5, were mixed and then divided into two groups, as follows: Group I, semen not exposed to virus, and Group II, semen exposed<br />
to 102,3 TCID50/50µL of a Brazilian strain of BoHV-5 virus, and then subjected to the freezing process. Ovarian follicles (2-7 mm in diameter)<br />
obtained from slaughtered cows were punctured and the selected oocytes were washed 3 times in PBS with 10% FCS (Nutricell, Campinas,<br />
Brazil). Groups of 15-20 oocytes were matured in TCM (GIBCO, Grand Island, USA) with 0.5 µg/mL FSH (Pluset ® , Calier, Spain), 50 µg/<br />
mL LH (Lutropin- V ® , Bioniche Inc., Canada), and 10% FCS, for 24 h. Then, semen the Group I and II was thawed and the sperm selected by<br />
centrifugation (700x g/20 min) in discontinuous Percoll gradient. Afterwards, the supernatant was discarded and the pellet was washed in TALP<br />
by centrifugation at 200x g for 5 min. The resulting pellet was diluted in TALP (with PHE and heparin) and used for IVF. After a 20 h coincubation<br />
period, the cumulus cells were partially removed and presumptive zygotes were transferred to drops of culture medium (m-SOF). The<br />
cleavage rates and percentages of oocytes that reached morula (M)/blastocyst (B), blastocyst/expanded blastocyst (EB) stages were recorded at<br />
72, 144 and 168 h post-insemination (h pi), respectively. The presence of BoHV-5 was investigated by PCR and in situ hybridization assay. Data<br />
were analyzed through use of Student’s t test with P < 0.05 considered as significant. There were no significant differences among treatment<br />
groups for cleavage, M/B, B/EB rates between Group I (86.4%, 44.3%, and 34.1%, respectively) and Group II (86.6%, 39.8%, and 31.2%,<br />
respectively). Viral DNA was found only in embryos from Group II (30; 100%) at 168 h pi. Thus, it can be conclude that, sperm that was<br />
infected with BoHV-5 can fertilize oocytes during IVF, and zygotes became contaminated without compromising their developmental competence.<br />
Keywords: bohv-5, fluorescent in situ hybridization, embryonic development.<br />
s411
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A151 OPU-IVP AND ET<br />
IN VITRO PRODUCTION OF OVINE EMBRYOS OF DORPER AND SANTA A INES BREED ASPIRATED BY VIDEOLAP<br />
APAR<br />
AROSC<br />
OSCOPY<br />
OPY<br />
AND TRANSPOR<br />
ANSPORTED OVER LONG DISTANCES<br />
Marcos Chalhoub 1 , Andrea Cristina Basso 2 , Janaína Silva Chacon 3 , Antônio de Lisboa Ribeiro Filho 4 , Marcus Vinícius Galvão Loiola 5 , Leandro Moureira de<br />
Castro Feitosa 6 , Bruno Henrique de Araújo Andrade 7 , Alexandra Soares Rodrigues 8 , Priscila Assis Ferraz 9 , Endrigo Adonis Braga de Araujo 10 , Sidnei Nunes<br />
Oliv<br />
liveir<br />
eira 11 & Ale<br />
lexandr<br />
xandre Far<br />
aria<br />
Tab<br />
abet<br />
12<br />
1,4,5,6,7,8,9,10,11<br />
UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR, BA, BRAZIL. 2 IN VITRO BRASIL SA, MOGI MIRIM, SP, BRAZIL. 3 AUTÔNOMO, SÃO PAULO, SP, BRAZIL. 12 AUTÔNOMO,<br />
SÃO PAULO, SP, BRAZIL.<br />
The sheep industry in Brazil is in a time of expansion, increasing thus the demand for biotechnologies of reproduction in this specie,<br />
amongst which stands out in vitro production of embryos (IVP), which allows the intensive exploration of the genetic and reproductive potential<br />
of females. However, this technology still has not been adequately explored in small ruminants, and several factors may influence results, for<br />
example, the breed of the donors. Aimed to compare the efficiency of IVP in Dorper and Santa Ines breed sheep after videolaparoscopic<br />
aspiration of oocytes and transport over long distances. We analyzed the number of oocytes retrieved and the rates of cleavage. We used 59<br />
donors ewes, single, with three years of age on average, created in an extensive way, being 34 Dorper and 25 Santa Ines ewes. All animals were<br />
previously synchronized and superstimulated according to Baldassare et al. (2004, Animal Reproduction Science 82-83, 255-266). At the<br />
beginning, follicular aspiration was performed by videolaparoscopy in Dorper and Santa Ines donors created in the microregion of the Chapada<br />
Diamantina in Bahia. The aspirated follicular fluid was evaluated under stereomicroscope and cumulus-oocyte complexes visualized were<br />
counted, sorted and transferred to cryogenic tubes containing maturation medium on temperature, humidity and CO2 concentration suitable.<br />
Later sent to a commercial laboratory in the state of São Paulo, which followed the steps of in vitro maturation, fertilization and culture, according<br />
to Basso et al. (2008, O embrião 38, 9-13). In order to compare the number of oocytes, embryo produced and cleavage rate in different breeds,<br />
was employed the statistical package SAS, version 6.0 (Procedure MEANS and GLM with P < 0.05). The average of total oocytes retrieved and<br />
embryo produced of Dorper ewes of 11.90±7.18 and 8.83±5.37 was higher (P < 0.05) to that obtained in Santa Ines ewes of 9.00±4.47 and<br />
4.62±2.48, respectively. The cleavage rate (embryos produced/oocytes recovered) in the Dorper ewes was of 76.09%, significantly higher<br />
compared to the Santa Ines ewes of 57.47%. These results suggest that exist a superiority in the production of oocytes by videolaparoscopy<br />
and in the cleavage rate of in vitro produced embryos and transported over long distances, in the Dorper in relation to Santa Ines breed.<br />
Evidenced the feasibility of transporting long distance oocytes in sheep.<br />
Keywords: ivep, videolaparoscopy, cleavage rate.<br />
A152 OPU-IVP AND ET<br />
IN VIVO EMBRYO PRODUCTION IN COWS SUPEROVUL<br />
OVULATED 1 OR 2 DAYS AFTER OPU<br />
Ricardo Silva Surjus 1 , Alexandre Barbieri Prata 1 , Marta Borsato 1 , Fernada C.S.Z. Mattos 2 , Mariana Curci Martins da Silveira 1 , Gerson Barreto Mourão 1 ,<br />
Alexandre Vaz Pires 1 & Roberto Sartori 1<br />
1<br />
ESALQ-USP, PIRACICABA, SP, BRAZIL. 2 UNESP, BOTUCATU, SP, BRAZIL.<br />
The aim of this study was to evaluate the superstimulatory and superovulatory responses and in vivo embryo production in<br />
cows treated with FSH starting at 1 or 2 days after OPU. Thirty two nonlactating Nelore cows, weighing 489.5±11.3 kg, handled in a two<br />
cows per stall system, were fed a diet with corn, sugarcane bagasse, urea and mineral salt, according to the NRC (2000). All cows were<br />
subjected to aspiration of follicles >2 mm for OPU. Recovered oocytes were used in another experiment. After OPU, the cows were randomly<br />
divided into two groups: D1 and D2, in which the follicle superstimulatory treatments with FSH started 1 or 2 days after OPU, respectively.<br />
The embryo donors received an intravaginal device (IVD) of progesterone release (Sincrogest ® , Ouro Fino, Ribeirão Preto, Brazil), soon after<br />
OPU. The cows received eight decreasing doses of FSH (100 mg, i.m., Folltropin-V ® , Bioniche, Ontario, Canada), and concomitant with the<br />
fifth and sixth treatments of FSH, PGF2α (150 µg each, i.m., d-cloprostenol, Sincrocio ® , Ouro Fino) was administered. The IVD was removed<br />
at the time of the last FSH injection. Twelve hours after IVD removal ovulation was induced with GnRH (0.01 mg, i.m., Buserelin acetate,<br />
Sincroforte ® , Ouro Fino). Twelve and 24 h later, cows were inseminated. Embryo collection was performed 8 days after GnRH injection. Data<br />
were analyzed using the GLIMMIX procedure of SAS and the results are presented as least squares means ± SE. On the fourth day of FSH<br />
treatment, cows in Group G1 had a greater superstimulatory (follicles =6 mm in diameter) response (21.1±3.0 follicles) than those in Group<br />
G2 (13.7±2.4 follicles, P = 0.08). In addition, there was a much greater proportion (P < 0.001) of follicles =8 mm compared to follicles =6 mm<br />
and 0.10). Nevertheless,<br />
the cows in Group D2 had more viable (3.3±1.2 vs.. 1.8±0.7, P = 0.09) and freezable (3.2±1.1 vs.. 1.3±0.5, P < 0.05) embryos than those in<br />
Group D1. In conclusion, to obtain a better superovulatory response and higher embryo production in superovulated cows after OPU, it is<br />
recommended to wait at least 2 days to start treatment with FSH. [Acknowledgements: We thank FAPESP, CNPq, and InVitro Brazil for<br />
financial and technical support].<br />
Keywords: embryos, superovatory, opu.<br />
s412
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A153 OPU-IVP AND ET<br />
HORMONAL PROTOC<br />
OCOLS OLS FOR OOCYTE RETRIEVAL AND IN VITRO PRODUCTION OF EMBRYOS IN BOVINE DONORS<br />
Tatimar<br />
timara a Mar<br />
aria Miy<br />
iyauchi<br />
1 , Tochimar<br />
chimara a Apar<br />
parecida Miy<br />
iyauchi<br />
1 , Miller Per<br />
ereir<br />
eira Palhão<br />
1 , Aline Sousa Camar<br />
amargos<br />
2 , Car<br />
arlos Antônio de Car<br />
arvalho alho Fer<br />
ernandes<br />
1 & Mar<br />
arilu<br />
Martins Gioso 1<br />
1<br />
UNIFENAS, ALFENAS, MG, BRAZIL. 2 FMVZ-UNESP, BOTUCATU, SP, BRAZIL.<br />
The in vitro embryo production (IEVP) can improve the genetic outcome of the zootechnical features in bovine specie. However,<br />
the efficiency of this technique is directly related to the number and the quality of the oocytes recovered after follicular aspiration (OPU). The<br />
control of the follicle development can improve the results of the IEVP. The aim of this study was to evaluate different protocols for<br />
synchronization follicle development in B. taurus and B. indicus donors. The animals were randomized in one of the four experimental groups:<br />
TI - Control (N = 81): OPU regardless the day of the estrous cycle with no previous hormone treatment; TII - (N = 97): synchronization of the<br />
follicular wave (2 mg BE1) five days before OPU; TIII - (N = 97): synchronization of the follicular wave and no corpus luteum (CL). Five days<br />
before the OPU, the follicular wave was synchronized with 2 mg of EB, an ear implant (Norgestomet) was placed and 0.150 mg of D-<br />
cloprostenol was given; TIV - (N = 69): the same protocol of the TIII plus stimulation of the follicular development. Two injections of FSH (each<br />
injection of the 50 UI for indicus and 75 UI for taurus) were performed 12 h apart four days after starting protocol. The OPU section was held<br />
12 to 16 h after last FSH injection. The oocytes were sent to PIV laboratory for a period until 6 h. Procedures were the same performed in routine<br />
of the commercial laboratory. The oocyte retrieval (total and viable) and embryo production were higher in indicus than in taurine (17.7 ± 0.9 vs.<br />
11.9 ± 0.6, 14.2 ± 0.7 vs.. 8.8 ± 0.5, 4.3 ± 0.3 vs. 2.1 ± 0.2, respectively, P < 0.05). There was an increase in number and quality of oocytes<br />
recovered from donors from TIII and TIV (16.8 ± 1.2 and 17.8 ± 1.4 vs. 10.7 ± 1.0, 13.2 ± 1.0 and 13.8 ± 1.1 vs.. 8.1 ± 0.8, respectively, P <<br />
0.05). The total embryo production was higher in all treatments when compared to control (P < 0.05). The FSH injections (TIV) to promote the<br />
ultimate follicle development and increased the average of the embryo production when compared to the other groups (4.6± 0.6 and 2.8± 0.3).<br />
In taurine, the OPU section from treatments III and IV provided more oocytes (total and viable) (12.7 ± 1.5, 14.3 ± 1.3 and 9.8 ± 1.2, 10.9 ± 1.1,<br />
P < 0 05). In Zebu, the embryo production was higher in treated groups when compared to control (5.1 ± 0.5 vs. 2.0 ± 0.4, P < 0.05). The<br />
synchronization treatments were efficient for oocyte retrieval in taurine and embryo production and zebu donors. [Support: FAPEMIG].<br />
Keywords: opu, gyr, follicle wave.<br />
A154 OPU-IVP AND ET<br />
TECHNICAL REPORT: OVUN PICK UP BY SINGLE FOR RECOVER<br />
OVERY OF OOCYTES IN MARES IN TRANSITION PERIOD<br />
André Gomier<br />
omiero o Rigo<br />
1 , Bruno<br />
Valen<br />
alente e Sanches<br />
anches, José Henr<br />
enrique For<br />
ortes Pon<br />
ontes<br />
es, Luc<br />
ucas Lop<br />
opes Moino<br />
oino, Rodr<br />
drigo Mendes Untur<br />
tura,<br />
Per<br />
erla Dagher Cassoli Fleur<br />
leury, Mar<br />
ario<br />
Martinez-Dias , Janaina Ferreira Nagao & Andrea Cristina Basso<br />
IN VITRO BRAZIL S.A., MOGI MIRIM, SP, BRAZIL.<br />
The most difficulty founded for practicing reproductive biotechnologies in horses as in vitro fertilization, intracytoplasmic sperm<br />
injection, oocyte transfer, gamete intrafallopian transfer and nuclear transfer is the difficulty of aspiration and oocyte retrieval in large quantities.<br />
For the present research we used 26 crossbreed mares, 4 to 16 years old, body scores from 3.0 to 3.5, average weight of 400 kg, daily subjected<br />
to gynecological examination. The mares presented multiple follicles with size between 5 and 22 mm and complete absence of uterine edema or<br />
diestrus featuring transitional period between March to April <strong>2011</strong>. For follicle aspiration the animals were treated with 0.5 mL of acepromazine<br />
2% EV. The aspiration was preceded by rectal cleaning using carboxy-methyl cellulose additioned of 10% lidocaine hydrochloride 2%. Epidural<br />
anesthesia was not performed. After cleaning the perineal area with water and 70% alcohol, the procedure was guided with ultrasound<br />
MINDRAY 2200, 5 MHz sector transducer. The transducer was connected to a single-way needle and positioned transvaginal, beside to the<br />
cervix and the ovary where the follicles were aspirated. The ovary was placed by transrectal manipulation to the follicular wall being juxtaposed<br />
to the transducer and the cannula guide. The needle was inserted through the cannula guide to perform the follicular puncture. The follicular fluid<br />
was aspirated without washing the follicle, using TERUMO 16 gauge needle (0.9 X 50 mm) and vacuum pump (Cook Veterinary Products,<br />
New Buffalo, MI, USA) using 200 mm Hg pressure. After follicular aspiration proceeded up the search and evaluation of oocytes in a petri dish<br />
with the aid of a binocular dissecting microscope with a magnification between 15 and 60 times. After completion of the total scan of the ovaries,<br />
298 follicles were aspirated, 84 oocytes were recovered (recovery rate 28%), yielding an average of 3.23 of total oocytes and 2.75 viable oocytes<br />
per mare. After complete aspiration of the ovaries, these mares were monitored daily for 21 days by ultrasound, all returned to a growth of new<br />
follicular wave 6-8 days after the first aspiration and there were no sequelae from the procedure. This research puts the follicular aspiration in<br />
mares receiving transitional or diestrus phase as an alternative to obtaining oocytes needed for the development and study of new biotechnologies,<br />
without affecting the commercial routine of an equine recipient center.<br />
Keywords: ovum pick up, mares, oocytes.<br />
N<br />
s413
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A155 OPU-IVP AND ET<br />
OVULATOR<br />
ORY RESPONSE OF CYCLING CLING MARES STIMULATED<br />
TED WITH AN ASSOCIATION OF PFSH AND EPE<br />
Natalia Claro de Abreu, Cezinande Meira , Fernanda Saules Ignácio & Gustavo Henrique Marques Araújo<br />
UNESP, BOTUCATU, SP, BRAZIL.<br />
It has been demonstrated by Krekeler et al., 2006 (Theriogenology, 66: Abstract No. 663) a satisfactory ovulatory response (2.28<br />
ovulations/mare) after 25mg i.m. of porcine FSH (pFSH; Folltropin-V ® ) twice a day. This result was not confirmed by following studies<br />
developed by Ignácio et al. in 2007 (Acta Scientiae Veterinariae 35, Supplement 3; Abstract:s1245) and 2008 (Acta Scientiae Veterinariae 36,<br />
Supplement 2; Abstract:s504), in which a mean of 1.4±0.26 and 1.29±0.49 ovulations/mare, respectively, were found. Even though, treatment<br />
contributed to an increased diameter of the second and third largest follicles. The present study aimed to reduce EPE length of treatment by<br />
verifying the ovarian answer of mares treated with EPE after a pre-stimulation of pFSH. A total of 22 cross-bred mares were randomly assigned<br />
into three groups: control (saline, n = 7), pFSH+EPE (n = 8) and EPE (n = 7). The hormonal treatment began at D7 (D0=ovulation) when<br />
follicles =23mm were detected to allow beginning of treatment until deviation. Concomitantly, luteolysis was induced with 7.5mg of dinoprost<br />
trometamine i.m. (Lutalyse ® ). In pFSH+EPE group, mares were treated with 25 mg i.m. BID of pFSH for four days followed by 12.5 mg i.m.<br />
BID of EPE. In EPE group or control, 12.5mg of EPE or an equivalent volume of saline 0.9% were injected i.m. every 12h. In all groups,<br />
treatments were maintained until the largest follicle achieved =35 mm, when ovulation was induced with hCG (Vetecor ® , 2500 UI, i.v.) followed<br />
by two inseminations (1x109 viable sperm cells) 12 and 36h after induction. Transrectal ultrasound was done daily for follicles measurements<br />
and ovulations detection. Analysis of variance followed by Student-Newman-Keuls test were used to compare number of follicles =25mm at<br />
induction ovulation moment, number of ovulations and length of treatments. Number of follicles =25mm was higher (P < 0,05) for EPE group<br />
(3.57±0.57) when compared to other groups and statistically similar between pFSH+EPE (2.0±0.38) and control (1.29±0.18), which reflected<br />
over number of ovulations: 3.0±1.73, 1.5±0.76 and 1.0±0.0 ovulations/mare for EPE, pFSH+EPE and control groups , respectively. Treatment<br />
length did not vary among groups but pFSH+EPE group needed less number of days of EPE (4.88±0.51) than EPE group (7.43±0.37).<br />
According to results, it is concluded that pFSH treatment followed by EPE, even requiring a shorter EPE treatment time, was not enough to<br />
significantly increase number of ovulations.<br />
Keywords: superovulation, mares, reproduction.<br />
A156 OPU-IVP AND ET<br />
PREGNANCY RATE IN EQUINE EMBRYO RECIPIENTS TREATED<br />
TED WITH FLUNIXIN MEGLUMINE<br />
Jhonnatha Paulo Oliveira, , Hélène Lacerda de Resende & Júlio César Ferraz Jacob<br />
UNIVERSIDADE FEDERAL RURAL DO RIO DE JANEIRO, SEROPEDICA, RJ, BRAZIL.<br />
Is already known that fixation of the embryo into the uterus of recipient mares elicits a transient inflammatory response of the<br />
endometrium. Although, in most cases this response remains subclinical, it may cause the release of prostaglandin (PGF2α) as part of an<br />
inflammatory. The PGF2α acts luteolysis corpus luteum, causing death or expulsion of the transferred embryo before maternal recognition of<br />
pregnancy. Non-steroidal anti-inflammatory drugs (NSAID) can then be used as anti-luteolytic. This study aimed to compare the rate of<br />
embryonic fixation between recipients treated and untreated with Flunixin meglumine - Banamine ® (Intervet / Schering-Plough, Cruzeiro,<br />
Brazil). Were performed 30 embryo transfers, in mares of Mangalarga Marchador breed, with age between four and 12 years, in the period from<br />
January to April 2010, where 15 recipients were treated of randomly with 1,1 mg Banamine ® / kg intramuscular at the time of transfer and 15<br />
recipients were not treated. We used only embryos considered as very good or excellent, with 8 or 9 days old, and synchronized recipients from<br />
D0 to D+5 in relation to ovulation of the donor (D0 = day of ovulation). For comparison the pregnancy rate (PR) between groups was submitted<br />
to the X2 test. Of embryo transfers performed, the PR was 64% (19/30). When comparing groups, the PR was 80% (12/15) between the<br />
recipients treated and 46.5% (7/15) for recipients untreated (P= 0,05), showing that, in these conditions, the NSAID is viable and increases the<br />
pregnancy rate.<br />
Keywords: nsaid, pregnancy rate, mares.<br />
s414
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A157 OPU-IVP AND ET<br />
PREGNANCY RATE IN MANGALAR<br />
ARGA MARCHADOR RECIPIENTS AT DIFFERENT AGES<br />
WITH OR WITHOUT PROGESTER<br />
OGESTERONE<br />
ONE<br />
SUPPLEMENTATION<br />
TION<br />
Sávio Oliveira Paiva, Carlos Gramatico Massoni de Oliveira, Paula Cardoso Almeida, Marcus Andre Ferreira Sá, Renato Guerra Castro Junqueira & Júlio<br />
César Ferraz Jacob<br />
UFRRJ, SEROPEDICA, RJ, BRAZIL.<br />
The Embryo Transfer (ET) in horses has been used due to the advantage of generating more than a product from high value<br />
livestock mares per year. However, several factors affect the success of the TE and the physiological moment of the recipientis a major<br />
determinant of success that depends on the degree of synchrony between donor and recipient during estrus, due to continuous secretion of<br />
progesterone, which is essential for the initiation and maintenance of pregnancy. This study aimed to evaluate the pregnancy rate of recipients<br />
in different age groups with and without the use of long acting progesterone (P4 LA). The project was conducted at a Central Commercial<br />
Embryo Transfer. A total of 52 Mangalarga Marchador recipients were used and alocated to four groups according to age and administration<br />
of progesterone: G1: 2 to 5 years without P4 (n = 18); G2: 2 to 5 years with P4 (n = 9); G3: 6 to 15 years without P4 (n = 44); and G4: 6 to<br />
15 years with P4 (n = 17). Embryos were recovered between 7 and 9 days after ovulation of the donor (D0); embryos classified as excellent<br />
or very good were transferred. Recipients were evaluated by rectal palpation and transrectal ultrasonography to evaluate the uterine condition,<br />
recipients that did not have good uterine tone and adequate uterine echotexture were treated with 1500 mg P4 LA IM on the day of ET.<br />
Pregnancy diagnosis was performed at 15 days after ovulation in the donors and repeated at 30 and 45 days of gestation to the early embryonic<br />
loss. The results were 55.6% (10/18), 22.2% (2/9), 38.6% (17/44), 58.8 (10/17) for G1, G2 , G3 and G4, respectively. The design was<br />
completely randomized using analysis of variance (ANOVA) and Tukey test, at 5% significance. There was no statistical difference between<br />
groups (P > 0.05). Therefore, we conclude that the use of progesterone in recipients, regardless of age, did not increase the pregnancy rate.<br />
Keywords: pregnancy rates, progesterone, different age.<br />
A158 OPU-IVP AND ET<br />
CONCEPTION RATES OF IN VITRO PRODUCED BOVINE EMBRYOS<br />
WITH CONVENTIONAL AND THAWED-SEXED SPERM OF<br />
NELLORE BULLS<br />
Julio Cesar Barbosa da Silva 1 , Wagner Kodato Okabe 1 , Maria Clara Costa Mattos 2 , Bruno Valente Sanches 2 , José Henrique Fortes Pontes 2 , André Gomiero<br />
Rigo 2 , Lucas Lopes Moino 2 , Rodrigo Mendes Untura 2 & Andrea Cristina Basso 2<br />
1<br />
EMBRYO SYS REPRODUÇÃO ANIMAL, OURO FINO, MG, BRAZIL. 2 IN VITRO BRAZIL, MOGI-MIRIM, SP, BRAZIL.<br />
Thawed-sexed sperm is a biotechnology that has been widely used on IVP bovine embryo routine. This process allowed sexing<br />
of sperm from frozen conventional doses. Especially, for bulls that have not produced semen or have died. However, conception rates of IVP<br />
embryos with thawed-sexed sperm are still variable. The aim of this report was to present the conception rates of IVP embryos using<br />
conventional and thawed-sexed sperm from Nellore bulls, on the same commercial farm, during January 2010 to April <strong>2011</strong>. The experiment<br />
was conducted at Eldorado´s farm, Itapetininga, SP, Brazil. Seven days after in vitro fertilization, embryos produced with conventional (n = 595)<br />
and thawed-sexed sperm (n = 184) from four Nellore bulls were transferred to synchronized recipients.The pregnancy diagnosis and fetal sexing<br />
were performed by trans-rectal ultrasound 30 and 60 days after transfer, respectively. The conception rates of embryos produced with<br />
conventional sperm were 50.8% and 47.5% at 30 and 60 days, respectively. The conception rates of embryos produced with thawed-sexed<br />
sperm were 51.1% and 44.3% at 30 and 60 days, respectively. Embryo loss was similar between IVP embryos with conventional (7.0%) and<br />
thawed-sexed (7.4%) sperm. Finally, embryos produced using conventional sperm resulted in 42% of females and 58% males. However,<br />
embryos produced using thawed-sexed sperm resulted in 94% of females and 6% of males. These results demonstrate that the use of thawedsexed<br />
sperm on IVP bovine embryos can be applicable in large commercial scale, because it presents a high sexing accuracy and it seems not<br />
compromise the conception rates and embryo loss. [Acknowledgements: Golin Group, Itapetininga, SP, Brazil].<br />
Keywords: embryo, sexed sperm, ivf.<br />
N<br />
s415
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A159 OPU-IVP AND ET<br />
USE OF RETINOIDS TO INHIBIT THE IN VITRO APOPTOSIS OSIS OF CAPRINE OOCYTES AND EMBRYOUS<br />
Juliana Costa Conceição 1 , Ricardo de Macedo Chaves 2 , Madson Atila Vidal Silva 3 , Edivaldo Rosas dos Santos Júnior 4 , Leopoldo Mayer Freitas Neto 5 , Fabíola<br />
Freitas de Paula-Lopes 6 , Paulo Bayard Dias Gonçalves 7 , Paulo Fernandes Lima 8 & Marcos Antonio Lemos de Oliveira 9<br />
1,4,8,9<br />
UNIVERSIDADE FEDERAL RURAL DE PERNAMBUCO, RECIFE, PE, BRAZIL. 2,3 UNIVERSIDADE ESTADUAL DO MARANHÃO, SAO LUIS, MA, BRAZIL. 5 FACULDADE PIO DÉCIMO,<br />
ARACAJU, SE, BRAZIL. 6 UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 7 UNIVERSIDADE FEDERAL DE SANTA MARIA, SANTA MARIA, RS, BRAZIL.<br />
The objective was to evaluate the apoptosis of goat oocytes and embryos through the activity of enzymes of group II caspases and<br />
DNA fragmentation by TUNEL assay, which underwent in vitro maturation (IVM) in medium with or without retinol (TR) and retinoic acid<br />
(RA). Ovaries were obtained from a slaughterhouse and transported to the laboratory in warm saline at 30°C. The oocytes were collected from<br />
follicles measuring 2-6 mm in diameter by aspiration with a syringe needle attached. The oocytes, tested in 10 replicates, were initially washed<br />
five times on medium itself, then screened and distributed in droplets of TCM 199 (Control Group - CG) and this medium supplemented with<br />
retinol (retinol group - GRT) or Retinoic Acid (Group Retinoic Acid - OHR), to finally being incubated at 39°C in a humidified atmosphere<br />
containing 5% CO2 for 24 h. After this period, a proportion of oocytes was processed through mDM and exposed to sperm for 18 h in the same<br />
atmospheric condition. Subsequently, presumptive zygotes were transferred to drops of KSOM medium containing the monolayer of cells in the<br />
oviduct, and finally incubated at 39°C in humid atmosphere with 5% CO2. After 48 h, the structures that were not cleaved and removed 30% of<br />
the medium was partially renovated and remained so for eight days. The other part of the oocytes and blastocysts in each experimental group were<br />
prepared to locate the changes characteristic of apoptosis, using the reagent PhiPhiLux-G1D2 and solutions of 4% paraformaldehyde and<br />
Phosphate-Buffered Saline + 1 mg/mL polyvinylpyrrolidone. For statistical analysis, variance analysis was performed by the method of least<br />
squares. The activity of caspase enzymes did not differ (P > 0.05) between oocytes in the CG (10.32%), GRT (8.08%) and the OHR (8.45%)<br />
and between GRT and the OHR. The oocytes and blastocysts positive for TUNEL assay were higher (P < 0.05), respectively, in the CG<br />
(11.46%, 9.22%) than in GRT (6.86%, 5.45%) and the OHR (7.41%, 6.12%), no difference (P > 0.05) between the GRT and the OHR. Zygotes<br />
GC had lower (P < 0.05) capacity development to the blastocyst stage (5.32 ± 0.81) than those of the GRT (7.94 ± 0.93) and GAR (7.36 ± 1.02),<br />
no difference (P > 0.05) between the GRT and the OHR. This result indicates that the addition of retinoids in the middle of oocyte maturation<br />
reduces cell apoptosis, and increases in vitro production of goat embryos.<br />
Keywords: retinoids, apoptosis, blastocyst.<br />
A160 OPU-IVP AND ET<br />
FSH IN PROTOC<br />
OCOLS OLS FOR FOLLICULAR<br />
WAVE SYNCHRONIZA<br />
ONIZATION AND EMBRYO PRODUCTION IN TAURIN AND ZEBU<br />
DONNORS<br />
Carlos Antônio de Carvalho Fernandes 1 , Ana Cristina Silva de Figueiredo 1 , Bruno Fernandes Ludgero Alves 2 , Eduardo Ramos de Oliveira 2 , Thais Camargo<br />
Rossi 2 , Adriana Agostini 1 & Ana Cristina Silva de Figueiredo 1<br />
1<br />
UNIV. DE ALFENAS, ALFENAS, MG, BRAZIL. 2 BIOTRAN LTDA, ALFENAS, MG, BRAZIL.<br />
The current knowledge in physiology of reproduction and hormone therapies can improve the results of the follicle aspiration<br />
(OPU) and the quality of the oocytes recovered. Including the FSH in protocols available reducing the atresia and the inconsistency of the oocytes<br />
retrieval after OPU, mainly for Taurus breeds. This study was designed to evaluate the inclusion of FSH in protocols for follicle synchronization<br />
and to compare the IVPE results between Taurus (Holstein, Jersey and Angus) and Zebu (Gir, Nelore and Guzerá) breeds. Twenty-one Taurus<br />
and 23 Zebu donators were included for this purpose. All animal were randomized in one of the three treatments: T1) Control - OPU without<br />
follicle synchronization; T2) Day 0 (D0) – implant of progesterone, 2 mg of Estradiol Benzoate (EB) and 0,530 mg of cloprostenol. Five days<br />
later (D5), two injections of FSH - 60UI intramuscularly (IM) during the morning and 40 UI IM 12 h later; T3) D0 - the same for T2, D4<br />
afternoon – two injections of FSH - 25 UI (IM) and 75 UI subcutaneously (SC) - given at the same time. The interval from previous aspiration<br />
was always greater than 30 days and all animals underwent all treatments. The cows were submitted to OPU six days after the beginning of the<br />
treatment. The procedures for IVEP were performed in the same laboratory with X sorted semen. Means among treatments for oocyte recovered<br />
and produced embryos were compared with Tukey test at 5% of probability. Zebu cows provided more oocytes and produced more embryos than<br />
Taurus (P < 0.05) in all treatments. Within subspecies (Zebu and Taurus), there were no statistical differences for oocytes (total and viable)<br />
recovered from different breeds. In Zebu donators, the total oocytes (19,9+8,3, 22,6+9,7 and 21,4+9,8) and those classified as viable (11,3+7,5,<br />
14,1+7,6 and 13,7+8,8) were similar for T1, T2 and T3, respectively. The same was observed in Taurus, 22,8+6,4, 15,6+5,2 and 14,4+6,2<br />
oocytes, and 7,6+6,6, 9,2+7,1 and 8,7+6,8 viable for T1, T2 and T3, respectively. In Zebu, the embryo production by donator was 4,3+2,6b,<br />
6,6+2,7a and 6,6+2,9a, and for Taurus it was 2,5+2,0b, 3,8+1,8a and 3,5+2,2a, respectively, for T1, T2 and T3. The treatments did not change<br />
the number and the quality of the recovered oocytes; however, the embryo production was improved. It seems that the inclusion of the FSH in<br />
protocols for follicle synchronization improved the intrinsic quality of the oocytes. Additionally, treatments 2 and 3 were similar and the injection<br />
of FSH given by SC could shorten the animal manipulation. [Support: FAPEMIG].<br />
Keywords: bovine, opu, Bos taurus.<br />
s416
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A161 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
USE OF β-MER<br />
-MERCAPT<br />
APTOETHANOL FOR IN VITRO MATUR<br />
TURATION TION AND CULTURE OF OVINE EMBRYOS<br />
Jorgea Pradieé 1 , Liziane Lemos Vianna 1 , Alexander Oliveira Gonçalves 1 , Elisa Caroline da Silva Santos 1 , Rafael Gianela Mondadori 1 , Thomaz Lucia Jr. 1 ,<br />
Arnaldo Diniz Vieira 1 & Ligia Margareth Cantarelli Pegoraro 2<br />
1<br />
UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS, RS, BRAZIL. 2 EMBRAPA, PELOTAS, RS, BRAZIL.<br />
One of the factors limiting the success of ovine IVP is the formation of reactive oxygen species (ROS) resulting from cell<br />
metabolism. The action of the ROS can be physiologically inhibited by antioxidants such as glutathione (GSH). As GSH’s concentration is<br />
reduced during most steps of the IVP, the media used for IVP are commonly supplemented with antioxidant substances. This study evaluated<br />
the development of ovine embryos after supplementation of the media for IVM and IVC with 20 µM β-mercaptoethanol (βME), a GSH<br />
precursor. Ovaries from pubertal sheep were collected in slaughterhouse and transported to the laboratory in a saline antibiotic solution at 30ºC.<br />
Cumulus-oocytes complexes (COC) were recovered and selected for IVM, which was conducted during 22-24 h, using the 199 TCM medium<br />
including estradiol, FSH, LH, pyruvate, estrus sheep serum and antibiotics. For this experiment 6 replicates were performed. Further comparisons<br />
were done between the IVM medium with (n = 328) or without (n = 320) βME. Sperm selection was conducted though swim-up method using<br />
fresh sperm and tris-citric acid-glucose medium. The IVF was done with sperm concentration of 1 x 106/mL in SOF medium with 2% estrus<br />
sheep serum. Thereafter, presumptive zygotes were denuded and cultured for 8 days in SOFaa medium and 2.5% FCS with or without âME.<br />
The IVM, IVF and IVC were conducted in an incubator with 5% CO2 and saturated humidity, at 39ºC. The rates of embryo development at D2<br />
(cleavage) and D8 (blastocyst) were compared between treatments through the chi-square test. Although cleavage rates were the same (70%) for<br />
medium with or without âME, the blastocyst formation rate was greater (P < 0.001) for the control (17%) than for the βME-supplemented<br />
medium (5%). Therefore, supplementation of IVM and IVC medium with âME at the tested concentration did not benefit cleavage rates and was<br />
associated with reduced blastocyst rate. Further studies are still needed to investigate potential benefits of supplementation with âME at distinct<br />
concentration for the development of ovine embryos.<br />
Keywords: sheep, embryo, antioxidant.<br />
A162 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
ADDITION OF NERVE GROW TH FACT<br />
CTOR ON MATUR<br />
TURATION TION AND DEVEL<br />
VELOPMENT MEDIA FOR IN VITRO<br />
PRODUCTION OF SHEEP EMBRYOS<br />
M. Vilar<br />
ilariño<br />
iño 1 , M. Crisp<br />
ispo 2 , P.C.<br />
dos San<br />
antos-N<br />
os-Net<br />
eto 1 , R. Wijma<br />
1 , N. Bar<br />
arrer<br />
era 1 , A. de León<br />
2 , L. Barb<br />
arbeit<br />
eito 2 & A. Menchac<br />
enchaca 1<br />
1<br />
FUNDACIÓN IRAUY, MONTEVIDEO, URUGUAY. 2 INSTITUTO PASTEUR DE MONTEVIDEO, MONTEVIDEO, URUGUAY.<br />
Nerve Growth Factor (NGF) is a neurotrophic factor that promotes survival and proliferation in different types of non-neuronal<br />
cells. In the present study we assessed whether NGF could improve the in vitro embryo production in sheep. A total of 1342 cumulus oocyte<br />
complexes (COCs) covered by at least two layers of granulosa cells and homogeneous cytoplasm were selected for in vitro maturation (IVM)<br />
after aspiration from ovine ovaries obtained in a local slaughterhouse. The selected COCs were incubated in groups of 25-30 in 100 µL drops<br />
of TCM199 supplemented with 10% estrous ovine serum, 10 µg/mL FSH, 10 µg/mL LH, 100 ìM cysteamine and antibiotics; covered with<br />
embryo tested mineral oil for 24 h, at 39°C and 5% CO2 in air. For in vitro fertilization (IVF), matured oocytes (25-30/100 µL drop) were<br />
incubated for 22 h in fertilization medium with 1x106 frozen-thawed spermatozoa selected by swim-up method. The fertilization medium<br />
consisted of synthetic oviductal fluid (SOF) supplemented with 2% estrous ovine serum, 10 ìg/mL heparin and 10 ìg/mL hypotaurine. In vitro<br />
development (IVD) was performed in groups of 25-30 zygotes in 100 ìL drops of SOFaa BSA covered with mineral oil at 39°C and 5% O2,<br />
5%CO2, 90% N2. NGF was evaluated using 0, 100 or 1000 ng/mL during IVM (0 ng/mL, n = 226; 100 ng/mL, n = 264; and 1000 ng/mL, n<br />
= 286), or IVD (0 ng/mL, n = 191; 100 ng/mL, n = 189 and 1000 ng/mL, n = 186). Cleavage rate (2 cell embryos/oocytes) and development/<br />
rate (morula and blastocysts/oocytes) were recorded after 48 h and 6 d in culture after fertilization, respectively. Statistical analysis was performed<br />
by logistic regression. For IVM, no differences were found in the cleavage rate (46%, 104/226; 54.5%, 144/264; and 50%, 143/286), the<br />
development rate (12.4%, 28/226; 17.8%, 47/264; and 13.6%, 39/286), and the developed/cleaved rate (26.9%, 28/104; 32.6%, 47/144; and<br />
27.3%, 39/143) for 0, 100 and 1000 ng/mL NGF, respectively. When NGF was used during IVD, no differences were found in the development<br />
rate (41.4%, 79/191; 45.5%, 86/189; and 46.8%, 87/186) and developed/cleaved rate (53.7%, 79/147; 57%, 86/151; and 56.5%, 87/154) for 0,<br />
100 and 1000 ng/mL NGF, respectively. These results suggest that the addition of NGF in the maturation or development media does not affect<br />
the number of in vitro produced sheep embryos.<br />
Keywords: ngf, in vitro production, embryos sheep.<br />
N<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A163 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
PHYSIOLOGICAL AND ENDOCRINE CHANGES OF BOVINE CORPUS LUTEUM AFTER CHALLENGES WITH SUB-DOSE OF<br />
CLOPROTENOL SODIUM<br />
Eduardo Trevisol 1 , Wolff Camargo Marques-Filho 2 , Flavia Caroline Destro 3 , Jair Camargo Ferreira 4 , Aline Sousa Camargos 5 , Jackson Barros Amaral 6 ,<br />
Cezinande Meir<br />
eira 7 , Fer<br />
ernada C.S.Z. Matt<br />
ttos<br />
8 , Rob<br />
ober<br />
erto Sar<br />
artor<br />
ori 9 & João C.P. Fer<br />
erreir<br />
eira 10<br />
1,2<br />
UNESP-BOTUCATU, AMERICANA, SP, BRAZIL. 2,6,7 UNESP-BOTUCATU, CAMPINAS, SP, BRAZIL. 3,4,5,7,8,10 UNESP-BOTUCATU, BOTUCATU, SP, BRAZIL. 4 UNESP-BOTUCATU, SÃO PAULO,<br />
SP, BRAZIL. 6 INSTITUTO DE ZOOTECNIA, NOVA ODESSA, SP, BRAZIL. 8 UNESP-BOTUCATU, PIRACICABA, SP, BRAZIL. 9 USP-ESALQ, PIRACICABA, SP, BRAZIL.<br />
The action of sub-doses of PGF2α does not always cause CL regression due to some factors, such as cycle phase, concentration<br />
and administration form of the product. In some situations in which luteolysis does not occur, it is observed within the early 12-24 h after PGF2α<br />
administration, the decrease in plasma progesterone (P4) concentration to values near to 1.0 ng/mL, followed by the subsequent increase of P4<br />
to values similar to those observed before treatment. Due to the importance of this phenomenon to understand the factors that regulate luteolysis,<br />
the objective of this study was to evaluate changes in P4 and luteal blood flow after administration of 83.33 µg of cloprostenol sodium on the sixth<br />
day of the estrous cycle. Nineteen Caracu mature cows were submitted the following synchronization protocol: Day -9 (D-9 - protocol<br />
beginning): 50 µg lecirelin (Gestran Plus ® - Tecnopec - São Paulo – Brazil; IM) and insertion of a P4 intravaginal device (DIB ® - Syntex -<br />
Argentina); D-2.5: 500 µg cloprostenol sodium (Sincrocio ® - Ouro Fino, São Paulo, Brazil, IM); D-2; 250 µg cloprostenol sodium (Sincrocio ® ,<br />
IM) and withdrawn of the intravaginal device; D0: 50 µg lecirelin (Gestran plus ® , IM). On D6, the cows were divided randomly in three<br />
experimental groups: G1 (2 mL of saline; IM, n = 6), G2 (two doses of 500 µg cloprostenol sodium 2 h apart; IM, n = 6) and G3 (83,33 µg<br />
cloprostenol sodium; IM, n = 7). Data collection were performed with mode B ultrasonography and Power-Doppler evaluations followed by<br />
blood collection for P4 measurement at 0, 8, 16, 24, 32, 40 and 48 h after treatment. Data was submitted to ANOVA and for the differences<br />
between means the TUKEY test with 5% probability was applied. Treatments influenced P4 concentration, blood flow and luteal volume. On<br />
G1, no changes were observed in any varible (P > 0.05). However, on G2, P4 decreased 16 h after treatment (P < 0.001) and maintained until<br />
the end of the evaluation period. Blood flow and luteal volume decreased 48 h after treatment in G2 (P = 0.049). On G3, there was a decrease<br />
in circulating P4 concentration from the time of treatment until 18 h later (P = 0.025), but after that time, circulating P4 increased and returned to<br />
initial values. Despite this observation, blood flow and luteal volume did not change (P > 0.05). Based on these results, we concluded that 1/6<br />
of cloprostenol sodium dose, cause a partial luteolysis.<br />
Keywords: bovine, luteolysis, corpus-luteum.<br />
A164 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
CHANGES IN BLOOD CHEMISTRY PROFILES AND ASSOCIATION<br />
WITH BIRTHWEIGHT<br />
THWEIGHTS IN IVF-DERIVED NEWBORN CAL<br />
ALVES<br />
Luiz Fer<br />
ernando Schütz 1 , Fabiano Car<br />
armina<br />
minatti Zago 2 , Luis Henr<br />
enrique Aguiar<br />
1 , P.C.<br />
dos San<br />
antos-N<br />
os-Net<br />
eto 1 , Jamir Machado Júnior 1 , Monic<br />
onica Urio<br />
1 , Leonar<br />
eonardo<br />
Tondello<br />
Martins 3 , Diego Fernandes 1 , Willian Braun 1 , Saul Gaudêncio Neto 3 , Cristiano Feltrin 3 , Luciana Relly Bertolini 3 , Ubirajara Maciel da Costa 1 , Fabiana Forell 1 &<br />
Marcelo Bertolini 3<br />
1<br />
UDESC / CAV, LAGES, SC, BRAZIL. 2 EPAGRI, LAGES, SC, BRAZIL. 3 UNIFOR, FORTALEZA, CE, BRAZIL.<br />
The aim of this study was to compare clinical and blood chemistry parameters between newborn calves produced either in vivo<br />
by superovulation (SOV) or in vitro by in vitro fertilization (IVF) during the immediate neonatal period, correlating possible changes in<br />
physiological profiles with risk of death in IVF animals. Parturition was pre-induced in nine bovine females on Day 268.6 ± 0.6 of pregnancy<br />
by the administration of 8 mg triamcinolone acetonide IM, followed by induction of parturition on Day 275.4 ± 0.6 days with 25 mg<br />
dexamethasone acetate and 0.5 mg PGF2á analogue (IM). Four IVF- and five SOV-derived Flemish calves, delivered on Day 276.3 ± 0.6 of<br />
gestation, were subjected to sequential blood sampling by jugular venipuncture during the first 6 h ex utero (5, 15, 30, 60, 120, 240, and 360<br />
min) for the analyses of blood gases and electrolyte concentrations, concurrent with the recording of vital clinical signs (rectal temperature, BT;<br />
respiratory rate, RR; and hearth rate, HR). Data for groups (IVF and/or SOV vs.. time) were analyzed by ANOVA (Minitab), Proc MIXED<br />
(SAS), and simple correlation (P
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A165 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
BREEDING SEASON ANTICIPATION TION IN MARES SUBMITTED<br />
TED TO A PROGESTER<br />
OGESTERONE ONE INTRAVAGINAL IMPLANT OR ARTIFICIAL<br />
LIGHTING<br />
Rob<br />
ober<br />
erta Pahim de Melo<br />
elo, Fer<br />
ernanda Saules Ignácio, Jair Camar<br />
amargo Fer<br />
erreir<br />
eira,<br />
José Nicolau P. Puoli Filho & Cezinande Meir<br />
eira<br />
FMVZ - UNESP, BOTUCATU, SP, BRAZIL.<br />
The present study was done aiming to evaluate the use of progesterone intravaginal implants on mares’ ovulation anticipation and<br />
to compare with the use of artificial lighting. For this use, 27 cross-breeding mares were randomly assigned into three groups: P4 (n = 7;<br />
progesterone intravaginal implant; Primer ® , 1g of progesterone with no estradiol), lighting (n = 10; artificial lighting program) and control (n =<br />
10; not treated). In P4 group, the progesterone intravaginal implant was introduced and kept for 12 days when transitional phase characteristics<br />
were detected. Animals of the lighting group were daily treated with five hours of artificial lighting (5 to 10 pm) for 60 days, beginning at June<br />
21st. For the comparison of the evaluated intervals among P4, lighting and control groups, analysis of variance followed by Tukey test were<br />
used. For the first ovulation frequency, a chi-square test was used. The progesterone intravaginal implant reduced the interval until detection of<br />
a follicle =35mm when comparing to control group and was similar to mares under artificial lighting program. The findings of =35mm follicles<br />
in P4 group indicate that implant had a higher efficiency on follicular growth stimulation. The interval between transitional phase detection to<br />
=35mm follicles were similar among groups, but the moment transitional phase were detected was different. All mares in P4 group showed<br />
follicles =35mm during treatment to five days post treatment. Even though the largest follicle achieved =35mm, the mean days from removal to<br />
ovulation was of 20.6 days, which was higher than 6.6 days found by Newcomb, 2002 (Journal of equine veterinary science. 22, 378-382). This<br />
difference is explained by the two mares that showed largest follicles regression. Considering the mean interval (days) between winter solstice<br />
and ovulation, lighting treatment was efficient compared to control. The use of progesterone implants showed regular efficiency and it was<br />
similar to both other groups. This fact probably happened because of two mares that ovulated in late December and the cause was not established.<br />
In relation to frequency of ovulation distribution along months, as lighting treatment as progesterone implant promote ovulations earlier in the<br />
breeding season when compared to control (P < 0,05). Concluding, lighting treatment and progesterone implants when used in transitional mares<br />
efficient to promote a higher frequency of ovulations early in the breeding season.<br />
Keywords: p4, lighting, ovulation.<br />
A166 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EVAL<br />
ALUATION OF HYPOTHAL<br />
THALAMIC-PITUIT<br />
AMIC-PITUITAR<br />
ARY RESPONSIVENESS DURING THE POSTPAR<br />
ARTUM NELLORE COWS<br />
Cir<br />
iro Mor<br />
oraes Bar<br />
arros<br />
os, José Rena<br />
enato Cur<br />
ury, Rafael August<br />
ugusto Satr<br />
trapa,<br />
Mar<br />
arcelo Pegor<br />
egorer<br />
er, Luzia Apar<br />
parecida<br />
Trinc<br />
inca & Vinicius Pinheir<br />
inheiro<br />
UNESP, BOTUCATU, SP, BRAZIL.<br />
The presence of calf, body condition score, energy balance, number of births (multiparous vs. primiparous) and breed are factors<br />
that influence the duration of postpartum anoestrus in beef cows. The objective of the present study was to evaluate, during early postpartum, the<br />
time of re-establishment of pituitary LH stocks, mensured by the hypothalamic-pituitary axis responsiveness to exogenous administration of<br />
GnRH or estradiol benzoate (EB). Multiparous lactating Nellore cows (Bos indicus, n = 90) were randomly allocated into eight groups,<br />
according to the hormonal treatment: EB group (1.0 mg of EB, i.m., n = 7), GnRH group (50 µg lecireline, i.m., n = 17). The EB-SUP (n = 9)<br />
and GnRH-SUP (n=11) groups received the same treatments above specified and were supplemented (SUP) with a balanced diet, based on<br />
cotton meal and ground corn (3.5 kg/cow/day). Additionally, animals from EB-CR (n = 4) and GnRH-CR (n = 11) received the same treatments<br />
of EB and GnRH group respectively and had their calf removed (CR) shortly after parturition. Moreover, two other groups were LH (LH 12.5<br />
mg, i.m., n = 14) and Control group (received saline instead of an ovulation-inducing agent, n = 11). The hormones were administered weekly,<br />
from seven days postpartum (±5 days) until the occurrence of the first ovulation, which was determined by the presence of CL during ovarian<br />
ultrasonography weekly performed. Blood samples were collected just before and 2 h (GnRH, LH and control groups) or 18 h (EB groups) after<br />
hormone or saline (control) administration, in order to determine LH concentration by RIA. Data were analyzed by ANOVA. Means values in<br />
days (±SEM) for the first post-partum LH surge were: EB (73.0±5.2); EB-CR (16.7±5.8); EB-SUPL (41.7±6.7); GnRH (32.3±3.0); GnRH-<br />
CR (11.0±3.5); GnRH-SUPL (15.6±2.8). There were significant differences (P, 0.05) between groups EB vs.. EB-CR; EB vs. EB-SUP; BE vs..<br />
GnRH; GnRH vs.. GnRH-CR; GnRH vs.. GnRH-SUP, and a tendency between EB-SUPL vs. EB-CR (P < 0.10). Results indicate that from<br />
the second week postpartum there is sufficient LH in the pituitary to induce ovulation after GnRH or EB administration. However, the cows from<br />
EB group ovulated later than animals from the other groups, possibly due to the sensitivity of the hypothalamus to negative feedback of<br />
estrogens, inhibiting the pre-ovulatory LH surge. Additionally, calf removal and food supplementation reduced in 2 to 4 weeks the time of the<br />
first postpartum LH surge induced by GnRH or EB in Nellore cows.<br />
Keywords: post-partum, lh, bovine.<br />
N<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A167 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
IMMUNOHISTOCHEMIC<br />
OCHEMICAL EVAL<br />
ALUATION OF COLL<br />
OLLAGEN I AND III IN CERVIX AND UTERUS US FROM BITCHES<br />
WITH OPEN OR<br />
CLOSED PYOMETRA<br />
Rodrigo Volpato, Ian Martin, Camila Louise Ackermann, Miriam Harumi Tsunemi, Reneé Laufer Amorim & Maria Denise Lopes<br />
UNESP, BOTUCATU, SP, BRAZIL.<br />
Canine Pyometra is defined as the accumulation of purulent secretions in the uterine lumen of adult and or older bitches. It is<br />
presented as a closed or open form and normally occurs in the luteal phase of the estrous cycle (Ângulo, 2009. Proceeding of the SEVC,<br />
Barcelona, available in www. ivis.org). Recently Chatdarong et al. (2010. <strong>Proceedings</strong> 9th Chulalongkorn University Veterinary Science the<br />
Animal Company. v.1, p.129) concluded that the uterus of cyclic bitches have a higher proportion of collagen fibers compared with smooth<br />
muscle, probably associated with E2 increased during estrus, causing relaxing of the cervix. Similar results were seen in dogs with open cervix<br />
pyometra, suggesting that the opening of the cervix in these cases is associated with an increased ratio of collagen and muscle fibers. Currently<br />
the origin of cervical relaxation in cases of open pyometra is unknown. The aim of this study was to evaluate the mechanism of cervical opening<br />
and closing in bitches with pyometra, by assessing immunohistochemistry for collagen I and III in the cervix and uterus of bitches with pyometra<br />
open or closed. Fragments of uterus and cervix from bitches with open (n = 25) and closed (n = 6) pyometra were collected after ovariohysterectomy,<br />
fixed in 10% buffered formalin for 24 h and kept in 70% alcohol until the time of inclusion in paraffin. The cuts and deparaffinization were<br />
performed according Volpato et al. (Anais do XIX CBRA, <strong>2011</strong>.). Antigen retrieval was performed with 1% pepsin pH 1.8 (Pepsin 1:1000 NF<br />
Nuclear-SP-Brazil), hatching in an incubator at 60°C and then at 37°C. Endogenous peroxidase and skimmed milk powder 3% was used as<br />
block. The incubation was carried out with primary antibody at a dilution of 1:2000 for collagen I and III (respectively Rabbit anti bonvine col<br />
I IgA Novotec. Ref. 20121; Rabbit anti bovine col III IgA Novotec. Ref. 2930 - Dako - USA ) and revelation chromogen DAB (3,3 ‘-<br />
diaminobenzidine - Liquid DAB Chromogen ® - Dako, USA). To evaluate the immunostaining, ten fields in the stroma and muscle tissues from<br />
both uterus and cervix were observed under light microscope. It was used a score from 1 to 4 to determine the percentage markup: (1 75%). Final score was the average of the scores of the 10 fields analyzed.<br />
For the statistical analysis the Mann-Whitnney test was performed (P < 0.05). There was no statistical difference in the immunostaining in any<br />
of the regions studied between the groups opened and closed pyometra.The cervical opening in cases of open pyometra is not associated with<br />
collagen receptor increase or decrease in the uterine horns and cervix of bitches.<br />
Keywords: immunohistochemical, collagen i and iii, pyometra.<br />
A168 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
ULTR<br />
TRASONOGR<br />
ASONOGRAPHIC APHIC EVAL<br />
ALUATION OF THE CRIOLLO O CLONED EQUINE PLACENT<br />
CENTA<br />
Andres Gambini 1 , Javier Jarazo 1 , Florencia Karlanian 1 , Adrian De Stefano 1 , Cesar Bergadá 2 & Daniel Salamone 1<br />
1<br />
FACULTAD DE AGRONOMÍA UBA, BUENOS AIRES, ARGENTINA. 2 HOSPITAL EQUINO KAWELL, BUENOS AIRES, ARGENTINA.<br />
Cloned pregnancies are less capable to continue development after implantation. Alterations in placental development of these<br />
animals are considered one of the main reasons of low parturition rates. The combined utero-placental thickness (CUPT) can be measured by<br />
transrectal ultrasonography to monitoring an ongoing placenta. An increase in the CUPT is considered one of the main signs of placentitis. The<br />
objective of this study was to record equine clones placental development by CTUP measurement, at a monthly interval, starting at the 4th month<br />
of gestation. Five Criollo pregnant mares (A, B, C ,D and E) with good perineal conformation derived from zona-free blastocyst produced by<br />
embryo aggregation at Day 0 (Gambini et al., 2010; Reprod Fertil Dev, 23, 166) were examined. All mares received oral progesterone since<br />
pregnancy diagnosis until day 320, unless abortion took place. At each examination, the CUPT values were obtained by transrectal ultrasonography<br />
(5 MHz linear array probe, Aloka 500) in three different sections at the placenta-cervical junction, and the largest measurement was recorded. A<br />
total of eight pregnancies were achieved, but only five could develop placenta and, therefore, were evaluated in this work. CTUP values were<br />
between 0.5 and 1.3 cm. In mares A, C, D and E, CTUP were below 1 cm. Although some measurements were larger than previous reports in<br />
Criollo mares, no other abnormal clinical sings were detected. The largest CTUP was found in mare B in the sixth month of gestation, suggesting<br />
the presence of placentitis, supported with premature udder development and post-abortion placenta examination. This mare aborted, even<br />
though the CUPT decreased due to the treatment with antibiotics, anti-inflammatory and pentoxifylline. Abortion also occurred in mare D (at<br />
month 8) and E (at month 5) without detectable changes in CUPT. Mare C CUPT increased until 8th month and then remained without greater<br />
changes until parturition. This pregnancy resulted in the birth of a healthy clone. The pregnancy of mare A is still ongoing with a expected CUPT<br />
at the 8th month. These preliminary results showed that CUPT values could help in monitoring cloned placenta development. An increase higher<br />
than 1 cm may suggest a sign of compromised pregnancy, allowing the beginning of an early therapy. Abortion could also have other causes<br />
without changes on CUPT and, therefore, others clinical and echografic evaluations are necessary.<br />
Keywords: equine, clone, placenta.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A169 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
CHIMERIC MOUSE BLAST<br />
ASTOCY<br />
OCYST<br />
STS S RECONSTR<br />
ONSTRUCTED BY INNER CELL MASS AND TROPHECT<br />
OPHECTODERM ODERM AGGREGA<br />
GGREGATION<br />
Isabele Picada Emanuelli, Caio Pontes Godói, Bruna Castilho Soto Campanha, Pablo Diego Moço, Bruno Cazari, Patrícia Villela Silva & Marcelo Fábio<br />
Gouveia Nogueira<br />
UNESP, BOTUCATU, SP, BRAZIL.<br />
The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, i.e. post-compaction,<br />
such as morula and blastocysts, are used1. To perform the inner cell mass (ICM) transfer to a recipient trophectoderm (TE) it is essential to use<br />
embryos at an advanced stage of development and with well-established blastocoels. In mice, few studies have tried the aggregation between<br />
ICM and TE and, so far, only microinjection methods have been used for such reconstructions 2,3. The aim of this study was to validate, in mice,<br />
the technique of blastocyst reconstruction using the method of ICM and TE aggregation. Swiss Webster females aged between 21 and 45 day,<br />
were superstimulated and placed to mate according to Mancini et al. (2008; Transgenic Research 17:1015). At 3.5 days post coitum (dpc),<br />
expanded blastocysts were recovered by uterine flush and were sectioned with microblade assisted by micromanipulators (NK2, Eppendorf,<br />
Germany) and mounted on an inverted microscope (Eclipse Ti, Nikon, Japan) in order to isolate ICM and TE. The section was designed to be<br />
tangential to ICM and to produce two fragments, one with just TE and another with the whole ICM and a minimum amount of the TE. The joining<br />
and subsequent aggregation were tested between pairs ICM+TE (n = 28) from different blastocysts, i.e. the ICM and TE of the same pair did<br />
not come from the same blastocyst. After joining, pairs were cultured in vitro for 24 h (37ºC, 5% CO2 and saturated humidity). The parameter<br />
used to detect a chimera in the post-culture structure was the image of a single and cohesive cell mass or a strongly aggregated mass spannning<br />
more than half of structure total diameter. The viability of the 56 sectioned structures was observed after 24 h of culture by the parameter of cellular<br />
reorganization and blastocoels re-expansion. The aggregation rate of the reconstructed blastocysts (chimerism rate) was 25% (7/28) and the<br />
viability of sectioned structures (ICM and/or TE) was 84% (47/56). Despite of low adhesion potential of embryonic cells after compaction, the<br />
proposed aggregation method for blastocyst reconstruction technique was considered feasible to the chimera production. (1) Nogueira et al.;<br />
2010. Transgenic Research, 19:344-345. (2) Zheng et al.; 2005. Zygote, 13:73-77. (3) Sotomaru et al.; 1997. Theriogenology, 48:977-984.<br />
[Financial support: FAPESP, Brazil].<br />
Keywords: chimeric blastocyst, embryo reconstruction, mouse.<br />
A170 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
CHARACTERIZA<br />
CTERIZATION TION OF SECONDAR<br />
ONDARY CORPOR<br />
ORPORA LUTEA FORMATION IN NON-CYCLIC CLIC AND CYCLIC CLIC EMBRYO<br />
RECIPIENT MARES (PAR<br />
ARTIAL RESULTS)<br />
S)<br />
Elisa Sant´Anna Monteiro da Silva 1 , Sandra Figueiredo Frade 2 & Cezinande Meira 1<br />
1<br />
UNESP-FMVZ-BOTUCATU, BOTUCATU, SP, BRAZIL. 2 FAZENDA SÃO SABASTIÃO, MIRA ESTRELA, SP, BRAZIL.<br />
There are few reports about the characteristics of secondary corpora lutea (SCL) formation in mares and how such characteristics<br />
interfere with gestation. Therefore, the aim of the present study is to describe the morphology, number and formation pattern of SCL in pregnant<br />
embryo recipient mares, considering as hypothesis the similarity of SCL formation between cyclic and non-cyclic mares. Non-cyclic mares that<br />
received embryos during spring transition (n = 5; non-cyclic group) and cyclic mares that received embryos during breeding season (n = 5; cyclic<br />
group) were used. Prior to embryo transfer, non-cyclic mares were given a single dose of estradiol benzoate (2.5 mg, IM; EstroginTM,<br />
Farmavet, Brazil) and had their uteri examined daily by transrectal ultrasonography (US) for observation of uterine edema. Twenty four hours<br />
after detection of satisfactory endometrial edema, daily administration of altrenogest began (0.044 mg/kg, PO; ProgestalTM, Pro-Ser, Argentina).<br />
Embryos were collected by non-surgical uterine flush 8 days after ovulation of the donor mare and transferred into non-cyclic recipients 4-<br />
6 days after the onset of altrenogest administration or into cyclic recipients 4-6 days post-ovulation. Pregnancy diagnosis was performed by US<br />
at 15 days. Non-cyclic recipients that became pregnant received altrenogest until day 120 of gestation. Additional pregnancy examinations were<br />
performed in all pregnant recipients on days 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85 and 100. Presence of ovarian follicles, as well as<br />
formation, number and morphologic characteristics of SCL were also monitored beginning on day 35. Emergence of SCL varied among mares,<br />
ranging from days 36-40 to 61-65, and was associated with ovulation of large follicles whereas later formation of SCL originated mainly from<br />
luteinization of follicles. At 100 days of gestation luteinized structures were still present on the ovaries of some animals. A total of 2 to 6 SCL that<br />
had emerged at different periods during pregnancy were observed per mare. Regarding morphology, types of SCL observed were clot-like, solid<br />
and spherical, with anechoic center or elongated SCL projecting towards the ovulation fossa. Secondary corpora lutea vary in number and<br />
morphology among animals and their formation seem to occur similarly in recipients that became pregnant during the ovulatory season and<br />
progestin-treated transitional recipients. Data regarding the production of progesterone by these glands are necessary to clarify their role during<br />
gestation in mares. [The authors thank Fapesp for financial support].<br />
Keywords: altrenogest, mares, non-cyclic.<br />
N<br />
s421
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A171 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
MORPHOLOGIC<br />
OGICAL CHARACTERIZA<br />
CTERIZATION TION OF THE PLACENT<br />
CENTA OF CURRALEIR<br />
ALEIRO/PÉ-DUR<br />
O/PÉ-DURO O COWS<br />
Heit<br />
eitor Castr<br />
astro Alv<br />
lves<br />
Teix<br />
eixeir<br />
eira,<br />
Paula Lor<br />
orena Grangeir<br />
angeira a Sout<br />
outo, Eleonor<br />
leonora Araújo Barb<br />
arbosa,<br />
Nathalia Hack Mor<br />
oreir<br />
eira,<br />
Arthur da Silv<br />
ilva Mar<br />
arian<br />
iante & Ale<br />
lexandr<br />
xandre Flor<br />
loriani<br />
Ramos<br />
EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL.<br />
Native or naturalized breeds, like Curraleiro/Pé-Duro cattle, are formed by domesticated animals that are already known to a long<br />
period under the action of natural selection, in certain environments. The Curraleiro/Pé-Duro cattle has as its main characteristic, adaptation to<br />
semi-arid climate, characterized by high temperatures and low food availability. Because these are animals of Iberian origin, brought by Spanish<br />
and Portuguese settlers, who have a small size and high degree of adaptability, it is necessary studies to evaluate the morphophysiological<br />
characteristics of these animals. The porpuse of this study was to evaluate the morphology of the placenta of the Curraleiro/Pé-Duro cows. The<br />
placenta of ten Curraleiro/Pé-Duro cows placenta were collected immediately after release. The placentas were evaluated for weight, number of<br />
cotyledons, width and length of the two bigger and two smaller cotyledons, length and width. The results are presented as mean ± standard<br />
deviation: weight (3.25 ± 0.5 kg), length (164.8 ± 19.42 cm), width (37.1 ± 5.93 cm), number of cotyledons (72 ± 14.56), length of the bigger<br />
cotyledons (12.3 ± 1.2 cm), width of the bigger cotyledons (10.4 ± 1.9 cm), length of the smaller cotyledon (3.6 ± 1 3 cm) and width of the<br />
smaller cotyledons (1.7 ± 1.2 cm). The weight, length, width and number of cotyledons of the placenta of Curraleiro/Pé-Duro cows were lower<br />
than those reported by Schlafer et al., 2000 (Animal Reproduction Science, v.60-61, pp.145-160) in cows of comercial breeds (Holstein and<br />
Hereford), possibly due to their smaller size and weight in the animals of the genetic group studied. However, the length and width of the biggest<br />
and smallest cotyledons were similar to those found by other authors with animals of commercial breeds. This may be suggestive of efficiency<br />
in maternal-fetal exchanges of the cotyledons in the placentome. These results allow to characterize the morphology of the placenta of Curraleiro/<br />
Pé-Duro cows, and may in future be used as reference for this genetic group.<br />
Keywords: cotyledon, placentome, genetic resources.<br />
A172 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
SODIUM CLOPR<br />
OPROSTENOL<br />
OSTENOL TREATMENT AND POSTPAR<br />
ARTUM UTERINE INVOL<br />
OLUTION IN DAIRY GOATS –<br />
PRELIMINARY RESULTS<br />
L eandro Bec<br />
ecalet<br />
alete e Rizz<br />
izzoni<br />
1 , Jose Ant onio D. Gar<br />
arcia<br />
1 , M iller Per<br />
ereir<br />
eira Palhão<br />
1 , To chimara a Apar<br />
parecida Miy<br />
iyauchi<br />
1 , S ilvio Dor<br />
oria de<br />
Almeida Ribeiro 2 , João Henrique Moreira Viana 3 & Carlos Antônio de Carvalho Fernandes 1<br />
1<br />
UNIFENAS, ALFENAS, MG, BRAZIL. 2 CAPRITEC, ESP. SANTO DO PINHAL, SP, BRAZIL. 3 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL.<br />
The endogenous prostaglandins have proved importance in process of uterine involution postpartum. The sodic cloprostenol, a<br />
synthetic analogue molecule, has been used with similarly results after calving in dairy cattle. The aim of this study was to investigate the<br />
effects of the sodic cloprostenol on uterine involution in dairy goats. The experiment was conducted in Sao Paulo state between January and<br />
April of <strong>2011</strong>. Twenty-one lactating dairy goats of Anglonubiana breed with normal calving were included in this experimental design. The day<br />
of parturition was considered as day 0 (D0) and the animals were randomly distributed in two groups: G1 (control) - 0.5 mL of saline (0.9%)<br />
and G2 - 0.133 mg of sodic cloprostenol. One intramuscular injection was performed on D1 and D4. The reproductive system of the animals<br />
were scanned (transrectal ultrasound with a linear transducer of 5.0 MHz) on days 1, 4, 10, 16, 22, 28, 34, 40 and 46 after parturition. The<br />
measured variables were: the size and position of the uterus, volume and echogenicity of the intra-uterine contents. Starting 16 days after<br />
parturition, blood samples were collected and plasma concentrations of progesterone will be further analyzed to evaluate the ovarian activity<br />
between groups. Data were subjected to analysis of variance and differences between means were assessed by Student’s test at 5%<br />
probability. The uterine involution were different between G1 and G2 (P < 0.05). The complete involution of the uterus were observed 6 days<br />
earlier in G2 (D22, uterine diameter of 3.5 ± 0.8 cm) compared to G1 (D28, uterine diameter of 3.5 ± 0.7 cm). Changes in uterine position<br />
among days after parturition were different between G1 and G2 (P < 0.05). For group 2, 100% of animals had shown the uterus in pelvic<br />
position at 28 days postpartum. However, the same observation was held only 12 days later (40 days postpartum) in control group. Similarly,<br />
the uterus was earlier empty in G2 (D40) compared to control (D46). The results suggest that sodic cloprostenol hastens the period of uterine<br />
involution after parturition in dairy goat. Further analysis of the blood samples could help to elucidate the effects of the treatment on<br />
postpartum ovarian activity in dairy goats. [Support: FAPEMIG].<br />
Keywords: goats, reproductive efficience, puerperium.<br />
s422
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A173 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
SODIUM CLOPR<br />
OPROSTENOL OSTENOL AND POSTPAR<br />
ARTUM<br />
TREATMENT<br />
MENTS OF DAIRY COWS<br />
Regis José de Car<br />
arvalho<br />
alho 1 , Miller Per<br />
ereir<br />
eira Palhão<br />
1 , Mar<br />
arilu Mar<br />
artins Gioso<br />
1 , Bruno Fer<br />
ernandes Ludger<br />
udgero Alv<br />
lves<br />
2 , Eduar<br />
duardo do Ramos de Oliv<br />
liveir<br />
eira 2 , Tatimar<br />
timara a Mar<br />
aria<br />
Miyauchi 2 , Anamara Pereira 1 & Carlos Antônio de Carvalho Fernandes 1<br />
1<br />
UNIFENAS, ALFENAS, MG, BRAZIL. 2 BIOTRAN, ALFENAS, MG, BRAZIL.<br />
The aim of this study was evaluate different strategies of PGF2á treatments on reproductive performance of the dairy cattle after<br />
parturition. The hypothesis is that splitting doses could be more effective than two doses of cloprostenol after parturition. The study was<br />
performed in a dairy farm with an intensive production system (free-stall) located in Southern of Minas Gerais (the city of Carmo do Rio Claro).<br />
Two hundred sixty-two Holstein cows according with delivery date were assembled in one of the four treatments: 1) Control (n = 61) - 2 mL<br />
of saline given intramuscular (IM) on days 2 and 5 after parturition; 2) Second group (Full dose, n = 69) - 2.0 mL of PGF2α (IM, 0.530 mg of<br />
cloprostenol) given on days 2 and 5; 3) Third group (1/2 dose, n = 63) - 1.0 mL PGF2α (IM, 0.265 mg of cloprostenol) given on days 2, 3, 4<br />
and 5; 4) Fourth group (1/4 dose, n = 69) - 0.5 mL PGF2α (IM, 0.1375 mg of cloprostenol) given twice daily on days 2, 3, 4 and 5 after<br />
parturition. The measured variables were the intervals from parturition to first artificial insemination (IA), and to first detected pregnancy,<br />
additionally the number of IA for conception was also compared among treatments. The data were first tested for normal distribution. Then,<br />
ANOVA was performed and statistically differences between each treatment and the control group were explored with Dunnet test. The chisquare<br />
was performed to detect differences in pregnancy rate among treatments. The Kaplan-meyer survey was performed to study the intervals<br />
from parturition to conception and to first IA. Probabilities < 5% were considered significant and those between 5 and 10% were considered<br />
approached of significance. Statistical differences among treatments were not detected for any of the variables analyzed: Parturition/AI interval<br />
(98.1±58.4, 89.2±33.2, 82.8±27.0 e 89.1±34.1 days), parturition/pregnancy interval (170.1±103.2, 163.2±89.8, 146.5±82.6 e 158.8±94.8<br />
days) and the number of AI per conception (2.4±1.6, 2.7±1.8, 2.4±1.9 e 2.4±1.8 for groups 1, 2, 3 and 4, respectively). Apparently, after 100<br />
days postpartum, the dairy cows of the group 2 became pregnant faster than those from control (Kaplan-meyer, P < 0.09). Thus, the pregnancy<br />
rate at 150 and 200 day post-partum had approached significance (P < 0.06 and 0.07, respectively) and was higher for group 2 (49.3 [34/69] and<br />
61.9% [43/69]) when compared to control (39.3 [27/61] and 50.8% [31/61]). Carefully interpretation may be taken to conclude this study. The<br />
results did not support the hypothesis and two full doses of prostaglandin given after parturition seem to improve fertility in dairy cows after 100<br />
day postpartum, however, the number of cows included in this study might not be enough to find the statistical differences. [Support:<br />
FAPEMIG].<br />
Keywords: conception rate, reproductive eficiency, artificial insemination.<br />
A174 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
PLASMA LEPTIN CONCENTR<br />
ONCENTRATION TION IN NELORE HEIFERS SUPPLEMENTED WITH PROTECTED FAT OR<br />
CARBOHYDR<br />
ARBOHYDRATE EXCESS<br />
Guilherme de Paula Nogueira 1 , Rafael Silva Cipriano 2 , Maria Carolina Villani Miguel 1 , Heni Falcão da Costa 1 , Leandro Mingroni<br />
Pavanello 1 , Juliana Stephani de Souza 1 , João Lucas Cânovas Delfino 1 , Pedro Vitor de Luna Freire Oliveira 1 , Emiliana de Oliveira<br />
Santana Batista 1 & Devani Mariano Pinheiro 1<br />
1<br />
FMVA-UNESP, ARAÇATUBA, SP, BRAZIL. 2 FMVZ-USP, SÃO PAULO, SP, BRAZIL.<br />
N<br />
Nellore is the main breed for beef in Brazil it is well adapted to tropical climates and extensive management. Heifers show the first<br />
ovulation at 24 months old, which is delayed compared to Bos taurus. A decrease in Nellore heifers puberty age can increase heard productivity<br />
up to 16%. It is well known that the fat mass percentage is important for first ovulation and leptin signaling play a role in this process. The aim<br />
of this study was to evaluate the effect of feeding protected fat and/or carbohydrate excess on plasma leptin level in prepubertal heifers. Heifers<br />
with 8 months of age and weighing 167.07 ± 15.66 kg were fed 2.2 kg/day of concentrate mixed with sugarcane bagasse in a feedlot system.<br />
Heifers were divided into three groups of energy supplementation: Heifers in the Corn group (n = 10) received 500 g of corn added to the diet,<br />
in the Fat Group (n = 10) were fed with 200 g protected fat (Megalac-E ® ) added to the diet and in the Energy Excess group (n = 10), received<br />
500 g corn plus 200 g of protected fat per animal. The supplementation was preceded by 60 days of adaptation to the diet. Venous blood samples<br />
were taken every 10 days for 120 days for plasma leptin quantification using a RIA Multi-species (Cat. # XL-85K) but using bovine recombinant<br />
leptin to make the standard curve. Samples were quantified from three animals in each of the Control and Fat Groups and from four animals in<br />
the Group Excess. Results were analyzed by the Instat (Graphpad ® ) program using repeated measures ANOVA. Before the diet supplementation,<br />
leptin concentrations did not differ (p = 0.5754) between Corn (0.87±0.19 ng/mL), Fat (0.79±0.14 ng/mL) and Excess (0.91±0.17 ng/mL)<br />
groups. After supplementation, the Energy Excess Group showed higher (P < 0.0001), leptin concentration (1.46±0.34 ng/mL) than the Corn<br />
(0.82±0.11 ng/mL) and Fat (0.874±0.16 ng/mL) groups. It was concluded that the animals treated with the Excess Energy diet had higher leptin<br />
concentration than those from Corn and Fat groups.<br />
Keywords: puberty, nutrition, leptin.<br />
s423
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A175 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
PROGESTER<br />
OGESTERONE ONE CONCENTR<br />
ONCENTRATION TION DURING ESTROUS CYCLE CLE IN TWO SEASONAL PERIODS IN JENNIES<br />
José Victor de Oliveira 1 , Elisa Sant´Anna Monteiro da Silva 2 & Cezinande Meira 2<br />
1<br />
POLO REGIONAL ALTA MOGIANA (APTA), COLINA, SP, BRAZIL. 2 FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA - UNESP, DEPTO REPROD ANIMAL E RADIOL.<br />
VETERINÁRIA, BOTUCATU, SP, BRAZIL.<br />
The aim of this study was to evaluate the plasmatic progesterone (P4) concentration profile during jennies estrous cycle at increasing<br />
luminosity periods, 01/08 a 15/09 (Phase 1), transitional periods for mares, and maximum luminosity periods, 01/12 a 20/01 (Phase 2) of the<br />
breeding season (latitude 20o43’05"S; longitude 48o32’38"W). Ten Brazilian Jennies were used, considering that one estrous cycle was<br />
monitored per animal at phase 1 (n = 10) and 2 (n = 10). Once ovulation was detected by ultrasound, blood samples were collected with 24 h<br />
intervals during interovulatory periods at phases 1 and 2. Blood sample was obtained by puncture of the jugular vein in heparinized tubes and,<br />
immediately after the collection, blood was centrifuged and plasma stored at -20ºC, for posterior determination of plasmatic P4 concentration. P4<br />
was measured by radioimmunoassay method in solid phase, using commercial KITs (DPC-Diagnostic Products Corporation, Los Angeles<br />
USA). The anti-serum was specific for P4, with low cross reaction with other steroid hormones. The intra and inter assay coefficient of variation<br />
was 8.7% e 16.7%, respectively. The results were analyzed by t-Student test matched to 5% significance for the two experimental phases. P4 data<br />
referring to two animals estrous cycle that presented respectively, diestrous ovulation on the 7th day of the cycle (Phase 2) and asynchronous<br />
double ovulation with maximal 24 h interval (Phase 1), were excluded of statistical analysis because they had additional CL, being analyzed<br />
separately. P4 plasmatic concentration was similar between phases 1 and 2 (P > 0.05). On ovulation day P4 values were 1 ng/mL), which duration was respectively 15.4±0.5 and 15.8±0.8 days for phases 1 and 2 (P > 0.05; by t-paired test). The data allow to<br />
conclude that P4 concentration are similar during periods of increasing luminosity (considered transitional phase for mares) and maximum<br />
luminosity, suggesting that jennies kept on that region did not show seasonal effects on estrous cycle. [Financial Support FAPESP and OEPAS].<br />
Keywords: jennies, progesterone, estrous cycle.<br />
A176 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
CULTURE OF INNER CELL MASS CELLS OBTAINED FROM BOVINE EMBRYOS PRODUCED<br />
IN VITRO AND IN VIVO<br />
IN THE PRESENCE OF LEUKEMIA INHIBITOR<br />
ORY FACT<br />
CTOR (LIF).<br />
Midyan Daroz Guastali, Mateus José Sudano, Daniela Martins Paschoal, Tatiana da Silva Rascado, Letícia Ferrari Crocomo, Isadora Arruda, Rosiara Rosária Dias<br />
Maziero, Luis Eduardo Vergara, Joao Ferreira de Lima Neto & Fernanda da Cruz Landim Alvarenga<br />
UNIVERSIDADE ESTADUAL PAULISTA, BOTUCATU, SP, BRAZIL.<br />
The application of biotechnologies involving different tissues for regenerative medicine has been possible due to a greater understanding<br />
of the biology of stem cells and embryo production in vitro and in vivo. The isolation of pluripotent cell lines from the inner cell mass (ICM) of<br />
blastocysts is already known and successful. Attempts to primary culture ICM cells from various species on the top of fibroblast monolayer<br />
(Feeder) in the presence of leukemia inhibitory factor (LIF) is controversial. Embryonic stem cells (ESC) of mice can be maintained undifferentiated<br />
when grown in culture medium with addition of LIF. Preliminary studies have shown that ICM cells of bovine blastocysts have receptors for<br />
LIF, however, the presence of LIF did not help the establishment and maintenance of ESC in other ungulates. In fact, Vejlsted et al. (Biol. Reprod,<br />
72: 678-686. 2005) demonstrated a deleterious effect of the addition of LIF to the culture of ESC in bovine. Thus, the objective of the present<br />
experiment was to evaluate the potential in vitro growth of cells derived from the ICM of bovine embryos produced in vitro and in vivo in the<br />
presence of LIF. Seven embryos obtained by IVF were cultured in vitro until day 9 when their ICM were extracted mechanically with the aid of<br />
two insulin needles and transferred to six wells plates containing a monolayer of bovine fibroblasts cultured previously (20x104 cels/1.9 cm 2 per<br />
well ) and inactivated with mitomycin C (0.5 mg/mL). The medium used was DMEM: F12 (Gibco ® ) supplemented with 20% FCS, penicillin<br />
G 10,000 IU, streptomycin sulfate 10.000 ìg, 1% nonessential amino acids, 1% essential amino acids, 0.1 mM 2-Mercaptoethanol and LIF 1000<br />
U for each 1 mL of medium. Also, three in vivo embryos (D7) produced by FTAI protocol, retrieved transcervicaly also had their ICMs isolated<br />
and cultured according to the same methodology. After 5 days of culture adherence and early colony growth was observed in 28.5% (2/7) of ICM<br />
obtained from in vitro produced embryos and 100% (3/3) ICM from in vivo produced embryos. At 12 days of culture the first passage was<br />
performed through mechanical dissociation. The results obtained so far, indicate that both the ICM of embryos produced in vitro and in vivo can<br />
potentially generate colonies similar to ESC in the presence of LIF. However the potential for development of ICM from in vivo produced<br />
embryos is superior. Future experiments will be conducted to characterize and confirm the potential strain of bovine embryonic stem cells.<br />
[Acknowledgement: FAPESP (Grant - 2010;0065-7)].<br />
Keywords: cell culture, embryonic stem cells, leukemia inhibitory factor.<br />
s424
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A177 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
REPRODUCTIVE PERFORMANCE OF TWO<br />
WISTAR RATS GENERATIONS SUPPLEMENTED WITH THE OMEGA-3 FATTY ACID<br />
Carolina Bespalhok Jacometo 1 , Simone Halfen 1 , Francielle Bado 1 , Fernanda Trindade da Rosa 1 , Eduardo Schmitt 1 , Luiz Francisco Machado Pfeifer 2 , Augusto<br />
Schneider 1 , Marcio Nunes Corrêa & Nelson José Laurino Dionello 1<br />
1<br />
UFPEL, PELOTAS, RS, BRAZIL. 2 EMBRAPA, PORTO VELHO, RO, BRAZIL.<br />
The long-chain polyunsaturated fatty acids are not strictly necessary in the diet, but are important during the embryonic period,<br />
acting on cell division, growth and differentiation. The aim of this study was to evaluate the effect of a diet enriched with omega-3 fatty acids<br />
on reproductive performance of successive generations. Diets were prepared in accordance to the requirements of AIN-93, being isoenergetic<br />
and isoproteic. Adult Rattus norvegicus - Wistar/UFPel female rats were used. In the F0 generation the animals received diets with linseed oil<br />
(Om Group, n = 18) or soybean oil (CTL Group, n = 18) throughout pregnancy. F1 generation was divided into three groups: females from<br />
Om Group of the F0 generation were supplemented with linseed oil (Om/Om Group, n = 16) or soybean (Om/CTL Group, n = 16) and<br />
females from CTL Group of the F0 generation were supplemented with soybean oil (CTL/CTL Group, n = 16). In both generations the<br />
females were mated in a male:female ratio of 1:3. From the F0 offspring, progenies were selected at weaning (day 21 postpartum) to compose<br />
the F1. The variables evaluated in both generations were: pregnancy rate, number of pups per litter and average weight at birth. Statistical<br />
analyses were performed using SAS. The pregnancy rate was compared among groups and generations by Chi-square, and number and weight<br />
of pups by ANOVA and Tukey Test was used to compare means. There was no difference for the pregnancy rate among groups (P > 0.05).<br />
Also, no difference was observed for the number of pups per female between group and generations (P > 0.05). However, in the F1 offspring,<br />
higher weight at birth was observed in the Om/Om Group than CTL/CTL Group (7.3±0.2g vs. 6.6±1.3g; P = 0.01). Previous studies<br />
demonstrated that pregnant rats subjected to diets enriched with omega-3 delivered higher weight at birth offspring. These effects could be<br />
associated to the reduction of gestational hypertension and intensity of inflammatory effects, due to the inhibition of PGF2α and E2<br />
synthesis (Church et al., Neurotoxicology and Teratology, 30: 107-117, 2008). Regarding performance between generations, the OmG<br />
maintained the mean weight at birth from F0 to F1. However the CTLG reduced the mean weight of the offspring (0.84 g, P = 0.01). In<br />
conclusion, a diet enriched with omega-3 fatty acids, in successive generations, did not affect the pregnancy rate nor the number of pups per<br />
female, despite the offspring had a higher birth weight.<br />
Keywords: generations, linseed oil, progenies.<br />
A178 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
THE ONSET OF PUBERTY IN EWE LAMBS AFTER BIOSTIMULATION TION AND MEDROXYPR<br />
YPROGESTER<br />
OGESTERONE ONE ACET<br />
CETATE TE OR LONG-<br />
ACTING PROGESTERONE<br />
Claudia Dias Mon<br />
onteir<br />
eiro Toma,<br />
Son<br />
ony Dimas Bicudo<br />
icudo, Hugo Shisei<br />
Toma,<br />
Car<br />
armo Emanuel Almeida Bisc<br />
iscar<br />
arde<br />
de, Tiago Matos Oliv<br />
liveir<br />
eira,<br />
Mar<br />
arcel Barb<br />
arbosa Falleir<br />
alleiros<br />
os,<br />
Luana de Cassia Bicudo & Leandro Rodello<br />
UNESP BOTUCATU, BOTUCATU, SP, BRAZIL.<br />
The aim of this study was to evaluate the response of prepubertal ewe lambs to exogenous administration of either<br />
medroxyprogesterone acetate (MAP) or long-acting progesterone (LAP) together with biostimulation under both male/female (MF) and<br />
female/female (FF) effect for eight weeks. Two Pool Dorset adult males and 75 mixedbreed prepubertal ewe lambs (average of 179 ± 1,20 daysold<br />
and 30.0 ± 0,124kg) were used. The females were randomly assigned to three different groups. Twenty five ewe lambs were submitted to<br />
the insertion of intravaginal sponges containing MAP (60 mg) for 12 days and were then biostimulated. Twenty five other females were<br />
submitted to a single dose of LAP (225 mg, IM) and then to biostimulation. The rest of the females (n = 25) were only submitted to<br />
biostimulation. The cycling was demonstrated by evaluation of plasma progesterone (P4) = 1ng/mL concentration exceeded 1.0 ng/mL in at<br />
least one of two consecutive blood samples taken within a 7-day interval (Shabankareh et al., 2009) in the pre and post-hormone treatment<br />
and final period of biostimulation in all lambs, a total of 450 samples was collected and assayed. Data are submitted to the variance analysis<br />
and compared by the Tukey test with 5% of probability at the SAS program System. After treatments 93.3% (70/75) of the females<br />
disregarding their group started their cyclicity and most of them (92.0% 69/75), remained cycling until the end of the period (63 days)<br />
biostimulation with or without the use of MAP or LAP. We conclude that employment at six months old, protocols with administration of<br />
MAP for 12 days or a single dose of LAP, followed by biostimulation trigger puberty and maintance of cyclicity in lambs. It can be deducted<br />
that the biostimulation MF and FF effects trigger puberty and maintance of cyclicity in untreated lambs.<br />
Keywords: puberty, ewe lambs, progesterone.<br />
N<br />
s425
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A179 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
DIMENSIONS, STEROIDOGENESIS AND VASCUL<br />
ASCULARIZA<br />
ARIZATION OF THE BOVINE CORPUS LUTEUM<br />
IN RESPONSE TO<br />
MANIPULATION OF THE PRE-OVULATOR<br />
ORY FOLLICLE GROWTH<br />
Fernando Silveira Mesquita 1 , Moana Rodrigues França 2 , Saara Carolina Scolari 3 , Valdir Pavanello Jr. 4 , Fabiana Fernandes Bressan 5 , Flávio Vieira Meirelles 6 ,<br />
Paula de Carvalho Papa 7 , Guilherme de Paula Nogueira 8 , Luciano Andrade Silva 9 , Claudia Maria Bertan Membrive 10 & Mário Binelli 11<br />
1,2,3,11<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL-FMVZ-USP, PIRASSUNUNGA, SP, BRAZIL. 4,7 DEPARTAMENTO DE CIRURGIA-FMVZ-USP, SÃO PAULO, SP, BRAZIL.<br />
5,6,9<br />
DEPARTAMENTO DE CIÊNCIAS BÁSICAS-FZEA-USP, PIRASSUNUNGA, SP, BRAZIL. 8 DEPARTAMENTO DE APOIO DE PRODUÇÃO E SAÚDE ANIMAL-UNESP, ARAÇATUBA, SP,<br />
BRAZIL. 10 FACULDADE DE ZOOTECNIA - UNESP, DRACENA, SP, BRAZIL.<br />
In cattle, fluctuations in plasma concentrations of estradiol and progesterone (P4), which characterize the peri-ovulatory phase of the<br />
estrous cycle, influence oocyte maturation, transport of gametes, fertilization and early embryonic development. This work aimed to study the<br />
effects of manipulations of the pre-ovulatory follicle (POF) growth on growth, P4 production and expression of steroidogenesis- and angiogenesisrelated<br />
genes of the subsequent corpus luteum (CL). Cyclic, non-pregnant Nelore cows received two injections of PGF2α (PGF; 0.5 mg; i.m.)<br />
14 days apart. Ten days later (day -10; D-10), cows received a P4-releasing device along with estradiol benzoate (2 mg; i.m.). In order to modulate<br />
the growth of the POF and alter post-ovulatory P4 production, on D-10 animals received PGF (high post-ovulatory P4 group; HP; n = 10) or<br />
not (low post-ovulatory P4 group; LP; n = 10). Progesterone-releasing devices were removed and PGF injected on D-2.5 for the HP group and<br />
on D-1.5 for the LP group. Ovulation was induced with GnRH (buserelin; 10 µg; i.m.) on D0. Growth and ovulation of the POF and CL<br />
formation and vascularization were assessed by Doppler ultrasonography from D-2 to D7. On D7, plasma was obtained for measurement of P4<br />
concentration, cows that ovulated were slaughtered (HP, n = 7 and LP, n = 6) and CLs were dissected and stored. Relative concentrations of<br />
transcripts related to steroidogenesis (STAR, CYP11A1, HSD3B) and angiogenesis (VEGF, FLT1, KDR) were determined by TaqMan qPCR,<br />
using tubulin as the endogenous control gene. Differences between group means were determined by student’s t test. Maximum diameter of the<br />
POF (mean ± standard error of the mean; 13.01±0.35 vs. 10.98±1.07 mm; P = 0.06), weight (3.10±0.32 vs. 1.91±0.33g), diameter (17.75±0.76<br />
vs. 13.75±1.14 mm), volume (1659.94±220.53 vs. 801.99±191.11 mm 3 ) and vascularization of the CL on D7 (80.00±3.54 vs. 56.25±8.00%)<br />
and P4 concentration (4.34±0.13 vs. 2.74±0.35 ng/mL) on D7 were greater in HP vs. LP (P < 0.05). Progesterone concentrations were<br />
positively correlated with diameter, volume and weight of the CL (r = 0.61, 0.56 e 0.5, respectively). Weight of the CL was positively correlated<br />
with its diameter (r = 0.84) and with POF diameter (r = 0.73). Luteal gene expression of CYP11A1, STAR (P < 0.05) and VEGF (P = 0.07)<br />
on the HP group was 3.2, 2.1 and 2.5 times greater than that of the LP group. In conclusion, stimulation of POF growth resulted in a larger CL<br />
that produced more P4. It is suggested that an increase in the differential gene expression of steroidogenic enzymes and angiogenesis-related<br />
molecules by the CL is associated with a higher functional capacity of this organ. [Supported by CNPq (481087/2010-9) and FAPESP (2010/<br />
01284-4)].<br />
Keywords: bovine, corpus luteum, progesterone.<br />
A180 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EFFECT OF DEXAMETHASONE AND TRIIODOTHYRONINE ON IN VITRO BOVINE EMBRYO<br />
Nathália Nogueira da Costa 1 , Marcela da Silva Cordeiro 2 , Thiago Velasco Guimarães Silva 2 , Priscila Di Paula Bessa Santana 1 , Rafael Vilar Sampaio 1 , Danuta<br />
Carolina Sastre 1 , Bruno Baraúna da Silva 1 , Andre Luiz Alves de Sá 1 , Carlos Leonardo de Aragão Araújo 1 , Simone do Socorro Damasceno Santos 1 , Moysés dos<br />
Santos Miranda 1 & Otávio Mitio Ohashi 1<br />
1<br />
UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2 INSTITUTO FEDERAL DE EDUCAÇÃO, CIÊNCIA E TECNOLOGIA DO PARÁ, ABAETETUBA, PA, BRAZIL.<br />
The embryos produced in vitro differ in many aspects from those in vivo produced and in most cases this difference is related to<br />
the conditions and composition of the culture media (Khurana & Niemann, 2000, Biology of Reproduction, 62: 847-856). Therefore, the aim<br />
of this study was to evaluate the effect of the hormone triiodothyronine (T3) and the synthetic glucocorticoid, dexamethasone (Dexa) on IVC,<br />
since they are important regulators of metabolism, cell growth and differentiation (Aranda & Pascual, 2001, Physiological Reviews, 81:1269-<br />
1304). For that COCs were selected and incubated for IVM at 38.5°C and high humidity for 18 h. For IVF, the sperm of a single bull was<br />
thawed and co-incubated with the COCs under the same conditions described for IVM. After approximately 30 h of incubation, presumptive<br />
zygotes were subjected to repeated pipetting to remove remaining cumulus cells. Then, they were randomly distributed among the following<br />
groups: Control, without hormones; Dexa (0.1 ug / mL), T3 (50 nM T3), T3 + Dexa (0.1 ug / mL Dexa and 50 nM T3). All groups were<br />
cultured in SOF medium supplemented with 6 mg / mL BSA and 10% FCS. Cleavage rate and the rate of blastocyst and kinetics of<br />
development were analyzed on the Day 2 and Day 8 of culture. The results were analyzed using the ANOVA test, adopting P < 0.05. The rate<br />
of cleavage and blastocyst (P > 0.05) were similar between groups, control (79% and 52% respectively), Dexa (75% and 49% respectively),<br />
T3 (80% and 46% respectively) and Dexa + T3 (81% and 53% respectively). There was no difference in the kinetics of development between<br />
the embryos cultured with or without hormones, and observed a predominance of hatched embryos in all groups (control group, 69.2%; Dexa,<br />
70%, T3 71% and T3 + Dexa 73%, P > 0.05). Thus, these results suggest that T3 and Dexa used alone and / or combined during IVC, showed<br />
no effect on in vitro development of bovine embryos.<br />
Keywords: in vitro production embryo, dexamethasone, triiodothyronine.<br />
s426
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A181 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EFFECT OF CELL QUANTIT<br />
ANTITY IN OBTAINING MICE CHIMERAS BY DEMI-BLAST<br />
ASTOCY<br />
OCYST<br />
STS S AGGREGA<br />
GGREGATION<br />
Caio Pontes Godói, Pablo Diego Moço, Bruno Cazari, Patrícia Tiemi Mihara, Patrícia Villela Silva, Isabele Picada Emanuelli & Marcelo Fábio Gouveia Nogueira<br />
UNESP, ASSIS, SP, BRAZIL.<br />
Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early<br />
stages, synthesize cell adhesion molecules that increase the aggregation chances among them (Vestweber et al., 1987; Developmental Biology<br />
124:451-6). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al.,<br />
2010; Transgenic Research 19:344-5). Based on this evidences, the aim of this study was to evaluate, in mice, the influence of cell quantity in<br />
the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and/or Swiss<br />
Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008; Transgenic Research<br />
17:1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona<br />
pellucida removed using pronase treatment (2 mg/mL for 15 min), while blastocysts (recovered 3.5 dpc) were split with microblade controlled<br />
by micromanipulator in an inverted microscope (NK2, Eppendorf, Germany and Eclipse Ti, Nikon, Japan, respectively). The aggregation<br />
groups were: a control (C) with two pre-compaction whole embryos 8C and/or PCM and two experimental with post-compaction embryos, i.e.,<br />
two (2DB) or four (4DB) demi-blastocysts. The structures (2 or 4) of the groups were sticked each other with the use of phytohemagglutinin<br />
(1 mg/mL) and cultured in vitro by 24 h (37ºC, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified<br />
by detection of a single, cohesive cell mass or a structure in “8 shape” with more than half of its total diameter aggregated. For the 4DB group,<br />
a successful aggregation was considered when, at least, two of four demi-blastocysts have aggregated. The results were analyzed using Chisquare<br />
test, Fisher’s exact test and Kruskal-Wallis (to comparison among groups, between groups and among median of group replicates,<br />
respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a;<br />
8.3b e 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in<br />
farm animals (e.g., bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as eight cells) and when only<br />
trophectoderm aggregation is desired. It was concluded that cell increasing (from 2 to 4 demi-blastocysts) improved the chimerism rate, but not<br />
enough to be similar to control group.<br />
Keywords: aggregation chimerism, demi-blastocyst, mice.<br />
A182 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EFFECT OF DIFFERENT MEDIA ON BONE MORPHOGENETIC PROTEIN 15 (BMP-15) AND GROWTH<br />
DIFFERENTIATION TION FACT<br />
CTOR 9 (GDF-9) MRNA EXPRESSION DURING OOCYTE IN VITRO MATUR<br />
TURATION TION (IVM)<br />
Paula Fernanda de Lima 1 , Mariana Fernandes Machado 1 , Ester Siqueira Caixeta 1 , Felipe Morales Dalanezi 1 , Christopher Price 2 &<br />
José Buratini Junior 1<br />
1<br />
UNESP, BOTUCATU, SP, BRAZIL. 2 UNIVERSITY OF MONTREAL, MONTREAL, CANADA.<br />
N<br />
BMP-15 and GDF-9 are oocyte secreted factors (OSF) that control cumulus-oocyte complex (COC) maturation. They bind to<br />
receptors in cumulus cells and regulate indirectly oocyte quality. Considering that the oocyte retains transcriptional activity during IVM<br />
(Mano et al. BMC Genomics <strong>2011</strong>, 12:151), we tested the hypothesis that different media currently used in IVM would determine different<br />
levels of BMP-15 and GDF-9 mRNA expression in the oocyte. More specifically, we tested the effects of the addition of fetal calf serum<br />
(FCS), bovine serum albumin (BSA) and BSA plus estradiol to the base medium on BMP-15 and GDF-9 mRNA expression during IVM.<br />
Immature COCs (grades 1 and 2) were obtained from 3-8 mm follicles from abattoir ovaries (predominantly Bos indicus). Four groups of 20<br />
COCs per treatment were cultured for 22 h in TCM 199 bicarbonate plus BSA (0.4%), or BSA (0.4%) plus estradiol (1µg/mL), or FCS (10%).<br />
After culture, oocytes were denuded and stored at -80°C. Four groups of 20 immature oocytes were also included in the analysis to verify the<br />
effect of maturation on gene expression. Total RNA was extracted from pools containing 20 oocytes with RNeasy ® (Qiagen). Reverse<br />
transcription was performed with Sensiscript ® (Qiagen). mRNA expression of both target genes was investigated by real time RT-PCR and<br />
normalized by the expression of cyclophilin A (CYC-A). Expression values were determined by the Pfaffl equation. Effects of treatments<br />
were tested by ANOVA and groups were compared by Tukey-Kramer HSD. Differences were considered significant when P < 0.05. mRNA<br />
abundance for both BM-15 and GDF-9 did not vary with different maturation media. Interestingly, GDF-9 mRNA expression was higher in<br />
the immature group compared with all others. In conclusion, addition of BSA or FCS determined the same levels of mRNA expression for<br />
BMP-15 and GDF-9 and the addition of estradiol did not alter the expression pattern. The suppressing effect of maturation on GDF-9 mRNA<br />
expression is interesting and deserves further investigation. [Financial support FAPESP].<br />
Keywords: bmp-15, gdf-9, bovine.<br />
s427
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A183 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EFFECT OF DRY AND WET SEASONS ON THE ABILITY OF OOCYTES DEVEL<br />
VELOPING AND IN VITRO PRODUCTION OF EMBRYOS<br />
IN SHEEP<br />
Mysa<br />
Tatiana Cor<br />
orrêa rêa Gonçalv<br />
onçalves<br />
1 , Filip<br />
ilipe Quierós Godim Bezer<br />
erra 2 , Ric<br />
icar<br />
ardo de Mac<br />
acedo Cha<br />
haves<br />
1 , Cristiano Rocha Aguiar Filho<br />
2 , Fabíola Freitas de Paula-L<br />
aula-Lop<br />
opes<br />
3 ,<br />
Paulo Fernandes Lima 2 & Marcos Antonio Lemos de Oliveira 2<br />
1<br />
UNIVERSIDADE ESTADUAL DO MARANHÃO, SÃO LUÍS, MA, BRAZIL. 2 UNIVERSIDADE FEDERAL RURAL DE PERNAMBUCO, RECIFE, PE, BRAZIL. 3 UNIVERSIDADE FEDERAL DE SÃO<br />
PAULO, DIADEMA, SP, BRAZIL.<br />
Exposure of female mammals to thermic stress increases embryonic mortality, especially in early development. Thus this study<br />
aimed to evaluate the effects of dry and rainy seasons in the capacity of developing oocytes and IVP ovine embryos. The ovaries of ewes during<br />
the dry season (October to March) and rainy (April-September) were collected at a slaughterhouse and transported to the Laboratory of<br />
Reproduction Biotechnology on UFRPE in thermos bottle containing saline solution at 30ºC, added 30 µg/mL gentamicin sulfate, occurring in<br />
12 replicas. Cumulus complexes (COCs) were recovered from follicles between 2 and 8 mm per slice with the help of chisel in a Petri dish<br />
containing medium collection (MC) for searching and selection of the COCs. After, the COCs were washed three times in MC and placed in<br />
drops of 100 µL to maturity under paraffin oil sterilized in basic medium maturity, and put to mature in incubator at 39ºC in a humidified<br />
atmosphere containing 5% CO 2<br />
for 24 h. After this period oocytes were evaluated for morphology and those showed good expansion of<br />
cumulus cells were submitted IVF using fresh sperm selected by Swim-Up at a concentration of 2 x 10 6 sperm/mL, and incubated under identical<br />
conditions the maturation for 18 h. The zygotes were denuded in mechanical agitator in the middle «Synthetic oviduct fluid» modified (SOFm)<br />
for two minutes and the structures transferred to drops of 100 µL of the medium SOFm supplemented with 10% fetal bovine serum under sterile<br />
paraffin oil. These were incubated under similar conditions to maturity added 5% O 2<br />
and 90% N 2<br />
, after 7 days of culture, we evaluated the cell<br />
number, cleavage rate and which reached blastocyst stages. Embryos were submitted to DNA fragmentation characteristic of apoptosis and<br />
analyzed by TUNEL assay. The percentage of cleaved oocytes was determined on day 3 (D-3) and that developed at levels of 8-16 cell, morula<br />
and blastocyst was determined on day 4 (D-4), 5 (D-5) and 8 (D-8) after fertilization, respectively. For statistical analysis, variance analysis was<br />
performed by the method of least squares. Significant difference was found (P < 0.05) in fertilization stages, D-3 and D-4. No significant<br />
difference was found (P > 0.05) to in vitro maturation stages, morula and blastocyst and DNA fragmentation (TUNEL). Under the conditions<br />
observed in this study, we conclude that there was no effect of season on the developmental capacity of oocytes and in vitro production of<br />
embryos in sheep.<br />
Keywords: oophorus cumulus complexes, fertilization, apoptosis.<br />
A184 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
UTERINE HEMODYNAMICS CHANGES AFTER SEMEN INFUSION IN MARES<br />
Jair Camargo Ferreira, Fernanda Saules Ignácio & Cezinande Meira<br />
UNESP-BOTUCATU, BOTUCATU, SP, BRAZIL.<br />
For provide anatomic details and immediately information about the hemodynamics of tissue and organs, the Doppler exam allows<br />
evaluating the uterine status of mares after the semen infusion. In consequence of this new knowledge, concepts before considered to be<br />
definitive regarding the physiology of reproduction are being reconsidered. The porpouse of the present stud were to describe the uterine<br />
vascular perfusion and quantify the mesometrial Doppler indices during the post-breeding stage in mares. Cycling mares (n = 18) with preovulatory<br />
follicles .35mm were inseminated with cooled semen (H=0). All animals were evaluated using Power-flow and Spectral-modes<br />
Doppler ultrassonography hourly through the 12 firsts h post-uterine infusion. Uterine vascularity was scored from 1 to 4 (minimum and<br />
maximum, respectively). Resistance (RI) and Pulsatility (PI) indices were measured using Spectral-mode and mesometrial arteries. Effect of<br />
time and difference between medians were analyzed by the Scott-Knot and Tukey. No effect of time was detected for mesometrial RI, while<br />
an increased uterine vascularity and decreased mesometrial PI were detected (P < 0.05) between H1 and 2. However, the uterine vascularity<br />
increase did not reached the mean value (score=2) during the data collection. The present study demonstrates for the first time the use of<br />
power-mode function and mesometrial arteries for spectral evaluation of the uterine hemodynamics during the post-breeding phase in mares.<br />
Apparently, hourly exams did not affect the reproductive hemodynamics of mares. The subjectively exam (uterine vascularity) was validated<br />
by objectively evaluation (mesometrial PI). The increased uterine blood-flow observed on the first 2 h after intrauterine infusion of semen may<br />
be associated with endometrial inflammatory factors and/or vasodilatory components of the seminal plasma. These findings suggest a prompt<br />
interaction between uterus and semen during the post-breeding phase in mares.<br />
Keywords: doppler, mare, uterus.<br />
s428
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A185 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EFFECTS OF OXYGEN<br />
TENSION AND GLUC<br />
UCOSE CONCENTR<br />
ONCENTRATION TION DURING IN VITRO MATUR<br />
TURATION TION OF BOVINE<br />
OOCYTE ON OOCYTE QUALIT<br />
ALITY AND MATUR<br />
TURATION TION AND EMBRYONIC DEVEL<br />
VELOPMENT<br />
Gisele Zoccal Mingoti 1 , France Filion 2 , Lawrence Charles Smith 2 & Patrick Vincent 2<br />
1<br />
UNESP, ARAÇATUBA, SP, BRAZIL. 2 UNIVERSITE DE MONTREAL, ST-HYACINTHE, CANADA.<br />
Aiming to avoid the production of free radicals during IVP of bovine embryos and consequently improve the production and quality<br />
of embryos, this study evaluated the effects of O2 tension and addition of glucose during IVM on bovine oocyte nuclear and cytoplasmic<br />
maturation (assessed by the cytoplasmic distribution and mitochondrial DNA quantification - mtDNA) and embryonic development. Oocytes<br />
were IVM in mSOF (Soto & Smith, Mol Reprod Dev 76:637-46, 2009) with hormones for 24 h at 38.5ºC. Depending on the experimental<br />
group, glucose was added to the medium at concentrations of 0.48 mM or 5.6 mM (high glucose concentration - 20 mM - was also tested during<br />
the nuclear and cytoplasmic maturation, but there was no difference from 5.6 mM and it was not used in subsequent experiments). Oocytes were<br />
IVM under high (~20% -atmospheric) or low (5%) O2 tension. Afterwards, oocytes (n = 248) were stained with 10 µg/mL of MitoTracker<br />
Green (to evaluate the mitochondrial distribution) and 10 µg/mL Hoechst 33342 (nuclear maturation assessment). The mtDNA number of copies<br />
was measured in individual oocytes (Soto & Smith, Mol Reprod Dev 76:637-46, 2009, n = 20). Other oocytes (n = 453) were IVF and the<br />
zygotes were IVC in mSOF; the blastocyst (Bl) rates were assessed at 168 hpi. Means were compared by ANOVA followed by Tukey’s test (P<br />
< 0.05). The proportion of MII oocytes was not affected by glucose concentration (P > 0.05), but it was affected by O2 tension (20%: 86.1%<br />
vs. 5%: 67.4; P < 0.05). The mitochondrial cytoplasmic redistribution (disperse = mature) was not affected by O2 tension (20%: 55.0% vs. 5%:<br />
56.2%; P > 0.05) or glucose (0.48 mM: 54.4% vs. 5.6 mM: 54.2%; P > 0.05). There was no effect of O2 tension (20%: 46.8×104 vs. 5%:<br />
43.2×104; P > 0.05) or glucose (0.48 mM: 39.2×104 vs. 5.6 mM: 49.2×104; P > 0.05) on mtDNA copy number. Embryonic development to<br />
Bl stage was unaffected by O2 tension (20%: 10.4% vs. 5%: 11.9%; P > 0.05) or glucose (0.48 mM: 11.2% vs. 5.6 mM: 11.0%; P > 0.05).<br />
However, there was effect of O2 tension (20%: 83.4% vs. 5%: 136.2; P < 0.05) on the total number of Bl cells, but no effect of glucose<br />
concentration was observed (0.48 mM: 109.3 vs.. 5.6 mM: 98.3; P > 0.05). There was no effect of O2 tension (20%: 7.2% vs. 5%: 5.5%; P ><br />
0.05) or glucose (0.48 mM: 7.2% vs. 5.6 mM: 5.8%; P > 0.05) on percentage of TUNEL+ cells in Bl. In conclusion, in vitro oocyte maturation<br />
under low O2 tension reduces the proportion of nuclear maturation, but does not affect the cytoplasmic maturation and embryonic development,<br />
and increases the total number of cells in the blastocysts.<br />
Keywords: in vitro maturation, mitochondria, embryo development.<br />
A186 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
FLORFENIC<br />
ORFENICOL OL AND SODIUM CLOPR<br />
OPROSTENOL OSTENOL FOR THE TREATMENT OF RETAINED PLACENT<br />
CENTA IN DAIRY COWS<br />
A namara a Per<br />
ereir<br />
eira 1 , M iller Per<br />
ereir<br />
eira Palhão<br />
1 , Br uno Fer<br />
ernandes Ludger<br />
udgero Alv<br />
lves<br />
2 , Eduar<br />
duardo do Ramos de Oliv<br />
liveir<br />
eira 2 , Ta timara a Mar<br />
aria Miy<br />
iyauchi<br />
2 ,<br />
Thais Camargo Rossi 1 , Jose Antonio D. Garcia 1 & Carlos Antônio de Carvalho Fernandes 1<br />
1<br />
UNIFENAS, ALFENAS, MG, BRAZIL. 2 BIOTRAN, ALFENAS, MG, BRAZIL.<br />
N<br />
Retained placenta (RP) is considered a pathologic condition when fragments or total fetal membranes remained attached for more<br />
than 12h after calving. A variety of risk factors include early or induced parturition, dystocia, hormonal imbalances and immunosuppression.<br />
This disease has several implications for reproductive efficiency in dairy farms. It is close related to postpartum uterine infection, to delay the<br />
ovarian activity and the uterine involution after parturition, and to decreased fertility after artificial insemination (AI). The aim of this study was<br />
to compare the reproductive efficiency in dairy cows with RP and treated with Florfenicol associated or not with Cloprostenol. Hundred-eight<br />
cows with RP, from dairy herds in southern of Minas Gerais and northern of São Paulo state, were included for this purpose. The experiment<br />
was arranged in factorial 2X2. The four treatments encompassed two levels of each factor: 1) Florfenicol - 40 mg/kg (IM) divided in two<br />
injections - 20 h after calving (20 mg/kg) and 48 h after the first (20 mg/kg) - or 40 mg / kg (SC) - one injection 20 h after calving; 2) Cloprostenol<br />
(0.530 mg) - one injection associated with each florfenicol treatment - or without cloprostenol. The variables assembled for this design were the<br />
intervals from parturition to AI (P/IA) and to conception (P/C), and the number of services per conception (S/C). The percentage of animals with<br />
corpus luteum (CL) between 25 and 40 days postpartum were also evaluated. The data were submitted to ANOVA and means of continuous<br />
variables were compared by Tukey test (P < 0.05). The distributions of frequency were compared by c2 (chi square). A total of 579 parturitions<br />
were evaluated to accomplish 108 animals with RP. Thus, the overall occurrence of RP was 18.6%. There were no significant differences<br />
between treatments for any analyzed variable. The overall mean for P/IA, P/C and S/C were 65.1±17.5 d, 3.0±1.0 and 154.6±42.0 d,<br />
respectively. The percentage of animals with CL did not differ between treatments (P > 0.3), and 55.6% (60/108) of cows had CL between 25<br />
and 40 days postpartum. Combining treatments in two groups according to cloprostenol injections – with cloprostenol or without cloprostenol<br />
– the interval from parturition to AI was different (P < 0.05) between groups (59.2 ± 16.9 vs. 70.8 ± 18.3 d, respectively). It is concluded that<br />
the route of administration of florfenicol did not influence the reproductive performance of postpartum dairy cows in postpartum, however,<br />
cloprostenol reduced the interval from calving to first AI. [Support: FAPEMIG].<br />
Keywords: reproductive eficiency, puerperium, uterine involution.<br />
s429
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A187 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
ESTROGEN SOURCES IN FEMALE SIRIO HAMSTERS (<br />
(MESOCRICETUS AUR<br />
URATUS<br />
TUS) ) AFTER OVARIET<br />
ARIETOMY<br />
Kelly Annes 1 , Marie Odile M. Chelini 2 , Nivea Lopes Souza 3 , Silvia Renata Gaido Cortopassi 3 & Marcella Pecora Milazzotto 1<br />
1<br />
CENTRO DE CIÊNCIAS NATURAIS E HUMANAS - UFABC, SANTO ANDRÉ, SP, BRAZIL. 2 INSTITUTO DE PSICOLOGIA - USP, SÃO PAULO, SP, BRAZIL. 3 FACULDADE DE MEDICINA<br />
VETERINÁRIA E ZOOTECNIA - USP, SÃO PAULO, SP, BRAZIL.<br />
The technique of doses of steroid hormones in feces is increasingly used in animals of small size, a characteristic that makes it<br />
difficult to obtain blood samples. In a previous work estrogen and progesterone were measured in feces and blood of non-treated females (IN)<br />
and ovariectomized (OV). Although quantitative differences between the methods (there was no difference between the levels of fecal estrogen<br />
groups, since the serum concentrations of estradiol were different), the possibility of detection of these metabolites in the feces is shown in the<br />
study of relevant reproductive physiology these animals. Based on this data, the hypothesis of this work is that although female Sirio hamsters<br />
(Mesocricetus auratus) OV produce estrogen in non-ovarian tissues which cannot be detected in sistemic circulation, these metabolites may be<br />
quantified in their feces. Therefore, our objective was to verify the presence and quantify estrogen metabolites in feces of non-treated females<br />
(IN) or OV and quantify the gene expression of the responsible for the synthesis of aromatase (ARO) in non-ovarian tissues (brain, liver, adrenal<br />
glands and gastric mucosa). For such, 11 adult females were randomly divided in two groups: OV (n = 6) and IN (n = 5). Estrogen<br />
concentrations from the OV and IN animals was followed for 7 months after the ovariectomia. After this period the females were euthanized for<br />
the target tissue gathering for the analysis of the ARO expression by RT-PCR. The collect of feces were made before the OV of 4 on 4 h for 4<br />
days, the day of OV and 8 days later (once a day), from 30 days and every 30 days (produced by 24 for 6 months ). Estrone, estriol, estradiol<br />
was extracted by suspension in methanol 80% and quantified by enzyme immunoassay. The analysis of the ARO expression showed that there<br />
was no difference on this enzyme expression in the brain in OV or in IN, there was expression in the liver and gastric mucosa, nevertheless, the<br />
expression in adrenals was greater in OV. Before surgery, the 11 females showed fecal metabolites curve exactly parallel the dosage blood, with<br />
6 h delay. The short and long term after surgery, OV females showed marked and steady decline of fecal estrogens. After challenge with ACTH<br />
dosage of the curve was similar of non-treated females obtained before surgery. We conclude that the adrenal has become a major source of<br />
estrogen in females OV, and these metabolites in the feces of these animals. However, further studies would be required for the determination of<br />
the majority of estrogen metabolite detected in feces. [Acknowledgments: FAPESP process 2009/ 52750-8].<br />
Keywords: hamster, oestrogen, ovariectomy.<br />
A188 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
IMMUNOLOC<br />
OCALIZA<br />
ALIZATION OF THE PIDERMAL GROW TH FACT<br />
CTOR (EGF), TRANSFORMING GROW TH FACT<br />
CTOR-<br />
ALPHA (TGF<br />
GFA) AND EGF RECEPTOR (EGFR) IN THE OVAR<br />
ARY OF THE BITCH<br />
Diogo José Cardilli 1 , José Félix Pérez-Gutiérrez 2 , Kellen De Sousa Oliveira 1 , Carolina Franchi João 3 , Fabiana Azevedo Voorwald 1 ,<br />
J oão Ademir Oliv<br />
liveir<br />
eira 1 & Gilson Hélio Toniollo<br />
1<br />
1<br />
FCAV/UNESP, JABOTICABAL, SP, BRAZIL. 2 UCM-MADRID, MADRID, ESPANHA. 3 UFP-CASTANHAL, CASTANHAL, PA, BRAZIL.<br />
The EGF and the TGFa, belong to the same family. They exhibit 40-50% structural homology; both bind to the EGFr and induce<br />
similar biological effects: cellular division, differentiation and survival. Previous studies had shown their role in a wide variety of physiological<br />
functions including oocyte maturation in different species such as: porcine, rodents, primates, bovine and canine; however their localization in<br />
the ovary of the bitch has not been examined. The aim of this study was to determine the distribution of EGF, TGFa and EGFr in the ovary of<br />
the bitch. Reproductive tracts from 34 bitches at the different phases of the estrous cycle were collected, fixed and processed with paraffin<br />
embedding. Sections were cut, mounted, dried, deparaffinized and rehydrated. Immunocytochemistry was performed using a 1:20 dilution of the<br />
following antisera: anti-EGF-pc (Ab-3, Calbiochem), Anti-TGFá-mc (Ab-2) and anti-EGFr receptor-mc (Ab-5). Samples were incubated<br />
overnight. Primary antibody binding was detected using a biotynilated secondary goat-anti-rabbit or rabbit anti-mouse IgG with avidin-biotinperoxidase<br />
complexing. The detection reagent was AEC (Vector). Immunohistochemistry revealed the presence of EGF, TGFα and EGFr in the<br />
ovary of the bitch. Within the cell, both ligands, EGF and TGFá were localized in the cytoplasm, while EGFr staining was nuclear. EGF<br />
immunolocalized in the follicular cells (FC) and in the oocytes of secondary and tertiary follicles, as well as, in the vascular endothelium,<br />
granulosa cells strings and in the luteal cells (LC). Positive reaction to TGF-α was found in the superficial epithelium, stroma, cortical tubules,<br />
vascular endothelium, LC, as well as, in the FC and oocytes of primary, secondary and tertiary follicles. EGFr was immunolocalized in the<br />
superficial epithelium, stroma, cortical tubules, vascular endothelium, LC and FC of primary, secondary and tertiary follicles. This study<br />
describes the localization of EGF system in the ovary of the bitch and suggest its participation in the regulation of ovarian functions such as<br />
follicular growth and luteinization.<br />
Keywords: stroma, immunocytochemistry, bitches.<br />
s430
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A189 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
INDUCTION OF PAR<br />
ARTURITION<br />
WITH AGLEPRIST<br />
GLEPRISTONE IN EWES<br />
Fernanda Machado Regazzi, Liege Garcia Silva, Gisele Almeida Lima Veiga, Cristina Fátima Lucio, Guilherme Cain de Oliveira,<br />
Daniel Souza Ramos Angrimani, Claudia Barbosa Fernandes & Camila Infantosi Vannucchi<br />
DEPARTAMENTO DE REPRODUÇÃO ANIMAL - FMVZ - USP, SÃO PAULO, SP, BRAZIL.<br />
The maintenance of pregnancy in mammals requires the binding of progesterone to its endometrial receptor, promoting the<br />
proliferation of epithelial and vascular uterine cells. In sheep, increases in production of progesterone are essentially of placentary origin after 50<br />
days of pregnancy. Placental progesterone is sufficient to maintain pregnancy in the absence of the corpus luteum. Antiprogestagens of indirect<br />
action, such as corticosteroids and analogs of the prostaglandin F2α, lead to luteolysis but interval between treatment and labor is variable and<br />
lengthy. The use of aglepristone for this purpose shows satisfactory results in some species such as cows and goats. The aglepristone is an active<br />
antiprogestagen that acts in late pregnancy, as a competitive inhibitor of the uterine progesterone receptor causing the end of pregnancy. So far,<br />
there are no reports of the aglepristone as an inducer of labor in sheep. The assessment of an efficient protocol for induction of parturition in sheep<br />
can provide appropriate medical intervention in cases of pregnancy toxemia, prolonged pregnancy or for the synchronization of delivery. The aim<br />
of this study was to assess the efficacy of aglepristone (Alizin ® ) to induce parturition in pregnant ewes, especially during the period of pregnancy<br />
in which placental progesterone synthesis is still intense, and observe possible side effects. Pregnant ewes were allocated into 2 groups: preterm<br />
group I that received aglepristone with 133 days of gestation (n = 4, 3 twin pregnancies) and preterm group II that received treatment with 143<br />
days of gestation (n = 4, 2 twin pregnancies). Ewes received two subcutaneous (SC) injections (0.33 mL / kg / day) of aglepristone 24 h apart.<br />
Means were compared by student’s “t” test (P = 0,05). For ewes of the group I, the first signs of labor were noted 44h±5h after the first injection<br />
of aglepristone. The group II showed interval between the first medical induction and labor of 40h±3h, with no statistical difference between<br />
groups (P= 0,25). No statistical difference (P = 0,07) was observed between labor induction and parturition in singleton pregnancies (38h ± 2<br />
h) compared to twins (44h ± 4h). It was observed that aglepristone effectively induced labor in all ewes, regardless of treatment. The complete<br />
elimination of fetal membranes was observed after 8h±2h of fetal expulsion in group I and after 4h±2h in group II. Only one animal from group<br />
I had retained placenta, with time of placenta expulsion of more than 12 h (12 h and 55 min). This study demonstrated, for the first time, that<br />
aglepristone can be used for the induction of parturition in sheep with satisfactory efficiency, both in single and twin deliveries, with no significant<br />
side-effects. [Support and acknowledgments Virbac Animal Health].<br />
Keywords: parturition, aglepristone, ewes.<br />
A190 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EVIDENCE OF THE NITRIC OXIDE IMPORTANCE TO IN VITRO DEVELOPMENT OF BOVINE EMBRYO<br />
Priscila Di Paula Bessa Santana 1 , Thiago Velasco Guimarães Silva 1 , Bruno Baraúna da Silva 1 , Nathália Nogueira da Costa 1 , Davi<br />
César Nascimento dos Santos 1 , Stefanne Dhullia Braga Conceicão 1 , Marcela da Silva Cordeiro 2 , Simone do Socorro Damasceno<br />
Santos 1 , Otávio Mitio Ohashi 1 & Moysés dos Santos Miranda 1<br />
1<br />
UFPA, BELEM, PA, BRAZIL. 2 IFPA, BELEM, PA, BRAZIL.<br />
N<br />
Nitric Oxide (NO) has multiple cellular functions by acting as a cellular messenger or reacting with oxigen reactive species for cell<br />
protection (2005, Molecular Aspects of Medicine 26, 3-31). The role of NO during bovine pre-implantational development is unknown. The<br />
goal of this work is to evaluate the importance of NO production during the in vitro culture of bovine embryos in an indirect way by using a NO<br />
sinthesis inhibitor (L-NAME; N-nitro-L-Arginine Metil Ester). Bovine COCs obtained from ovaries of the slaughterhouse were in vitro<br />
matured in TCM199 supplemented with 0.5 µg/mL of FHS, 50 µg/mL of LH, 50 µg/mL of gentamicin, 10 mg/mL of pyruvate and 10% of FBS<br />
for 24h. Groups of 20 mature oocytes were fertilized with 2X106 of spermatozoa/drop for 24h. The presumptive zygotes were cultivated for 8<br />
days in SOF medium (suplemmented with 50 µg/mL of gentamicin, 0,6 mg/mL of BSA, 5% de FBS) in an incubator at 38.5°C and 5% CO2<br />
in air. For the experiment 10 mM of L-NAME and 200 mM of Glutamine (Gln), an aminoacid which leads to NO production (1999, Journal of<br />
Surgical Research 86, 213-219) were added to SOF medium according to the experimental groups: SOF medium alone (SOF), SOF medium<br />
with L-NAME (SOF-NAME), SOF medium with Gln (SOF-Gln), and SOF medium with Gln and L-NAME (SOF-Gln-NAME). Cleavage<br />
and blastocyst rates of the groups SOF (n = 120), SOF-NAME (n = 131), SOF-Gln (n = 119), e SOF-Gln-NAME (n = 127) were evaluated<br />
at 2nd and 8th day of culture, respectively. The results of 6 repetitions were analyzed by ANOVA and Bonferroni post-hoc test and significance<br />
level of 5%. Cleavage rates were similar between the treatments (P > 0.05). Treatment with L-NAME impaired bovine embryo production only<br />
in the absence of Gln (rates of 22,16% and error of median of 5,27 vs. 33,75% ± 6,06 vs. 38,80% ± 4,13 for SOF-NAME, SOF and SOF-Gln-<br />
NAME; P< 0,05). Also, supplementation of SOF medium with Gln only had no effect on the embryo production rate (rates of 49,11% and error<br />
of median of 6,44 vs. 33,75% ± 6,06 to SOF-Gln and SOF respectively; P > 0,05). The results suggest that the NO production it seems to be<br />
important to in vitro development of bovine embryos, and the addition of Gln can reverse the effect of L-NAME on the embryo culture .<br />
Complementary studies has been doing to evaluate directly the production and the importance of NO at specific moments during bovine embryo<br />
development in ivtro. [Acknowledgements: CNPq, FAPESPA and UNOPAR].<br />
Keywords: bovine, ivc, nitric oxide.<br />
s431
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A191 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
INFLUENCE OF FOLLICLE STIMULATING TING HORMONE ON GENES INVOL<br />
OLVED IN THE REGULATION OF MATUR<br />
TURATION TION AFTER<br />
CULTURE OF BOVINE CUMULUS-OOCY<br />
US-OOCYTE COMPLEXES<br />
Pedro Ratto Lisboa Pires, Kátia Regina Lacelotti Schwarz, Lígia Garcia Mesquita, Marcos Roberto Chiaratti, Laís Vicári Figueredo Pessôa, Tiago Henrique<br />
Câmara De Bem, Paulo Fantinato Neto, Fabiane Gilli Zaffalon & Cláudia Lima Verde Leal<br />
USP, PIRASSUNUNGA, SP, BRAZIL.<br />
Oocyte maturation is a process involving several changes at ultrastructural, molecular and biochemical levels, adapting the oocyte<br />
for fertilization and embryo development. Such changes occur both in the oocyte and its companion cumulus cells, which are intimately involved<br />
in the process. The present study aimed to evaluate the effect of FSH on relative abundance of mRNA coding for genes of signaling pathways<br />
related to maturation in bovine oocytes in vitro, the nitric oxide pathway, represented by its synthesis enzymes (NOS2 and NOS3) and the cAMP<br />
pathway represented by synthesis (ADCY6 and ADCY9) and degradation enzymes (PDE3A, PDE4D and PDE8A). Cumulus-oocytes<br />
complexes were aspirated from slaughterhouse ovaries and cultured for 24h in maturation medium (TCM-199 with 0.2 mM pyruvate, 10 µg/<br />
mL gentamycin and 0.1% polyvinylalcohol) with or without recombinant human FSH (0.05I U/mL rhFSH) at 38.5°C and 5% CO2 in air and.<br />
After maturation, the oocytes and their respective cumulus cells were separated and then evaluated for transcripts relative abundance for the<br />
following genes: NOS2, NOS3, ADCY6, ADCY9, PDE3A, PDE4D and PDE8A. For oocytes, only NOS2 and PDE4D transcripts underwent<br />
changes on relative abundance (P > 0.05). NOS2 was downregulated after maturation with or without FSH, but downregulation was more<br />
intense in the presence of FSH (P < 0.05). However, PDE4D transcripts were downregulated only after maturation without FSH. For culumus<br />
cells NOS2, NOS3, ADCY9, PDE4D and PDE8A, also showed changes in transcripts relative abundance (P < 0.05). NOS2, NOS3, and<br />
ADCY9 presented similar behavior and underwent downregulation when cultured with FSH PDE4D and PDE8A also were downregulated<br />
when cultured without or with FSH, respectively. Thus, we can conclude that under the conditions of this study, rhFSH influences the expression<br />
of genes from the nitric oxide and the cAMP signaling pathways during in vitro maturation of bovine cumulus-oocyte complexes.<br />
Keywords: oocyte, FSH, in vitro maturation.<br />
A192 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
INFLUENCE OF THE BULL AND THE GRADE OF MORPHOLOGIC<br />
OGICAL QUALIT<br />
ALITY OF CUMULUS-OOCY<br />
US-OOCYTE<br />
COMPLEXES<br />
IN VITRO BOVINE EMBRYONIC DEVEL<br />
VELOPMENT<br />
OPMENTAL<br />
Rafaella Curvelano Lemes 1 , Fernanda Prado Elias 1 , Adriana Renzi 1 , Reginaldo Aparecido Vila 1 , Claudia Cristina Paz 2 & Raysildo<br />
Barbosa Lôbo 1<br />
1<br />
FACULDADE DE MEDICINA DE RIBEIRÃO PRETO, UNIVERSIDADE DE SÃO PAULO, RIBEIRAO PRETO, SP, BRAZIL. 2 INSTITUTO DE ZOOTECNIA - APTA/SAA, RIBEIRAO PRETO, SP,<br />
BRAZIL.<br />
During folliculogenesis, there is a bidirectional communication in cumulus-oocyte complex (COC) that enables the oocyte<br />
maturation through the organelles rearrangement and mRNAs and proteins accumulation that will allow the full competence of the gamete to<br />
be fertilized and progress on embryonic development. These COCs have different grades of morphological quality, which can influence the<br />
embryo quality, as well as the male gamete. The aim of this work was to establish a relationship between the grades of morphological quality<br />
of the COCs, the influence of the bull and embryo production. Grades I, II and III of morphological quality of COCs were extracted from<br />
ovaries of slaughtered cows, being collected 1244, 1283 and 1321, respectively. They were matured in modified TCM199 by 24h to 38.8°C<br />
and 5% CO2 and were fertilized with semen from five bulls, in triplicate (2.106 sperm/mL). After 12h, the embryos were cultured in modified<br />
CR2 medium by 168h to 38.8°C and 5% CO2. In the fourth and seventh days after IVF, the number of embryos at stage 16-32 cells and viable<br />
embryos cells was counted, respectively. ANOVA results showed that in the fourth day after IVF, the number of embryos produced in relation<br />
the initial number of COCs undergoing IVP was influenced by the grade of morphological quality (P < 0.0001), with higher number of embryos<br />
for COCs I and less for COCs III. By excluding the factor bull, the influence of grade of morphological quality persisted (P = 0.0006), showing<br />
that in this phase can analyze better the intrinsic competence of COCs in progress in embryonic development, since the embryos that did not<br />
activate the embryonic genome were discarded. The analysis of the relationship between the number of embryos on the seventh day by the<br />
number of embryos on the fourth day after IVF showed the bull factor’s influence (P= 0.0004) in the continuation of embryo development<br />
after embryonic genome activation. That is, there is a sperm-oocyte interaction, which is the occurrence of variations where the fertility rate<br />
of a given set of COCs is lower when the mating is done with a particular bull in comparison to others. The analysis showed that the kinetics<br />
of development is influenced by the grade of morphological quality in intermediate stages, blastocyst and expanded blastocyst, while the bull<br />
influence in the final stages, expanded blastocyst and hatched blastocyst. Thus, it’s possible to conclude that the cumulus cells actively<br />
interact with the oocyte, possessing an important role during folliculogenesis, fertilization and preimplantation embryo development.<br />
Keywords: cumullus oocyte complex, morphological quality, bovine.<br />
s432
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A193 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
BOVINE EMBRYO MODULATES GENE EXPRESSION OF UTERINE GROWTH FACT<br />
CTORS<br />
Alan Diego Bezerra Lira 1 , Abimael Estevam Silva Júnior 1 , Luciana Alves Fátima 2 , Lawrence de Oliveira Barros 3 , Paula de Carvalho Papa 2 & Danila Barreiro<br />
Campos 1<br />
1<br />
UFPB, AREIA, PB, BRAZIL. 2 USP, SAO PAULO, SP, BRAZIL. 3 UFRPE, RECIFE, PE, BRAZIL.<br />
In most species the molecular basis of maternal-fetal communication are not well defined, and studies related to the modulation of<br />
uterine events by the embryo are limited. Kashiwagi et al. (2007, Kashiwagi, A., Endocrinology 148, 4173-4184) showed that the mouse<br />
embryo has an active role in establishing the uterine environment during implantation, regulating development and differentiation of maternal<br />
endometrium. The observation that caruncles of the pregnant uterine horn are more developed than those from the non-pregnant horn in cattle,<br />
and that in pregnancies from in vitro-produced embryos several anomalies are detected, including insufficient caruncular development and<br />
vascularization, led us to hypothesize that not only the ovarian hormones control caruncular development, but also fetal factors are involved. This<br />
study aimed to investigate the role of bovine embryo in the modulation of VEGF and bFGF system gene expression in uterine caruncles.<br />
Caruncles from pregnant and non-pregnant uterine horns were collected at 35 days of gestation and total RNA was isolated using Trizol ® reagent<br />
(Invitrogen, Carlsbad, USA) and specific columns for RNA extraction. Samples were treated with DNase and reverse-transcription was<br />
performed using the Superscript III kit (Invitrogen, Carlsbad, USA) and 1µg of RNA. Expression of VEGF and its receptors Flt-1 and KDR,<br />
and bFGF and its receptors R1, R2 and R4 was determined by real-time PCR, using tubulin as the endogenous control. Data were expressed<br />
relative to tubulin and calculated by ÄÄCt method with correction by the amplification efficiency. Results showed that expression levels of bFGF<br />
and its R2 receptor were significantly lower in the caruncles of the pregnant horn when compared with caruncles of the non-pregnant horn, with<br />
no variation in the abundance of receptors R1 and R4, as well as VEGF and its receptors Flt-1 and KDR. VEGF and bFGF growth factors are<br />
described as the most important factors controlling angiogenesis in the placenta, however, Mingju et al. (2006, Mingju, C., Matrix Biology 25,<br />
342-354) have demonstrated that bFGF does not alter levels of cellular proliferation and inhibits expression of genes related to tissue remodeling<br />
in granulosa cells. Thus, the reduced expression of bFGF and its receptor R2 in caruncles of pregnant uterine horn could be associated with<br />
intense tissue remodeling occurring during placentation. Results suggest that the bovine embryo may be involved in the process of caruncular<br />
development through modulation of uterine growth factors gene expression.<br />
Keywords: growth factors, caruncle, embryo.<br />
A194 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
NEW MEDIUM FOR THE USE OF BRILLIANT CRESYL BLUE IN OOCYTE SELECTION FOR IN VITRO SWINE<br />
EMBRYOS PRODUCTION - PRELIMINARY DATA<br />
Elisa Caroline da Silva Santos 1 , Jorgea Pradieé 1 , Alexander Oliveira Gonçalves 1 , Rafael Gianela Mondadori 1 , Thomaz Lucia Jr. 1 ,<br />
Arnaldo Diniz Vieira 1 & Ligia Margareth Cantarelli Pegoraro 2<br />
1<br />
UFPEL, PELOTAS, RS, BRAZIL. 2 EMBRAPA/CPACT, PELOTAS, RS, BRAZIL.<br />
N<br />
Selection of competent oocytes improves the in vitro embryo production outcomes. Among the available methods of selection,<br />
Brilliant Cresyl Blue (BCB) is a cell viability dye, which determines the presence of glucose-6-phosphate dehydrogenase. This enzyme is active<br />
in growing oocytes and inactive in the fully grown oocytes. Thus, BCB stained oocytes are considered more competent, because they already<br />
reached their full growth. This selection method was previously tested in several species, however, the results of its efficiency remains<br />
contradictory. Some studies in which exposure to the dye resulted in a decrease of oocyte viability suggested that the maintenance medium (PBS),<br />
and time of exposure during the staining process may be relevant for the decrease of oocyte viability. This study tested the modified “porcine<br />
zygote medium” (mPZM-4) for the staining of porcine oocytes with BCB as well as reducing the dye exposure time from 90 to 60 min. We used<br />
388 cumulus oocyte complexes (COCs) obtained from abattoir ovaries, distributed on media containing 26 µM BCB on mPZM-4 (n = 133),<br />
PBS (n = 142) and control (mPZM-4 without BCB, n = 113). The COCs were maintained for 60 min in BCB solution at 39°C. Subsequently,<br />
they were separated in five groups according to the dye reaction: mPZM-4 dyed and not-dyed, PBS dyed and not-dyed and control. After the<br />
exposure to the dye and selection, all COCs were submitted to in vitro maturation. In the first 24 h IVM was conducted in mPZM-4 medium<br />
containing EGF, LH, FSH, c-AMP and porcine follicular fluid. The final 24h maturation period was conducted in the same medium, without<br />
hormones and cAMP. The nuclear maturation was evaluated with Hoechst (Sigma ® H33342) using epifluorescence microscopy. Metaphase II<br />
(MII) oocytes were considered matured. Data were compared using Chi-square test. The rate of MII was 38% in the control group (n = 113),<br />
23% in dyed mPZM-4 (n = 91), 7% in not dyed mPZM-4 (n = 42), 24% in dyed PBS (n = 82) and 19% in not dyed PBS (n = 60). The results<br />
show that mPZM-4 and PBS can be used for BCB staining. The rate of IVM in the not dyed mPZM-4 group was lower than control (P < 0.05).<br />
These preliminary results show that the BCB diluted in mPZM-4 and an exposure period of 60 min, can be used to select more competent<br />
structures to in vitro swine embryo production. To confirm the data more repetitions will be done.<br />
Keywords: brilliant cresyl blue, porcine oocytes, maintenance médium.<br />
s433
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A195 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
THE WNT SIGNALING-ANTAGONIST GONIST DKK1 DOES NOT IMPAIR DEVEL<br />
VELOPMENT OF BOVINE EMBRYOS<br />
TO THE BLAST<br />
ASTOCY<br />
OCYST<br />
STAGE<br />
Kyle B. Dobbs 1 , Anna Carolina Denicol 2 & Peter J. Hansen 1<br />
1<br />
UNIVERSITY OF FLORIDA, GAINESVILLE, FL, USA. 2 EMBRAPA GADO DE LEITE - CAMPO EXPERIMENTAL SANTA MONICA, VALENÇA, RJ, BRAZIL.<br />
The WNT signaling pathway has been identified to be crucial for mammalian development during embryonic and fetal stages.<br />
WNT signaling activation and inactivation by antagonists such as Dickkopf-related protein 1 (DKK1) work in balance to regulate embryo<br />
development (Lieven et al., Dev Biol 340, 256-258, 2010). Previous findings from our lab demonstrated that activation of WNT signaling in<br />
bovine embryos at day 5 after insemination decreased blastocyst development by day 8. Here we tested whether DKK1 would improve<br />
development and developmental dynamics of blastocysts. If so, it is likely that WNT agonists are produced by the embryo in culture. Beginning<br />
at day 5 or 6 after insemination, embryos were cultured with 0, 50, 100, 200 or 400 ng/mL DKK1. DKK1 is an antagonist of WNT co-receptors<br />
LRP5/6 and a concentration of 100 ng/mL was shown previously to block WNT signaling in bovine embryos. The experiment was performed<br />
in 3 replicates using 10-63 embryos/treatment. The percent of embryos that reached the blastocyst stage and the percent of blastocysts in<br />
advanced stages (expanded, hatching or hatched) was assessed at day 8 after insemination. Data was analyzed by ANOVA (P < 0.05 considered<br />
significant). When DKK1 was added to culture at day 5, the percent of embryos that were blastocysts was not affected by treatment. At day 8,<br />
percent blastocysts were 58.6, 33.1, 48.5, 31.2 and 41.8% (SEM=7.6). DKK1 also did not affect the percentage of blastocysts that were<br />
advanced [Day 8: 89.2, 90.7, 97.2, 77.6 and 94.4% (SEM=7.7) for 0, 50, 100, 200 and 400 ng/mL]. Similar results were obtained when DKK1<br />
was added at day 6. The percent embryos that were blastocysts at day 8 was 32.7, 33.9, 39.3, 31.4 and 41.0% for 0, 50, 100, 200 and 400 ng/<br />
mL, respectively (SEM=5.0) and the percent of blastocysts that were advanced at day 8 was 72.9, 75.6, 65.4, 79.5 and 72.6% (SEM=8.4). In<br />
conclusion, inhibition of WNT signaling by DKK1 did not affect development of bovine embryos cultured in vitro to the blastocyst stage or the<br />
continued growth to advanced stages. Results suggest that there is little signaling through LRP5/6 mediated by WNT agonists of embryonic<br />
origin at this developmental stage. [Support: USDA-AFRI Grant No. 2009-65203-05732].<br />
Keywords: wnt signaling, dkk1, embryo.<br />
A196 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
THE EFFECT OF INSULIN-LIKE GROW TH FACT<br />
CTOR-I ON MITOCHONDRIAL ACTIVIT<br />
CTIVITY OF BOVINE OOCYTES<br />
EXPOSED TO HEAT SHOCK<br />
Jéssica Ispada 1 ; Rafaela Sanchez Lima 2 , Pedro Henrique Bugallo Risolia 2 , Mayra Elena Assumpção 3 , José Antonio Visintin 3 &<br />
Fabíola Freitas de Paula-Lopes 1<br />
1<br />
UNIFESP, DIADEMA, SP, BRAZIL. 2 UNESP, BOTUCATU, SP, BRAZIL. 3 USP, SÃO PAULO, SP, BRAZIL.<br />
Maternal heat stress compromises fertility of lactating dairy cows causing economical loss for the dairy industry. It has been<br />
shown that the oocyte and the preimplantation bovine embryo are major targets of the deleterious effects of heat stress (Hansen, Philos Trans<br />
R Soc Lond B Biol Sci. 2009 27; 364:3341-50). However the cellular mechanisms triggered by elevated temperature as well as the molecular<br />
events involved in oocyte survival and death in response to stress are not well known. Previous studies indicated that insulin-like growth<br />
factor-I (IGF-I) exerts a thermoprotective effect reducing heat-induced apoptosis in bovine oocytes (Ispada et al., III <strong>International</strong> Symposium<br />
on Animal Biology of Reproduction, 2010 7:333). Considering that many cellular stresses activates the mitochondrial pathway of apoptosis,<br />
the objective of the present study was to determine the effect of heat shock during IVM on bovine oocyte mitochondrial activity, and to<br />
evaluate the thermoprotective role of IGF-I in this context. Cumulus-oocyte complexes (COCs) were subjected to Control (38.5°C for 22 h)<br />
and Heat Shock (41°C for 14 h followed by 38.5°C for 8 h) treatments in the presence of 0 or 100 ng/mL IGF-I. Oocytes were mechanically<br />
denuded 22 h after IVM and subjected to the MitoTracker Red CMX-Ros assay (Invitrogen M-7512, Oregon, USA) to identify and quantify<br />
active mitochondria. Oocytes were incubated in TCM-199-Hepes containing 50 µg/mL polyvinyl alcohol and 50 nM MitoTracker at 37º C<br />
for 15 min, evaluated under fluorescence microscope and digital images stored as tiff files. Mitochondrial activity was quantified using the free<br />
software Image J 1.43. This experiment was replicated six times using 97-204 COCs/treatment. Data were analyzed by least-squares analysis<br />
of variance using the General Linear Models procedure of SAS. In the absence of IGF-I heat shock reduced (P < 0,001) mitochondrial activity<br />
from 64.31±1.91 in the control group to 56.74±1.26 arbitrary units in the heat shock group. Addition of IGF-I to maturation medium did not<br />
affect mitochondrial activity in the control group (66,25±1,56). However IGF-I improved (Temperature x IGF-I P < 0,001) mitochondrial<br />
activity of bovine oocytes subjected to heat shock (70,32±1,32). In conclusion, heat shock reduced bovine oocyte mitochondrial activity,<br />
suggesting activation of mitochondrial cascade of apoptosis. Moreover, IGF-I exerted a termoprotective role reducing such deleterious effect.<br />
[Fapesp 2007/53323-0; CNPq 478558/2008-2].<br />
Keywords: oocyte, heat shock, IGF-I.<br />
s434
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A197 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
THE ROLE OF INSULIN-LIKE GROWTH FACT<br />
CTOR-I ON DEVEL<br />
VELOPMENT<br />
OPMENTAL COMPETENCE OF BOVINE OOCYTES EXPOSED TO<br />
HEAT SHOCK<br />
Pedro Henrique Bugallo Risolia 1 , Rafaela Sanchez Lima 1 , Jéssica Ispada 2 , Mayra Elena Assumpção 3 , José Antonio Visintin 3 & Fabíola Freitas de Paula-<br />
Lopes 2<br />
1<br />
UNESP, SAO PAULO, SP, BRAZIL. 2 UNIFESP, DIADEMA, SP, BRAZIL. 3 USP, SÃO PAULO, SP, BRAZIL.<br />
Adverse environmental conditions such as elevated temperature and humidity compromise fertility of lactating dairy cows. The<br />
events associated with oocyte growth and maturation are susceptible to disruption by elevated temperature compromising the ability of the oocyte<br />
to undergo adequate fertilization and embryonic development. Such events are regulated by a variety of growth factors and dynamic communication<br />
between the oocyte and its surrounding cumulus cells. Previous studies indicated that insulin-like growth factor-I (IGF-I) exerts a thermoprotective<br />
effect reducing heat-induced apoptosis in bovine oocytes (Ispada et al., III <strong>International</strong> Symposium on Animal Biology of Reproduction, 2010<br />
7:333). Therefore, the objective of the current study was to determine the role of IGF-I on developmental competence of bovine oocytes exposed<br />
to heat shock. Cumulus-oocyte complexes (COCs) were exposed to control (38.5°C for 22h) or heat shock (41°C for 14h followed by 38.5°C<br />
for 22h) during in vitro maturation (IVM) in the presence of 0 or 100 ng/mL IGF-I. Oocytes were then subjected to in vitro fertilization and<br />
culture at 38.5°C. The percentage of oocytes that cleaved and developed to the blastocyst stage was evaluated on days 3 and 9 after fertilization,<br />
respectively. This experiment was replicated 7 times using 266-280 COCs/treatment. Day 9 blastocyst total cell number was determined with<br />
Hoechst 33342 (5 ìg/mL) labeling. Data were analyzed by least-squares analysis of variance using the General Linear Models procedure of SAS.<br />
There was no effect of Temperature, IGF-I or Temperature x IGF-I interaction on cleavage rate and blastocyst total cell number. However there<br />
was a Temperature effect on blastocyst development. In the absence of IGF-I, exposure of bovine oocytes to heat shock reduced (P < 0,05)<br />
blastocyst development from 33.0 + 4.93 to 16.0 + 4.93% for 38.5 and 41ºC, respectively. Similarly, in the presence of IGF-I, heat shock reduced<br />
(P < 0,05) blastocyst development from 19.44 + 4.93% to 14.32 + 4.93% for 38.5 and 41ºC. In conclusion, exposure of bovine oocytes to<br />
physiological heat shock of 41ºC during the first 14h-IVM reduced preimplantation embryonic development. Supplementation of IVM medium<br />
with 100 ng/mL IGF-I did not stimulated subsequent oocyte development and did not exert a thermoprotective effect. [Research supported by<br />
FAPESP 2007/53323-0 and 2010/07673-2; CNPq 478558/2008-2].<br />
Keywords: oocyte, heat stress, igf-i.<br />
A198 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EXPRESSION PROFILE OF HISTONE DEACETYLASE 2 (HDAC2) DURING DEVELOPMENT OF BOVINE EMBRYOS<br />
PRODUCED IN VITRO<br />
Allice Rodrigues Ferreira 1 , Grazieli Marinheiro Machado 2 , Isabela Rebouças Bessa 2 , Rosana Camargo Nishimura 2 , Roberto Sartori 1 ,<br />
Margot Alves Nunes Dode 3 & Maurício Machaim Franco 3<br />
1<br />
UNESP, BOTUCATU, SP, BRAZIL. 2 UNIVERSIDADE DE BRASILIA, BRASÍLIA, DF, BRAZIL. 3 EMBRAPA CENARGEN, BRASILIA, DF, BRAZIL.<br />
N<br />
During the pre-implantation embryo development in mammals, an extensive epigenetic reprogramming occurs. Histone acetylation<br />
is an essential epigenetic mechanism for normal embryo development being a dynamic and reversible process. This epigenetic event is controlled<br />
by two main classes of enzymes, histone acetyltransferases and histone deacetylases (HDAC). HDAC2 is responsible for the process of histone<br />
deacetylation, generally resulting in gene repression. The aim of this study was determine and quantify the expression pattern of HDAC 2 gene<br />
in 4-cell, 8-16 cell and morula bovine embryos produced in vitro. Cumulus oocyte complexes (COCs) were isolated from slaughterhouse ovaries<br />
and were matured in vitro and inseminated using semen from a Nellore bull. Embryos of each developmental stage: 4-cell (44 h p.i.), 8-16 cell<br />
(72 h p.i.) and morula (144 h p.i.) were produced. Three pools of 10 embryos for each stage were frozen at -80ºC until RNA extraction. Total<br />
RNA was isolated using the Invisorb RNA Spin Cell Mini Kit (Invitek ® ) according to the manufacturer’s protocol. cDNA was done using Oligo<br />
dT primers (Invitrogen ® ) and SuperScript III reverse transcriptase enzyme (Invitrogen ® ). Relative expression of target gene was assessed by<br />
qPCR using the Power Sybr ® Green PCR Master Mix (Applied Biosystems ® ) in an ABI Prism 7500 Fast Sequence Detection System (Applied<br />
Biosystems ® ). GAPDH gene was used as endogenous control. Relative abundance of HDAC 2 was calculated using the ÄÄ Ct method with<br />
efficiency correction. The identity of PCR products was confirmed by the amplicon size in agarose gel and by melting curve in qPCR. Gene<br />
quantification were compared among treatments using one-way analysis of variance (ANOVA) and Tukey test, in the Prophet program, version<br />
5.0 (BBN Systems and Technologies, 1996). Results showed significant difference (P < 0.01) among morula (13.08 ± 3.61) compared to 4-cell<br />
(0.97 ± 0.47) and 8-16 cell (1.64 ± 0.47) stages, showing a higher amount of HDAC 2 transcripts in morula stage. We speculated that this<br />
increase in transcription or the higher transcript accumulation of HDAC 2 gene during development is due to the necessity for subsequent histone<br />
deacetylation activity in the embryonic genome. [Acknowledgements: Projeto Rede de Inovação em Reprodução Animal (01.07.01.002).Financial<br />
support: FAPESP and EMBRAPA].<br />
Keywords: epigenetic, gene expression, bovine embryo.<br />
s435
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A199 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
LEPTIN GENE POLYMORPHISMS AND THEIR ASSOCIATIONS<br />
WITH PUBERTY IN NELLORE CATTLE<br />
TLE<br />
Luiz Lehmann Coutinho<br />
1 , Gusta<br />
ustavo Gaspar<br />
asparin<br />
1 , Fer<br />
ernanda Savalio<br />
alio 1 , Gerson Bar<br />
arret<br />
eto Mourão<br />
1 , Jose B.S.<br />
Fer<br />
erraz<br />
2 , Luis F.P<br />
.P. Silv<br />
ilva 2 & Joanir P. Eler<br />
2<br />
1<br />
USP/ESALQ, PIRACICABA, SP, BRAZIL. 2 USP, PIRASSUNUNGA, SP, BRAZIL.<br />
In the past few years, the leptin gene (LEP) has been subject of intense investigation in cattle due to its association with economic<br />
traits such as energy metabolism, efficiency and food intake, fat deposition and reproductive characteristics. Previous studies suggest that this<br />
gene may be involved in the onset of puberty in animals and humans. Given the importance of this gene for the livestock, the objectives of this<br />
study were to identify single nucleotide polymorphisms (SNPs) in the leptin gene and its promoter (PLEP) and evaluate the effects of these SNPs<br />
on puberty. DNA was extracted from subcutaneous adipose tissue samples collected from 28 Nellore breed heifers aged around 25 months, 14<br />
pre-pubertal and 14 pubertal, and DNA extracted from peripheral blood of 96 female Nellore, from another herd, 48 of them with high expected<br />
progeny difference (EPD) for probability of pregnancy at 14 months (PP14) and 48 that have low DEP for PP14. In the 28 heifers, sequencing<br />
of three regions of the leptin gene and three regions of the leptin gene promoter allowed the identification of 37 polymorphisms, 20 of them had<br />
not been previously reported, which formed 10 distinct haplotypes. In the 96 Nellore females, only a fragment of 715 base pairs was sequenced,<br />
on which 11 SNPs were identified. None of the SNPs and haplotypes formed from the polymorphisms was associated with puberty in the 28<br />
heifers. However, in the 96 females, the haplotype-substitution effect had suggestive association (P = 0.09) with DEP for PP14, with a haplotipic<br />
substitution effect of -9.51. Homozygous animals for haplotype 2 transmitted for the progeny less 19.02 percentile units of chance to conceive<br />
at 14 months, than animal homozygous for haplotype 1. This study identified new polymorphisms in LEP and PLEP and presented initial results<br />
of polymorphisms association with puberty in Nellore.<br />
Keywords: dna, leptin, puberty.<br />
A200 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
PRODUCTION OF BOVINE EMBRYONIC CHIMERAS BY AGGREGA<br />
GGREGATION OF DIPLOID (<br />
(BOS<br />
TAUR<br />
URUS<br />
US) ) AND<br />
TETRAPLOID EMBRYOS (<br />
(BOS INDICUS)<br />
Eduardo Montanari Razza 1 , Rafael Augusto Satrapa 1 , Isabele Picada Emanuelli 1 , Ciro Moraes Barros 1 & Marcelo Fábio Gouveia<br />
Nogueira 2<br />
1<br />
DEPARTAMENTO DE FARMACOLOGIA, IB, UNESP, BOTUCATU, SP, BRAZIL. 2 DEPARTAMENTO DE CIÊNCIAS BIOLÓGICAS, FCL, UNESP, ASSIS, SP, BRAZIL.<br />
The embryonic chimeras production by aggregation allows embryonic cells from two fertilizations to be randomly distributed in<br />
order to form a single individual. However, the formation of tetraploid embryos (4n) by electrofusion and their subsequent chimerism with a<br />
diploid embryo (2n) must result in a chimeric conceptus, whose inner cell mass (ICM) is entirely 2n. Hence, the aggregation of a zebu embryo<br />
(4n, thermotolerant) with a taurine embryo (2n, thermosensitive) would result in an exclusively taurine MCI, but with trophectoderm (future<br />
extraembryonic components, i.e. the chorion) mostly from zebu embryo, which could support taurine embryo/fetus to adapt - during pregnancy<br />
- in tropical climate. Thus, the purpose of this study was to standardize the production of 4n Nelore embryos (Bos indicus) and the production<br />
of embryonic chimeras by aggregation of Bos taurus (2n) with Bos indicus (4n) embryos. Oocytes from Nelore and Holstein cows from abattoir<br />
were matured, fertilized with semen from Nelore and Holstein bulls, respectively, and cultured in SOF (synthetic oviduct fluid). Two-cell stage<br />
Nelore embryos (30 h post insemination, hpi), with a well defined inter-blastomeric axis, were selected for electrofusion procedure (ECM 830-<br />
BTX, Harvard Apparatus) to produce tetraploid embryos. For this procedure, some parameters were tested according to the number of pulses<br />
(1 or 2), voltage (40, 50, 75, 100, 140, 500 V) and duration of electroshock (20, 25, 50, 60 µs). Nelore tetraploid embryos produced after<br />
electrofusion and diploid taurine embryos, both on 8 to 16 cells stage (72 hpi) were subjected to protease (Pronase ® ) treatment to remove the zona<br />
pellucida, and subsequently treated with the agglutinant agent phytohemagglutinin. Bos indicus (4n) and Bos taurus (2n) embryos were added<br />
in pairs (4n+2n) into individual wells (WOW) for culture until the blastocyst stage to validate the chimeric embryos formation. Among the tested<br />
parameters, the best fusion results (92%) and rates of cleavage after-fusion (66%) were obtained with a single pulse of 75 V for 60 µs. The<br />
production of embryo chimeras by aggregation (4n+2n) is on its final stage of standardization. The formation of bovine embryonic chimeras<br />
[Bos indicus (4n) + Bos taurus (2n)], with a non-random pattern of distribution of their cell aggregates, will enable the validation of this technique<br />
in applied research, by producing exclusively taurine calves, but with placental elements from Bos indicus breed, following transfer of these<br />
chimeras into recipient cows. [We thank FAPESP for financial support and scholarships granted].<br />
Keywords: chimeric embryo, tetraploid embryo, bovine.<br />
s436
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A201 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
EMBRYO PRODUCTION BY PAR<br />
ARTHENOGENETIC ACTIV<br />
CTIVATION TION AND IVF IN CAPUCHIN MONKEYS<br />
Sheyla Farhayldes Souza Domingues 1,2 , Julianne Silva de Lima 1,2 , Danuza Leite Leão 1,2 , Adriel Behn de Brito 1,2 , Rafael Vilar Sampaio 3 , Moysés dos Santos<br />
Miranda 1 , Otávio Mitio Ohashi 1 & Regiane Rodrigues dos Santos 4<br />
1<br />
UFPA, CASTANHAL, PA, BRAZIL. 2 UFPA, BELÉM, PA, BRAZIL. 3 UFOA, BELÉM, PA, BRAZIL. 4 UU, UTRECHT, HOLANDA, NL.<br />
The aim of the study was to evaluate the ability to produce in vitro Cebus apella embryos from in vitro matured<br />
oocytes submitted to IVF or parthenogenetic activation. Oocytes were obtained from 6 adult females of C. apella and<br />
submitted to IVM in TCM199 supplemented with 0.5 µg/mL FSH (Animal Bioniche Health, Bellevile, Canada) and 50 µg/mL<br />
LH (Bioniche Animal Health) for 40 h at 38.5°C in a humidified atmosphere of 5% CO2. Semen collection and liquefaction of<br />
seminal coagulum were carried according Oliveira et al. (<strong>2011</strong>, Animal Reproduction Science 123, 75-80). The semen was<br />
cooled in ACP-118 ® (Biotecnologia, Fortaleza, Brazil) using a decreasing curve of temperature of 36 °C for 4°C, in an interval<br />
of 90 min (- 0,4 °C/min) and remained in refrigerator until the moment of the IVF. For the IVF, 5 ìL of the semen (5x106 sptz/<br />
mL) in drops were added (~10 oocytes) contends 80 µL of Fert-TALP. The oocytes destined to the parthenogenetic activation<br />
had been distributed in treatments using 5 µM of ionomycin (Sigma Chemical Co., St. Louis, USA) for 5 min in association<br />
with 2 mM of 6-DMAP (Sigma Chemical Co.) or 50 µM of roscovitine (Sigma Chemical Co.) for 3 h. Embryo culture was carry<br />
out in SOF medium at 38.5°C in a humidified atmosphere of 5% CO2 for up to 7 days. All data were reported as mean ± SEM.<br />
Approximately 83% of the oocytes had reached metaphase MII (MII) after 40 h of IVM. Collected semen presented 80% of<br />
motility and vigor 4 after dilution in ACP-118 ® (Biotecnologia, Fortaleza, Brazil) and cooling for 6 h at 4°C. After 30 h-IVF,<br />
extrusion of the 2PB was observed in 40 ± 12% of the oocytes, and 20 ± 4% of formed zygotes cleaved to 4-cells. Cleavage after<br />
parthenogenesis was obtained only when oocytes were activated by 6-DMAP (8 ± 4%). Roscovitine treated oocytes formed<br />
haploid zygotes (15 ± 9%), but no cleavages were observed. This is the first study describing in vitro embryo production in C.<br />
apella. The results of the IVF indicate that cooling did not affect the viability of spermatozoa, suggesting that the energy of the<br />
spermatozoa was conserved until the moment of the fertilization. As well as the zygotes produced by IVF, the parthenotes<br />
activated with ionomycin/6-DMAP cleaved until 4-cells. The use of 6-DMAP resulted in better embryonic production that<br />
specific inhibitors of the MPF (roscovitine).<br />
Keywords: Cebus apella, ivf, parthenogenetic activation.<br />
N<br />
A202 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
SELECTION FOR POST WEANING DOES NOT CHANGE AGE AT PUBERTY IN HEIFERS<br />
Joaquim Mansano Gar<br />
arcia<br />
1 , Fabio Mor<br />
orato o Mon<br />
onteir<br />
eiro 2 & Angelo<br />
Tor<br />
orres Arevalo<br />
1<br />
1<br />
FCAV/UNESP, JABOTICABAL, SP, BRAZIL. 2 INSTITUTO DE ZOOTECNIA, SERTÃOZINHO, SP, BRAZIL.<br />
The evaluation of female´s reproductive parameters subjected to intense genetic selection for post weaning weight is an important<br />
information for the design of breeding programs for meat production improvement. The objective of this study was to evaluate the effect of post<br />
weaning weight selection on age at puberty in Nelore heifers based on ovaries and uterine horns ultrasound evaluation. Nelore heifers from<br />
selected herds (NeS; Nelore herd with greater differences for weight) and Nelore control herd (NeC: Nelore herd with close to zero differential<br />
for weight), from the Zebu breeding project of Instituto de Zootecnia of Sertãozinho/SP were utilized. A total of 125 heifers were used, being 47<br />
of the NeC and 78 of the NeS groups. We evaluated by transrectal ultrasound the endometrial thickness, ovary area, largest follicle and corpus<br />
luteum (CL) diameters (when present) in both ovaries, each 18 to 21 days, between 12 and 24 months of age (total of 18 evaluations). Heifers<br />
were considered pubertal after showing visible CL and concomitantly plasma progesterone levels above 1,5ng/ml quantified by radioimmunoassay.<br />
Statistical analysis was performed in SAS PROC MIXED using the REPEATED command. The first heifers ovulated (from NeS) at 18,4<br />
months. However, at the end of the evaluations (at 24 months), there were no difference between herds in percentual of puberty manifestation<br />
(NeC: 52% and NeS: 48%). The endometrial thickness and ovaries area average did not differ between NeC and NeS. At the last evaluation (at<br />
24 months of age) the NeC and NeS herds presented 11,74 mm and 11,80 mm of endometrial thickness and ovaries area of 3,88±0,09 cm2 and<br />
4,10±0,07 cm2, respectively (P > 0,05). The largest follicle diameter of heifers from NeS was higher in 8 of 18 evaluations compared to the NeC,<br />
but at the last evaluation (24 months), this measure did not differ between herds (NeC: 10,70 mm and NeS: 10,45 mm; P > 0,05). It was observed<br />
in the present study that genetic selection for weaning weight, made since 1981, did not influence the percentage of pubertal heifers at 24 months<br />
in a grazing management system. At 24 months of age, approximately 50% of heifers ovulated, however, no difference in the onset of puberty<br />
was observed between the analyzed herds. It was suposed that this high non pubertal heifers rate at 24 months was consequence of feed<br />
restriction after weaning, mainly due to low forage availability and the lack of feed supplementation for the heifers under development during the<br />
dry season.<br />
Keywords: Nellore, heifers, puberty.<br />
s437
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A203 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
SUPPLEMENTATION TION OF MEDIA FOR IN VITRO MATUR<br />
TURATION TION OF OOCYTES OF RABBIT<br />
ABBITS<br />
Tássia Mangetti Gonçalves, , Juliano Rodrigues Sangalli, Marcos Roberto Chiaratti & Flávio Vieira Meirelles<br />
FZEA- USP, PIRASSUNUNGA, SP, BRAZIL.<br />
In vitro maturation is an important tool for biotechnologies. The importance of rabbits in this studies is due to the later embryonic<br />
genome activation in this species, that turns it into an efficient model for dedifferentiate of somatic cell nuclei from many species (eg, pandas,<br />
chickens, monkeys, cats and humans), supporting the development of interspecies nuclear transfer (WEN et al., 2005, Journal of Experimental<br />
Zoology, 303:689-697). This study aimed to test supplementations of media for in vitro maturation of oocytes donated by immature rabbits using<br />
a protocol commonly used with bovine oocytes. During in vitro maturation (IVM) of rabbit oocytes it was tested addition of 0.01% PVA or<br />
0.003% BSA or control (no addition) in TCM 199 (10 mg/mL of FSH and 10 mg/mL of LH). After 20 h of IVM, oocytes denuded of cumulus<br />
cells by gentle pipetting in 0.2% hyaluronidase were analyzed for the presence of first polar body (1st PB). Due to the low rate of 1st PB<br />
extrusion, 1st PB-selected oocytes from the three experimental groups were activated together (5 mM ionomycin for 5 min and 2 mM 6-DMAP<br />
for 4 h) (Tao et al., 2008, Journal of Animal Physiology and Animal Nutrition, 92:438-447). Presumptive zygotes were cultured in CR2aa in a<br />
humidified atmosphere of 5% CO2 and 5% O2 for a maximum period of 4 to 5 days. Embryos were evaluated regarding their development to<br />
morulae or blastocyst stages and the number of nuclei. Four independent experiments were carried out. The extrusion rate of 1st PB was<br />
compared between groups using Tukey (P < 0.05). A significant increase in the rate of 1st PB extrusion was found when supplementation with<br />
BSA (13.6%, 40/294) or PVA (12.6%, 34/270) was performed in comparison to no supplementation (6.3%; 21/333). When 1st PB oocytes<br />
were activated, it was found that 69.5% (66/95) of them degenerated before first cleavage, 20% (19/95) blocked at 2-4 cell stage, 7.4% developed<br />
into morulae, and 3.1% (3/95) reached the blastocyst stage. Thereafter, a positive effect of supplementation with BSA or PVA during maturation<br />
was seen. However, the number of oocytes that extruded the 1st PB was very low. Furthermore, the rate of blastocyst formation was also very<br />
low as well. These results suggest that the use of media commonly employed for bovine embryo production is not suitable for embryo production<br />
using rabbit oocytes. Alternatively, the poor developmental rates found may have been caused by the use of oocytes from immature rabbits and,<br />
therefore, with lower developmental potential.<br />
Keywords: supplementation, rabbits, ivm.<br />
A204 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO VALIDA<br />
ALIDATE<br />
THE TECHNIQUE OF MICROINJECTION IN<br />
EMBRYONIC CHIMERA PRODUCTION<br />
Daniela Motta Souza, , Hugo Fernandes, Patrícia Villela Silva, Bruno Cazari, Pablo Diego Moço, Bruna Castilho Soto Campanha,<br />
Isabele Picada Emanuelli & Marcelo Fábio Gouveia Nogueira<br />
UNESP, ASSIS, SP, BRAZIL.<br />
Among the techniques to produce chimeras, microinjection (MI) of embryonic stem cells (ESC) into blastocysts - or in the<br />
perivitelline space (PVS.) of the embryos with 4-8 cells - is one of most popular. A well-established training model for this technique could be<br />
very useful when ESC were not available and that was able to identify the injected cellular components and their subsequent aggregation within<br />
the embryo. Hence, we aim to validate, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived<br />
from testis of C57BL/6/EGFP strain. Embryos were recovered from prepubertal females SW (n = 20) superstimulated and mated according to<br />
Mancini et al. (2008; Transgenic Res 17:1015). As an alternative to the classical MI in blastocysts (Nagy et al., 2003; Manipulating the Mouse<br />
Embryo 3rd edn., CSHL), in this study were used 4 to 8 cells embryos (collected at 2.5 days post coitum). Embryos from the same female were<br />
randomly allocated to three groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation<br />
by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS. injection with 6 to 8 cells from<br />
EGFP testis. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO 2<br />
and saturated humidity). The<br />
viability and quality of the embryos (according to the general and specific criteria; IETS Manual, 1998 and Nagy et al., 2003, respectively) and,<br />
in group MI, the fluorescence of testicular cells, were evaluated pre and post-culture (bright field and under UV light for group MI). The results<br />
were analyzed by X 2 test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P<br />
< 0.05. There was no difference among mortality rates of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively). The percentage of<br />
embryos that have retained the quality, after 24 h of culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%,<br />
respectively). It was obtained one chimeric blastocyst in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training<br />
of MI of EGFP testicular cells in the PVS. was feasible and practical to acquire skills, when ESC are available. The method allows easy<br />
identification of injected and, eventually, aggregated cellular components. The source for EGFP testicular cells could be obese and senile males<br />
(scheduled to be sacrificed) from local animal house or refrigerated testis sent from a distant animal facility.<br />
Keywords: microinjection, mice, egfp testicular cells.<br />
s438
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A205 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION<br />
ALTER<br />
TERATIONS TIONS IN METHYLATION TION PROFILES OF LYSINE RESIDUES 4 AND 27 OF HISTONE H3 IN BOVINE BLAST<br />
ASTOCY<br />
OCYST<br />
STS<br />
FOLLOWING CRYOPRESER<br />
OPRESERVATION<br />
Daniel Robert Arnold, Bianca Maria Campanelli Faccio, Roberta Vantini, Carlos Valério da Rocha Jr., Carolina Alves Pereira Corrêa, Isabella Campanelli dos<br />
Santos, Clara Slade Oliveira, Joaquim Mansano Garcia & Flavia Lombardi Lopes<br />
FCAV-UNESP, JABOTICABAL, SP, BRAZIL.<br />
Cryopreservation is an important part of the IVF industry often due to a limited number of suitable recipients for embryo transfer.<br />
However, pregnancy rates from cryopreserved embryos remain lower than their non-cryopreserved counterparts, even though these embryos<br />
appear morphologically normal upon visual inspection. Epigenetics, or chromatin regulation such as histone modifications, are known to control<br />
gene expression and are vital for embryo development. Specific histone modifications can either repress or allow gene expression. The current<br />
study aimed to evaluate the effect of conventional freezing on subsequent histone modifications commonly associated with gene expression,<br />
methylation of histone H3 at lysine 4 (H3K4me3), or gene repression, methylation of histone H3 at lysine 27 (H3K27me3), of bovine<br />
blastocysts. Embryos were produced by IVM/IVF. At day 7 of in vitro culture, blastocyst stage embryos were either frozen by slow-freeze<br />
method (-0.5ºC/min) in 1.5M ethylene glycol (F/T group) or remained in culture for an additional 18 h (Ctrl), then fixed in 4% paraformaldehyde<br />
and stored in PBS+0.1% tritonX at 4ºC. Frozen embryos were stored in liquid N2 for a minimum of 10 days, then thawed and placed in culture<br />
for 36 h for recovery. Expanded blastocysts were then fixed as described above. Embryos were used for immunofluorescence utilizing<br />
antibodies against H3K4me3 and H3K27me3. Images were analyzed with ImageJ software (NIH) and results are shown as percentage of total<br />
DNA. Blastocyst development rate at day 7 was 35.6% (352/990) with blastocyst recovery 36 h post-thawing at 54.23% (77/142). Total cell<br />
numbers per blastocyst were not different amongst groups (117.8+12.49 and 116.1+14.69, F/T and Ctrl groups respectively). Global staining<br />
for the active mark, H3K4me3, was lower in F/T blastocysts compared to Ctrl (17.24+ 2.80% vs. 34.95+ 3.77%; P < 0.01). However, staining<br />
for the inhibitory mark, H3K27me3, was nearly 2-fold higher in F/T blastocysts (40.41+ 3.83% vs. 21.29+ 3.92%; P < 0.01). These results<br />
suggest that bovine blastocysts, subjected to conventional freezing methods, have altered histone modifications that may play a role in poor<br />
pregnancy rates. We are currently investigating possible differences in expression of enzymes responsible for these particular histone modifications,<br />
in bovine embryos subjected to cryopreservation protocols, as well as the effects of alternate freezing methods. [Funded by FAPESP 09/50381-<br />
5 and 09/50603-8].<br />
Keywords: cryopreservation, histone modifications, blastocysts.<br />
A206 CLONING, TRANSGENESIS AND STEM CELLS<br />
MESENCHYMAL STEM CELLS ISOLATED FROM SUBCUTANEOUS FAT OF A MARE DISPLAY CHONDROGENIC,<br />
OGENIC,<br />
OSTEOGENIC AND ADIPOGENIC POTENTIAL<br />
A ngelo Tor<br />
orr es Are v alo, Jo el Cab<br />
abe zas Salazar<br />
alazar, Lleretn<br />
etny Ro drigue<br />
iguez & Fidel Ovidio Castr<br />
astro<br />
UNIVERSIDAD DE CONCEPCION, CHILLAN, CHILE.<br />
Differentiation crosswise mesoderm lineages are a feature of adult mesenchymal stem cells. The ability of such cells to differentiate<br />
in vitro and ultimately in vivo into specific tissues can be of great help in designing therapeutic approaches for horses. The aim of this work was<br />
to isolate adult stem cells from horse fat and induce it to differentiation into mesoderm lineages in vitro. Biopsy-primary culture: 15 grams of<br />
subcutaneous fat was taken surgically from the upper back quart of a mare. Several washes in PBS+ antibiotics, mincing and collagenase<br />
digestion were performed. Isolated cells were cultured in DMEM:F12 +(10% FCS;10 ng/mL EGF) at 39ºC in 5%CO2. Primary culture termed<br />
YMG was established and frozen. Differentiation: YMG cells in passage 3 were seed in 6-well dishes allowed to get confluent and changed to<br />
appropriate differentiation media (DM) based on DMEM low glucose to induce to: osteogenic lineage [OL]; (DM + 10% SFB, 10-7M<br />
dexamethasone, 0.01 mM beta glycerophosphate, 0.3 mM vitamin C), chondreogenic lineage [CL]; (DM + 1X insulin-selenium-transferrin<br />
mix; Gibco, NY, USA, 35 ug/mL vitamin C, 100 nM dexamethasone, 10 ng/mL TGF beta), adipogenic lineage [AL];(DM + 10% FCS, 1 uM<br />
dexamethasone, 10 ug/mL insulin, 0.25 mM 3-isobutyl-1-methylxanthine, 100 uM indomethacine). Control time-mated cells were cultured in<br />
DMEM low glucose + 10% FCS. In all cases, media was changed every third day. At days 0,7,14 and 21 cells were stained for the appropriate<br />
lineage (alizarin red for OL, alcian blue for CL, oil red for AL and RNA extracted for RT-PCR analysis in 10 uL reactions using Quantance<br />
(Berlin, Germany) reverse transcription and PCR kits. Markers of stem cells (CD44; telomerase), osteogenesis (osteonectin; runX2) and<br />
chondrogenesis (collagen 2a1; Sox9) were studied. YMG cells expressed mesenchymal stem cell markers CD44 and telomerase, the later at very<br />
low levels though. After induction, YMG cells differentiated into osteogenic, chondrogenic and adipogenic lineages as judged by both<br />
histological staining and PCR. The kinetics of differentiation varied: osteogenic differentiation was observed at day 7, while adipogenic was<br />
obvious only at day 21 and chondrogenic differentiation around day 14 of induction. PCR analysis showed that specific gene markers were<br />
activated in the cultures and correlated with the histological staining. Here we showed that fat cells isolated from a mare harbored adult<br />
mesenchymal stem cells able to differentiate in vitro into osteogenic, chondrogenic, and adipogenic cell lineages, and thus might be used<br />
therapeutically in vivo for high value sprint horses with recurrent lesions in their limbs.<br />
Keywords: mesenchymal stem cells, differentiation, horses.<br />
N<br />
s439
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A207 CLONING, TRANSGENESIS AND STEM CELLS<br />
CLONED EMBRYOS FROM TWO CELL TYPES OF ENDANGERED PIG BREEDS<br />
Lain Uriel Ohlweiler 1 , Joana Cláudia Mezzalira 1 , Daniela dos Santos Brum 2 , José Cristani 1 , Aury Nunes Moraes 1 ; Martielo Ivan Gehrcke 1 , Gustavo Zemke 1 ,<br />
Alysson Macedo Silva 1 , Norton Klein 1 , Fábio Gallas Leivas 2 , Vilceu Bordignon 3 & Alceu Mezzalira 1<br />
1<br />
LAB. DE REPRODUÇÃO ANIMAL PROF. ASSIS ROBERTO DE BEM, UNIVERSIDADE DO ESTADO DE SANTA CATARINA, (CAV/UDESC), LAGES, SC, BRAZIL. 2 BIOTECH, LAB. DE<br />
BIOTECNOLOGIA DA REPRODUÇÃO, UNIVERSIDADE FEDERAL DO PAMPA (UNIPAMPA), URUGUAIANA, RS, BRAZIL. 3 DEPARTAMENT OF ANIMAL SCIENCE, MCGILL UNIVERSITY,<br />
STE. ANNE DE BELLEVUE, CANADÁ.<br />
Pig cloning is of great interest for the creation of animal models for biomedical research due to close similarities between<br />
physiological and metabolic functions in pigs and humans. In addition, somatic cell cloning can be used to replicate animals of zootechnical or<br />
preservationist importance. Due to the intensification of industrial crossbreeding, a number of breeds of native pigs from Brazil have disappeared<br />
or are currently endangered. It is crucial to maintain these animals genotypes for biodiversity reasons and to prevent the lost of important genetic<br />
traits that might be explored in the future. The main objective of this study was to assess development of somatic cell cloned embryos from one<br />
Moura breed pig (Mo) and one Mulefoot pig (Mf) using either fibroblasts (Fib) or adipocyte-derived mesenchymal stem cells (Adi) as nuclear<br />
donor cells. Fib cell cultures were established through the explantation technique whereas Adi cells were obtained through enzymatic digestion<br />
of adipose tissues. Oocytes were obtained from gilts, maturated according to Martinez Diaz et al. (2010, Cel. Reprog. 12, 85), and then used as<br />
recipient cytoplasts. Handmade cloning steps for somatic cell nuclear transfer were performed according to Ohlweiler et al. (2009, Reprod. Fert.<br />
Dev. 22, 194–5). Oocyte activation was induced with ionomycin (10 µM/5 min) followed by the exposure to 6-dimethylaminopurine (2 mM/<br />
3h). Reconstructed embryos were cultured into micro-wells in PZM-3 at 38.8°C with 5% CO 2<br />
, 5% O2 and 90% N2 for 7 days. Cleavage and<br />
embryo development rates (Morula + Blastocyst) were recorded and analyzed using the Chi-square test (where P < 0.05 was considered<br />
significant). Cleavage (91.1%; n = 112/123 vs.. 93.4%; n = 113/121) and embryo development (30.6% vs.. 41.2%) rates were not statistically<br />
different between MoFib and MoAdi embryos, respectively. The cleavage rate was higher in the MfFib group (95.5%; n = 128/134) compared<br />
to the MfAdi (88.2%; n = 105/119) group, but embryo development was not statistically different between MfFib (30.6%) and MfAdi (43.8%)<br />
embryos. Reconstructed embryos from the MoAdi (n = 29) and the MfAdi (n = 45) groups were cultured for 18 h and then transferred to the<br />
oviducts of an estrus synchronized gilt at approximately 22 h prior to the presumptive ovulation time. This gilt was confirmed pregnant by<br />
ultrasound examination performed 25 days after embryo transfer. This study shows that somatic cell cloned embryos from endangered native pig<br />
breeds can be generated by transferring nuclei from different cell types into in vitro matured host oocytes derived from industrial crossbreeding<br />
gilts and that these (Adi) embryos can develop beyond the blastocyst stage. [Financial Support: FAPESC 17.418/2009-1].<br />
Keywords: genetic conservation, hmc, pig cloning.<br />
A208 CLONING, TRANSGENESIS AND STEM CELLS<br />
ESTABLISHMENT OF A HG-CSF TRANSGENIC GOAT LINE ORIGINATED FROM A MALE FOUNDER AND<br />
DETECTION OF AGE AT PUBERT Y IN F1<br />
R ibrio io Ivan<br />
Tav ares Per<br />
ereir<br />
eira Ba tista, Joanna Mar<br />
aria Gonçalv<br />
onçalves de Souza,<br />
Car<br />
arla Ro zilene Guimarães Silv<br />
ilva Oliv<br />
liveir<br />
eira,<br />
Talles Mon<br />
ont e de<br />
Almeida, Maiara Pinheiro Vieira, Carlos Henrique Sousa de Melo, Érica Souza Albuquerque, Maria Claudia dos Santos Luciano,<br />
Juliana Bar<br />
arroso Félix, Luciana Magalhães Melo<br />
elo, Ale<br />
lexsandr<br />
sandra a Fer<br />
ernandes Per<br />
ereir<br />
eira,<br />
Dárcio Ítalo Alv<br />
lves<br />
Teix<br />
eixeir<br />
eira & Vic<br />
icen<br />
ente e José de Figueirêdo Freitas<br />
UNIVERSIDADE ESTADUAL DO CEARÁ (UECE), FORTALEZA, CE, BRAZIL.<br />
The growing worldwide demand for human Granulocyte Colony Stimulating Factor (hG-CSF) stimulated our laboratory to<br />
produce transgenic goats harboring this gene (Freitas et al., 2010; Transgenic Res., 19, 146). From the birth of founders, obtained by pronuclear<br />
microinjection, the imminent aim was the establishment of a transgenic herd for a commercial scale production of the protein. The objectives of<br />
this study were: a) to obtain transgenic goats from a male founder and b) to determine the age at puberty of the progeny (F1). For this, seven nontransgenic<br />
Canindé females with the synchronized estrus were inseminated with semen from the transgenic founder male. The characterization<br />
of F1, for the presence of foreign gene was performed by conventional PCR. All animals received the suckling kids received supplementation<br />
with commercial concentrate and Tifton hay until three months of age (weaning). Thereafter, the detection of puberty was performed on both<br />
males and females. In males, sexual behavior was evaluated weekly, using a female in estrus until the onset of the first ejaculate containing<br />
spermatozoa. In females, blood samples were taken weekly to determine serum progesterone (P4). A total of 12 kids were born, with a foreign<br />
gene transmission rate of 54.5% (6/11) and the same ratio sex (three females and three males). Concerning the non-transgenic animals born,<br />
80.0% (4/5) were males. An embryonic loss and four stillborn offspring of which was a transgenic were observed in this work. The offspring<br />
remains healthy until now (180 days). Both transgenic males had motile spermatozoa in the ejaculate for the first time at 144 days of age, with<br />
17.2 and 15.8 kg, corresponding to 42.2 and 38.7% of the adult weight of an adult Canindé male, respectively. Similar results were observed in<br />
non-transgenic founders, the same found occurred at 119, 119 and 165 days, with 15.8, 12.6 and 13.6 kg, respectively. The transgenic females<br />
showed serum levels of P4 > 1 ng/mL (suggestive of ovulation) at 119 and 150 days of age, being 48% (14.9 kg) and 58% (17.9 kg) of the adult<br />
weight of an adult Canindé female, respectively. These results are consistent with the average age at puberty of naturalized breeds in Northeast<br />
Brazil. Moreover, indicate that the presence of foreign gene in F1, transmitted from the transgenic male thorough Mendelian inheritance, does<br />
not compromise the age at puberty in both males and females. Additional studies with a higher number of animals derived from line, as well as<br />
verification of fertility, are still needed.<br />
Keywords: transgenesis, goats, puberty.<br />
s440
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A209 CLONING, TRANSGENESIS AND STEM CELLS<br />
IN VITRO CULTURE AND OSTEOGENIC DIFFERENTIATION TION OF CRYOPRESER<br />
OPRESERVED CANINE ADIPOSE-DERIVED STEM CELL<br />
Isadora Arruda, Camila Chavier Macedo, Amanda Jeronimo Listoni, Midyan Daroz Guastali, Margarita Pardo, Bruna de Vita, Leandro Maia & Fernanda da<br />
Cruz Landim Alvarenga<br />
UNESP - FMVZ, BOTUCATU, SP, BRAZIL.<br />
The utilization of canine adipose-derived stem cell (adMSC) has raised great interest in the scientific community due to its amazing<br />
regenerative potential, especially for the treatment of osteogenic and tendinous lesions. The adMSC had the advantage of been easily collected,<br />
appearing in abundance in the tissue of origin. However, the protocols of isolation, culture and cryopreservation are not well established in all<br />
domestic animals. The objective of the present study was to verify the potential of in vitro culture and osteogenic differentiation of cryopreserved<br />
adMSC visceral fat obtained from dogs. Fragments from visceral fat were obtained from the mesentery during cesarean sections and the cells<br />
were obtained after enzymatic digestion with 0.4% colagenase (Gibco ® , NY/USA). Isolation and expansion were performed in DMEM-F12<br />
with fetal calf serum (FCS) supplemented with antibiotics and antimycotics (Gibco ® , NY/USA) at 37°C with 5% CO2. The adMSC in 1st and<br />
3rd passage were cryopreservated in media with 10% DMSO (Sigma ® , St. Louis/USA) at -80°C. After 8 days, the samples were thaw in water<br />
bath at 37°C for 2 min, centrifuged, diluted and seeded in new bottles. The differentiation of the samples in P3 was performed as soon as the<br />
culture reached 80% confluence but the cells in P1 were kept in culture until the 3rd passage and then differentiated. For differentiation, the media<br />
was replaced by the Mesenchymal Stem Cell Osteogenesis Kit (Chemicon ® , MA/USA) and StemPro Mesenchymal Osteogenesis Kit (Gibco ® ,<br />
NY, USA). The media was changed every two days and the confirmation of the osteogenic differentiation was performed on day 9 by staining<br />
the samples with Alizarin Red (Sigma ® , St. Louis/USA), who stains the extracellular deposit of calcium in red. All samples were positive for<br />
calcium deposit. So, it was possible to conclude that the culture and osteogenic differentiation of adMSC obtained from visceral fat from dogs<br />
was efficient even after cryopreservation at -80°C for 8 days. Moreover, the both commercial kits used in this experiment were efficient in<br />
inducing osteogenic differentiation in the dog cells utilized.<br />
Keywords: stem cell, cryopreservation, canine.<br />
A210 CLONING, TRANSGENESIS AND STEM CELLS<br />
EFFECT OF FIBROBL<br />
OBLAST CELL PASSA<br />
ASSAGE ON PRODUCTION OF GYR EMBRYOS BY NUCLEAR TRANSFER<br />
TECHNIQUE<br />
C arolina Cap<br />
apobiango R. Quin<br />
uintão<br />
1 , M ichele Munk Per<br />
ereir<br />
eira 1 , Na tana Cha<br />
hav es Rab<br />
abelo<br />
1 , Jorje R. Toledo Alonso<br />
2 , Lilian Tam<br />
amy Iguma 1 João<br />
Henrique Moreira Viana 1 & Luiz Sérgio Almeida Camargo 1<br />
1<br />
EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2 UNIVERSIDAD, DE CONCEPCIÓN, CHILE.<br />
The success of the somatic cell nuclear transfer (SCNT) depends, among other factors, on donor cell competence to undergo nuclear<br />
reprogramming and generate clone embryos. Such competence can be influenced by the cell passage (Kubota et al. 2000; PNAS 97:990-995).<br />
This study aimed to evaluate the effect of cell passage of fibroblasts from Gyr cow to produce clone embryos. Recipient oocytes were obtained<br />
from abattoir ovaries and matured in TCM 199 (Invitrogen, California, USA) supplemented with 10% estrous cow serum (ECS) under 5% CO2<br />
and 95% humidity at 37°C in air. After maturation, a portion of oocytes was denuded and enucleated for SCNT and the remainder was used<br />
for in vitro fertilization (IVF). Bovine fibroblasts collected from Gyr cows were cultured for several passages in DMEM supplemented with<br />
10% fetal calf serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity. Three experimental groups were established as follow: G1-<br />
enucleated oocytes fused with bovine fibroblasts at 3rd - 4th cell passagel; G2- enucleated oocytes fused with bovine fibroblasts at 6th - 8th cell<br />
passage; and G3- oocytes fertilized in vitro with 2 x 106 sperm/mL for 21h. For G1 and G2, the fibroblasts were cultured for 48h in total<br />
confluence before being trypsinized and used for SCNT. Fibroblasts from G1 and G2 were fused with enucleated oocytes using two pulses of<br />
2.4 kV/cm for 30 msec and chemically activated with ionomycin (Sigma, St. Louis, USA) followed by DMAP (Sigma). Zygotes from all groups<br />
were cultured in CR2aa medium with 2.5% FCS under 5% CO2, 5% O2 and 90% N2 at 38.5ºC. Cleavage was evaluated at 72h and blastocyst<br />
at 168h post-activation (G1 and G2) or post-fertilization (G3) and the data were analyzed by chi-square. No significant differences (P > 0.05)<br />
in cleavage between G1 (84.74%) and G2 (84.31%), but the blastocyst rate was higher (P < 0.05) for G2 (39.2 %) than for G1 (26.2%). The<br />
cleavage (66.67%) and blastocyst (13.70%) rates were lower for G3 than for other groups (P < 0.05) in. In conclusion, bovine fibroblasts at<br />
earlier passages (3rd-4th passage) of cell culture are less effective in producing Gyr embryos by nuclear transfer when compared to later passages<br />
(6th-8th passage). However, it is noteworthy that embryos produced with deriving cells of a long period of culture may have affected their quality<br />
(Merighe et al. 2010). [Financial support: CNPq – FAPEMIG and Innovation Network Project on Animal Reproduction].<br />
Keywords: clone, somatic cell, cell passage.<br />
N<br />
s441
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A211 CLONING, TRANSGENESIS AND STEM CELLS<br />
EFFECT OF TRICHOSTAIN A ON PRODUCTION OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER<br />
AND SUBSEQUENT GESTATIONS<br />
TIONS<br />
Luiz Sérgio Almeida Camar<br />
amargo<br />
go, Car<br />
arolina Cap<br />
apobiango R. Quin<br />
uintão<br />
tão, Anna Car<br />
arolina Denic<br />
enicol,<br />
Lilian Tam<br />
amy Iguma, Bruno Camp<br />
ampos Car<br />
aravalho<br />
alho, Luiz Gusta<br />
ustavo Bruno<br />
Siqueira & João Henrique Moreira Viana<br />
EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL.<br />
Histone deacetylation is associated to a compact and less permissive chromatin, which may impair an efficient nuclear reprogramming<br />
of somatic cell nucleus. The inhibition of histone deacetylation can be achieved by histone deacetylases inhibitors, as Trichostatin A. This study<br />
evaluated the effect of zygotes treatment with trichostatin A on production and quality of nuclear-transferred bovine embryos. Oocytes were<br />
matured in vitro, denuded and exposed to Hoechst 33342 (Sigma, St Louis, USA) and cytochalasin (Sigma) before enucleation. Zygotes were<br />
reconstructed with somatic cells from adult Gyr cow, fused with double electric pulse of 2.4 kV/cm for 30 µsec and activated with ionomycin<br />
(Sigma) for 5 min. After ionomycin activation the zygotes were randomly separated in two groups: Trichostatina (TRICHO, n = 132) - zygotes<br />
were cultured for 4h in 6-DMAP followed by 7h in CR2aa medium plus with 2.5% fetal calf serum, both supplemented with 50nM Trichostatin<br />
A (Sigma); Control (CONT, n = 116) - zygotes were cultured in the same conditions described above but without Thichostatin A supplementation.<br />
Parthenogenetic embryos (PART group, n = 287) were used as control for activation and embryo culture. Embryos from all groups were cultured<br />
in CR2aa supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5%O2 and 90%N2. Embryos at 168h postactivation<br />
(hpa) from TRICHO and CONT groups were transferred to synchronized recipients. Analyses of cleavage at 72hpa, blastocyst at<br />
168hpa, total cell number and apoptotic cell index were performed by ANOVA and means compared by SNK test. Data are shown as<br />
mean±SEM. Gestation and birth rates were analyzed by Fischer’s Exact Test. No difference on cleavage (87.7±4.1%, 83.3±4.0% and 79.3±4.1%)<br />
and blastocysts (38.4±4.7%, 34.2%±6.0% and 30.5±3.7) rates was found among TRICHO, CONT and PART groups, respectively. Embryos<br />
from TRICHO group presented lower (P < 0.05) index of apoptotic cells (0.065±0.009; n = 17) than embryos from CONT group (0.129±0.021;<br />
n = 13), but similar (P > 0.05) to those from PART group (0.083±0.011; n = 19). No difference (P > 0.05) on total cell number was found among<br />
groups. Although it is not statistically significant (P > 0.05), pregnancy rate at 60 days with embryos from TRICHO group (64.2%; 9/14) was<br />
numerically higher than those ones from CONT group (45.4%; 5/11). One pregnancy from TRICHO group went to term (7.1%; 1/14) but the<br />
calf died in the second day after birth. No offspring was obtained with embryos from CONT group. In conclusion, exposure of clone zygotes<br />
to 50 nM of trichostatin A decreases apoptosis in embryos, which may favor pregnancy rate. [Financial support: Embrapa Project 01.07.01.002,<br />
CNPq 403019/2008-7 and Fapemig].<br />
Keywords: cloning, histone deacetylases inhibitor, apoptosis.<br />
A212 CLONING, TRANSGENESIS AND STEM CELLS<br />
EFFICIENCY OF NUCLEAR TRANSFER IN EQUINE: DIRECT INJECTION OF THE DONOR CELL NUCLEUS INTO THE<br />
RECIPIENT CYTOPL<br />
OPLAST<br />
ASTS<br />
Claudia Barbosa Fernandes 1 , Camila Gabriela Pereira Gonçalves 1 , Lilian Rigatto Martins 2 & Fernanda da Cruz Landim Alvarenga 3<br />
1<br />
USP FMVZ, SAO PAULO, SP, BRAZIL. 2 UFMT, MATO GROSSO, MT, BRAZIL. 3 UNESP FMVZ, BOTUCATU, SP, BRAZIL.<br />
The aim of this study was to evaluate the efficiency of NT with donor cell nucleus direct injection in equine recipient cytoplasts.<br />
There were used 150 equine compact and expanded oocytes in vitro matured (IVM) for 30h or kept for 24h under the roscovitine action before<br />
the period of IVM. After 30 h of IVM equine oocytes were denuded and incubated during 30 min in H-MEM medium, with FBS, Hoechst<br />
33342 and cytochalasin B. The oocytes enucleation and the recipient cytoplasts reconstitution with equine fibroblasts were performed between<br />
32 and 34 h after the beginning of IVM. The metaphaseI (MI) and metaphaseII (MII) oocytes enucleated and reconstituted successfully were<br />
incubated for one hour until the artificial activation, using Roscovitine + Ionomycin (I+R) and Ionomycin + 6 DMAP (I+6DMAP). After the<br />
activation period, the oocytes were cultured in DMEM/F12 with FBS, myoinositol and gentamicin at 39ºC in 5% CO2, 5% O2 and 90% N2<br />
during 3 days. The assessment of the activation and nuclear decondensation formation rates were performed with Hoechst 33342 in inverted<br />
microscopy. There was used the Analysis of Deviance to select the best logistic regression model to explain the percentage of equine oocytes with<br />
nuclear decondensed formation after NT. The equine oocytes classified as compact that were exposed or not to roscovitine, in MI and MII at the<br />
time of enucleation, had 0%, 20% and 37.5%, 25% of nuclear decondensation rate after activation with I+R and 0% 30% and 22.2%, 50% after<br />
I+6DMAP, respectively. The equine oocytes classified as expanded that were exposed or not to roscovitine in MI and MII at the time of<br />
enucleation, had 14.2%, 30% and 0%, 14.2% of nuclear decondensation rate after activation with I+R and 12.5 %, 20% and 20%, 33.3% after<br />
I+6DMAP respectively. We can observe that the only significant effect in chromatin decondensation rates was the stage of nuclear maturation,<br />
regardless of the oocyte classification, the artificial activation protocol and the exposure or not to roscovitine, the percentage of oocytes with the<br />
decondensed nucleus formation on the third day of culture was significantly higher for MII (P = 0.033) compared to MI. There was no<br />
significant interaction among any other factors, however, for the equine the technique when the donor cell nucleus was directly injected into the<br />
recipient cytoplast resulted in unsatisfactory cloned embryos production. [Acknowledgement: FAPESP].<br />
Keywords: cloning, artificial activation, nuclear maturation.<br />
s442
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A213 CLONING, TRANSGENESIS AND STEM CELLS<br />
NEW STRATEGY<br />
TEGY TO INDUCE TRANSGENESIS IN IN VITRO FERTILIZA<br />
TILIZATION TION (IVF) BOVINE EMBRYOS<br />
Natalia Gabriela Canel, Romina Jimena Bevacqua, María Inés Hiriart & Daniel Salamone<br />
FACULTAD DE AGRONOMÍA U.B.A., BUENOS AIRES, ARGENTINA.<br />
Direct plasmid injection into the cytoplasm of IVF zygotes is a simple and efficient method to induce transgenesis in mammals,<br />
however it still gives transient expression from extrachromosomal plasmid DNA. To solve this problem, we employed a new transgenesis<br />
method (Ogino H et al., Nat. Prot., 1, 1703-10) only employed in X. laevis, consisting in intracytoplasmic injection of I-SceI meganuclease<br />
(Mega) plus a plasmid that contains its recognition sites flanking Pax6 promoter-GFP cassette (IS-EGFP). IS-EGFP (gifted by Dr. Ogino H)<br />
was digested by 0.5 UI of Mega at 37ºC for 45 min. Fragment digestion was confirmed by agarose electrophoresis. Digestion mixture or plasmid<br />
alone were transferred to 10% PVP at a final concentration of 25 or 50 ng/µL IS-EGFP (groups IS-EGFP 25/50 +Mega, for digestion mixture,<br />
and IS-EGFP 25/50 for plasmid alone) and were microinjected into IVF presumptive zygotes. A control group injected with 50 ng/µL of pCX-<br />
EGFP plasmid was included. Embryos were cultured in SOF medium and rates of blastocysts were evaluated on day 7. Expression of egfp was<br />
determined at days 4, 5 and 7. Data was analyzed by Fisher’s test. All treatments showed no differences in blastocyst rates [from 8/40 (20%) to<br />
19/51 (37.3%)]. Expression of egfp at day 4 was lower (P < 0.05) for all treatments compared to control group (from 0 to 8.2% vs. 30.2%),<br />
except for IS-EGFP 50 (22.5%). The same pattern was observed at day 5 for IS-EGFP 25 +Mega or alone groups (8.2 and 7.9% respectively),<br />
but not (P < 0.05) for IS-EGFP +Mega or alone groups (13.7 and 47.5% respectively), which did not differed from control (30.2%). At day 7,<br />
all groups showed egfp expression levels not different from control (from 23.8 to 35% vs. 32.6%), except for IS-EGFP 25 +Mega, which<br />
showed lower (P < 0.05) expression levels (14.3%). Embryos with IS-EGFP 25 +Mega or alone and IS-EGFP 50 +Mega tended to show lower<br />
expression rates than those with IS-EGFP 50 alone or control. Blastocyst rates of embryos expressing egfp did not differed among groups.<br />
However, at all evaluations, groups with Mega showed a tendency to express egfp at lower levels than the groups treated with plasmid alone.<br />
Conclusions: IS-EGFP has a delayed expression dynamics compared with pCX-EGFP. The presence of the enzyme keeps the EGFP fragment<br />
released, possibly preventing concatemer formation. Our results show that the use of IS-EGFP + Mega influences the transient transgene<br />
expression in IVF bovine embryos.<br />
Keywords: transgenesis, ivf, bovine.<br />
A214 CLONING, TRANSGENESIS AND STEM CELLS<br />
EXPRESSION OF XIST, G6PD AND HSPA1A GENES IN BOVINE BLAST<br />
ASTOCY<br />
OCYST<br />
STS S RECONSTITUTED BY NT FROM<br />
RECIPIENT OOCYTES PRODUCED BY CHEMICALL<br />
ALLY ASSISTED ENUCLEATION<br />
N aiara a Zo ccal Sar<br />
araiv<br />
aiva 1 , Clar<br />
lara a Slade Oliv<br />
liveir<br />
eira 1 , Ta tiane Almeida Dr ummond Tetzner<br />
1 , Mar<br />
arina Ragagnin de Lima 1 , Simone Cr istina<br />
Méo 2 & Joaquim Mansano Garcia 1<br />
1<br />
FCAV-UNIVERSIDADE ESTADUAL PAULISTA - UNESP, JABOTICABAL, SP, BRAZIL. 2 EMBRAPA PECUÁRIA SUDESTE-CPPSE, SÃO CARLOS, SP, BRAZIL.<br />
N<br />
The aim of this work was to study the effect of enucleation (EN) technique on the expression of XIST, G6PD and HSPA1A genes<br />
on bovine blastocysts produced by IVF, parthenogenetic activation and reconstitution by NT of somatic cells after conventional EN or chemically<br />
assisted EN. Bovine oocytes were in vitro matured in TCM 199 + FCS + hormones for up 24 h. IVF embryos were produced with female<br />
sexed sperm because the cell line of donor nuclei for NT was female. Parthenogenetic embryos were produced by activation with 5 ìM<br />
ionomycin for 5 min and 10 µg/mL cycloheximide for 4 h. In conventional EN, after IVM for 18 h, MII oocytes were denuded, incubated with<br />
Hoechst 33342, and enucleated by removing the 1 st PB and the adjacent cytoplasm. In chemically assisted EN, after 19 h of IVM, the oocytes<br />
were denuded, exposed to 0.05 ìg/mL demecolcine for 2 h and underwent to enucleation, through the removal of the protrusion formed in the<br />
cortical region. After in vitro culture in SOFaa with 2.5% FCS and 0.5% BSA for seven days at 38.5°C, 5% CO 2<br />
in air under saturated<br />
humidity, blastocysts were individually submitted to RNA extraction with Trizol and reverse transcription with Reverse Transcriptase<br />
ImPron-II kit. Relative transcript quantification of XIST, G6PD e HSPA1A genes was performed in real-time PCR with SYBR Green ®<br />
(Invitrogen), employing GAPDH as endogenous control, in triplicate, with five to ten samples per group and final volume of 20 ìL. The measn<br />
amplification efficiency was estimated using a linear regression on the log fluorescence per cycle. After applying the comparative CT method<br />
(ÄÄCT) with the SDS software v1.4, data was analyzed by the pairwise fixed reallocation randomization test. All expression ratios were<br />
calibrated by IVF group and shown in log 10 scale. No significant differences in XIST and G6PD genes were detected between control groups<br />
(IVF: 0 and 0, and parthenogenetic: 4.088 and 1.398, respectively) and reconstituted groups. However, a higher expression (P < 0.05) of these<br />
genes was seen in chemically assisted EN group (6.690 and 3.851) compared to conventional EN (-0.643 and -0.794). HSPA1 gene expression<br />
was similar (P > 0.05) between groups. Tthe expression of the G6PD gene has been associated with high embryo respiratory activity rates,<br />
suggesting that the increased expression of this gene is indicative of embryo quality. Also, higher levels of ROS generated by increased oxygen<br />
consumption may trigger G6PD activation. The expression pattern of more genes related to stress response should be investigated to conclude<br />
if the difference in expression between the EN techniques relates to the quality of embryos obtained. [Funded by FAPESP 06/51481-5 and<br />
07/55969-5].<br />
Keywords: demecolcine, enucleation, gene expression.<br />
s443
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A215 CLONING, TRANSGENESIS AND STEM CELLS<br />
DEVEL<br />
VELOPMENT OF BOVINE CLONE EMBRYOS USING FIBROBL<br />
OBLAST<br />
ASTS S CULTURED IN SOF SUPPLEMENTED WITH CONJUGA<br />
ONJUGATED<br />
LINOLEIC ACID (C-9,T-11)<br />
Norton Klein 1 , Lain Uriel Ohlweiler 1 , Fábio Gallas Leivas 2 , Daniela dos Santos Brum 2 , Maicon Gaissler Lorena Pinto 1 , Fernanda Gemelli 1 , Nerissa Albino 1 ,<br />
Laura Tais Nickelle Sasse<br />
1 , Joana Cláudia Mezzalir<br />
zzalira 1 & Alc<br />
lceu Mezzalir<br />
zzalira 1<br />
1<br />
LAB. DE REPRODUÇÃO ANIMAL PROF. ASSIS ROBERTO DE BEM, UNIVERSIDADE DO ESTADO DE SANTA CATARINA, (CAV/UDESC), LAGES, SC, BRAZIL. 2 BIOTECH, LAB. DE<br />
BIOTECNOLOGIA DA REPRODUÇÃO, UNIVERSIDADE FEDERAL DO PAMPA, (UNIPAMPA), URUGUAIANA, RS, BRAZIL.<br />
The standard protocol for cell culture is the one using DMEM-based media, whereas the standard medium for bovine embryo<br />
culture is SOF. The culture of fibroblasts anticipates one step of the cloning procedure as it might enhance the further embryo culture, with cells<br />
already adapted to SOF conditions. The Conjugated Linoleic Acid (CLA c-9, t-11) plays a role on reducing the anabolism of certain cell types,<br />
then it might contribute for the reprogramming process. This study evaluated the development of clone embryos in which the donor (fibroblast)<br />
cell was previously cultured or not with CLA prior to the reconstruction process. For that, cells on second passage were cultured in DMEM<br />
added of 10% fetal calf serum (FCS) at 38.5ºC, in atmosphere containing 5% CO 2<br />
and saturated humidity during 72 h. In a previous experiment,<br />
we observed the viability of cells cultured in SOF. Six hours prior to cell fusion, DMEM medium was replaced by SOF added of 5% (G1) and<br />
supplemented with 100 µM of c9, t11 CLA (Matreya, ref.001249) and 0.1% ethanol (G2). The SCNT cloning was performed with MII oocytes<br />
as recipient cytoplasts according to Mezzalira et al. (<strong>2011</strong>, Cell. Reprog., 13, 65-76), being cleavage and blastocyst rates (D7), in 3 replications.<br />
Data were analyzed through the Chi-square test (where P < 0.05 was considered significant). Group G2 presented better cleavage rate (94.5%,<br />
104/110) in comparison to G1 (81.3%, 100/123), however blastocyst rates were not statistically different between G1 (31.7%) and G2 (36.4%).<br />
Aiming to obtain at least one pregnancy for each treatment,as an additional evaluation step, D8 blastocysts were transferred, in pairs, to<br />
synchronous recipient cows. Ultra-sound guided pregnancy diagnosis was performed on day 30, 60 and 90 after inovulation. Five cows were<br />
transferred with G1 embryos and were detected pregnant (100%) on day 30, being rates respectively 40% at day 60 and 20% (n = 1) at day 90.<br />
The only recipient cow transferred with fresh G2 embryos was detected pregnant on day 30 and maintains pregnancy up to day 80. In conclusion,<br />
the use of CLA for cells treatment prior to reconstruction on SCNT enhances embryo development performance, at least during the first cell<br />
divisions. Embryos produced in both experimental groups showed to be efficient on in vivo early development, but additional investigations are<br />
needed in order to evaluate the nucleus donor treatment with CLA effect in the viability of viable offspring. [Financial support: UDESC, CNPQ<br />
471330/2009-4].<br />
Keywords: HMC, Reprogramming, c9 and t11 cla.<br />
A216 CLONING, TRANSGENESIS AND STEM CELLS<br />
PREGNANCY OF CLONED EMBRYOS FROM A FREEMARTIN COW OF AN ENDANGERED BREED<br />
Joana Cláudia Mezzalira 1 , Maicon Gaissler Lorena Pinto 1 , Fabiano Carminatti Zago 2 , Alysson Macedo Silva 1 , Nerissa Albino 1 ,<br />
Norton Klein 1 , Vilceu Bordignon 3 , Lain Uriel Ohlweiler 1 & Alceu Mezzalira 1<br />
1<br />
UDESC - UNIVERSIDADE DO ESTADO DE SANTA CATARINA, LAGES, SC, BRAZIL. 2 EPAGRI-EMPRESA DE PESQUISA AGROPECUÁRIA E EXTENSÃO RURAL DE SANTA CATARINA,<br />
LAGES, SC, BRAZIL. 3 MCGILL UNIVERSITY, STE. ANNE DE BELLEVUE, QUEBEC, CANADÁ.<br />
The Rouge Flamand (Flemish) cattle were introduced into Brazil in 1945. As a dual-purpose breed, it had easily adapted to the<br />
regional climate conditions and several herds of Rouge Flamand cattle were established. However, during the last years the number of herds has<br />
been considerably reduced due to specialized breeds import. There are about fifty remaining animals at the experimental station Epagri (Empresa<br />
de Pesquisa Agropecuária e Extensão Rural do Estado de Santa Catarina) located in Lages, Santa Catarina, but they are threatened with extinction<br />
due to imminent lack of genetic variability. One of these remaining animals is a freemartin cow, which is about 17 years old and extremely wellconformed,<br />
though infertile. Over the last years, a number of animals of different species have been produced by somatic cell nuclear transfer<br />
(SCNT) for different purposes. The main objective in this work is to use SCNT to rescue the genotype of a Rouge Flamand freemartin cow and<br />
produce a fertile animal. Somatic cells were obtained from an ear skin biopsy through the explantation technique. Up to the third passage and after<br />
obtaining at least 90% confluence, cells were cryopreserved in 0.25mL straws in DMEM + 10% fetal calf serum and 10% DMSO. The<br />
handmade cloning steps were performed according to Mezzalira et al. (<strong>2011</strong>, Cell. Reprog, 13, 65-76). Cleavage and blastocyst rates were<br />
respectively 87% (204/233) and 34% (80/233). Recipient cows of tricross European breeds were used as recipient females at day seven after<br />
estrus and D8 blastocysts were transferred. Part of the SCNT blastocysts were vitrified/thawed according to Mezzalira et al. (2010, Reprod. Fert.<br />
Dev, 22, 210). The remaining blastocysts were maintained fresh and were transferred in pairs to each recipient. From the six recipient cows that<br />
received fresh embryos, 6 (100%) established pregnancy; 3 (50%) were pregnant up to 60 days and 2 (33%) are still pregnant at 80 and 180 days.<br />
Vitrified/thawed embryos were transferred to two recipient cows, which were both confirmed pregnant but embryos died by day 45 after transfer.<br />
For the first time, this study shows that SCNT embryos derived from a freemartin animal can develop into blastocyst and establish pregnancies<br />
after it transfer to recipient cows.<br />
Keywords: cell reprogramming, freemartin cattle, genetic biodiversity.<br />
s444
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A217 CLONING, TRANSGENESIS AND STEM CELLS<br />
AGE INFLUENCE OF CYTOPLST DONOR IN BOVINE CLONE PRODUCTION BY NUCLEAR TRANSFER<br />
Emivaldo de Siqueira Filho 1 , Tiago Omar Diesel 1 , Edson Ramiro Júnior 1 , Andrei Antonioni Fidelis 2 & Rodolfo Rumpf 3<br />
1<br />
GENEAL-GENÉTICA ANIMAL ANÁLISE, PESQUISA E LABORATÓRIO S/A, UBERABA, MG, BRAZIL. 2 PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS ANIMAIS-UNIVERSIDADE<br />
FEDERAL DE UBERLÂNDIA (UFU), UBERLÂNDIA, MG, BRAZIL. 3 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL.<br />
The Brazilian Ministry of Agriculture, Livestock and Supply issued, in 2009, guidelines for clone production by nuclear transfer<br />
(NT). This document requires that both cytoplast donors (CD) and the animal to be cloned be of the same breed. Thus, laboratories should have<br />
a group of animals to OPU sessions. These animals are selected by the largest follicle population, however they are of different ages. It has been<br />
reported that the number and quality of oocytes decrease with aging and consequently, the rate of in vitro embryo production decreases as well.<br />
The aim of this study was to evaluate the influence of CD age on the rates of NT embryo production, pregnancy and birth. Two CD group were<br />
used. Group 1: 5-6 year-old animals and Group 2: 11-14 year-old animals. The OPU was perfomed in both groups in the same day and a total<br />
of eight sessions were conducted. Viable oocytes were matured in vitro in plates separated by donor. After 18 h of maturation, oocytes were<br />
denuded and only those which extruded the first polar body (PB) were enucleated. All cytoplast were reconstructed with nucleus from a single<br />
donor. Reconstructed structures were activated using ionomycin and 6-DMAP. Zygotes were cultured in SOFaaci and evaluated after 2 days for<br />
cleavage (Clea) and 7 days for blastocysts production (BP). The blastocysts were transferred to synchronized recipients and pregnancy<br />
diagnosis was performed at 30 days (P30). Pregnancy was evaluated again in the 60th (P60) and 150th (P150) days and accompanied until the<br />
birth. The birth rate was assessed by Fisher’s Test and other parameters were tested using chi-square. In Group 1, 237 structures were fused, 111<br />
cleaved (46.8%) and 51 blastocysts were produced (21.5%). In Group 2, 305 structures were fused, 147 (48.2%) cleaved, resulting in 48<br />
blastocysts (15.7%). A total of 36 embryos were transferred in Group 1 and 24 in Group 2, with 11 (30.56%) and 6 (25%) pregnancies (P30).<br />
At the 60th day, 3 pregnancies were maintained in each group. At the 150th day, Group 1 had 3 pregnancies whereas the Group 2 had 2. All<br />
parameters assessed were not significantly different. Two viable births were obtained in Group 1 and none in Group 2. In conclusion, based on<br />
the present work, senile cows can be used as cytoplast donors in NT.<br />
Keywords: nuclear transfer, cytoplast donor, age.<br />
A218 CLONING, TRANSGENESIS AND STEM CELLS<br />
ISOLATION AND IMMUNOPHENOT YPING OF MULTIPO<br />
TIPOTENT TENT MESENCHIMAL STEM CELLS FROM BOVINE<br />
EMBRYOS<br />
YOLK SAC<br />
Celina Almeida Furlanetto Mançanares 1 , Lilian Jesus Oliveira 1 , Rafael Vilar Sampaio 1 , Rodrigo Silva Nunes Barreto 1 , Ana Carolina<br />
Fur<br />
urlanett<br />
lanetto Mançanar<br />
ançanares<br />
2 , V anessa Cr istina Oliv<br />
liveir<br />
eira 1 , Juliano Ro drigues Sangalli<br />
1 , Alicia Gre y ce Tor<br />
ora tti Pessola<br />
essolato 2 , A na Fla<br />
lavia de<br />
Carvalho 3 , Flávio Vieira Meirelles 1 , Maria Angélica Miglino 2 & Carlos Eduardo Ambrosio 1<br />
1<br />
FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOS-FZEA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL. 2 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO,<br />
SP, BRAZIL. 3 UNIFEOB FUNDAÇÃO DE ENSINO OCTÁVIO BASTOS, SÃO JOÃO DA BOA VISTA, SP, BRAZIL.<br />
Yolk sac (YS) plays an important role on the embryo survival by giving the nutritional support to the developing embryo until<br />
a functional placenta is completely formed. Moreover, the YS may be a promising source of for stem cell research due to the niches of<br />
hematopoietic and mesenchymal cells present in this structure along the embryo/fetus development. Our study aimed to establish a primary<br />
culture of bovine YS (bYS) mesenchymal cells and characterize them through their expression of specific cell surface markers. A total of 6 yolk<br />
sacs from bovine fetuses at 38 SD ±3 days were collected at local abattoir. 2 SV were designated to establish the primary mesenchymal cell<br />
culture (PMC) and flow cytometry analysis and 4 were frozen for immunohistochemistry analysis. Briefly, the PMC were established after<br />
collagenase type IV cellular desaggregtion and culture in standard culture medium (supplemented 10% of fetal bovine serum) at 37 o C and 5%<br />
CO2 atmosphere for 24 h. The adherent cells were maintained in culture for 9 passages until the flow cytometry analysis for leucocytes markers<br />
(CD3); monocytes/macrophages (CD14); adhesion molecules (CD44); hematopoietic and T-cells (CD45); lymphocyte differentiation (CD4;<br />
CD73) and B-cell receptor (CD79). In parallel, cryosections of 4 µm were stained for CD14; CD45 and additional mesenchymal cells markers<br />
(CD90 – a cell-T precursor; CD105 - endoglin; OCT4 – a pluripotent cells marker). The flow cytometric analysis showed that over 80% of the<br />
putative mesenchymal stem cells were positive for CD44, which plays a role on cell-to-cell and cell-to-matrix interaction, at the 9o passage of cell<br />
culture, whereas 95% of the cells were negative for markers expressed by already differentiated cells (CD3, CD4, CD14, CD45, CD73 and<br />
CD79). Furthermore, immunohistochemistry analysis showed that all cells were positive for OCT4 at all analyzed stages of embryo development.<br />
Additionally, positive cells for CD90 and CD105 were found in the mesenchymal region, characterizing the niches of stem cells in the YS and<br />
negative for CD14 and CD45. Our results suggest that bYS is a promising source of mesenchymal stem cells, which falls in 2 of 3 criteria (the<br />
ability of adhere to the plastic and expression of surface cell markers) established for by the <strong>International</strong> Stem Cell Society (ISCS). Our future<br />
studies will be focused on the of the bYS putative mesenchymal cells analysis to differentiate in vitro and in vivo as it is the third criteria<br />
established by the ISCS.<br />
Keywords: yolk sac, mesenchimal stem cells, bovine.<br />
N<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A219 CLONING, TRANSGENESIS AND STEM CELLS<br />
THE EXPRESSION OF PLURIPOTENCE MARKERS IN DONOR CELLS INCREASED CLONING EFFICIENCY IN BOVINE<br />
José Reinaldo Manriquez, Alejandra Estela Velasquez, Fidel Ovidio Castro & Lleretny Rodriguez<br />
UNIVERSIDAD DE CONCEPCION, CHILLÁN, CHILE.<br />
Nuclear transfer (NT) in mammals is a complex process in which a differentiated cell is reprogrammed to induce embryo<br />
development. NT is associated with different degrees of nucleus epigenetic reprogramming of the donor cells. Expression of certain genes in<br />
these cells facilitates its expression in the embryo produced after NT. We observed that certain somatic cell lines express pluripotency genes (e.g<br />
OCT4 and SOX2). Based on these facts we postulate that expression of pluripotency genes in donor cells increase cloning efficiency expressed<br />
as good quality blastocysts. To test this, we correlate the developmental potential (DP) of NT bovine embryos created from 5 different cells lines<br />
(termed CWA, HAR, HCH, HB12, HAN) with the expression level of OCT4 and SOX2 in each cell line. DP was assessed on the basis of<br />
blastocysts rate; grading and total cell counts at day 7. Donor fibroblast cell lines were isolated from adult cattle after collagenase digestion of<br />
tissue biopsies. For NT cells (passages 3-7) were used at least 1d after reaching confluence. Cloned embryos were produced by zona-free NT,<br />
cultured individually for 7d in SOFaci in 5%O2; 5%CO2 and 90%N, 100% humidity at 39ºC in sealed aluminium foiled bags, in well of the well<br />
system. Gene expression analysis: total RNA was extracted (4 biological replicates per cell line), converted to cDNA and subjected to real time<br />
PCR with specific primers; semiquantification was conducted using the standard curve method. Data were analysed with non-parametric test<br />
using Infostat (Buenos Aires, Argentina). Day-7 embryos were produced from all cell lines (CWA:64.3%; HAR:62.2; HCH:57.2; HB12:49.4;<br />
HAN:27.9). The total percentage of blastocyst, including grade I and total cell number obtained with HAN was significantly lower compared<br />
with the other 4 cell lines, while HAR-derived embryos showed the best grading and cell number counts. OCT4 and SOX2 expression was<br />
evaluated in all cell lines (only OCT4 was quantified). The highest expression was obtained in HAR and the lowest in HAN. There was a<br />
correlation between expression level of OCT4 in the cell lines and the blastocysts rate (r = 0,92; P = 0,02), rate of grade I blastocysts (r = 0,96;<br />
P = 0,01) and total cell number (r = 0,98; P = 0,002). The expression of SOX2 was determined in three of the cell lines, but it did not correlated<br />
with any of the parameters mentioned earlier. Conclusions: expression of OCT4 in the cell lines used for NT might increase embryo quality at<br />
blastocyst stage and could be a useful marker to select cell lines and to improve cloning efficiency. [This work was partially supported by<br />
Fondecyt grant No. 11100082 from the Ministry of Education of Chile].<br />
Keywords: nuclear transfer, oct4, cell line.<br />
A220 CLONING, TRANSGENESIS AND STEM CELLS<br />
IN VITRO CULTURE OF BOVINE EMBRYOS IN MURINE ES CELL CONDITIONED MEDIA NEGATIVEL<br />
TIVELY AFFECTS<br />
EXPRESSION OF PLURIPO<br />
URIPOTENCY RELATED MARKERS OCT4, SOX2 AND SSEA1<br />
C lara a Slade Oliv<br />
liveir<br />
eira,<br />
Mar<br />
arc ela Mar<br />
aria Souza,<br />
Naiar<br />
aiara a Zo ccal Sar<br />
araiv<br />
aiva,<br />
a, Tatiane Almeida Dr ummond Tetzner<br />
etzner, Mar<br />
arina Ragagnin de Lima,<br />
Flavia Lombardi Lopes & Joaquim Mansano Garcia<br />
UNESP, JABOTICABAL, SP, BRAZIL.<br />
Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that<br />
culture conditions for in vitro produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their<br />
undifferentiated state. Therefore, the aim of this work was to evaluate the effect of culture medium on pluripotency related markers expression<br />
of IVP blastocysts, in order to produce suitable ICM for further culturing. We tested KSR (5%) and FCS (5%) supplements in SOF 0.5% BSA<br />
medium, and ES cell conditioned medium (CM) on IVC (Groups: KSR, KSR CM, FCS and FCS CM). After, the expression of pluripotencyrelated<br />
markers was assessed by immunocytochemistry of OCT3/4 (Sigma), NANOG (Sigma), SOX2 (Sigma) and SSEA1 (Invitrogen), and<br />
mean fluorescence intensity from nuclei area was measured using Adobe Photoshop CS3 software (0 to 255 scale). No difference was detected<br />
between KSR and FCS groups. On FCS supplemented groups, we detected down-regulation of pluripotency markers in the inner cell mass<br />
(OCT3/4: FCS 70.40a, FCS CM 49.29b; SOX2: FCS 132.43a, FCS CM 93.72b; SSEA1: FCS 59.39a, FCS CM 49.19b), except for NANOG<br />
(FCS 29.00, FCS CM 24.24) (One way ANOVA and Tukey post test, P = 0.05). On KSR supplemented groups, no statistic difference was<br />
observed, although there was a numeric decrease in KSR CM group (NANOG: KSR 32.80, KSR CM 29.60; OCT3/4: KSR 66.82, KSR CM<br />
56.87; SOX2: KSR 135.11, KSR CM 113.29; SSEA1: KSR 65.8, KSR CM 54.21). Then we evaluated SOX2 gene expression by real-time<br />
PCR, using SYBR Green detection system (Applied Biosystems). SOX2 relative gene expression revealed lower levels on KSR CM<br />
blastocysts (KSR 0.44±0.08a, KSR CM 0.27±0.04b), and a remarkable variation in SOX2 mRNA levels on FCS supplemented blastocysts<br />
(SFB 0.53±0.25, SFB CM 0.47±0.29) (Unpaired T test, P = 0.05). In conclusion, pluripotency-related markers tend to decrease after<br />
supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not<br />
differ from FCS, but FCS replacement by KSR would be interesting in order to produce blastocysts with stable SOX2 gene expression levels.<br />
[Financial Support: FAPESP 07/58506-6, 08/58370-0 and 09/54510-4].<br />
Keywords: pluripotency, in vitro fertilization, embryonic stem cells.<br />
s446
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A221 CLONING, TRANSGENESIS AND STEM CELLS<br />
TECHNICAL REPORT: FIRST PREGNANCY OF A EQUINE CLONE IN BRAZIL<br />
Bruno Valente Sanches 1 , André Gomiero Rigo 1 , José Henrique Fortes Pontes 1 , Rodrigo Mendes Untura 1 , Lucas Lopes Moino 1 , Andrea Cristina Basso 1 , Perla<br />
Dagher Cassoli Fleury 1 , Janaina Ferreira Nagao 1 , Mario Martinez-Dias 1 & Vilceu Bordignon 2<br />
1<br />
IN VITRO BRAZIL S/A, MOJI-MIRIM, SP, BRAZIL. 2 MACGILL, MONTREAL, CANADÁ.<br />
Equine cloning by somatic cell nuclear transfer (SCNT) has enormously evolved in the last 10 years after the birth of the first<br />
clones in France and then United States in 2003. However, cloned horses have not yet been produced in Brazil. In this study we describe for<br />
the first time the in vitro development and the establishment of the first equine pregnancy in Brazil from SCNT embryos. SCNT embryos<br />
were produced using in vitro matured oocytes and skin fibroblast cells. Equine oocytes were obtained from slaughterhouse ovaries by<br />
follicular aspiration and curettage of the internal follicular wall. Collected oocytes were matured for 26 h in DMEM F12 supplemented with<br />
LH, FSH, estradiol, IGF, EGF, FCS, ITS and antibiotics, under mineral oil in a humidified atmosphere of 5% CO2 and 95% air at 38.5°C. After<br />
maturation, the oocytes were freed from their cumulus cells by pipetting into a solution of (440 units/mL) hyaluronidase in BO medium. Cell<br />
lines were established from skin biopsies of adult animals using standard methods of cell culture. Once achieving confluence in culture, cells<br />
were frozen and stored in liquid nitrogen until use. Before use in SCNT, cells were thawed and cultured for 4-5 days until achieving complete<br />
confluence. Mature (MII stage) oocytes were enucleated by micromanipulation under ultraviolet light, and then an average cell size was<br />
injected into the perivitelline space of enucleated oocyte. Cell fusion was induced by electrical stimulation into a (0.25 M) sorbitol solution.<br />
The reconstructed oocytes were parthenogenetically activated with ionomycine and DMAP, and then were cultured in C4 medium for 24 h<br />
in 5% CO2, 5% O2 and 90% N2. They were then transferred to DMEM F12 supplemented with (5 mg/mL) BSA and (25 ug/mL) gentamicine<br />
and cultured for 7-8 days. A total of 484 oocytes were cultured in vitro and 243 (50.2%) extruded the first polar body and were considered<br />
mature. Using these oocytes a total of 100 embryos were reconstructed and 17 developed into blastocysts at the end of the in vitro culture<br />
period. The blastocysts were transferred to 17 recipients. Pregnancy diagnosis was performed after 14 days from SCNT (day 0). Seven<br />
females were confirmed pregnant and 4 of them remained pregnant between 30 and 45 days after SCNT. This confirms the successful<br />
application of SCNT for the production of equine embryos in vitro, which have the potential to develop beyond the blastocyst stage. Studies<br />
are currently been conducted using oocytes collected in vivo by follicular aspiration, which are expected to improve the development of SCNT<br />
embryos.<br />
Keywords: equine clone, embryo, nuclear transfer.<br />
A222 CLONING, TRANSGENESIS AND STEM CELLS<br />
TOXICITY OF CARBON NANOTUBES IN BOVINE FIBROBLAST CELLS<br />
Michele Munk Pereira 1 , Carolina Capobiango Romano Quintão 2 , Humberto de Mello Brandão 2 , Nadia Barbosa Rezende Raposo 1 , Natana Chaves Rabelo 3 ,<br />
Savana Giacomini Brito 4 , João Henrique Moreira Viana 2 , Luiz Orlando Ladeira 5 & Luiz Sérgio Almeida Camargo 2<br />
1<br />
UNIVERSIDADE FEDERAL DE JUIZ DE FORA, JUIZ DE FORA, MG, BRAZIL. 2 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 3 CENTRO DE ENSINO SUPERIOR DE JUIZ DE<br />
FORA, JUIZ DE FORA, MG, BRAZIL. 4 UNIVERSIDADE PRESIDENTE ANTÔNIO CARLOS, JUIZ DE FORA, MG, BRAZIL. 5 UNIVERSIDADE FEDERAL DE MINAS GERAIS, JUIZ DE FORA, MG,<br />
BRAZIL.<br />
Carbon nanotubes (CNT) are promising nanomaterials to carry exogenous DNA and transfect cells in order to generate transgenic<br />
animals. However, little is known about the toxicity of CNT in bovine fibroblasts. The aim of this study was to evaluate the toxicity of CNT in<br />
bovine fibroblasts cultured in vitro. Bovine fibrobrasts were cultured in DMEM medium supplemented with 10% fetal calf serum and incubated<br />
at 37.5°C, 5% CO2 and 95% humidity. After reaching 60% of confluence, the cells were exposed to multi-walled carbon nanotube (MWNT;<br />
diâmetro: 10-20nm; comprimento: 1-4 µm) for 24h with different concentrations: 0 µg/mL (Control); 5 µg/mL; 25 µg/mL; 50 µg/mL; and 100<br />
µg/mL. Cells were stained with propidium iodide (50 µg/mL) and subsequently evaluated by flow cytometry to assess their viability. A flow<br />
cytometry (FACScalibur; Becton Dickinson, San Jose, CA) equipped with a laser (488nm) was used and data collected using a 585±42 nm filter.<br />
Three repetitions with three replicate each one were performed. The analysis was performed by counting 10.000 cells for every cell culture.<br />
Statistical analysis was performed by ANOVA and means were compared by Student Newman Keus. The concentration of 5 µg/mL did not<br />
affect the cell viability (92.13±0.88%) compared to control (93.80±1.12%). However, cell viability was reduced (P < 0.001) in the concentrations<br />
of 25 µg /mL (88.21±0.96%), 50 µg/mL (84.78±0.82%) and 100 µg/mL (65.88±1.77%). In conclusion, MWNT may be used in bovine<br />
fibroblast cell culture at 5 µg/mL without detriment to cell viability. [Financial support: Embrapa – Project Animal Genomics Network<br />
(01.06.9.01.01.00) and Project Innovations in Animal Reproduction Network (01.07.01.002), CNPq,CAPES and Fapemig].<br />
Keywords: flow cytometry, cell viability, gene transfection.<br />
N<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A223 CLONING, TRANSGENESIS AND STEM CELLS<br />
NUCLEAR TRANSFER<br />
WITH APOPTOTIC TIC BOVINE FIBROBL<br />
OBLAST<br />
ASTS:<br />
S: CAN PROGR<br />
OGRAMMED CELL DEATH BE REPROGR<br />
OGRAMMED?<br />
Moysés Dos Santos Miranda 1 , Fabiana Fernandes Bressan 2 , Tiago Henrique Câmara de Bem 2 , Giovana Krempel Merighe 2 , William Allan King 3 , Otávio Mitio<br />
Ohashi 1 & Flávio Vieira Meirelles 2<br />
1<br />
UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2 FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOSC, PIRASSUNUNGA, SP, BRAZIL. 3 UNIVERSITY OF GUELPH,<br />
GUELPH, CANADÁ.<br />
Apoptosis is a programmed cell death considered irreversible. Reports indicate that reversibility is possible if the cells undergoing<br />
apoptosis have not reached the “point of no return” (Span et al. 2002, Cytometry 47, 24-31). However, characterization of this point has not been<br />
well documented so far. Objective this experiment was to add new information on this subject by using apoptotic cells as nuclear donors in<br />
somatic cell nuclear transfer (SCNT). Adult bovine fibroblasts cultivated in DMEM were treated with 10 µM of staurosporine (STS) for 3 h and<br />
analyzed for the phosphatidylserine externalization at plasma membrane (annexin V assay), presence of active caspase9 (FLIC Apoptosis Kit,<br />
Bloomington, USA), mitochondrial membrane potential (ÄØm; JC-1) and nuclear fragmentation (TUNEL). Bovine oocytes obtained from<br />
slaughterhouse were matured in TCM199 with 0.5 ìg/mL of FSH and 50 µg/mL of LH. SCNT was performed as described by Miranda et al.<br />
(2009 Cloning and Stem Cells 11, 565-575). Annexin-positive (Anx+) and Caspase9-positive (Casp9+) fibroblasts were sorted by flow<br />
cytometry in PBS and immediately used for SCNT. Untreated, flow cytometricaly sorted fibroblasts served as controls. Successfully reconstructed<br />
embryos were cultured in SOF in incubator at 38.5°C and 5% CO 2<br />
in air. Fusion, cleavage and blastocyst rates were analyzed by Chi-square test<br />
(P < 0.05). Data from the Annexin V Caspase9 and JC-1 assays were calculated by flow cytometer and compared to control cells by Student’s<br />
“t” test (P < 0.05). TUNEL result was qualitatively analyzed by fluorescence microscopy. After treatment with STS for 3 h, 89.9% of cells were<br />
Anx+ (4.6% in Control cells; P < 0.01), 24.9% were Casp9+ (2.4% in Control cells; P < 0.01), ÄØm was reduced in 70% compared to control<br />
cells (P < 0.05) and the no signal of TUNEL assay was detected in any cell. Fusion and cleavage rates were not affected by the use of apoptotic<br />
cells as nuclear donors (P> 0.05). Also, use of Anx+ cells did not affect blastocyst production compared to Control (26.4 vs. 22.9%, respectively,<br />
P > 0.05). However, the use of Casp9+ cells for SCNT significantly reduced blastocyst formation (12.3%, P < 0.05). These findings contribute<br />
with new evidence on the apoptosis reversibility, especially at early stages, since cells with typical symptoms of the onset of apoptosis (loss of<br />
ÄØm and phosphatidylserine exposure) were efficiently reprogrammed to form embryos after SCNT. Moreover, considering that only few<br />
Casp9+/TUNEL negative cells were reprogrammed by SCNT, we speculate that the “point of no return” of apoptosis may be located around the<br />
activation Caspase9, before nuclear fragmentation. Further experiments are necessary to confirm this hypothesis.<br />
Keywords: nuclear transfer, apoptosis, reversibility.<br />
A224 CLONING, TRANSGENESIS AND STEM CELLS<br />
USE OF EMBRYO TRANSCER<br />
ANSCERVIC<br />
VICAL COLLECTION IN A FOUNDER TRANSGENIC GOAT FOR HUMAN<br />
GRANUL<br />
ANULOCY<br />
OCYTE COL<br />
OLONY-STIMUL<br />
ONY-STIMULATING TING FACT<br />
CTOR (HG-CSF)<br />
R aylene Ramos Mour<br />
oura 1 , Joanna Mar<br />
aria Gonçalv<br />
onçalves de Souza<br />
1 , Talles Mon<br />
ont e de Almeida<br />
1 , Maiar<br />
aiara a Pinheir<br />
inheiro V ieira 1 , Car<br />
arlos Henr<br />
enrique<br />
S ousa de Melo<br />
1 , Ribr<br />
ibrio io Iv an Tav ares Per<br />
ereir<br />
eira Ba tista 1 , Ale<br />
lex sandra a Fer<br />
ernandes Per<br />
ereir<br />
eira 1 , Dárcio Ítalo Alv<br />
lves<br />
Teix<br />
eixeir<br />
eira 1 , Luciana<br />
Magalhães Melo 1 , Jeferson Ferreira Fonseca 2 & Vicente José de Figueirêdo Freitas 1<br />
1<br />
UECE, FORTALEZA, CE, BRAZIL. 2 EMBRAPA CAPRINOS E OVINOS, SOBRAL, CE, BRAZIL.<br />
Goats are used as model of transgenic animals secreting recombinant proteins in their milk. After the founder production, its<br />
multiplication is indispensable in order to obtain a transgenic herd, which can be achieved by the transfer of in vivo produced embryos. However,<br />
embryo collection in goats is usually done by surgery, which can promote adhesions, limiting the number of times that it can be applied in the<br />
same animal. Thus, the study aimed to use the embryo transcervical collection in a founder goat obtained by our group (Freitas et al., 2010,<br />
Transgenic Res, 19, 146) for embryo transfer into synchronized recipients. This study was approved by UECE biosafety and ethic committee.<br />
One transgenic founder Canindé doe as a donor, two non-transgenic bucks of the same breed and four u breed recipients were used. The embryo<br />
donor goat was submitted to estrus synchronization and superovulation using progestagen (Progespon ® , Buenos Aires, Argentina), pFSH<br />
injections (Folltropin ® , Ontario, Canada) and cloprostenol (Prolise ® , Buenos Aires, Argentina). In the prevention of premature regression of<br />
corpora lutea (CL), flunixin-meglumine (Flumedin ® , Varginha, Brazil) was used. Donor fertilization was performed at estrus onset and 24 h later.<br />
The number of CL was assessed by laparoscopy before embryo collection. Embryo transcervical collection was performed seven days postestrus.<br />
Twelve hours before collection, the donor received 37.5 µg cloprostenol for cervix dilatation. The embryo collection was performed with<br />
a circuit and catheter for small ruminants (Circuit/catheter to collect embryos for sheep and goats ® , Embrapa, Brasília, Brazil). It was possible to<br />
recover almost all the collection medium at the end of process. The transgenic donor goat had 11 CLs and eight structures were recovered, with<br />
a collection rate of 72.7%. Five blastocysts grade I, one grade II and one compacted morula were transferred. The recovered embryos were<br />
transferred by semi-laparoscopy into recipients that received progestagen and cloprostenol associated with an eCG injection (Novormon ® ,<br />
Buenos Aires, Argentina). Pregnancy diagnosis was assessed by ultrasonography 30 and 45 days post-estrus. Identification of transgenic<br />
offspring was performed by PCR. On day 30 post-estrus, pregnancy rate was 75.0% (3/4) and dropped to 50.0% (2/4) at 45 days. From the two<br />
pregnant recipients, one showed twin pregnancy, achieving a total of three kids. One transgenic female was identified by PCR. In this study, 33%<br />
of kids born were transgenic. In conclusion, embryo transcervical collection showed to be an efficient method for propagation of founder<br />
transgenic goat for hG-CSF.<br />
Keywords: transgenesis, goat, hg-csf.<br />
s448
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A225 CLONING, TRANSGENESIS AND STEM CELLS<br />
SUCCESS CESS ON ISOLATION,<br />
IMMUNOPHENOTYPIC<br />
YPICAL CHARACTERIZA<br />
CTERIZATION TION AND DIFFERENTIATION TION IN OSTEOGENIC,<br />
ADIPOGENIC AND CHONDROGENIC LINEAGES OF MESENCHYMAL STEM CELLS (MSCS) FROM THE EQUINE AMNIOTIC<br />
LIQUID.<br />
Bruna De Vita 1 , Ian Martin 1 , Loreta Leme Campos 1 , Amanda Jeronimo Listoni 1 , Natália Pereira Paiva Freitas 1 , Leandro Maia 1 , Isadora Arruda 1 , Ilusca Finger 2 ,<br />
Bruna da Rosa Curcio 2 , Fernanda da Cruz Landim Alvarenga 1 , Reneé Laufer Amorim 1 & Nereu Carlos Prestes 1<br />
1<br />
FMVZ-UNESP, BOTUCATU, SP, BRAZIL. 2 UFPEL, PELOTAS, RS, BRAZIL.<br />
The bone marrow is the major source of MSCs in equine medicine, however as the amount and differentiation capacity of these<br />
cells decrease with age, its therapeutic potential also decrease across the time. So, over the last decades it was observed an increase interest in<br />
investigate new sources of MSCs, mainly cells with origin in fetal annexes, which had already demonstrate the capacity of maintain the<br />
pluripotency characteristics of the tissues from which its originate (Cremonesi, <strong>2011</strong>, Theriogenology, 75, 1400-1415). The aim of the present<br />
study was to isolate, characterize immunophenotipically and differentiate the MSCs from equine amniotic liquid (AL) in osteogenic, adipogenic<br />
and chondrogenic lineages. Samples were collected from gravidic uterus in slaughterhouse and transported at 5 o C to the laboratory in Botutainer ®<br />
(Botupharma, Botucatu/BR) transport system; where it were centrifuged and pellets were resuspended in culture medium containing DMEM,<br />
F12, bovine fetal serum, antibiotic and antimycotic (Gibco ® - NY/USA). The culture system was maintained at 37 o C with 5% carbon dioxide<br />
in air. After the second passage, a portion of the cells were fixed in paraformaldehyde 4% in 24 “well” culture plates. Immunocytochemistry was<br />
performed with antibodies anti-vimentine, -cytokeratin, -CD44, -PCNA and -OCT 4. The rest of the cells were maintained in culture system and<br />
after the third passage the differentiation process was induce by replacement of support media by another media compound by the differentiation<br />
kits (Gibco ® -NY/USA). This process was verified by the use of following staining, Alizarin Red (osteogenic strain), Oil Red O (adipogenic<br />
strain), Toluidine Blue and Alcian Blue (chondrogenic strain, Sigma ® , St. Louis/USA). The results were a positive immunostaining for<br />
vimentine, CD44 and PCNA and negative for cytokeratin, confirming the mesenchymal origin of these cells and their proliferation capacity. The<br />
absence of OCT-4 immunostaining differs from the results observed in the literature; however a quantitative evaluation was not performed and<br />
once the AL population was very heterogeneous the percentage of expression of this marker could vary within the samples. The<br />
immunocytochemistry performed directly in the culture plate was efficient to characterize the studied cells population maintaining their fibroblastoid<br />
morphology. The staining showed the presence of calcium mineralization, intracytoplasmic lipid and glycosaminoglycans confirming the<br />
differentiation potential of the cells obtained from the AL on the three cited lineages. So, we could concluded that the isolation, characterization<br />
and differentiation of MSCs from equine AL was successful done. [Acknowledgements: Capes, Fundunesp e FAPESP].<br />
Keywords: mesechymal stem cells, amniotic fluid, equine.<br />
A226 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
ALTERED EXPRESSION OF GENES RELATED<br />
TO GLUC<br />
UCOSE METABOLISM IN BOVINE OOCYTES MATUR<br />
TURATED<br />
TED IN<br />
VITRO OR IN VIVO<br />
Sabine Wohlres-Viana 1 , Michele Munk Pereira 1 , José Nelio S. Sales 2 , Agostinho Jorge dos Reis Camargo 3 , Carolina Capobiango R.<br />
Quintão 1 , Natana Chaves Rabelo 5 , Anna Carolina Denicol 4 , Luiz Gustavo Bruno Siqueira 4 , João Henrique Moreira Viana 4 , Luiz<br />
Sérgio Almeida Camar<br />
amargo<br />
4 , M arta Fonsec<br />
onseca M. Guimarães<br />
4 &Lilian Tam<br />
amy Iguma 4<br />
1<br />
UFJF, JUIZ DE FORA, MG, BRAZIL. 2 USP, SÃO PAULO, SP, BRAZIL. 3 PESAGRO, NITERÓI, RJ, BRAZIL. 4 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 5 CES/JF, JUIZ DE FORA,<br />
MG, BRAZIL.<br />
One of the main challenges of reproductive biotechnology is to understand the metabolic pathway in normal embryo development.<br />
Molecular biology methodologies allow the evaluation of metabolic modifications in in vitro culture conditions, which could induce epigenetic<br />
responses that would finally result in altered gene expression patterns. The aim of this study was to evaluate the expression of genes related to<br />
glucose transport and metabolism (GLUT1 – Glucose Transporter; IGF1R – Insulin-Like Growth Factor 1 Receptor, IGF2R – Insulin-Like<br />
Growth Factor 2 Receptor) in bovine oocytes obtained from Gyr cattle (Bos indicus) maturated in vivo (M1 group) and in vitro (M2 group). The<br />
protocol used to recover the M1 oocytes was: Day 0 (D0) – 2 mg of estradiol benzoate + norgestomet auricular implant; D4- D7 – four<br />
administrations of 200mg FSH given every 24h, in decreasing dosages; D6 (morning) – 150 µg of cloprostenol; D7 (afternoon) – auricular<br />
implant removal; D8 (12:00h) – 25 mg of LH; D9 (morning) – follicular aspiration (OPU). In the M2 group, the follicular wave was<br />
synchronized as follows: D0 – 2 mg of estradiol benzoate + auricular implant + 150 µg of cloprostenol; D5 – auricular implant removal; D6 –<br />
OPU. The in vitro maturated oocytes (IVM) were cultured in TCM-199 media (Invitrogen, CA, USA) added with 10% of estrous cow serum<br />
and 2µg of FSH (Pluset, Callier, Spain) for 24h, at 38.5ºC, 5% of CO2 and saturated humidity. Both in vivo and in vitro maturated oocytes were<br />
denuded and stored in 3 pools of 10 oocytes, snap-frozen in liquid N2 and kept at -80ºC until use. RNA extraction was performed using the<br />
commercial kit RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s specifications. The reverse transcription<br />
and the cDNA preamplification were performed using the commercial kit TransPlex Complete Whole Transcriptome Amplification Kit (WTA2<br />
– Sigma Aldrich) according to the manufacturer’s specifications. The cDNA was submitted to Real-Time PCR, using the β-actin gene as<br />
endogenous control and the commercial kit Power SYBR ® Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s<br />
specifications, for expression analysis in the REST ® software. The genes GLUT1 (1.37±0.07) and IGF1R (2.01±0.14) were up-regulated (P<br />
< 0.05) in the in vitro group when compared to the in vivo group, while in the IGF2R gene (1.29±0.11) there was no difference (P > 0.05). In<br />
conclusion, the IMV environment can alter the expression pattern of GLUT1 and IGF1R genes. [Financial support: Embrapa – Project Rede<br />
Genômica Animal (01.06.9.01.01.00) and Project Rede de Inovação em Reprodução Animal (01.07.01.002), CNPq,CAPES and Fapemig].<br />
Keywords: mRNA, development, gamete.<br />
s449<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A227 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
COMP<br />
OMPAR<br />
ARATIVE ANALYSIS OF MEMBRANE INTEGRITY METHODS OF OVINE CRYOPRESER<br />
OPRESERVED SEMEN<br />
Aline Matos Arrais, Carla Sobrinho Paes de Carvalho, Bruno Pena Carvalho, Fernanda Queiroz Costa, Edgar Maurício Mogollon-Waltero & Angelo José<br />
Burla Dias<br />
UENF, CAMPOS DOS GOYTACAZES, RJ, BRAZIL.<br />
Plasmatic membrane integrity is very important in assessing the viability of frozen sperm, due to the detrimental actions during<br />
freezing. There are several techniques used for such analysis. Among the techniques used to evaluate the plasma membrane, the hypoosmotic<br />
swelling test (HOST) and propidium iodide (PI) are the most used. The aim of this work was to conduct a comparative analysis of these methods<br />
for the evaluation of cryopreserved ovine spermatozoa. Twenty straws of semen were used (10 with ethylene glycol - EG and 10 with glycerol<br />
- Gc). The thawed semen was mixed with 2 mL of TALP and centrifuged. 10 µL of the pellet was removed, and added to the 300 µL of a solution<br />
100 mOsm during 30 min to make the HOST. Sperm cells were evaluated in a phase contrast microscope and classified those sperm cells with<br />
linear tails as injured, and those with coiled tail as intact. The remaining semen was resuspended in 1 mL TALP, with 150 µL removed. A total<br />
of 3 µL of PI (10 µg / mL in PBS) was added to this volume . The evaluation was performed on epifluorescence microscope, with sperm cells<br />
labeled in red classified as injured, and not labeled as intact. In both tests, 200 cells were analyzed for each treatment, in ten replicates. Data were<br />
analyzed by the t test (LSD), with a significance level of 5%. The mean number of viable cells detected by the HOST was 135.4 ± 21.2 (Gc) and<br />
140.6 ± 13.4 (EG) and was significantly different among the values obtained by staining with PI (Gc = 87.7 ± 23.0; EG = 114.7 ± 12.3). The<br />
data differ from results reported by Brito et al. (2003, Theriogenology 60:1539-1551) for cattle semen, which found more intact sperm with IP<br />
than with HOST. We concluded that the cryoprotectant used for freezing semen did not alter the pattern of response to the HOST and IP tests,<br />
and there are differences among the methods for detection of plasmatic membrane integrity of ram semen, still needing to correlate these data with<br />
fertility.<br />
Keywords: sperm cell, membrane, cryopreservation.<br />
A228 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
PEPTIDE FINGERPRINTING ANALYSIS OF BOVINE EMBRYOS OF NELORE (<br />
(BOS INDICUS) ) AND RED ANGUS (<br />
(BOS<br />
TAUR<br />
URUS<br />
US) ) RACES OBTAINED<br />
THROUGH<br />
IN VITRO FERTILIZA<br />
TILIZATION<br />
TION<br />
R aquel Cellin Ro chetti 1 , Yeda Fumie<br />
Wa tanabe 2 , Felip<br />
elipe e Per<br />
erecin<br />
ecin 3 , Juliano Ro drigues Sangalli<br />
4 , Flávio<br />
V ieira Meir<br />
eirelles<br />
elles 5 , C hristina<br />
Ramires Ferreira 6 , Marcos Nogueira Eberlin 7 , Fabio Cesar Gozzo 8 , Eduardo Jorge Pilau 9 , Ricardo Pimenta Bertolla 10 & Edson<br />
Guimarães Lo Turco 11<br />
1,10,11<br />
UNIFESP, SÃO PAULO, SP, BRAZIL. 2 VITROGEN, CRAVINHOS, SP, BRAZIL. 3,4,5,7,8,9,10,11,12 USP/FZEA, PIRASSUNUNGA, SP, BRAZIL. 6,7,8,9 UNICAMP, CAMPINAS, SP, BRAZIL.<br />
Fingerprinting studies performed by mass spectrometry (MS) using MALDI (Matrix – Assisted Laser Desorption/Ionization) are<br />
becoming frequent because it turns the analysis faster and enables determination of specific biological characteristics. With the expansion of<br />
livestock, studies related to molecular biomarkers of breeds have an important role in animal reproduction. In this context, an important feature<br />
for improvement of reproductive response is to identify and define goups of breeds. This study aimed to introduce embryos peptide fingerprinting<br />
technique for molecular identifications and develop applications for characterization of two bovine breeds for futures preimplantational diagnostics.<br />
We used bovine embryos from Nellore (Group 1) and Red Angus (Group 2) breeds. Embryos were produced according to the protocol of<br />
Vitrogen Ltda. (Ripamonte, P.; Genet. Mol. Res. Vol.4(4), pg. 726-33) located in Cravinhos, SP. For the study of proof of concept of this<br />
methodology, five embryos from Group 1 and seven from Group 2 were used. Lipids and salts were removed from embryos through serial<br />
washes in isopropanol (70 and 95%). Then, they were individually transferred to the MALDI plate (Waters) and covered with trypsin (20µg/mL)<br />
during 12 h under moist chamber in 37°C. Afterwards, the samples were covered with matrix (sinapinic acid 15mg/mL) and the spectra was<br />
acquired using the MALDI-QTOF (Synapt G1) in positive model with mass ranging from 800 to 3500 m/z. Statistical analysis was<br />
performed online (www.metaboanalyst.ca). Data standardization was performed by z score. Ions were only considered different if they<br />
showed a minimum of 4 - Fold Change and P = 0.05 regarding the Student-t test (Volcano). Considering the multivariate analysis, it was<br />
performed regarding the principal component analysis (PCA) followed by the method of summation of partial least squares (PLS-DA). It was<br />
observed 5300 ions from which 223 showed difference in the Volcano analysis. The first three PCs explained 78.2% of data variance (PC1<br />
66.7%, PC2 7.8%, 3 PC3, 7%). These preliminary results showed that the analysis of peptide profile from individual embryos through<br />
MALDI is fast, sensitive and it makes possible separation between zebu (Group 1) and taurus (Group 2) embryos. Thus, the peptide<br />
fingerprinting may become a powerful tool to distinguish biological conditions, such as precocity, fertility, disease resistance, among others.<br />
Keywords: peptide fingerprinting, embryos, mass spectrometry.<br />
s450
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A229 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
PROTEOMIC ANALYSIS OF PRIMITIVE GUT IN BOVINE EMBRYOS<br />
Ana Carolina Furlanetto Mançanares 1 , Lilian Jesus Oliveira 2 , Erica Zimberknopf 3 , Felipe Perecin 4 , Gustavo Henrique Martins Ferreira Souza 5 , Christina<br />
Ramires Ferreira 6 , Jerusa Simone Garcia 7 , Celina Almeida Furlanetto Mançanares 8 , Ricardo Pimenta Bertolla 9 , Carlos Eduardo Ambrosio 10 , Flávio Vieira<br />
Meirelles 11 , Marcos Nogueira Eberlin 12 & Maria Angélica Miglino 13<br />
1,13<br />
USP-FMVZ, SÃO PAULO, SP, BRAZIL. 2,4,8,10,11 USP-FZEA, PIRASSUNUNGA, SP, BRAZIL. 3,4,5 UNIFEOB, SÃO JOÃO DA BOA VISTA, SP, BRAZIL. 5 WATERS CORPORATION, SÃO PAULO,<br />
SP, BRAZIL. 6,12 UNICAMP, CAMPINAS, SP, BRAZIL. 7 UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL. 9 UNIFESP, SÃO PAULO, SP, BRAZIL.<br />
The development of embryo biotechnology in farm animals is hampered by embryo losses between 30 and 60 days of gestation in<br />
cattle. Most of embryonic losses in cattle occur around the transition period from vitellinic to placental nutrition of the developing embryo, thus<br />
the proteomic study of bovine primitive gut (PG), and its crosstalk with the yolk sac, during this period may increase the knowledge on the<br />
molecular pathways involved in normal embryo development and in embryo losses as well. PG from bovine embryos on day 39 SD± 4 of<br />
development (ranging from 33 to 45 days) were collected at a local slaugtherhouse. The samples were processed and pooled for label-free<br />
shotgun proteomics analysis using MudPit (Multidimensional Protein Identification Technology) tandem MSE acquistion. Functional and<br />
pathway analysis using FatiGo (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress) and Ingenuity Pathway<br />
Analysis (www.ingenuity.com) were used to identify relevant ontologies and canonical or noncanonical pathways represented by the expressed<br />
proteins in the PG. A total of 74 protein sequences were identified corresponding to 30 unique proteins expressed by the bovine PG. Out of 30<br />
unique proteins, 21 proteins were used on the ontology and pathway analysis. The ontology analysis showed an enrichment of ontologies related<br />
to binding (n = 5); catalytic activity (n = 6); intracellular organelle (n = 6). There was an enrichment of ontologies associated to cytoskeleton<br />
modifications; cell differentiation process (n = 3); cellular migration (n = 4) and cell metabolism (n = 6). Furthermore, the pathway and the<br />
network analysis showed an enrichment of cell-to-cell communication pathways such as gap and tight junction, and focal adhesion pathways.<br />
In addition, pathways involved in cellular movement (regulation of actin cytoskeleton and leukocyte transendothelial migration) were extremely<br />
enrichment in the group of proteins expressed by the bovine PG. Our results suggested that the cells from primitive gut have high migratory<br />
profile and are composed of not fully differentiated cells with high cellular metabolism. The proper migration and differentiation of these cells<br />
would dictate the fate of the developing embryo. Moreover, understanding the function and interaction of proteins expressed by normal embryo<br />
will give clues of the impact of the reproductive biotechnologies in embryo development during the window between implantation and<br />
placentation.<br />
Keywords: proteomic, primitive gut, embryo.<br />
A230 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EVAL<br />
ALUATION OF DIFFERENT TIMES OF EQUILIBRIUM ON INTENSIVE PROGRESSIVE MOTILIT<br />
TILITY AND SPERM<br />
PATHOL<br />
THOLOGIES OGIES OF CRYOPRESER<br />
OPRESERVED RAM SEMEN<br />
R aimundo Teles Per<br />
ereir<br />
eira & Osni Luis Garszar<br />
arszareck<br />
CESCAGE, PONTA GROSSA, PR, BRAZIL.<br />
This work aimed to study the influence of different equilibrium times during the cryopreservation of ovine semen on motility<br />
intensive phase (MIP) and pathology sperm after thawing. Samples of semen from sheep aged 2-3 years of Texel, Dorper, White Dorper, Ile de<br />
France and Santa Ines proven fertility, and performed five repetitions for each player studied. Samples were collected with the support of an<br />
artificial vagina. The samples were diluted in Tris-yolk-glycerol 7% and packed in 0.25 mL straws, sealed with polyvinyl alcohol at a final<br />
concentration of sperm per straw 200 x 10 6 . The doses were lotted to five groups. For all groups, a cooling curve of 0.4°C/ min was used until<br />
they reached 5° C, when it was applied to the different equilibrium times (TE). Therefore, the experimental groups were assembled according<br />
to the duration, in which the straws were kept at this temperature, i.e., GI = 60min, GII = 90min, GIII = 120min, GIV = 150min and GV =<br />
180min. Then, the straws were placed 4 cm from the surface level of liquid N 2<br />
for 12 min. And thus held the negative slope. After this period,<br />
the straws were plunged into liquid N 2<br />
. The samples were thawed at 37°C for 20 s and evaluated for MIP and then stained with eosin-nigrosin.<br />
For the evaluation of sperm pathology, held in a 1000x magnification, two hundred cells were examined per sample. The sperm pathologies<br />
were classified as major, minor and total defects. The data were statistically analyzed by Tukey test at 1% significance. It was observed that<br />
the samples from groups GIV and GV had better rates of MIP (60 and 63%, respectively) did not differ statistically among themselves, but<br />
showed difference from the other groups. There was no statistical difference in the percentage of defects, minor and total in the groups<br />
evaluated. We conclude that the equilibrium times of 150 and 180 min showed better preservation of the motility characteristics of ram<br />
cryopreserved semen. Whereas both groups had similar results, we recommend the TE 150 min running for reasons of convenience in<br />
conducting the activity. The sperm pathologies suffered no influence of the different times of equilibrium.<br />
Keywords: semen, ram, time of equilibrium.<br />
N<br />
s451
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A231 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
ENDOMETRIAL BIOPSY IN MARES IN HEAT REPEATER:<br />
IMMUNOHISTOCHEMIC<br />
OCHEMICAL ANALYSIS OF T LYMPHOCY<br />
YMPHOCYTES AND<br />
MACROPHAGES<br />
Franciele Basso Fernandes Silva, , Joana Fernandes Eigenheer Moreira, Juliana da Silva Leite & Ana Maria Reis Ferreira<br />
UFF, NITEROI, RJ, BRAZIL.<br />
Heat repeaters mares bring large economic losses, so the endometrial biopsy has been a major procedure in the evaluation of<br />
uterine health in horses, besides being easy, safe, cheap and with minimum distress to the animal (SNIDER et al. <strong>2011</strong>, Theriogenology 75,<br />
1567–1581; EIGENHEER-MOREIRA et al. 2007, Pesquisa Veterinária Brasileira 27, 506-512). The aim of this study was to evaluate the<br />
presence of T lymphocytes and macrophages in the endometrium of heat repeaters mares during estrus and diestrus. Endometrial biopsies<br />
were performed in ten heat repeaters mares, five in estrus and five in diestrus. For the control group were selected and examined four mares<br />
sound. After collection, samples were preserved in 10% buffered formaldehyde and subsequently subjected to routine histologic processing<br />
to perform the immunohistochemistry (IHC). The IHC technique for identification of T lymphocytes and macrophages was performed with<br />
antibody rabbit polyclonal anti-CD3 (A 0452, DAKO, Denmark) at 1:200 dilution and mouse antibody monoclonal anti-antigen myeloid/<br />
histiocytic IgG1 Clone MAC 387 kappa (M0747, DAKO, Denmark) at dilution 1:400, respectively. For analysis of the results was made<br />
count of immunostained cells in five random fields and the average number of cells per field in two groups and control group was compared<br />
to significance level α = 0.05, through the Student’s t statistic for independent groups. The control group had at the time of diestrus, an average<br />
of 9.1 immunostained cells for antibody anti-CD3 and 8.1 to antibody anti-antigen myeloid/histiocytic at the time of estrus an average of 13.2<br />
and 1.6 respectively. Heat repeaters mares assessed during estrus showed a mean of 20.4 cells immunostained for the antibody anti-CD3 and<br />
10.0 for the antibody anti-antigen myeloid/histiocytic and animals assessed during the diestrus had an average of 11.3 and 8.3, respectively.<br />
When compared with the control group, the group of heat repeaters mares in diestro showed no statistically significant difference for T<br />
lymphocytes (Student’s t-test: t =0.434; g.l.=6; p-value = 0.680) and macrophages (Student’s t-test: t = 0.061; g.l. = 6; p-value = 0.953),<br />
however the group observed on estrus time showed statistically significant difference for macrophages (Student’s t test: t = 2.814; g.l. = 4;<br />
value-p = 0.048) which was not repeated for T lymphocytes (Student’s t-test: t = 2.068; g.l. = 4; p-value = 0.107). This way, we can conclude<br />
that heat repeaters mares in estrus period have increased infiltration of macrophages than observed in healthy mares.<br />
Keywords: endometrial biopsy, cd3, mac 387.<br />
A232 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
MICRO-FTIR BIOCHEMICAL CHARACTERIZA<br />
CTERIZATION TION OF BULL SPERMATOZ<br />
OZOA IRRADIA<br />
ADIATED BY LOW POWER LASER<br />
Thiago Revers ers Dreyer<br />
er 1 , Adr<br />
driano Felip<br />
elipe e Siqueir<br />
iqueira 2 , Taciana Deprá Magr<br />
agrini<br />
3 , Mayr<br />
yra Elena Assumpção<br />
4 , Her<br />
erculano Silv<br />
ilva Mar<br />
artinho<br />
5 & Mar<br />
arcella Pec<br />
ecor<br />
ora<br />
Milazzotto 6<br />
1,3,5,6<br />
UNIVERSIDADE FEDERAL DO ABC, SANTO ANDRÉ, SP, BRAZIL. 2,4 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL.<br />
Low level laser therapy increases ATP production and consequently the energy supply to the cell. In sperm cells it could reflect<br />
in increased motility, acrosomal reaction and consequently in the fertilizing potential. The present study aimed the biochemical characterization,<br />
by Fourier Transform infrared micro-spectroscopy, of bovine sperm cells irradiated by low power laser under different fluences. Semen<br />
straws were thawed in a water bath at 37°C for 30 s, washed and diluted in TALP medium to a concentration of 7x10 6 spermatozoa/mL.<br />
Samples were placed in Petri dishes for irradiation and during incubation times. Irradiation were done with a diode laser in the red region (ë<br />
=633nm) with fluences of 30, 150 and 300mJ.cm -2 (obtained due to sample irradiation with laser power of 5mW for 1, 5 and 10 min,<br />
respectively); 45, 230 and 450mJ.cm -2 (7.5mW for 1, 5 and 10 min); and of 60, 300 and 600mJ.cm -2 (10mW for 1, 5 and 10 min). Micro-FTIR<br />
analyses were held with samples air dried in a platinum slide after irradiation and 30 min after irradiation. Spectra were acquired in a Varian 620-<br />
IR FTIR Spectrochemical Imaging Microscope system, in the range of frequencies 400-4000 cm -1 . For statistical analysis, acquired spectra<br />
were divided in three different regions, in the range 900-1300cm -1 (nucleic acids region), 1300-1800cm -1 (proteins and phospholipids) and<br />
2820-3000 cm -1 (lipids). The spectra were baseline corrected and normalized. Statistical analysis were held by principal component analysis<br />
(PCA) and by t-student test (P < 0.05) from intensities of the spectral frequencies. Treatments with higher fluencies (300 and 600mJ.cm -2 )<br />
resulted in low intensity DNA bands (1083 cm -1 ), which could be due to the negative effect of higher fluences in the molecule integrity. For the<br />
fluence of 300 mJ.cm -2 , spectral intensities showed no statistically significant deviation between the used laser powers. Phospholipids<br />
(1740cm -1 ), proteins (1655cm -1 ) and lipids (2852cm -1 ) bands were higher for 600mJ.cm -2 fluence compared to 30, 45 and 60mJ.cm -2 , which<br />
showed no statistically significant deviation by the applied methodology. Based on these data, we can conclude that irradiation with fluence<br />
of 600mJ.cm -2 lead to alterations of the structure of cellular molecules which may be reflected in sperm metabolism. [Acknowledgment:<br />
FAPESP, UFABC, Capes, Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São<br />
Paulo].<br />
Keywords: micro-ftir, low power laser irradiation, bovine.<br />
s452
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A233 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
COMP<br />
OMPARISON OF MRNA EXPRESSION OF STAT3 T3 AND AKR2 IN BOVINE EMBRYOS<br />
WITH 7 AND 11 DAYS AFTER IN VITRO<br />
FERTILIZA<br />
TILIZATION<br />
TION<br />
Tatiana da Silv<br />
ilva Rasc<br />
ascado<br />
ado, Mid<br />
idyan Dar<br />
aroz Guastali,<br />
Luis Eduar<br />
duardo<br />
do Ver<br />
ergar<br />
gara,<br />
a, Rosiar<br />
osiara a Rosár<br />
osária Dias Mazier<br />
aziero, Mateus José Sudano, Daniela Mar<br />
artins Paschoal,<br />
Bianca Andriolo Monteiro & Fernanda da Cruz Landim Alvarenga<br />
UNESP- BOTUCATU, BOTUCATU, SP, BRAZIL.<br />
The aim of this study was to evaluate the expression of Stat 3 and Akp2, markers of pluripotency in blastocysts (D7) and hatched<br />
blastocysts (D11) with seven and eleven days after in vitro fertilization. The oocytes were matured for 24 h in 5% CO2 in air, after they were<br />
fertilized and cultured in SOFaaci for seven and eleven days. The RNA of polls of seven embryos of each developmental stage was extracted<br />
with Rneasy Micro Kit (Qiagen) and reverse transcriptase IMPROM II (Promega, MA, USA) was used for the synthesis of complementary<br />
DNA (cDNA). The polymerase chain reaction (PCR) was performed with the qPCR Master Mix Gotaq (Promega, MA, USA) using as<br />
endogenous control genes SDHA and YWHAZ (Goosens et al., 2005) with 130 and 180 base pairs respectively. For each gene was performed<br />
negative control in which cDNA was replaced by nuclease-free water. Were performed 10 repetitions with RNAs from different groups of<br />
embryos. For statistical analysis of data from 10 replicates was used the analysis of variance with a significance level of 5%. Stat 3 and Akp2 were<br />
expressed in blastocysts (D7) and hatched blastocysts (D11) exhibiting 130 and 80 bp respectively. From the results we concluded that Akp2<br />
and Stat 3 are present in embryos indifferenced, but also in the hatched blastocysts in which MCI has already become a fully differentiated<br />
epithelium, the hypoblast and epiblast (VEJLSTED et al., 2006).<br />
Keywords: pluripotency, gene expression, PCR.<br />
A234 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
CORREL<br />
ORRELATION BETWEEN STALLION FERTILIT<br />
TILITY AND DNA FRAGMENT<br />
GMENTATION TION OF FROZEN SPERM ANALYSED BY<br />
THE ACRIDINA ORANGE TEST<br />
Camila de Paula Freitas Dell´Aqua 1 , José Antonio Dell ´Aqua Junior 1 Marco Antonio Alvarenga 2 & Frederico Ozanam Papa 1,2<br />
1<br />
FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA-UNESP, SÃO PAULO, SP, BRAZIL. 2 FMVZ-UNESP BOTUCATU, BOTUCATU, SP, BRAZIL.<br />
Analysis of DNA fragmentation from sperm has helped to identify men with infertility but showing spermiogram apparently<br />
normal. Regarding that some stallions do not show normal conception rate when considering frozen sperm, but have normal semen evalution.<br />
Thus, the objective of this work was to evaluate the relathionship betweens stallion fertility and the DNA fragmentation index or the tests<br />
routinely used in frozen semen analysis, such as: computer analysis of sperm movement (CASA), plasma membrane integrity and morphology N<br />
sperm. Thirteen animals of different breeds, nine animals Quarter horses, two Lusitano and two Brazilian Equestrian, were considered. From<br />
each animal, a pool of straws was thawed and used for the following analyses: sperm movement assessed by CASA, plasma membrane integrity<br />
assessed using the association of two fluorescent probes - carboxyfluorescein and propidium iodide (Harrison and Vickers, 1990, Journal of<br />
Reproduction and Fertility, 88: 343-52), morphology by Karras staining, and rate of DNA fragmentation assessed by the Acridine Orange test<br />
(Naves et al., 2004. Bioscience Journal, 20:117-124). Statistical analyses were performed by Pearson’s test for analysing the correlation between<br />
sperm parameters and fertility rates, with significance level P < 0.05. No correlation was found between sperm movement or morphology and<br />
fertility. On the other hand, plasma membrane integrity correlated positively(r = 0.8047, P = 0.0016) with fertility whereas the rate of DNA<br />
fragmentation correlated negatively (r = -0.78, P = 0.0025). In conclusion, the acridine orange test for evaluation of DNA fragmentation and the<br />
plasma membrane integrity using fluorescent probes showed strong relationship with fertility, and thus are useful for assessing quality of sperm<br />
frozen. [The authors thank FAPESP for financial support].<br />
Keywords: DNA fragmentation, frozen semen, fertility.<br />
s453
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A235 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
CRYOPRESER<br />
OPRESERVATION OF SOMATIC CELLS IN 0.5 ML OR 0.25 ML STRAWS S AND PROPYLENE GLYCOL OR ETHYLENE GLYCOL<br />
Monica Urio 1 , Claudia Regina Appio Duarte 1 , Renata Cristina Fleith 1 , Maria Luiza Munhoz 1 , Fabiano Carminatti Zago 1 , Fabiana Forell 1 Alceu Mezzalira 1 ,<br />
Marcelo Bertolini 2 & Ubirajara Maciel da Costa 1<br />
1<br />
CAV - UDESC, LAGES, SC, BRAZIL. 2 UNIFOR, FORTALEZA, CE, BRAZIL.<br />
The standard protocol for the cryopreservation of somatic cells makes use of cryovials as classic containers for the storage of<br />
cells, and dimethyl sulfoxide (DMSO) as the cryoprotectant agent of choice. However, cryovials take significant space into LN2 tanks, and<br />
DMSO can be highly toxic and induce differentiation in cells in culture. Thus, the aim of this study was to establish a protocol for<br />
cryopreservation of somatic cells in straws comparing the efficiency of three cryoprotectant agents: DMSO, Ethylene Glycol (EG) and<br />
Propylene Glycol (PG). Primary cell cultures of bovine fibroblasts were maintained in DMEM + 10% FCS in an incubator at 5% CO2 in air<br />
and saturated humidity. Such cells were used for nine experimental groups varying the cryoprotectant agent (DMSO, EG, PG) and the<br />
container (0.25 and 0.5 mL straws and cryovials). Cells were frozen in a solution containing 10% of one of the cryoprotectants in culture<br />
medium. For this, cells were loaded into the container and exposed to the cryoprotectant at 4° C for 15 min before freezing. Straws were kept<br />
in LN2 vapor for 5 min and then immersed in LN2, whereas cryovials were cooled at -1°C/min down to -80°C, using Mr. Frosty ® (Nalgene),<br />
and then transferred to LN2. The experiment was separated in two steps for the survival study, evaluated by the Trypan blue staining. In the<br />
first step, the survival rate was evaluated immediately after thawing (seven replications). In the second step, cell survival was evaluated after<br />
24 h of culture (only for the groups usning straws, in nine replications). Results were evaluated by the ÷2, for P < 0.05. The rate of cell survival<br />
immediately after thawing showed no significant differences between the cryoprotectants and containers, with survival rates for DMSO, EG<br />
and PG being 83%, 86% and 87% in 0.5 mL straws ; 81%, 83% and 87% in 0,25 mL straws; and 87%, 90% and 91% in cryovials, respectively.<br />
Survival rates after 24 h of culture also showed no significant difference between cryoprotectants (DMSO, EG or PG), with survival rates<br />
being 91%, 90% and 91% for 0.5 mL straws, and 91%, 91% and 93% for 0.25 mL straws, respectively. In summary, the freezing of somatic<br />
cells in straws was proven practical and feasible, facilitating storage of larger batches of cells in LN2 tanks, also providing the same cell viability<br />
after freezingas cryovials. Moreover, EG and PG can be readily used as cryoprotectant agents for somatic cell freezing with the same cell<br />
survival efficiency as for DMSO.<br />
Keywords: somatic cell, cryoprotectants, straws.<br />
A236 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
OVARIAN<br />
TISSUE CRYOPRESER<br />
OPRESERVATION FROM BITCHES:<br />
MORPHOLOGIC OGIC EVAL<br />
ALUATION OF PRE-ANTRALS ALS FOLLICLES<br />
José Luiz Jivago de Paula Rôlo 1 , Michelle Silva Araújo 2 , Fernanda Paulini 1 & Carolina Madeira Lucci 1<br />
1<br />
PPG BIOLOGIA ANIMAL, IB, UNB, BRASILIA, DF, BRAZIL. 2 FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA, UNB, BRASILIA, DF, BRAZIL.<br />
The ovarian tissue can be used as an storable source of oocytes, especially enclosed in pre-antral follicles (PAF), and its<br />
cryopreservation is well established in some species, with few reports in dogs (ISHIJIMA et al., 2006, J. Reprod. Dev. 52, 293-299). The slow<br />
freezing (SF) is the most used technique, but the vitrification (V) is an alternative that offers advantages in convenience. There are no studies<br />
comparing the two methods in bitch ovarian tissue. The aim of this study was to compare the effects of SF and V techniques in the bitch<br />
ovarian tissue in the morphology of PAF. Ten ovaries from five healthy bitches were collected after elective ovariohysterectomy and<br />
transported to the lab in buffered saline solution at 37°C. The ovaries were cut in fragments of 1x1x3 cm and randomly assigned to the<br />
procedures of SF, V or immediate fixation (control, C). For SF, the tissue samples were equilibrated in a programmable freezer (Biocom) for<br />
20 min at 10°C in a solution of 1.5M DMSO, 0.4% sucrose, 10% FBS in MEM. Then submitted to the following freezing curve: -1°C/min<br />
until -7°C, when seeding was performed, and -0.3°C/min until -30°C. At the end, the samples were stored in liquid nitrogen. The V method<br />
was based on ISHIJIMA et al. (2006), but the vitrification was done in a solid surface. After 7 days the samples were placed at 37°C and<br />
equilibrated in 0.4% sucrose, 10% FBS and MEM, and washed 3 times with decreasing sucrose concentration. Thawed fragments and the C<br />
were fixed in Carnoy and processed for light microscopy with HE staining for the morphological evaluation of PAF. The percentages of normal<br />
follicles (NF) in the different treatments were analyzed by ANOVA and Tukey´s test. The total percentage of normal PAF was 93.66±6.81%<br />
for C, 86.16±11.05% for SF and 68.14±12.75% for V. For primordial follicles the percentage of NF was 96.69±4.72% for C, 89.51±10.39%<br />
SF and 75.32±9.23% in V. For primary and secondary follicles the values were, respectively, 94.80±6.91% and 87.62±17.12% for C,<br />
86.8±12.15% and 76.35±26.34% for SF and 61.53±14.78 and 52.25±22.13% for V. The percentage of NF in V was significantly lower (P <<br />
0.05) than in the other two treatments, except for the secondary follicles, where no significant difference between SF and V was observed.<br />
There was no significant difference between SF and C for any follicular class. In conclusion, SF is the best cryopreservation method for PAF<br />
in bitches ovarian tissue, and the V protocol needs improvement for a better preservation of the morphological characteristics of the follicles.<br />
Keywords: slow freezing, vitrification, oocyte.<br />
s454
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A237 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
CRYOTOLER<br />
OLERANCE OF BOVINE IN VITRO-PR<br />
-PRODUCED EMBRYOS SUBJECTED TO CONJUGA<br />
ONJUGATED LINOLEIC ACID<br />
Luciana Simões Rafagnin Marinho 1 , Joana Cláudia Mezzalira 1 , Lain Uriel Ohlweiler 1 , Fabiana Forell 1 , Dimas Estrasulas Oliveira 2 & Alceu Mezzalira 1<br />
1<br />
UDESC, LAGES, SC, BRAZIL. 2 UDESC, CHAPECÓ, SC, BRAZIL.<br />
In vitro production (IVP) of bovine embryos is an efficient biotechnology to multiply genetically superior animals, being applied<br />
on a commercial scale worldwide. Nevertheless, it is limited by the low embryo survival rates after cryopreservation, which restricts the use<br />
of surplus embryos obtained in routines. The low cryotolerance of bovine IVP embryos is attributed to excessive accumulation of lipids in the<br />
cytoplasm. The isomer of conjugated linoleic acid trans-10, cis-12 CLA is targeted to reduce the lipid content in different cells, including<br />
embryos. The aim of this study was to evaluate the effect of trans-10, cis-12 CLA on bovine IVP embryos cryotolerance subjected to vitrification<br />
or conventional freezing. Bovine oocytes were selected and subjected to in vitro maturation and fertilization. For in vitro culture, presumptive<br />
zygotes were allocated into two groups: CLA group, with 100 µM of trans-10, cis-12 CLA, and control group, free of CLA. Cleavage (D2) and<br />
blastocyst (D7) rates were evaluated. Grades I and II expanded blastocysts (n = 156) were subjected to vitrification (20% propanediol + 20%<br />
ethylene glycol) or conventional freezing (1,5 M ethylene glycol). After warming, re-expansion and hatching rates were evaluated using the chisquare<br />
test with 5% of significance. There was no difference between cleavage rates of control (81.3%) and CLA groups (77.9%) or blastocyst<br />
rates between control (30.4%) and CLA groups (28, 5%). After cryopreservation, there was no difference between hatching rates of vitrified<br />
control (67.4%) and vitrified CLA groups (65.8%). Both rates were higher than those of frozen control group (28.6%) and frozen CLA group<br />
(13.3%), which did not differ among each other (P > 0.05). It can be concluded that, under the conditions of this study, the addition of trans-10,<br />
cis-12 CLA to culture medium does not improve cryotolerance of bovine IVP embryos and that vitrification provides greater embryo viability<br />
after warming than conventional freezing.<br />
Keywords: vitrification, freezing, cla.<br />
A238 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
BVD<br />
VDV V DETECTION BY A POLYMER<br />
YMERASE CHAIN REACTION-B<br />
CTION-BASED ASSAY IN BOVINE FOLLICULAR FLUID<br />
A ndrea Giannotti Galupp<br />
aluppo, Ma theus Nunes<br />
Web<br />
eber<br />
er, Rena<br />
enata Fon<br />
ont oura Budasz<br />
udaszew<br />
ewsk<br />
ski,<br />
A ngela Oliv<br />
liveir<br />
eira Corb<br />
orbellini,<br />
Lis San<br />
ant os Mar<br />
arques<br />
ques,<br />
José Luis Rigo Rodrigues & Claudio Wageck Canal<br />
UFRGS, PORTO ALEGRE, RS, BRAZIL.<br />
Because of its broad distribution among populations of cattle and association with cells and fluids from infected animals, bovine<br />
viral diarrhea vírus (BVDV) represents a potential problem in applications of assisted reproduction (Gard et al. Theriogenology 68: 434-442, N<br />
2007). The use of ovaries and oviducts harvested in abattoirs from unknown health status animals is already an established practice in the<br />
production of in vitro embryos. The aim of this study was detect BVDV by RT-PCR from bovine follicular fluid (FF) obtained from in vitro<br />
embryo production procedure. The collects were performed between February and December 2010. Bovine ovaries were collected immediately<br />
after slaughter at a commercial abattoir and washed with modified phosphate-buffered saline (PBSm). FF was aspirated from ovarian follicles<br />
range 2-8 mm in diameter and a 2 mL sample was stored at -80ºC until the moment of use. A total of 22 FF pools were analyzed, from 1844<br />
animals (range 43-136 animals/pool). For the isolation of total RNA was used TRIzol ® LS Reagent (Invitrogen, USA) according to manufacture<br />
recommendation. The cDNA was synthesized using the SuperScript ® Reverse Transcriptase Kit (Invitrogen, USA) according to manufacture<br />
recommendation. PCR was performed using the primers 324 and 326 (Vilcek et al. Archives of Virology 136: 309–323, 1994). The PCR<br />
products were separated by electrophoresis carried out in 2% agarose gel dyed with Blue green loading dye I (LGC Biotecnologia, Brazil).<br />
Visualization of amplification products was performed using UV light. It was obtained 3 positive BVDV FF pools (13.63 %). The presence of<br />
BVDV has been determined to be detrimental in culture systems, causing a reduction in rates of maturation, fertilization and/or development.<br />
Considering that, the use of FF BVDV PCR-assay is an important tool to assure the embryos quality production in vitro.<br />
Keywords: bvdv, folicular fluid, bovine.<br />
s455
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A239 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
ECHOGENICITY OF TESTES OF BUFF<br />
UFFAL<br />
ALO CROSSBREDS CREATED IN THE STATE TE OF PAR<br />
ARA,<br />
A, AMAZON,<br />
BRAZIL<br />
Henr<br />
enry Daniel Manr<br />
anrique Ayala,<br />
Sebastião<br />
Tavar<br />
ares Rolim Filho<br />
ilho, Haroldo Francisc<br />
ancisco Loba<br />
obato o Rib<br />
ibeir<br />
eiro, Kim Borb<br />
orbor<br />
orema Nunes<br />
unes, Ellen<br />
Yasmin Eguchi Mesquita &<br />
Keitiane Colares Sousa<br />
CURSO DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL, UNIVERSIDADE FEDERAL DO PARÁ (UFPA), BELÉM, PA, BRAZIL.<br />
The aim of our study was to evaluate the changes in testicular ultrasonographic echotexture of nineteen male crossbred buffaloes<br />
(Murrah x Mediterranean) from 12 to more than 60 months in pre-puberty, puberty and sexual maturity, extensive creations in the period May-<br />
November 2010. The results mean ± standard deviation of echogenicity (pixels) in animals from 12 to 13 months was 78.67 ± 6.36 pixels, 14<br />
and 15 months was 94.22 ± 3.40 pixels, 16 and 17 months was 88.16 ± 3.95 pixels, 18 to 19 months was 96.09 ± 3.40 pixels, 20 to 21 months<br />
was 103.12 ± 3.86 pixels between 22 and 23 months was 98.4 ± 5.87 pixels, 24 to 29 months was 114.05 ± 2.42 pixels, of the 30 at 35 months<br />
was 109.24 ± 3.13 pixel, from 36 to 41 months was 98.67 ± 3.5 pixels, from 42 to 47 months was 99.33 ± 01.02 pixels, 48 to 59 months was<br />
96.17 ± 1.90 pixels and more than 60 months 90.13 ± was 1.77 pixels, showing statistical differences between the different categories of age,<br />
by Duncan test (P < 0.05). Thus we can infer that ultrasonography is a useful tool in assessing andrological bull buffalo.<br />
Keywords: buffalo, echogenicity, testes.<br />
A240 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EFFECT OF DIFFERENT CRYOPR<br />
OPROTECT<br />
TECTANT SOLUTIONS IN THE BOVINE SPERMATOGONIAL CELL VIABILITY<br />
SUBMITTED TO TWO COOLING CURVES<br />
Andrielle Mendes Cunha 1 , Heidi Christina Bessler 2 , Elisa Ribeiro da Cunha 3 , Carolina Gonzales da Silva 4 , George Henrique Lima<br />
Martins 5 , Carlos Frederico Martins 6 & Luiz Osvaldo Fonseca Rezende 7<br />
1,3,4<br />
BOLSISTA CNPQ, EMBRAPA CERRADOS-CTZL, BRASÍLIA, DF, BRAZIL. 2,5,6,7 EMBRAPA CERRADOS-CTZL, BRASILIA, DF, BRAZIL. 4 UNB-EMBRAPA CERRADOS -CTZL, BRASILIA, DF,<br />
BRAZIL.<br />
The cryopreservation of bovine spermatogonial cell has been considered an important strategy of storaging prior to ICSI (Barbosa,<br />
et al., <strong>2011</strong>, Arch. Zootec., 60, 293-296). However, the freezing of immature germinal cells, such as spermatids, may cause greater damages<br />
when compared to freezing of mature spermatozoa (Ogonuki, et al., 2006, PNAS, 103, 13098-13103). Therefore, the objective of this study was<br />
to test four different cryoprotectant molecules and two freezing curves to preserve the spermatogonial cells. Testicles from six bulls slaughtered<br />
in a local slaughterhouse were used. The testicular parenchyma was macerated, filtered and centrifuged in Percoll column 20/30%. The isolated<br />
spermatogonial cells pool was cryopreserved in Dulbecco’s Modified Eagle Medium (DMEM) with fetal bovine serum and antibiotics, added<br />
to one of the four different cryoprotectants: dimetilsulfoxide (DMSO) 10%, glycerol (GLY) 7%, ethylene glycol (ETIL) 7% and<br />
dimethylformamide (DMF) 5%. The cells were frozen by using a non-controlled cooling curve at -80ºC for 24h followed by liquid nitrogen<br />
plunge and an automated controlled cooling curve (decreasing 0,25ºC/min until 5ºC, followed by a dropping of 20ºC per minute until - 120ºC).<br />
The cellular viability was assessed by using 1:1 Trypan blue staining 0.4%. The results were analyzed by ANOVA and Tukey test for comparison<br />
between groups (P < 0.05). The non-controlled temperature curve showed that 56.91±9.99%; 56.75±11.33%; 51.05±12.29% and 62.08±15.21%<br />
cells remained intact when using DMSO, DMF, GLY and ETIL, respectively. In the automatic controlled curve, 57.25±10%; 50.91±7.85%;<br />
50.00±9.18% and 55.08±9.82% cells were viable, respectively in the same media order. Apparently the glycerol solution has shown less viable<br />
cells than other treatments, it wasn’t possible to detect statistical significance differences among the tested solutions. The same happened to the<br />
cryopreservation system, neither one has differently influenced the cellular viability, showing no statistical difference among the methods tested.<br />
These results show that irrespective of the cryoprotectant solution, the cellular viability is kept above 50%. Despite the freezing system using -<br />
80ºC be an uncontrolled temperature dropping, it has the same efficiency as the monitored curve, which exempts from the acquiring of an specific<br />
equipment.<br />
Keywords: cryoprotectant, spermatogonial, viability.<br />
s456
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A241 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EFFECT OF ANTIOXIDANT SUBSTANCES IN CRYOPRESER<br />
OPRESERVED SEMEN FROM BUFF<br />
UFFAL<br />
ALOS (<br />
(BUB<br />
UBAL<br />
ALUS BUB<br />
UBALIS<br />
ALIS)<br />
Arnaldo Algaranhar Gonçalves 1 , Sâmia Rubielle Silva de Castro 2 , Alexandre Rossetto Garcia 3 , Alessandra Ximenes Santos 4 , Geanne Rocha Silva 5 & Daniel<br />
Vale Barros 6<br />
1,4,5<br />
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA ANIMAL UFPA/EMBRAPA/UFRA, BELEM, PA, BRAZIL. 2 FACULDADES INTEGRADAS DO TAPAJÓS (FIT), SANTARÉM, PA, BRAZIL.<br />
3<br />
LABORATÓRIO DE REPRODUÇÃO ANIMAL, EMBRAPA AMAZÔNIA ORIENTAL, BELÉM, PA, BRAZIL. 6 UNIVERSIDADE FEDERAL RURAL DA AMAZÔNIA (UFRA), BELEM, PA, BRAZIL.<br />
Cryopreservation causes damage to sperm plasma membrane due to the increase in oxygen use by cells, leading to increased<br />
production of free radicals. Therefore, the objective was to evaluate the effect of two antioxidants, alone or in combination on parameters of<br />
cryopreserved semen of buffalo. The work was performed at Embrapa Eastern Amazon, Belém-PA. Five bulls previously selected and with<br />
similar semen quality had semen collected by artificial vagina fortnightly and are used six ejaculates from each bull (n = 30). Each ejaculate was<br />
frozen under four distinct treatments. The control group (CONT) comprised the freezing extender with TES-TRIS, as Vale (2002, Buffalo<br />
Symposium of Americas 1, 156-171). The other groups followed the protocol of the control, but were enriched with 2.5 mM vitamin C (VIT<br />
Group), as recommended by Singh et al. (1996, <strong>International</strong> Journal Animal Science 11, 131-132) or with 3.5 mM pentoxifylline (Group<br />
PENTOX), according to Marques et al. (2002 Theriogenology 58, 257-260) or with the combination of 2.5 mM vitamin C + 3.5 mM<br />
pentoxifylline (VIT Group/PENTOX). The motility and plasma membrane integrity of sperm after thawing and after thermal resistance test<br />
(TRT) execution, as Rota et al. (1997, Theriogenology 47, 1093-1101). After testing the normality of distribution of variables, means were<br />
compared by ANOVA. If contrasts were significant, the Tukey test and t-Test were applied (P < 0.05). Raw semen presented progressive motility<br />
of 77.7 ± 6.1% and integrity of plasme membrane of 85.4 ± 8.3% before freezing. After thawing progressive motility was 39.5 ± 23.8%<br />
(CONT), 41.9 ± 22.3% (VIT), 44.6 ± 19.9% (PENTOX) and 46.9 ± 19% (VIT/PENTOX) P > 0.05. After the TTR, motility was 15.1 ± 15.3%<br />
(CONT), 25.6 ± 16.3% (VIT), 23.3 ± 14.6% (PENTOX) and 28.6 ± 16, 2% (VIT/PENTOX) with P < 0.05. The integrity of plasma membrane<br />
staining performed with Eosin-Nigrosine observed was 62.2 ± 15.9% (CONT), 63.5 ± 13.3% (VIT), 57.9 ± 15.7% (PENTOX) and 57.6 ±<br />
15.9% (VIT/PENTOX), P > 0.05. After the TRT was 58.0 ± 13.5% (CONT), 58.3 ± 13.3% (VIT), 54.0 ± 14.9% (PENTOX) and 51.8 ± 15.5%<br />
(VIT/PENTOX) P > 0.05. The significant increase in motility parameters after the TTR may be related to antioxidant effects, by decreasing the<br />
action of reactive oxygen species and the injuries on the sperm cells. Thus, the vitamin C, used alone or associated with pentoxifylline, caused<br />
significant shifting on progressive motility after TRT, which is worthy since this test simulates the stress that spermatozoa undergo after semen<br />
takes place into the female reproductive system.<br />
Keywords: acid ascorbic, pentoxifylline, spermatozoa.<br />
A242 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
BULL EFFECT IN THE EMBRYO SURVIV<br />
VIVAL PRODUCED IN VITRO AFTER SUBMISSION TO SLOW FREEZING<br />
M auricio Bar<br />
arr os Fer<br />
ernandes<br />
1 , A nderson Mior<br />
ioranza<br />
2 , V ivian Taís Fer<br />
ernandes Cipr<br />
ipriano<br />
iano 2 , R eginaldo Apar<br />
parecido<br />
V ila 2 , M arli Apar<br />
parecida<br />
Galerani 2 , Claudia Cristina Paz 2 & Raysildo Barbosa Lôbo 2<br />
1<br />
PRO-FIV, SÃO JOSÉ DO RIO PRETO, SP, BRAZIL. 2 FMRP-USP, RIBEIRÃO PRETO, SP, BRAZIL.<br />
N<br />
In the programs of embryo transfer, cryopreservation has fundamental importance on storage, transport, trade and sanitary control<br />
of embryos. One of the bottlenecks in the application of this technology on in vitro-produced embryos (IVP), it is the high sensitivity to freezing.<br />
Thus, the aim of this study was to assess the influence of the bull effect on survival of embryos that were submitted to freezing process. To<br />
evaluate that, 2856 cumulus-oocyte complexes (COCs) recovered from slaughterhouses were matured in TCM199 for 24 h at 38.5°C and 5%<br />
CO2. The COCs were fertilizated in vitro (IVF) using frozen semen from 15 Nelore bulls. For each bull, it was observed embryonic<br />
development at 168 h after IVF, considering viable the embryos in the stages of blastocyst and expanded blastocyst. The selected blastocysts<br />
were growth in the medium containing cryoprotectants, loaded into straws, refrigerated and freezed. Then, the cryoprotectant was removed and<br />
the embryos were washed and growth in another medium. To evaluate the effect of freezing, we analyzed two different groups of blastocysts:<br />
the control group (C), composed by blastocysts maintained under cultivation for 72 h, and the freezing group (FG) composed by blastocysts<br />
that were frozen and subsequently submitted to cultivation for 72 h. The data were analyzed using SAS statistical software using Chi-square<br />
and, when necessary, Fisher’s exact test, considering the significance of 5%. The embryos that passed through the freezing process had a<br />
significantly decreased in their survival rate when compared with the control group. From the bulls submitted to the freezing process, 13 had<br />
a statistically significant (P < 0.05) in hatching blastocysts. Among the bulls with statistically differences, some were more contrasting,<br />
reaching to 83% of embryos in the C group and 0% in the CG group. More interesting, two of the analyzed bulls did not show statistical<br />
differences between the groups (CG and FG), presenting similar amounts of embryos. Our results revealed a possible influence of the bull on<br />
the survival of embryos submitted to freezing process and will be important to commercial applications. Further investigations are needed to<br />
confirm this relationship and to elucidate the components responsible for promoting this change in the survival of frozen embryos.<br />
Keywords: embryos, freezing, in vitro fertilization.<br />
s457
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A243 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EFFECT OF POLYUNSA<br />
YUNSATUR<br />
TURATED TED FATTY ACIDS DURING IN VITRO CULTURE OF BOVINE EMBRYOS ON THE SUCCESS CESS OF<br />
VITRIFICATION TION - PRELIMINARY RESULTS<br />
Nathália Alves de Souza Rocha 1 , Beatriz Caetano da Silva Leão 2 , Gisele Zoccal Mingoti 3 & Mônica Ferreira Accorsi 3<br />
1<br />
UNESP-JABOTICABAL, RIBEIRÃO PRETO, SP, BRAZIL. 2 UNESP-JABOTICABAL, RIO VERDE, GO, BRAZIL. 3 UNESP-ARAÇATUBA, ARAÇATUBA, SP, BRAZIL.<br />
Studies report that bovine embryos produced in vitro have worse quality, lower viability and higher cryosensitivity in relation to<br />
that obtained in vivo, which can be attributed to the excessive content and/or lipid composition. Supplementation with polyunsaturated fatty<br />
acids (PUFAs) during IVC appears to increase cryotolerance of bovine embryos. The aim of this study was to evaluate the effects of IVC<br />
medium supplementation with the PUFAs conjugated linoleic acid (CLA, Omega-6), decosahexaenoic acid (DHA, omega-3), both or none of<br />
these on the embryonic development and cryotolerance. COCs (n = 932) were in vitro matured in TCM-199 supplemented with 0.2 mM<br />
pyruvate, 25 mM sodium bicarbonate, 75 g/mL gentamicin, 10% FCS and hormones for 24 h at 38.5°C and 5% CO2. After, they were<br />
fertilized and presumptive zygotes were cultured in SOFaa medium supplemented with 100 µM CLA (CLA), or 100 µM DHA (DHA), or<br />
100 µM of CLA + 100 µM DHA (CLA+DHA) or no fatty acid (Contr) for 7 days at 38.5°C and 5% CO2. The cleavage was evaluated 48<br />
hpi and the embryonic development 168 hpi, when embryos were vitrified by the Vitri-Ingá ® protocol (Ingámed ® , Maringá-PR, Brazil).<br />
Subsequently, blastocysts (n = 54) were warmed and cultured for 24 h to assess the rate of re-expansion. Data were analyzed by Chi-square<br />
test (P < 0.05). The cleavage rates were 79.8% a (Contr), 77.8% a (CLA), 75.3% a (DHA) and 81.5% a (CLA+DHA). The embryonic development<br />
rates were 46.5% a (Contr), 46.0% a (CLA), 39.0% ab (DHA) and 37.9% b (CLA+DHA). The rate of re-expansion after 3h and 24h of culture<br />
were, respectively, 66.7% a and 77.8% a (Contr), 57.1% a and 71.4% a (CLA), 50.0% a and 60.0% a (DHA) and 78.9% a and 73.7% a (CLA+DHA).<br />
Addition of CLA or DHA to IVC medium did not impair embryonic development, but their association was detrimental. There is no previous<br />
reported utilization of the association CLA+DHA in the IVC, so it is necessary to evaluate whether such detrimental effect is due to an<br />
undesirable direct effect of PUFAs or if it may be due to the increase in the total concentration of PUFAs in the culture medium. The present<br />
preliminary data showed no difference in the rates of embryo re-expansion after heating of vitrified embryos. Based on the results we can<br />
conclude that supplementation with CLA or DHA of IVC medium, but not their association, is suitable for in vitro embryo development and<br />
successful cryopreservation. [Acknowledgements: Ingámed and Alta Genetics Brazil].<br />
Keywords: criopreservation, pufas in in vitro culture, bovine embryo.<br />
A244 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EFFECTS OF POLYUNSA<br />
YUNSATUR<br />
TURATED TED FATTY ACIDS OMEGA 3 AND 6 DURING IN VITRO MATUR<br />
TURATION TION AND CULTURE ON BOVINE<br />
EMBRYO DEVELOPMENT AND CRYOTOLERANCE<br />
Beatriz Caetano da Silva Leão 1 , Nathália Alves de Souza Rocha 1 , Mônica Ferreira Accorsi 2 & Gisele Zoccal Mingoti 2<br />
1<br />
UNESP-JABOTICABAL, RIO VERDE, GO, BRAZIL. 2 UNESP-ARAÇATUBA, ARAÇATUBA, SP, BRAZIL.<br />
The use of polyunsaturated fatty acids (PUFAs) on IVP of bovine embryos aims to reduce the accumulation of cytoplasmic<br />
lipids, particularly triacylglycerols, and improve embryo cryotolerance. The aim of this study was to evaluate the effect of adding conjugated<br />
linoleic acid (CLA, Omega-6), docosahexaenoic acid (DHA, Omega-3) and the association of both (CLA + DHA) during IVM or IVM/IVC<br />
culture on development and cryotolerance of in vitro produced embryos. COCs (n = 1689) were in vitro matured during 24h in B199 medium<br />
(TCM-199 supplemented with bicarbonate, hormones and 10% FCS; CONTR group) which was supplemented with 100 µM CLA, or 100<br />
µM DHA or 100 µM CLA + 100 µM DHA. After IVF, zygotes were in vitro cultured in SOFaa medium (5% BSA + 2.5% FCS in a 5% CO 2<br />
atmosphere) supplemented or not with the same PUFAs used on IVM. The cleavage was evaluated at 48 hpi and blastocysts (Bl) at 168 hpi,<br />
when they were vitrified by the Vitri-Ingá ® protocol (Ingámed ® , Maringá-PR, Brazil). Later, the Bl were heated (n=101) to evaluate the rate<br />
of re-expansion after 3h of IVC. Data were analyzed by Chi-Square test. Cleavage rate was 79.8% ab (CONTR), 85.1% a (CLA/SOF), 75.3% ab<br />
(CLA/CLA), 81.1% ab (DHA/SOF), 72.2 % b (DHA/DHA), 74.6% ab (CLA+DHA/SOF) and 73.8% ab (CLA+DHA/CLA+DHA). The Bl rate<br />
was 46.5% ab (CONTR), 40.4% ab (CLA/SOF), 40.7% ab (CLA/CLA), 49.2% a (DHA/SOF), 38.3% b (DHA/DHA), 39.2% b (CLA+DHA/SOF)<br />
and 41.1% ab (CLA+DHA/CLA+DHA). The rate of re-expansion was 66.7% a (CONTR), 75.0% ab (CLA/SOF), 87.5% ab (CLA/CLA),<br />
81.0% ab (DHA/SOF), 85.7% ab (DHA/DHA), 75.0% ab (CLA+DHA/SOF) and 100.0% b (CLA+DHA/CLA+DHA). The IVM or IVM/IVC<br />
medium supplementation with CLA and/or DHA did not impair the cleavage rate and embryo development compared to the control group (P<br />
> 0.05); the only difference observed was the reduction in the Bl production on DHA/DHA and CLA+DHA/SOF groups in relation to the<br />
DHA/SOF group (P < 0.05). The re-expansion rate of the CLA+DHA/CLA+DHA group was higher than the control (P < 0.05), and no<br />
differences were found among the remaining groups. However, numerically, the addition of PUFAs resulted in better embryo re-expansion<br />
rates. In conclusion, the association CLA+DHA during IVM/IVC improved the cryotolerance of in vitro produced embyos and the addition<br />
of CLA or DHA on IVM or IVM/IVC showed a tendency to increase cryotolerance. [Acknowledgements: Ingámed and Alta Genetics Brazil].<br />
Keywords: embryo in vitro production, pufa, cryopreservation.<br />
s458
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A245 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
STUDY OF FOLICULAR<br />
VASCUL<br />
ASCULARIZA<br />
ARIZATION USING TRIDIMENSIONAL IMAGES:<br />
A NEW APPROACH<br />
Eduardo Kenji N Arashiro 1 , Miller Pereira Palhão 2 , Sabine Wohlres-Viana 3 , Luiz Gustavo Bruno Siqueira 4 , Marc Roger Jean Marie Henry 1,5 & João Henrique<br />
Moreira Viana 4<br />
1<br />
UFMG, JUIZ DE FORA, MG, BRAZIL. 2 UNIFENAS, ALFENAS, MG, BRAZIL. 3 UFJF, JUIZ DE FORA, MG, BRAZIL. 4 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 5 UFMG, BELO<br />
HORIZONTE, MG, BRAZIL. 6 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL.<br />
The vascularization is important to the follicle development and it is directly related to fluid follicular composition, which in turns is<br />
critical to oocyte maturation. Because of the reduced dimension of the blood vessels the assessment of vascularization by pulsatility and<br />
resistance indexes is frequently unfeasible, and quantification is represented by a percentage subjectively obtained by visualization. The aim of<br />
the present study was to develop a new methodology to assess the follicular vascularization using color Doppler technology and a software for<br />
image analysis (Image J) to recreate tridimensional images of the blood vessels found on the follicle. For tridimensional images generation it is<br />
important to determine the number of frames necessary to maintain the spatial resolution of the structure. Therefore, latex spheres of different<br />
diameters (12, 11, 10, 9, 8, 7, 6, 5 and 4 mm) were filled up with a volume of PBS similar to the expected volume of follicle of same diameters<br />
(approximately 900, 700, 520, 380, 270, 180, 110, 70 and 30 mm 3 , respectively). Subsequently, sequences of ultrasonographic images of these<br />
mimetic follicles were recorded and evaluated with Image J to calculate the volume. The number of frames necessary to maintain the spatial<br />
resolution was 120, 11, 99, 78, 70, 60, 50 and 31 for the mimetic follicles of 12, 11, 10, 9, 8, 7, 6, 5 and 4 mm in diameter, respectively. With<br />
these numbers of frames, the volume calculated by Image J varied less than 5% compared to the expected value (921.55; 712.70; 514.16; 385.99;<br />
277.34; 182.93; 112.73; 69.74 e 29.71 mm 3 , respectively). Thereafter, the follicular wave was synchronized (beginning of protocol = D0) in two<br />
cows (Gyr and Holstein). The growth of dominant follicle was daily evaluated by ultrasonography since its emergence, and its vascularization<br />
visualized using color doppler technology. Tridimensional images were generated using the number of frames previously determined, and the<br />
volume of vascularization was calculated. In both cows, the presence of vascularization was first detected on D5 (2.2 vs. 9.9 mm 3 , Gyr and<br />
Holstein respectively), and progressively grew until dominance phase (54.9 vs. 83.2 mm 3 , Gyr and Holstein respectively). After dominance a<br />
sharp decrease on vascularization volume was observed (4.1 vs. 49.3 mm 3 , Gyr and Holstein respectively). Although this is a preliminary study<br />
with a limited number of observations, the results obtained are consistent with the expected variation during the follicle development, showing<br />
the potential of this new approach as an alternative and less subjective methodology to assess follicular vascularization. [Acknowledgment: To<br />
FAPEMIG and MP1 of Embrapa (01.07.01.002)].<br />
Keywords: ultrasonography, color-doppler, ovary.<br />
A246 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
QUALIT<br />
ALITATIVE TIVE STUDY OF THE INTERACTION OF BSPS (BINDER OF SPERM PROTEINS)<br />
WITH THE SPERM CELLS<br />
IN RUMINANTS<br />
Maurício Fraga Van Tilburg 1 , Ítalo Cordeiro Lima 1 , Veronica Gonzalez Cadavid 1 , Airton Alencar Araújo 2 , David Ramos da Rocha 1 ,<br />
Carlos Eduardo Azevedo Souza 1 , Magno José Duarte Candido 1 & Arlindo Alencar Moura 1<br />
1<br />
UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.<br />
N<br />
BSPs (Binder of Sperm Protein) are the most abundant proteins of the seminal plasma of several ruminants and play important roles<br />
during sperm capacitation and interaction between sperm cells and the oviductal epithelium. In the ram, RSVP 14 and 22 kDa represent the BSP<br />
family and BSP 1 and 5 are their homologues in the bovine. Thus, the present study was conducted to evaluate the expression of BSPs in fluids<br />
of the reproductive tract and their interactions with sperm of ruminants. Seminal plasma and sperm were obtained by centrifugation of semen<br />
from Morada Nova rams and cauda epididymal sperm, collected from slaughtered animals. After the first centrifugation of semen samples, sperm<br />
were washed three times in PBS, homogenized and the resulting pellet was washed three more times in PBS. The pellet was resuspended in PBS<br />
1% triton X-100 and kept a 4°C for two h, with homogenizations every 15 min. The solution was sonicated at 4°C for 30 min and centrifuged<br />
for one h (5.000 x g, 4°C). The supernatant and seminal plasma were then precipitated with acetone and resuspended in a buffer (urea, thiurea,<br />
chaps, dtt, Ipg buffer). Samples containing 400 µg of total protein were subjected to 2-D electrophoresis and the maps, analyzed using<br />
PDQuest software (BioRad, USA). A similar protocol was used to process seminal plasma and sperm membrane proteins from Saanen goats<br />
and Holstein bulls. Two-dimensional maps were also constructed using fluid from the cauda epididymis (CEF) and accessory sex glands<br />
(AGF). In rams, we detected RSVP 14 as the major component in seminal plasma maps and in gels of proteins extracted from membranes of<br />
ejaculated sperm, but not from epididymal sperm. However, RSVP 22 did not appear in gels of ejaculated sperm as the same pattern detected<br />
for the RSVP 14. In goats, proteins with kDa and pI similar to those of RSVP 14 were detected in the seminal plasma and in gels containing<br />
proteins from ejaculated sperm. It is possible, thus, that 14-kDa BSPs, in comparison with 22-kDa BSPs, have stronger affinity for sperm<br />
membranes after ejaculation. In the case of bulls, BSP1 was also detected as the major component of seminal plasma, AGF and in gels of<br />
membrane proteins extracted from ejaculated sperm, but absent in the CEF. In conclusion, we suggest that there is a conserved mechanism of<br />
secretion of BSPs and of interaction of these proteins with sperm cells in different ruminant species.<br />
Keywords: proteomics, semen, ruminants.<br />
s459
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A247 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
GENE EXPRESSION OF EMBRYOS<br />
IN VITRO PRODUCED<br />
WITH SORTED SPERM BY DENSITY GRADIENT CENTRIFUGATION TION AND<br />
FLOW CYTOMETR<br />
OMETRY<br />
Aline Costa de Lucio<br />
FCAV, JABOTICABAL, SP, BRAZIL.<br />
The purpose of this work was to compare, using real-time quantitative RT-PCR, the relative mRNA abundance of several<br />
developmentally important gene transcripts of bovine embryos produced in vitro using unsexed semen (control group) sorted by density gradient<br />
centrifugation and by flow cytometry. Blastocysts at Day 7 and 8 of in vitro culture were used to examined the relative mRNA expression of eight<br />
genes: AKR1B1, COX2, IGF2R, PLAC8, MnSOD, GPX1, SLC2A1 and TP53. Poly(A) RNA was extracted using the Dynabeads mRNA<br />
Direct Extraction KIT (Dynal Biotech, Oslo, Norway). After extraction, the reverse transcription (RT) were performed using poly(T) primer,<br />
random primers and MMLV reverse transcriptase enzyme (Bioline, Ecogen, Madrid, Spain) in a total volume of 40µL to prime the RT reaction<br />
and to produce cDNA. Tubes were heated to 70ºC for 5 min to denature the secondary RNA structure and then the RT mix was completed with<br />
the addition of reverse transcriptase. They were then incubated at 42ºC for 60 min to allow the reverse transcription of RNA, followed by<br />
incubation at 70ºC for 10 min to denature the RT enzyme. Quantification of all mRNA transcripts was performed by real-time quantitative (q)<br />
RT-PCR. For qRT-PCR, three groups of cDNA per experimental group, each obtained from 10 embryos, were used with two repetitions for all<br />
genes. Experiments were conducted to compare relative levels of each transcript with those of the housekeeping histone H2AFZ in each sample.<br />
The PCR was performed by adding a 2µL cDNA of each sample. The comparative cycle threshold (CT) method was used to quantify expression<br />
levels. Fold changes in the relative gene expression of the target gene were determined using the formula 2 CT, with control group like reference.<br />
Data were analysed using one-way ANOVA with 5% of significance. Flow cytometry methodology resulted in a significant reduction in the<br />
expression level of expression of AKR1B1 (P = 0.023) and COX2 (P = 0.016). Density gradient centrifugation increased the transcripts levels<br />
of PLAC8 (P = 0.007) and SLC2A1 (P = 0.028). Both sorting process reduced the mRNA of TP53 (P < 0.05), compare to a control group.<br />
Flow cytometry reduced the expression of genes involved in pregnancy recognition and placenta formation. Density gradient methodology<br />
affected the expression pattern of genes related to metabolism and increased the expression of a gene related with pregnancy establishment. These<br />
results suggest that density gradient centrifugation produce better quality embryos to pregnancy induction than flow cytometry.<br />
Keywords: bovine, mRNA, real time quantification.<br />
A248 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
DNA EXTR<br />
TRACTION BY ALKALINE ALINE LYSIS FOR MOLECULAR DIAGNOSIS:<br />
A FAST<br />
AST, SIMPLE AND EFFICIENT METHOD<br />
FOR THE PCR-BASED SEXING OF IN VITRO-PRODUCED SHEEP EMBRYOS<br />
K aio Cesar Simiano<br />
Tavar<br />
ares<br />
1 , Cr istiano Feltr<br />
eltrin<br />
1 , Igor Sá Car<br />
arneir<br />
neiro 1 , Débor<br />
ora Barb<br />
arbosa Rios<br />
1 , Renna Kar<br />
aroline Costa<br />
1 , M aurício Barb<br />
arbosa<br />
Salviano 1 , Luiz Fernando Schütz 1,2 Felipe Jesus Moraes Junior 1,3 , Jamir Machado Junior 1 , Raquel Mello Pinho 1 , Juliana Lopes<br />
,<br />
A lmeida 1 , Saul Gaudêncio Net<br />
eto 1 , Leonar<br />
eonardo<br />
Tondello Mar<br />
artins<br />
1 , Ana Kar<br />
arolina Fr eire 1 , Mar<br />
arc elo Ber<br />
ert olini 1 & Luciana Relly Ber<br />
ert olini 1<br />
1<br />
UNIFOR, FORTALEZA, CE, BRAZIL. 2 UDESC, LAGES, SC, BRAZIL. 3 UFPI, TEREZINA, PI, BRAZIL.<br />
The molecular diagnosis of pre-implantation embryos, as for sex determination, is a biotechnological tool that can be extremely<br />
useful when applied to animal selection. However, traditional DNA extraction methods for PCR-based sex determination in embryos are either<br />
time consuming or all DNA obtained must be used in the reaction. The aim of this study was to verify the efficiency of the sodium hydroxide<br />
(NaOH)-based alkaline lysis procedure for DNA extraction for use in the sex determination of in vitro-produced (IVP) sheep embryos.<br />
Cumulus-oocyte complexes from sheep ovaries obtained at a local slaughterhouse were subjected to IVM for 24 h, followed by IVF with frozen<br />
ram semen for 24 h, and IVC for 6 days. The IVP media used were from a commercial source (Nutricell ® , São Paulo). A total of 57 in vitroproduced<br />
compact morulae and expanded blastocysts were subjected to gender diagnosis. A group of 41 embryos were subjected to zona<br />
pellucida digestion in 0.5% protease, being individually transferred to sterile microtubes containing 5 µL ultrapure water. Embryo biopsies<br />
performed on 16 embryos were also individually transferred to microtubes containing 5 µL ultrapure water. All samples were stored at -80ºC<br />
until analyses. The embryo lyses were performed by adding 5 µL of a 400-mM NaOH solution to each microtube. Then, microtubes were<br />
heated for 10 min at 68°C, followed by the addition of 10 µL of a 1-M Tris-HCl solution at pH 8.5. The PCRs were performed using PCR<br />
Mastermix (Quatro G Pesquisa e Desenvolvimento, Porto Alegre, Brazil), according to the manufacturer’s instructions; 5 mM of each primer<br />
specific to a polymorphic region of the amelogenin gene on the sex chromosomes (AME-F 5´-CAGCCCAAACCTCCCTCTGC-3´ and<br />
AME-R 5´-CCCGCTTGGTCTTGTCTGTTGC-3´; Chen et al., 1999; Mol Reprod Dev 54:209-214); and 2 µL of DNA from each sample.<br />
PCR products were subjected to electrophoresis on a 2% agarose gel for 1 h at 10 V/cm2. Sex was successfully determined on 98.2% of all<br />
analyzed embryos or embryo biopsies (56/57), with 29 males (50.9%) and 27 females (47.4%). No associations between sex and embryo<br />
quality or stage of embryo development were observed. The amplification of DNA extracted by the NaOH-based alkaline lysis procedure was<br />
proven fast and reliable, with high repeatability and efficiency (98.2%) for the sex determination of sheep embryos and embryo biopsies. This<br />
method has also been effectively tested for bovine and caprine embryos and cells in our laboratory. By using minimal amounts of the extracted<br />
DNA (10% of total), the alkaline lysis methodology has the advantage of preserving significant amounts of DNA in stock that can be used for<br />
further molecular analysis of the same biological sample.<br />
Keywords: embryos, sexing, alkaline lysis.<br />
s460
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A249 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
MULTIP<br />
TIPAR<br />
AROUS BUFF<br />
UFFAL<br />
ALOES SUBMITED TO EXOGENOUS MELATONIN IN LONG PHOTOPERIOD<br />
OPERIOD<br />
Abisai de Oliveira Sousa<br />
UEMA/CCA/CMV, SAO LUIS, MA, BRAZIL.<br />
This work objective was to evaluate the effect of melatonin subcutaneous implants containing 18 mg (Melovine) on reproductive<br />
function of buffaloes during long photoperiod. The efficiency of treatments was tested evaluating estrus behavior, using teaser bulls with<br />
reproductive device markers. The experiment included 100 females distributed in three groups: G1 = control group (n = 38), G2 = one melatonin<br />
implant (n = 36) and G3 = three melatonin implants (n = 26). Eighty six (86%) females were in cyclical activities in the long photoperiod. They<br />
were mated and 71 (82.6%) became pregnant. It was verified that 18 animals repeated heat totalizing 20 repetitions. Fourteen females stayed in<br />
anestrous during all the experimental period, being 6 of group G1 (15.8%), 6 of group G2 (16.7%) and 2 of group G3 (7.7%). Only 32 (84.2%),<br />
showed estrus in G1, and 10 (26.3%) were mating in the beginning and the end of the long photoperiod and 22 (57.9%) of them were during<br />
the greatest luminosity. In the G2, 30 buffaloes were mating (83.3%), wherever 10 (27.8%) at beginning and the end of the long photoperiod and<br />
the 20 remaining (55.6) during the greatest luminosity. And in the G3, 24 were mating (92.3%), but only 04 (15.4%) at beginning and the end<br />
of the long photoperiod and the 20 remaining (76.9) were mating during the period of greatest luminosity. The data showed a service rate (cows<br />
in heat/total number of cows) in G1=84.2%, G2=83.3% and G3=92.3% (P > 0.05), conception rate of: G1=78.1%, G2=83.3% and,<br />
G3=87.5% (P > 0.05) and, pregnancy rate of: G1=65.8%, G2=69.4% and, G3=80.8% (P > 0.05). The results showed reproductive activity in<br />
86% of buffaloes cows, with no differences (P > 0.05) between groups, indicating that reproductive behavior of this species prolongs almost<br />
until photoperiod by 13 h approximately. Consequently, reproductive behavior was observed during the long photoperiod months, indicating a<br />
pattern of reproductive behavior is different for this species. Moreover, the administration of melatonin exogenous did not affect the fertility<br />
during this period.<br />
Keywords: melatonin, seasonality, buffaloes.<br />
A250 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EQUINE EPIDIDYMAL SEMEN FERTILITY AFTER THE ADDITION OF SEMINAL PLASMA<br />
Cely Marini Melo e Oña, Luis Carlos Oña Magalhães, Frederico Ozanam Papa, Fernanda da Cruz Landim Alvarenga, Ian Martin,<br />
Eduardo Gorzoni Fioratti & Marco Antonio Alvarenga<br />
DRARV-UNESP, BOTUCATU, SP, BRAZIL.<br />
Post-mortem recovery of viable spermatozoa from epididymides is a very important technique to obtain gene pool of genetically<br />
valuable animals or even males of endangered species. Spermatozoa harvested from epididymides had no seminal plasma, which increased the<br />
volume of the ejaculate and also contained substances that increase the sperm life inside the female genital tract. The aim of the present study was<br />
to verify the influence that the addition of seminal plasma may have on equine epididymal sperm before freezing. Nineteen Brazilian Jumping<br />
colts were castrated and their epididymis were stored for 24 h at 5 º C. The cauda epididymides were dissected and then flushed using 20 mL of<br />
Botu-Semen ® then gathered into 5 pools before freezing: 4 pools composed of the sperm of 4 stallions and one pool composed of the sperm of<br />
3 stallions. Each pool was divided into 2 samples and diluted 1:1 (v/v) with the following extenders: Botu-Semen ® (control) or seminal plasma<br />
(Plasma) then incubated for 15 min, centrifuged at 600 x g for 10 min. The pellets were resuspended in Botu-Crio ® . Samples were cooled at 5ºC<br />
for 20 min and frozen at 6 cm above the level of liquid nitrogen, for additional 20 min, then plunged into liquid nitrogen. The samples were thawed<br />
at 46oC/20” and evaluated by computer-assisted semen motility analysis (CASA HTM IVOS 12). Twenty mares were used being randomly<br />
inseminated with epididymal sample incubated with either Botu-Semen (control) or seminal plasma. The inseminations were performed 36 and<br />
42 h after the induction of ovulation, using 400x10 6 total motile sperm, pre and post ovulation. The ovulation was induced using 1mg of<br />
deslorelin acetate. Sperm parameters were analyzed by ANOVA (SAS, Institute, Inc., Cary, NC) followed by Tukey’s test to identify the<br />
significant differences. Treatments were considered different if P < 0.05. Fertility rates were evaluated by X 2 (P < 0.05). Sperm parameters of<br />
the samples incubated with Botu-Semen and Seminal Plasma were 44.6±13.2 vs. 46.2±12.9 and 19.2±5.7 vs. 17.7±5.1 for total motility and<br />
progressive motility, respectively. Pregnancy rates were 80 and 50% for Botu-Semen ® and Seminal Plasma, respectively. Based on results of<br />
the present study we can conclude that seminal plasma is not necessary to obtain pregnancy with equine epididymal sperm. [Financial<br />
support: FAPESP].<br />
Keywords: equine, epididymides, seminal plasma.<br />
N<br />
s461
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A251 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
EMBRYO GENOTYPING FROM BIOPSIED BOVINE EMBRYOS USING A 50 K SNP CHIP<br />
D. Le Bourhis 1 , E. Mullaart 2 , Patrice Humblot 1 , H. Knijn 2 , B. Le Guienne 1 & Claire Ponsart 1<br />
1<br />
UNCEIA, R&D, MAISONS-ALFORT, FRANCE. 2 CRV, R&D, AL ARNHEM, NETHERLANDS.<br />
Genomic tools are now available for most livestock species and are used routinely for marker assisted selection (MAS) in cattle.<br />
Recently, multiple markers detection has been achieved from biopsies of pre-implantation stage embryos, thus allowing to select embryos before<br />
transfer.This strategy provides the opportunity to estimate some traits of particular interest and/or presence of genetic abnormalities. The present<br />
work aimed to assess the efficiency of MAS evaluation from biopsied bovine embryos using the bovine 50 K SNP Illumina chip. A biopsy of<br />
5 to 10 cells was realized under laboratory conditions using a microblade under a stereomicroscope from 30 in vitro-cultured morulas and<br />
blastocysts. Biopsies were transferred individually as dry samples in tubes and sent frozen (n = 13) or at room temperature (n = 16) to the<br />
genotyping laboratory. Genomic DNA of each biopsy was amplified using a whole genome amplification (WGA) kit according to the<br />
manufacturer instructions (WGA; QIAGEN REPLI-g Mini Kit). Following WGA, DNA concentration was determined using picogreen. For<br />
subsequent genotyping the custom CRV 50K Illumina chip was used. Call rates were calculated from 50905 SNPs. Percentage of Allele dropout<br />
(%ADO), which was estimated from the number of heterozygous markers (%ADO= (Calculated # hetero - Observed # hetero) / Calculated<br />
# hetero). Parentage error was estimated from 12 embryos using the genotypes of the parents of the embryos. Both groups of transport<br />
conditions were compared using a t test of Student. Results are presented as mean±SEM. A higher quantity of DNA was amplified when<br />
biopsies were sent as frozen to the lab when compared to room temperature (7.0±1.9vs. 5.0±0.4; P < 0.05). However, the SNPs call rate (87±7<br />
vs. 90±6), %ADO (18±16 vs. 17±10) and parentage error (0.26 vs. 0.32) did not differ between frozen and room temperature groups<br />
respectively. These results indicate that genotyping from embryo biopsies following WGA can be achieved with a good efficiency when using<br />
high density markers chips. In order to validate the use of MAS from early embryos in breeding schemes, a larger number of in vivo embryos<br />
are currently genotyped under field conditions. This will allow to assess the reliability of this method and quantify the correlation between<br />
embryo and calf genetic evaluation with the current WGA efficiency.<br />
Keywords: marker assisted selection, genotyping, bovine.<br />
A252 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
ACR<br />
CROSOME INTEGRITY OF RAM SPERMATOZ<br />
OZOA SUBMITTED<br />
TED TO CRYOPRESER<br />
OPRESERVATION:<br />
DYNAMICS RELATED<br />
TO<br />
PLASMA<br />
ASMATIC MEMBRANE CRYORESISTENCE<br />
Hymerson Costa Azevedo 1 , Sony Dimas Bicudo 2 , Marciane Silva Maia 3 , Daniel Bartoli Sousa 2 , Leandro Rodello 2 & Carmen Cecília<br />
Sicherle 2<br />
1<br />
EMBRAPA TABULEIROS COSTEIROS, ARACAJU, SE, BRAZIL. 2 FMVZ-UNESP, BOTUCATU, SP, BRAZIL. 3 EMBRAPA SEMI-ÁRIDO, PETROLINA, PE, BRAZIL.<br />
This work aimed to study the dynamics of acrosomal integrity in ram spermatozoa submitted to cryopreservation in different levels<br />
of semen freezability. Semen from 25 Santa Inês rams were cryopreserved, thawed and incubated (37ºC, 2 h) in synthetic oviduct fluid. Samples<br />
of semen were removed to evaluate acrosomal integrity (ACI) in the following moments: fresh semen (FR-S, n = 25); refrigerated semen (RE-<br />
S, n = 125); frozen-thawed semen (FT-S, n = 125) and incubated semen (IN-S, n = 125). Based on the plasmatic membrane integrity (X=22.1%)<br />
measured in FT-S by propidium iodide (PI - 5 mg/mL in PBS), rams were grouped in three cryoresistence levels (CRYO): 1 (X=10.3%), 2<br />
(10.3%>X=29.9%) and 3 (X>29.9%). To ACI evaluation semen samples were diluted in glutaraldeyde (0.2%) and examined under phase<br />
contrast microscope sperm with acrosome wrinkled. It was used the variance analysis (ANOVA) and Tukey method (P < 0.05). The differences<br />
among them were identified by letters: capital letters for differences among moments in each CRYO, lower cases for differences among CRYO<br />
in each moment. The averages of ACI were the following to FR-S, RE-S, FT-S and IN-S, respectively: 100.0aA; 89.6bB; 85.1bC and 73.1%cD<br />
for CRYO 1, 99.9aA; 94.7aB; 87.3bC and 78.5%bD for CRYO 2 and 99.6aA; 95.2aB; 92.5aB and 86.8%aC for CRYO 3. A gradual and sharp<br />
decline of ACI was verified as the semen was submitted to cryopreservation and incubation and was higher after thawing (P < 0.05). In spite of<br />
this, in CRYO 3 a significant fall (P < 0.05) was verified only between FR-S and RE-S as well as FT-S and IN-S. In CRYO 1 and 2 a progressive<br />
ACI reduction was observed along each evaluation (P < 0.05). In general, ACI was successively higher (P < 0.05) in CRYO 1, 2 and 3.<br />
Differences among CRYO were only significant from the RE-S and they became higher and higher along moments until the isolated superiority<br />
of CRYO 3 in IN-S (P < 0.05). It is concluded that acrosome morphologic damages of spermatozoa increase along the cryopreservation process<br />
and that this enhance is more pronounced with decreasing of plasmatic membrane integrity level or ram semen freezability.<br />
Keywords: sêmen, freezing, freezability.<br />
s462
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A253 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
IN VITRO MATUR<br />
TURATION TION OF IMMATURE OOCYTES<br />
VITRIFIED IN DIFFERENT CONDITIONS<br />
Fabiana For<br />
orell<br />
ell, Ner<br />
erissa Albino<br />
lbino, Jamir Machado Junior<br />
unior, Fabiano Car<br />
armina<br />
minatti Zago & Alc<br />
lceu Mezzalir<br />
zzalira<br />
CAV/UDESC, LAGES, SC, BRAZIL.<br />
Vitrification of immature oocytes is an important tool for use in both the IVF and cloning, though the efficiency of this technique<br />
is still low, limiting its application in commercial scale. For application in nuclear transfer, besides of survival of oocytes, vitrification of a large<br />
number of structures is required. The vitrified oocytes undergo various morphological and molecular changes during cooling and warming,<br />
which may potentially require another maturation conditions than those for non-cryopreserved oocytes. Thus, this study aimed to compare<br />
survival rates after in vitro maturation and embryo development of vitrified oocytes and matured under different conditions. Bovine oocytes were<br />
obtained from bovine ovaries and vitrified in groups of 15 COCs, loaded in a 5 µL vitrification solution microdrop in beveled-cut straws (Forell<br />
et al. Rep. Fert. Dev, 2009, 21 (1) 115). After warming all recovered COCs were submitted to IVM for 24 h at 38.8°C with 5% CO2 in air.<br />
Groups of 40 COCs allocated in 400 uL of medium, were subjected to three treatments: 1) ESS M199 +10% +0.5 ug/mL FSH +5 ug/mL LH<br />
(standard laboratory system) 2) M199 +10 % ESS + FSH + LH +10 ng/mL EGF, or 3) M199 +10% FCS +10 ng/mL EGF. After IVM, cumulus<br />
cells were mechanically removed and the surviving oocytes were parthenogenetically activated (5 min in ionomycin, and 4 h in 6DMAP) and<br />
in vitro cultured in SOFaaci with atmosphere of 5% CO2 +5% O2 in N2, for 7 days. Oocytes non-vitrified (control) were also subjected to the<br />
same treatments. The results were analyzed by chi-square test with significance level of 5%. The survival rate after the denudation did not differ<br />
between experimental groups, 44% (266/315), 49% (157/321) and 52% (165/320) respectively for groups 1, 2 and 3. There was no difference<br />
in cleavage rates of groups 1 - 36% (48/133), 2 - 45% (59/132) and 3 - 39% (65/165). The blastocyst rates were 3.8%, 3.8% and 9.7%<br />
respectively for groups 1 (n = 133), 2 (n = 132) and 3 (n = 165) and the rates of group 3 was higher (P < 0.05) than others. In fresh control groups<br />
was not statistically significant difference between the experimental groups in the cleavage rate (83%, 84% and 76%) or blastocyst rate (43%,<br />
42% and 48%) for groups 1 (n = 121 ), 2 (n = 125) and 3 (n = 121) respectively. Thus, among the tested systems, the maturation medium<br />
composed of FCS and EGF improves blastocyst rates for vitrified immature oocytes. [Funded by PNPD/CAPES].<br />
Keywords: vitrification, in vitro maturation, EGF.<br />
A254 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
DNA METHYLATION TION PAT TERN OF THE IGF2 AND XIST GENES IN OOCYTES OBTAINED OF THE PREANTRAL<br />
AL<br />
FOLLICLES FROM NELLORE COWS (<br />
(BOS<br />
TAUR<br />
URUS<br />
US INDICUS)<br />
Luís Fernando Soares Gomes 1 , Isabela Rebouças Bessa 2 , Fernanda Castro Rodrigues 3 , Pablo José Silva Rua 4 , Otávio Bravim 5 ,<br />
Juliana Mayumi Azevedo 6 , Margot Alves Nunes Dode 7 & Maurício Machaim Franco 8<br />
1,3,4<br />
FACULDADE DE MEDICINA VETERINÁRIA, UNIVERSIDADE FEDERAL DE UBERLÂNDIA, UBERLÂNDIA, MG, BRAZIL. 2,6 FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA,<br />
UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL. 5 UNIVERSIDADE DE BRASÍLIA, BRASÍLIA, DF, BRAZIL. 7,8 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF,<br />
BRAZIL.<br />
This study focus on evaluating the methylation pattern in a differentially methylated region (DMR) of the last exon of IGF2 gene<br />
in oocytes of 65-90 µm, and in a region of exon 1 of the XIST gene in oocytes =20 µm, and 65-90 µm obtained of the preantral follicles from<br />
nellore cows. To do this work, ovaries from abattoir were used and the preantral follicles were isolated with aid of tissue chopper. The isolation<br />
of oocytes enclosed in preantral follicles was performed by treatment with collagenase type II (Sigma, St. Louis, USA). Next the oocytes were<br />
photographed to measure their diameters. After, the oocytes were separated based on their size for molecular analysis. Genomic DNA was<br />
extracted from 65 oocytes =20 µm and 62 oocytes of 65-90 µm and it was treated with sodium bisulfate using the EZ DNA methylation kit ®<br />
(Zymo Research) and amplified by PCR nested to the genes IGF2 and XIST. PCR products were recovered from agarose gel and purified by<br />
GeneClean III kit (MP Biomedicals, LLC). The purified products were cloned using pGEMT-Easy vector kit (Promega) and transformed into<br />
Escherichia coli (XL-1 Blue). The clones were sequenced by the dideoxy method using an ABI 3130xl sequencer. Only clones sequences with<br />
=90% of conversion efficiency were used. There were sequenced and analyzed 28 clones for IGF2 65-90 µm group, 19 clones for XIST =20<br />
µm group and 27 clones for XIST 65-90 µm group. Methylation pattern was calculated with BiQ Analyzer program (Bock et al., 2005) and<br />
statistical analysis was performed by the Mann-Whitney test using the Prophet program, version 5.0 (BBN Systems and Technologies,<br />
1996). For IGF2, the oocytes of late secondary follicles of 65-90 µm presented 31,09 ± 31,01% of methylation. For XIST, the oocytes of<br />
primordial follicles =20 µm and the oocytes of late secondary follicles of 65-90 µm presented 15,17 ± 32,82% and 4,6 ± 3,46% (P = 0,0901)<br />
of methylation respectively. It is concluded that the genomic region of the XIST gene studied undergoes epigenetic reprogramming during<br />
gametogenesis.<br />
Keywords: bovine, methylation, oocyte.<br />
N<br />
s463
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A255 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
ULTR<br />
TRASONOGR<br />
ASONOGRAPHIC APHIC PROFILE OF EMBRYONIC/FET<br />
ONIC/FETAL GROWTH OF TRANSGENIC GOATS FOR HG-CSF<br />
Dárcio Ítalo Alv<br />
lves<br />
Teix<br />
eixeir<br />
eira,<br />
Car<br />
arla Rozilene Guimarães Silv<br />
ilva Oliv<br />
liveir<br />
eira,<br />
Car<br />
arlos Henr<br />
enrique Sousa de Melo<br />
elo, Antônio Car<br />
arlos de Albuquer<br />
lbuquerque<br />
Teles Filho<br />
ilho, Ribr<br />
ibrio io Ivan<br />
Tavar<br />
ares Per<br />
ereir<br />
eira Batista,<br />
Joanna Mar<br />
aria Gonçalv<br />
onçalves de Souza,<br />
Ale<br />
lexsandr<br />
sandra a Fer<br />
ernandes Per<br />
ereir<br />
eira,<br />
Luciana Magalhães Melo<br />
& Vic<br />
icen<br />
ente e José de Figueirêdo Freitas<br />
UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL.<br />
Transgenic goats should be evaluated by their embryonic/fetal growth, what could be done by real time ultrasonography. The aim<br />
of the study was to evaluate the morphological and morphometric development of transgenic goat embryos and fetuses for human Granulocyte<br />
Colony Stimulating Factor (hG-CSF). The project was approved by the ethics committee and biosafety of UECE. Four non-transgenic goats<br />
pregnant after mated by the transgenic Canindé male were used. Ultrasonographic exams (Falco 100 ® , Pie Medical, Maastricht, Netherlands)<br />
were performed at 30 and 40 days (transrectal via, linear array transducer, 6.0/8.0 MHz) and at 50, 60, 90 and 120 days of pregnancy<br />
(transabdominal via, convex transducer, 3.5/5.0 MHz). Images were recorded for posterior evaluation of gestational development and embryonary<br />
and fetal parameters such as Heart Rate (HR), conceptus movement, Embryonic Vesicle Diameter (EVD), Crown-Rump Length (CRL),<br />
Diameter of Thorax (DT), Abdome (DA), Umbilical Cord (DUC) and Placentomes (DP). Images were measured by the Image J software<br />
(National Institutes of Health, Millersville, EUA), with prior calibration for each frequency. Morphological aspects such as formation of skeleton<br />
and heart were also assessed. Four transgenic goats (two males and two females) besides two non-transgenic males were obtained after PCR<br />
analysis. After this moment, transgenic and non-transgenic sonograms were separated. In all conceptus the HR decreased during pregnancy. The<br />
movements of fetus were present in all exams, being less intense in the beginning and more evident after 50 days of pregnancy. EVD and CRL<br />
were evaluated at 30, 40, 50 and 60 days. It was observed that conceptus gradually grew, and the evaluation of other parameters was necessary<br />
to better follow the growth in the final stage. After the 50 initial days it was also possible to evaluate the DT, DA, DUC and DP. During the<br />
pregnancy, all fetuses grew as expected, with a higher development on the final third of pregnancy. Bone structures development occurred at 60<br />
days, with hiperechoic areas forming the skull, thorax, spine and long bones. At 30 and 40 days of pregnancy, the heart appeared as a small<br />
anechoic area and with intense movements. After 50 days it was already possible to evaluate the cardiac chambers, consisting in four anechoic<br />
well defined areas. Transgenic goats embryo and fetuses showed a morphologic development similar to the non-transgenic ones and remained<br />
viable during all period evaluated.<br />
Keywords: transgenic, goat, hG-CSF.<br />
A256 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
PROTEOME-B<br />
TEOME-BASED INSIGHTS S ON OOCYTE COMPETENCE FROM A RETROSPECTIVE ANALYSIS OF BOVINE<br />
FOLLICULAR FLUID<br />
Felipe Perecin 1 , Lilian Jesus Oliveira 2 , Juliano Rodrigues Sangalli 3 , Tiago Henrique Câmara de Bem 4 , Gustavo Henrique Martins<br />
Ferreira Souza 5 , Jerusa Simone Garcia 6 , Ricardo Pimenta Bertolla 7 , Christina Ramires Ferreira 8 , Marcos Nogueira Eberlin 9 & Flávio<br />
Vieira Meirelles 10<br />
1,2,3,4,6,10<br />
DEPTO. CIÊNCIAS BÁSICAS-FZEA-USP, PIRASSUNUNGA, SP, BRAZIL. 5 WATERS CORPORATION, LABORATÓRIO DE DESENVOLVIMENTO DE APLICAÇÕES EM<br />
ESPECTROMETRIA DE MASSAS, SÃO PAULO, SP, BRAZIL. 6 INSTITUTO DE CIÊNCIAS EXATAS, UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL. 7 UNIVERSIDADE FEDERAL<br />
DE SÃO PAULO (UNIFESP/EPM), SÃO PAULO, SP, BRAZIL. 8,9 LABORATÓRIO THOMSON DE ESPECTROMETRIA DE MASSAS, INSTITUTO DE QUÍMICA-UNICAMP, CAMPINAS, SP,<br />
BRAZIL.<br />
The proteome composition of follicular fluid produced during folliculogenesis reflects the metabolism of the ovarian follicle and the<br />
competence of the oocyte to finish maturation and to support embryo development. In order to study the proteome of bovine follicular fluid<br />
(bFF), 46 ovarian follicles (3.14+0.56 mm diameter) were dissected so that the oocyte and follicular fluid could be individually recovered. The<br />
bFF samples were stored at -80°C while oocytes were individually submitted to in vitro maturation and parthenogenetic activation. Oocyte<br />
maturation and embryo development were assessed through first polar body (1.PB) extrusion and development until blastocyst stage, respectively,<br />
generating three groups: (1) Non-matured (NM) – oocytes that did not extruded 1.PB; (2) Matured (M) – oocytes that extruded 1.PB but did not<br />
reached blastocyst stage and (3) Blastocyst (BL) – oocytes that reached blastocyst stage. Bovine FF of these groups were pooled and<br />
retrospectively used for label-free comparative shotgun proteomics analysis using MudPit (Multidimensional Protein Identification Technology)<br />
tandem MSE acquisition to investigate differential protein expression. Functional and pathway analysis using FatiGo (www.babelomics.org);<br />
Pathway Express (http://vortex.cs.wayne.edu/ontoexpress) and Ingenuity Pathway Analysis (www.ingenuity.com) were used to identify<br />
relevant ontologies and canonical or noncanonical pathways represented by the expressed proteins in the bFF. A total of 105 proteins or randomic<br />
sequences were identified corresponding to 59 unique proteins present in the bFF. Out of 59 proteins, 11 were exclusively expressed in the bFF<br />
from NM oocytes, 13 in the bFF from M oocytes and 10 in the bFF of BL oocytes whereas were 18 proteins commonly expressed among<br />
groups. There was a co-expression of 2 proteins in the bFF from NM and M, 3 proteins in the bFF from NM and BL groups and 2 proteins in<br />
the bFF from M and BL groups. The ontology and pathway analysis showed the overrepresentation of proteins from extracellular space, acute<br />
inflammatory response, vitamin D and single-stranded RNA binding process in all groups. Among the unique proteins present in the BL group<br />
we found Apolipoprotein A1 (APOA1), involved in the metabolism of cholesterol and steroid, and the Secretogranin-1 (CHGB) involved in<br />
vesicle sorting and exocytosis. Our results bring new insights about the proteins present in the bovine follicular environment and involved in the<br />
establishment of oocyte competence.<br />
Keywords: follicular fluid, oocyte competence, proteomic.<br />
s464
Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A257 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
RELATIVE ABUNDANCE OF MATERNAL<br />
TRANSCRIPT<br />
ANSCRIPTS S IN OOCYTES<br />
WITH AND WITHOUT POLAR BODY AFTER IN VITRO<br />
MATUR<br />
TURATION<br />
TION<br />
Lilian Tam<br />
amy Iguma 1 , Michele Munk Per<br />
ereir<br />
eira 2 , Sabine<br />
Wohlr<br />
ohlres-V<br />
es-Viana<br />
3 , José Nelio S. Sales<br />
4 , Car<br />
arolina Cap<br />
apobiango R. Quin<br />
uintão<br />
5 , Isab<br />
sabella Silv<br />
ilvestr<br />
estre e B. Pin<br />
into 6 , Raquel<br />
Varella Serapião 7 ; Savana Giacomini Brito 8 , Bruno Campos Caravalho 9 , João Henrique Moreira Viana 10 , Luiz Sérgio Almeida Camargo 11 & Marta Fonseca M.<br />
Guimarães 12<br />
1,5,6,9,10,11,12<br />
EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2,3,6 UNIVERSIDADE FEDERAL DE JUIZ DE FORA, JUIZ DE FORA, MG, BRAZIL. 4 UNIVERSIDADE DE SÃO PAULO, SÃO<br />
PAULO, SP, BRAZIL. 7 PESAGRO-RIO, NITERÓI, RJ, BRAZIL. 8 UNIVERSIDADE PRESIDENTE ANTÔNIO CARLOS, JUIZ DE FORA, MG, BRAZIL.<br />
The oocyte ability to undergo maturation and early cleavages during the maternal–zygotic transition (MZT) is associated to transcript<br />
levels stored in its cytoplasm. The extrusion of the polar body is an indicative of oocyte ability to undergo maturation. However, to our<br />
knowledge, no data comparing mRNA levels among bovine oocytes maturated in vitro presenting polar body (PB) and oocytes maturated in vitro<br />
without the presence of polar body (NPB) are available. The aim of the present study was to compare the relative abundance of maternal<br />
transcripts of Maternal Antigen That Embryo Requires (MATER), Zygote Arrest (ZAR1), TEA domain family member 2 (TEAD2) and High<br />
Mobility Group N (HMGN1) genes between oocytes with or without PB (PB and NPB groups, respectively) following in vitro maturation.<br />
Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 199 (Gibco Life Technologies, New York, USA)<br />
containing 10% estrus cow serum and 20 µg/mL FSH (Pluset, Serono, Italy), for 24 h under 5% CO2 in air at 38.5°C. Oocytes separated<br />
according to the presence or absence of PB and then they were denuded and frozen in liquid nitrogen. Three pools of ten oocytes for each group<br />
were subject to RNA extraction. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole<br />
Transcriptome Amplification Kit (WTA2 – Sigma Aldrich) according to the manufacturer’s instructions. Relative abundance of the target<br />
transcripts was performed by real-time PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, EUA) using the betaactin<br />
gene as the endogenous reference. The relative expression of MATER (0.81±0.11), ZAR1 (0.82±0.09), TEAD2 (0.83±0.07) and<br />
HMGN1 (0.90±0.08) was dowregulated (P < 0.05) for oocytes NPB after in vitro maturation. We conclude that the presence of polar body may<br />
be related to abundance of maternal mRNA and can contribute to predict quality of in vitro maturation conditions.[Financial support: Embrapa<br />
– Animal Genomics Network Project (01.06.9.01.01.00) and Innovations in Animal Reproduction Network Project (01.07.01.002), CNPq,<br />
CAPES and Fapemig].<br />
Keywords: gene expression, oocytes, in vitro maturation.<br />
A258 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
RELATIONSHIP BETWEEN ACR<br />
CROSSOME REACTION,<br />
POST-THAW SPERM EVAL<br />
ALUATION AND TOTAL PROTEIN IN<br />
MOXOTÓ GOAT SEMEN<br />
R ober<br />
erta<br />
V ianna do Valle<br />
alle, Ângela Mar<br />
aria Xavier Elo<br />
loy, Nadiana Mar<br />
aria Mendes Silv<br />
ilva,<br />
Fr ancisco W ilson Venâncio Silv<br />
ilva & João Ric<br />
icar<br />
ardo<br />
Furtado<br />
UVA/EMBRAPA CAPRINOS E OVINOS, SOBRAL, CE, BRAZIL.<br />
Even been able to fertilize, sperm cells from epididymis can present alterations when in contact with the seminal plasma during the<br />
ejaculation, losing its capacity of suffering the acromosse reaction (AR) and to penetrate the oocyte. This study aimed to evaluate the relation<br />
between (AR) with the post-thaw semen evaluation and the total proteins (TP) in the seminal plasma of Moxotó bucks in Brazilian Northeast.<br />
The semen was collected from five males from January to March/<strong>2011</strong>, totalizing three samples per animal, being one week of the month used<br />
for semen freezing and the other for total protein analysis. For semen freezing, Tris-glycerol at 2% was used, being the sperm stored in 0.25 mL<br />
straws , using the portable system Tetakon, TK 3000. The post-thawed spermatic motility (%) was evaluated and the TP analysis followed the<br />
Bradford method (1976, M Analyt Biochem 72:248-254). In the AR evaluation, the Naftol yellow/erythrosine B was used aiming to identify the<br />
percent of post-frozen/thawed sperm reaction. The Pearson correlation was run between post-thawed spermatic motility and total plasma protein.<br />
The mean spermatic motility and the post-thawed AR were 20% and 35%, respectively. It was verified a positive and high intensity correlation<br />
(r= 0.40) between AR and progressive post-thaw motility, according to studies that are in agreement with these results (Correa et al., 1997,<br />
Theriogenology 48:721-731). It was not observed a correlation (P > 0.05) between AR and seminal TP. It is concluded that probably Moxotó<br />
animals that showed higher spermatic motility can also be able to suffer good rate of AR and, in consequence, can probably show higher<br />
fertility. However, it is recommended more studies on this subject.<br />
Keywords: cryopreservation, fertilization, proteomics.<br />
N<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A259 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
MOURA PIG SPERM VIABILITY AFTER CRYOPRESER<br />
OPRESERVATION<br />
Mar<br />
ariana Grok<br />
oke Mar<br />
arques<br />
ques, Almir<br />
lmiro Dahmer<br />
ahmer, Vit<br />
itor Hugo Grings & Elsio Antonio Per<br />
ereia eia de Figueir<br />
igueiredo<br />
edo<br />
EMBRAPA SUÍNOS E AVES, CONCORDIA, SC, BRAZIL.<br />
The Moura pig breed was introduced soon after the discovery of Brazil, and possibly descended from Iberian breeds. In the first<br />
decades of the 20th century, the breed was widespread in southern Brazil. The main characteristics of this breed are rusticity, disease resistance<br />
and meat production with a higher rate of marbling (Favero et al., 2007, Rev Bras Agroecology 2, 1662-65). On the other hand, the breed<br />
selection has reduced genetic variability, limiting the Moura pigs to only 29 animals registered in the ABCS (Brazilian association of swine<br />
producers). This data demonstrates the importance of the formation of a germplasm bank for this breed. The objective of this study was to<br />
evaluate the effect of cryopreservation on the quality of Moura pig semen. The rich fraction of the ejaculate of 9 males was frozen by the method<br />
of Hulsenberg (Westendorf et al., 1975, Dtsch Tierärztl Wochenschr 82, 261-67) with 5 x 10 9 spermatozoa/straw. After collection and thawing,<br />
semen samples were evaluated for sperm motility, plasma membrane integrity (using the eosin/nigrosine dye test), acrosomal integrity (using<br />
Pope dye), and mitochondrial activity (Hrudka 1987, Int J Androl 10, 809-28). The data were compared using a Sign test (Campos 1983,<br />
Statistical experimental non parametric, ed. 4, 349). Average losses of 52.91% of motile spermatozoa (78.33 ± 3.22% and 25.42% ± 3.56,<br />
collection and thawing, respectively, P < 0.0001), and 40.67% in membrane integrity (74.00 ± 3.81% and 33.33% ± 3.47 collection and thawing,<br />
respectively, P < 0.0001) were observed. A milder decrease (12.61%) was observed in acrosomal integrity (63.50 ± 2.64% and 51.33% ± 3.25,<br />
collection and thawing, respectively, P= 0.0193). Regarding mitochondrial activity after thawing, 82% of sperm mitochondrial activity in grades<br />
1 or 2 was observed, indicating the predominance of active sperm. Rath et al. (2009, Soc Reprod Fertil Suppl 66, 51-66) describe the successful<br />
results of fertility using deep intra-uterine insemination (DUI) with 1-2 x 10 9 viable sperm after thawing. Although semen cryopreservation in<br />
pigs is challenging, and ejaculates of Moura pigs were not previously selected due to few available boars, the data shown here demonstrates that<br />
the semen of Moura pigs has good quality, and consequently can be used in DUI. Banking of Moura pig semen would serve a useful purpose<br />
in expansion of the breed, both genetically and in number.<br />
Keywords: cryopreservation, semen, moura.<br />
A260 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
VITRIFICATION TION OF IN VITRO PRODUCED BOVINE EMBRYOS AFTER CHEMICAL LIPOLYSIS<br />
D aniela Mar<br />
artins Paschoal<br />
1 , Mat eus José Sudano 2 , Tatiana da Silv<br />
ilva Rasc<br />
ascado<br />
ado 1 , Luis Car<br />
arlos Oña Magalhães<br />
1 , Letícia Fer<br />
err ari i Cro c omo 1 ,<br />
Joao Ferreira de Lima Neto 1 , Midyan Daroz Guastali 1 , Rosiara Rosária Dias Maziero 1 , Alicio Martins Júnior 2 & Fernanda da Cruz<br />
Landim Alvarenga 1<br />
1<br />
FMVZ/UNESP BOTUCATU, BOTUCATU, SP, BRAZIL. 2 FMVA/UNESP ARAÇATUBA, ARAÇATUBA, SP, BRAZIL.<br />
The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma) which is an activator<br />
agent of adenylate cyclase and is related to cAMP levels (Seamon et al., 1981; Proc Natl Acad Sc USA 78, 3363-67). Presumptive zygotes were<br />
cultured with SOFaa+BSA in the FCS presence or absence until D7. In D6 was added forskolin (F) in the median resulting in 4 groups: FCS<br />
(2.5% FCS and without F), 0%FCS (without FCS and F), FCS+F (2.5% FCS + 10 µM F) and 0%FCS+F (without FCS + 10 µM F). The<br />
cleavage was analyzed on D3 and the blastocyst rate on D7. On seventh day was performed: viability and ultra structural evaluation and embryos<br />
vitrification followed by viability evaluation. The IVP embryos were compared to in vivo-produced embryos. Data were analyzed with ANOVA,<br />
using the general linear model (GLM) SAS (SAS Inst Inc, Cary, NC, USA). Sources of variation in the model included FCS concentration,<br />
forskolin addition and first order interactions; all factors were considered fixed effects. The arcsine transformation (vy/100) was applied to<br />
percentage data. If the ANOVA was significant, means were separated by a Tukey’s test. There were no differences on cleavage, blastocyst<br />
production rates and the number of cells/embryo between in vitro and in vivo produced groups (P > 0.05). A higher rate of damaged cells was<br />
observed in 0%FCS group (11.3 ± 11.3 b ) comparison with FCS and FCS+F groups (P < 0.01). In vivo-produced embryos (5.1 ± 1.4ab) were<br />
similar to IVP embryos (FCS: 1.1 ± 1.3 a , 0%FCS: 11.3 ± 11.3 b ; FCS+F: 1.6 ± 3.2 a , 0%FCS+F: 6.8 ± 6.9 ab ). With the ultrastructural evaluation,<br />
the same characteristics were found between IVP and in vivo groups. The FCS group had higher amounts of lipid droplets, which decreased after<br />
the F addition. Just as the 0%FCS group had a higher rate of damaged cells, it showed a greater presence of cellular debris (cellular degeneration)<br />
being reduced with F addition. After vitrification there was no difference on re-expansion rate between the groups (P > 0.05), but the in vivo<br />
group (124.2 ± 12.2 b ) had a greater number of cells/embryo in relation to IVP groups (FCS: 42.6 ± 17.2 a ; 0%FCS: 65.1 ± 34.7 a ; FCS+F: 59.9<br />
± 46.2 a ; 0%FBS+F: 40.1 ± 12.6 a ; P < 0.05). When the damaged cells rate was assessed, the FCS group showed the highest value (69.7 ± 20.7 a )<br />
compared to 0%FCS (20.3 ± 22.7 b ), FCS+F (39.4 ± 33.0 b ), 0%FCS+F (27.0 ± 26.7 b ) and in vivo (14.1 ± 20.1 b ; P < 0.05). The serum presence<br />
was harmful to embryo cryopreservation, but this effect was reversed when forskolin was added.<br />
Keywords: ivp, forskolin, vitrification.<br />
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Acta Scientiae Veterinariae, <strong>2011</strong>. 39(Suppl 1): Abstracts - <strong>25th</strong> <strong>Annual</strong> <strong>Meeting</strong> <strong>SBTE</strong>-Brazil. August <strong>2011</strong>.<br />
A261 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
VITRIFICATION TION OF IN VITRO PRODUCED BOVINE EMBRYOS SUPPLEMENTED WITH TRIIODOTHYR<br />
THYRONINE (T3) AND<br />
THYROXINE (T4)<br />
Maria Clara Costa Mattos, Emerson Roberto Siqueira, Flávia Spessopoto Pavan Rochelle, Ana Carolina Freitas Pereira, Bruno Valente Sanches, José<br />
Henrique Fortes Pontes, André Gomiero Rigo, Lucas Lopes Moino, Rodrigo Mendes Untura & Andrea Cristina Basso<br />
IN VITRO BRAZIL S/A, MOGI-MIRIM, SP, BRAZIL.<br />
The aim of this study was to evaluate the triiodothyronine (T3) and thyroxine (T4) supplementation on in vitro early embryonic<br />
development and cryotolerance of these vitrified IVP embryos. Oocytes from ovaries of slaughterhouse were fertilized with conventional sperm<br />
from a single bull. After 18 to 22 h, presumptive zygotes were denuded and divided into two groups: In T3T4 Group (n = 322) were added 50<br />
mg/mL of T3 (Sigma 6397) and 50 ng/mL de T4 (Sigma 2502) to the culture media and in the Control Group (n = 392) in vitro embryo culture<br />
(IVC) was performed without T3 and T4 addition. Embryos were cultured for seven days in 38.5°C and atmosphere with 5% of CO2, 5% of<br />
O2 and 90% of N2. The cleavage and blastocyst production were assessed from the total number of oocytes on Days 3 and 7, respectively. On<br />
Day 7, embryos were vitrified at grade 1 blastocyst stage by Cryotop method with cryoprotectants solutions of Ethylene Glycol (EG) + Dimethyl<br />
sulfoxide (DMSO). Embryos of both groups were thawed and placed in culture for 48 h to assess hatching rates. The cleavage rate in T3T4<br />
Group (79.8%) was higher than the Control Group (72.3%, P = 0.03). However, the blastocyst rate was not different between groups (27.3%<br />
in T3T4 Group and 31.4% in Control Group, P = 0.24). The percentage of vitrified blastocysts did not differ between groups (44.3% in T3T4<br />
Group and 43.3% in Control Group, P = 0.86). Finally, the hatching rates after 48 h of culture were also similar between T3T4 Group (87%, n<br />
= 39) and Control Group (85%, n = 53; P = 0.85). Based on these results, we conclude that T3 and T4 supplementation did not increase<br />
blastocyst production. However, it seems not affect the quality and cryotolerance of IVP embryos. Therefore, future studies should also consider<br />
the mechanisms of action of these hormones during early in vitro embryonic development and its impact on the conception rates of cryopreserved<br />
embryos. [Acknowledgements: In vitro Brazil SA.].<br />
Keywords: bovine embryo, vitrification, t3 and t4.<br />
A262 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”<br />
BOVINE OOCYTE<br />
VITRIFICATION:<br />
TION: EFFECTS ON FERTILIZA<br />
TILIZATION TION RATES<br />
José Felipe Warmling Sprícigo 1 , Rodolfo Rumpf 2 & Margot Alves Nunes Dode 2<br />
1<br />
UNIVERSIDADE DE BRASÍLIA, BRASILIA, DF, BRAZIL. 2 EMBRAPA-CENARGEN, BRASÍLIA, DF, BRAZIL.<br />
Although vitrification has been reported as the most promissory method for cryopreservation of oocytes, it is quite inefficient,<br />
N<br />
resulting in low rates of cleavage and blastocyst formation. This information suggest that vitrification effects can already be observed in the<br />
beginning of embryonic development. The aim of this study was to evaluate the fertilization rates of bovine oocytes submitted to vitrification by<br />
cryotop at different moments, during in vitro maturation (IVM). Cumulus-oocyte-complexes were obtained from slaughterhouse ovaries, were<br />
selected and distributed into four groups: non-vitrified, used as control group (CG), vitrified immediately after selection (V0), vitrified at 8 h (V8)<br />
and vitrified at 22 h (V22) of IVM. After warming the four groups were returned to the incubator to complete the 24 h of maturation. At the end<br />
of IVM, the four groups were transferred to fertilization drops and incubated for 18 h with sperm selected by Percoll gradient. At the end of the<br />
period of fertilization the oocytes of the four groups were denuded, fixed and stained with lacmoid for the assessment of fertilization. Zygote that<br />
presented sperm head, decondensed sperm head, male and females pronuclei, cleavage or polyspermy were classified as fertilized. And those<br />
with unidentifiable chromatin, were classified as degenerated. Data were compared using Chi-square test (P < 0.05). Oocytes of V0 group (n =<br />
112) showed the lowest percentage of fertilization (44%) among the vitrified groups, while V8 (n = 119) and V22 (n = 106) showed lower<br />
percentages compared to CG (n = 143) with rates 63%, 66% and 81% respectively. The rate of unfertilized oocytes was similar among all groups,<br />
but groups of vitrified oocytes showed a higher percentage of polyspermy CG (11%, 3%), V0 (9%, 6%), V8 (12%, 7%) and V22 (7%, 12%).<br />
The most discrepant difference between GC (7%) and vitrified treatments was observed for the percentages of degenerated oocytes, where V0<br />
(46%) was the most affected (P < 0.05), followed by V8 (24%) and V22 (25%), which were similar. The results suggest that vitrification reduces<br />
the fertilization rate and induces an increase in the rate of degenerated oocytes, especially in the stage of germinal vesicle. Therefore, the reduction<br />
in fertilization rate is due to loss of oocyte viability that occurs as a result of vitrification procedure, and immature oocytes in GV are the most<br />
affected ones.[Financial support: CNPq, CAPES and EMBRAPA].<br />
Keywords: vitrification, bovine, oocyte.<br />
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