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VIII'h International Conference on
Pig Reproduction
Program and Abstract Book
Banff Centre, Banff
Alberta, Canada
May 31't
-
June 4th, 2a0g

INTERNATIONAL ORGANIZING COMMITTEE:
Cheryl Ashworth (Chair)
Nicoline Soede (Vice-Chair)
George Foxcroft (Local Organizing Committee Chair)
Randall Prather (Treasurer)
Heiner Niemann (Secretary)
Heriberto Rodriguez-Ma rtinez (Program Chair and Co-editor)
Jeff Vallet (Program Committee and Co-editor)
Adam Ziecik (Program Committee and Co-editor)
LOCAL ORGANIZII\G COMMITTEE:
George Foxcroft (Chair)
Susan Novak (Secretary)
Michael Dyck (Treasurer)
Walter Dixon (Co-editor)
Sue Charlton (Executive Secretary)
Front cover layout: Brian Klein, University of Alberta

WELCOME FROM THE INTERNATIOIYAL ORGANIZING
COMMITTEE
It is a pleasure to welcome you, on behalf of the International Organising Committee (IOC),
to the 81h International Conference on Pig Reproduction in the fabulous venue of the Banff
Conference Centre. We hope that you will find the meeting scientifically stimulating, that you
will enjoy interacting with friends and colleagues (old and new!) and that you will leave inspired
affer a very worthwhile meeting.
As programme chair, Heriberto Rodríguez-Martínez together with the rest of the IOC put
together a scientific programme that brings together state-of-the-art approaches to reproductive
biology in the pig with an awareness of current knowledge gaps and potential solutions. The
structure of the meeting, with parallel sessions addressing both contemporary approaches and
practical applications, reflects this focus. We would like to thank the invited speakers for
accepting our invitation and for the timely production of abstracts and manuscripts. Another
depafiure from the previous format of these meetings is that the authors of some abstracts
describing particularly novel research were invited to extend their abstract and to give a short
oral presentation. Again, we would like to thank these authors for agreeing to speak at the
meeting and for providing extended abstracts in a timely manner. Huge thanks are due to the
editors of the supplement: Heriberto Rodríguez-Martínez, Jeff Vallet, and Adam Ziecik for their
commitment to the timely publication of 'Control of Pig Reproduction VIII', which will be
published by Nottingham University Press as a supplement of the Society for Reproduction and
Fertility.
We would also like to thank all the commercial sponsors who have contributed to this
meeting. The generosity and foresight of organisations who invest in a meeting such as this is
much appreciated.
This conference would not be taking place without the superb efforts of the Local Organising
Committee, chaired by George Foxcroft. I am confident that their careful planning and attention
to detail will ensure that we all enjoy a memorable and inspiring conference.
Cheryl J. Ashworth
Chair, International Organizing Committee

WELCOME FROM THE LOCAL ORGANIZING COMMITTEE
On behalf of the other members of the Local Organizing Committee (LOC), Walter Dixon,
Mike Dyck, Sue Novak, and our tireless Executive Secretary Sue Charlton, it is my great
pleasure to welcome you to the VIII ICPR in Banff, Canada. We are confident that the scientific
program of the 2009 ICPR will live up to your expectations and that you will find Banff a
stimulating and uplifting venue for the meeting.
Although the ICPR began as the 34th Easter School on 'Control of Pig Reproduction' at the
University of Nottingham nearly 30 years ago, the fascinating insights that studies in the pig
continue to provide in the field of comparative mammalian reproduction are no less exciting
today. A key focus for the LOC in planning for the Banff meeting has been to work with the
International Organizing Committee to create a program that more directly links the science of
pig reproductive biology to implementation in the swine industry. The move to afternoon
parallel sessions provided the flexibility to achieve this. We have also tried to provide more
profile to our younger generation of research colleagues through inclusion of some "short-talks",
selected from abstracts submitted to the conference, as part of the afternoon sessions. We
appreciate the support from the IOC in implementing these changes and hope they are seen as a
worthwhile development.
It is a great disappointment that the VIII ICPR in Banff coincides with the worst ever
economic crisis in the Canadian pork industry. 'We are, therefore, especially grateful to those
industry and agency sponsors who supported the VIII ICPR and who are listed separately in this
program booklet. Please join us in thanking them for this essential support of the Banff meeting.
Hopefully, some of our discussions can provide the basis for a profitable and sustainable pork
sector in Canada and world-wide.
Welcome to the Canadian Rockies - enjoy!
George Foxcroft,
Chair, Local Organizing Committee

___--:|
WELCOME FROM THE DEPARTMENT OF AGRICULTURAL,
FOOD AND NUTRITIONAL SCIENCE (AFNS) AND THE
FACULTY OF AGRICULTURAL, LIFE AND
"^i1i3W,Tffi ',f"'f-\iJåki
As Chair of the Department of Agricultural, Food and Nutritional Science (AFNS), and on
behalf of the Faculty of Agricultural, Life and Environmental Sciences at the University of
Alberta, I would like to extend a warm welcome to all of the delegates to the 8th International
Conference on Pig Reproduction (ICpR).
As the home department of the Local Organizing Committee, we, in AFNS, are very pleased
to be hosting such a high profile international conference. We are proud of the work of ihè LOC,
expertly chaired by Dr. George Foxcroft, and their contribùtions to the eff'orts of the
Intemational Organizing Committee in assembling an outstanding scientific programme that I
am sure will engage and challenge all conference participants. In fulfilling the tenets of the
cunent University Plan - Dare to Deliver - that identifies areas of commitments such as:
D_iscovery Learning, and Incubating Scholarship, and Community Engagement Near and Far,
ICPR 2009 should provide an ideal forum for the exchange of scientifió information on leading
edge research in pig reproduction as well as permit ãmple opportunities for professional
networking and the development or renewal of acquaintãnces with international scientific
colleagues.
I have no doubt that the splendid backdrop of the Canadian Rockies in Banff wiu add to the
memories that you will take away from this conference and trust that you will thoroughly enjoy
your time with us in Alberla.
Erasmus Okine,
Chair, Department of AFNS,
Faculty of ALES
University of Alberta

TABLE OF CONTENTS
WELCOME FROM THE INTERNATIONAL ORGANIZING COMMITTEE ..................3
WELCOME FROM THE LOCAL ORGANIZING COMMITTEE............ .......4
\ryELCOME FROM THE DEPARTMENT OF AGRICULTURAL, FOOD AND
NUTRITIONAL SCIENCE (AFNS) AND THE FACULTY OF AGRICULTURAL, LIFE
AND ENVIRONMENTAL SCIENCES (ALES) AT THE UNIVERSITY OF ALBERTA..5
spoNSoRS ................ ....................9
suPpoRTERS.......... ...................10
GENERAL rNFORMATION........ ................13
MAP OF BANFF
MAp OF BANFF CENTRE..
LOCATTON OF EVENTS
...................1s
.......................1s
THE MAX BELL BUILDING .................16
CONFERENCE PROGRAM ........................17
INDEX OF INVITED pApERS................
.......................2s
INDEX OF SHORT ORAL PRESENTATIONS... .........27
INDEX OF POST8RS............. .......................29
ABSTRACTS OF INVITED PAPERS .........37
ABSTRACTS OF SHORT ORAL PRESENTATIONS... .................81
ABSTRACTS OF POSTERS ....119
POSTER INDEX BY FIRST AUTHOR. .....................187

SPONSORS
Platinum
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SUPPORTERS
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GENERAL INFORMATION

MAP OF BANFF
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LOCATION OF EVENTS:
Registration
Marché Breakfast and Dinners
Buffet Lunch
Conference Banquet
Plenary Sessions
Breakout Sessions
Professional Development Centre Foyer
Vistas Dining Room - Sally Borden Building
Donald Cameron Hall Dining Room
Donald Cameron Hall Dining Room
Max Bell Auditorium
Max Bell Auditorium OR
The TransCanada Pipeline Pavilion
Auditorium
Poster Receptions Max Bell Rooms 251,252,253, and, Foyer
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Main Floor
Lower Floor
Walkway lo Corbett Hall
THE MAX BELL BUILDING
MB 251
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MB 253
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CONFERENCE PROGRAM
suNDAY, MAY 31.t
16:00 - 20:00 Registration
Professional Development Centre Lobby
17:30 - 19:30* Marché Dinner (Vistas Dining Room)
xlf your arrival is later than 7:30 pm, a credit may be applied towards dinner at the Raven's Restaurant
Poster Presentations
Poster displays may be set up anytime from 16:00 _ 20.00 on Sunday, May 3l'tand from 08:00
- 10:00 on Monday, June 1't. Posters will be displayed in Rooms 25I,252, and 253 of the Max
Bell Building. Abstract numbers in this booklet refer to the room where your poster will be set
up followed by the abstracr number (e.g.252-13).
We request that you be present to answer questions during the Poster Receptions on Monday
and Tuesday (15:30 - 17:00).
t1

07:00 - 09:00
08:00 - 08:15
Marché Breakfast (Vistas Dining Room)
Chair's Welcome/Opening Ceremony: Cheryl Ashw orth
Max Bell Auditorium
Morning Plenary Session I
'hysiological Roles of the Boar Ejaculate"
Chair: Janice Bøiley (University of Laval, Cønada)
Max Bell Auditorium
Heriberto Martínez-Rodríguez (Sweilísh University of Agricultural Sciences,
Sweden)
The Physiological Roles of the Boar Ejaculate
MONDAY, JUNB l't
08:15 - 09:00
09:00 - 09:45
09:45 - 10:15
Break (Max Bell Foyer)
l0:15 - 11:00
11:00 - 11:45
1l:45 - 12:00
General Discussion
12:00 - 13:30
13:30 - 14:00
14:00 - 15:00
Bart Gadella (Utrecht University, The Netherlands)
Molecular Kinetics of Fertilization
Inmqculøds Purrilla (University of Murcía, Spøin)
Optimal Characteristics of Spermatazoa for Assisted Reproductive
Technologies
Detlef Rath (FAL, Neustadt, Germany)
Recent Advances in Boar Semen Cryopreservation
Lunch (Donald Cameron Hall Dining Room)
Breakout Session #1
"Future Developments in Swine AI"
Chaír: Mary Buhr (Universíty of Guelph, Canødø)
Max Bell Auditorium
William Flowers (North Carolína State (Jniversity, IISA)
Selection for Boar Fertility and Semen Quality - the Way Ahead
4 x 15 min Short Communications selected from submitted abstracts
H. Henning, A.M. Petrunkína, R.A.P. Hørrison, und D. Waberski
Changes in Responsiveness to Bicarbonate under Capacitating Conditions in
Liquid Preserved Boar SpermaÍozoa In Vitro
A.K. Olesen øndC. Hunsen
Intrauterine Insemination of Sows by Using a Two Chamber Semen Bag
System
U. Tøylor, H. Zerbe, H.M. Sefert, D. Rath, und H.J. Schuberth
Binding of Porcine Spermatazoa to Uterine Epithelial Cells Modulates the
Female Immune Response and Might Indicate the Formation of a Pre-
Oviductal Sperm Reservoir
J.M. MorreII, F. Søravia, H. Rodríguez-Martínez, and M. Wøllgren
Sperm Survival Following Colloid Centrifugation Varies According to the
Part of the Sperm-Rich Fraction Used
18

l5:00 - 15:30
General Discussion
Breakout Session #2
"State of the Art in -omic Biotogy of Swine"
Chair: Heiner Niemann (Mariensee, Germøny)
TransCanada Pipeline pavilion Auditorium
13:30 - i4:00
Msx Rothschíld (Iowa State (Jniversity, USA)
Gene Markers, Gene Microarrays and eTL Analyses in pig Breeding
14:00 - 74:30
Peter Sutovsþ (tlniversity of Missouri, USA)
Proteomic Analysis of Sperm and Oocyte Interactions
14:30 - 15:00
2 x 15 min short communications selected from submitted abstracts
S.C. Hernandez, H.A. Fínlayson, C.J. Ashworth, C.S. Haley, an¡t A.L.
Archibalil
Mapping Quantitative Trait Loci for Reproduction in pigs
!. P_øradis, H. Moore, S. Novøk, M.K.byck,IV.T. Dixon, anil G.R. Foxcroft
Global Protein Profiling of porcine cumulus cells in Response to Native
Oocyte Secreted Factors In Vitro
15:00 - l5:30
General Discussion
15:30 - 17:00
Poster Reception
Max Bell Foyer and Rooms 251,252 , and253
17:30 - 19:30 Marché Dinner (Vistas Dining Room)

TUESDAY, JUNE 2".I
07:00 - 09:00 Marché Breakfast (Vistas Dining Room)
Morning Plenary Session II
"Maturation of the Pre-Ovulatory Follicle"
Chair'. Bruce Murphy (CRRA, Université de Montreal, Cønadø)
Max Bell Auditorium
08:15 - 09:00
Helene Quesnel (INRA, Frønce)
Nutritional and Lactational Effects on Follicular Development in the Pig
09:00 - 09:45
K.Kikuchí (Nøtionøl Institute of Agrobiologicul Sciences, Tsukuba, Japøn)
In Vitro Oocyte Maturation and Oocyte Quality in the Pig
09:45 - 10: l5
Break (Max Bell Foyer)
10:15 - 11:00 Morag Hunter (University of Nottínghøm, UK)
Intra-Follicular Regulatory Mechanisms in the Pig Follicle
11:00 - 11:45
Randy Prather (University of Missouri, USA)
The Transcriptome and Epigenome of the Oocyte and Early Embryo
17:45 - 12:00
General Discussion
12:00 - 13:30 Lunch (Donald Cameron Hall Dining Room)
Breakout Session #3
"Management of Ovarian Activity in Swine"
Chair: Míchael Dyck (Universíty of Alberta, Cønada)
TransCanada Pipelines Pavilion Auditorium
13:30 - 14:00
Nic oline So ede (Wageníngen Univ ersitlt, The Netherlands)
Variable Ovarian Responses to Lactation Management Strategies
14:00 - 14:30
Klqus-Peter Brüssow (FBN Research Institute for the Biology of Førm
Animal s, D umm e rs torf, G ermøny )
Studies on Fixed-Time Ovulation Induction in the Sow
14:30 - 15:00
2 x l5 min Short Communications selected from submitted abstracts
J.J.J. van Leeuwen, S.Williams, B. Kemp, und N.M. Soede
Post-weaning Altrenogest Treatment in Primiparous Sows; The Effect of
Duration and Dosage on Follicular Development and Consequences for Early
Pregnancy
R. Knox, M. Altmyer, J. TaíbI, S. Breen, ønd D. Canadøy
Assessment of Follicle Population Changes in Sows at Estrus Using Real-
Time Ultrasound
l5:00 - 15:30 General Discussion
20

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l3:30 - 15:00
Breakout Session #4
"Breaking Science in pig Reproductive Biology"
Chair: Ina Dobrinski, (University of Calgøry, Caiaila)
Max Bell Auditorium
6 x l5 min short communications selected from submitted abstracts
S. Novak, H.S. Moore, F. paradß, G. Murdoch, M.K. Dyck,.W.T. Dixon,
snd GR Foxcroft
Temporal candidate Gene Expression patterns in the sow placenta During
Early Gestation and the Effect of Maternal L-arginine supplementation.
E.M. Kímbøll, G. Wyman, D.R. Stein, J.W. Rols, U.n. Àsnworth, R.D.
Geisert, and F. J.'White
Estrogen And Interleukin-1p, Trophinin, osteopontin, cycrooxygenase-r,
cyclooxygenase-2, and And Interleukin-1p System In The porðine uterus.
J.Bijttebier, K.TiIIeman, D. Deforce, M. Dhaenens, A.Vøn Soom, and D.
Maes
Proteomic study to Identify Factors in Follicular Fluid and/or Serum Involved
in in vitro Cumulus Expansion of porcine Oocytes
B.A. Freking, J.R. Miles, S.R. Bischoff, S. Tøi, N. Hardison, y. Xia, D.J.
Nonneman, J.L. Vallet, and J.A. píedrøhita
Impact of selection for uterine capacity on the placental rranscriptome
E. Behboodi, S. Mohan, J.R. Roilriguez-Sosa, y. Li, S. Megee, aid I.
Dobrinskí
Enrichment of Porcine Spermatogonia by Differential Culture
D.R. Eborn, D.L. Davis, und D.M. Grieger
Cloning and Expression of pluripotent Factors Around the Time of
Gastrulation in the Porcine Conceptus
15:00 - 15:30
15:30 - 17:00
17:30 - 19:30
General Discussion
Poster Reception
Max Bell Foyer and Rooms 251,252, and,253
Marché Dinner (Vistas Dining Room)

WEDNESDAY, JUNE 3"¿
07:00 - 09:00 Marché Breakfast (Vistas Dining Room)
Morning Plenary Session III
"Control of Prenatal Development"
Chair: CheryI Ashworth (The Roslin Institute, Uníversity of Eilinburgh, UK)
Max Bell Auditorium
08:15 - 09:00
George Foxcroft (University of Alberta)
Prenatal Programming of Postnatal Development in the Pig
09:00 - 09:45
Anne Croy (Queen's Universíty, Ontørio, Canøda)
Immune Regulation of Fetal-Maternal Interactions in the Pig
09:45 - 10:15
Break (Max Bell Foyer)
10:15 - 11:00
Jorge Piedrahita (North Carolinø Staie University, USA)
Application of Functional Genomics in Placental and Foetal Development in
the Pig
11:00 - 11:45
Jeffrey Vallet (USMARC, USA)
Development of the Pig Placenta
Il:45 - 12:00
General Discussion
12:00 - 13:30 Lunch (Donald Cameron Hall Dining Room)
Breakout Session #5
"Breeding Management Programs for the Future"
Chair Steve Webel (JBS Uniteil, USI¡
TransCanada Pipelines Pavilon Auditorium
13:30 - 14:00 Fernsndo Bortolozzo (Federal University of Porto Alegre, Brøzil)
Growth, Body State, and Breeding Performance in the Gilt and the Sow
14:00 - 14:30 Archie Clutter (Newshøm Choice Geruetics, USA)
Genetic Selection for Lifetime Reproductive Performance
14:30 - 15:00
2 x 15 min Short Communications selected from submitted abstracts
l5:00 - i5:30 General Discussion
J. P(ttterson, G. Foxcroft, A. Cameron, T. Smith, A. Kammer, R. Schott, L.
Greíner, J. Conner, und C. Francisco
The Effect of PG600 at Weaning on Sow Performance
L.J. Zak, J. Patterson, J. Hancock, D. Hockley, D. Roga, and G.R. Foxcroft
Benefits of Synchronizing Ovulation with Porcine Luteinizing Hormone
(pLH) in a Fixed Time Insemination Protocol in Weaned Multiparous Sows
22

Breakout Session #6
"state-of-the Art in conceptus-uterus InteractionsÆarly pregnancy signalling"
Chair: Adum Ziecik (Olsztyn, Poland)
Max Bell Auditorium
13:30 - 14:00
Agníeszka waclawík (Institute of Animal Reproduction and Food Reseorc,.
Olsztyn, Poland)
Antiluteolytic Mechanisms and the Establishment of pregnancy in the pig
14:00 - 14:30
Greg Johnson (Texas A&M, USA)
Conceptus - Uterus Interactions
14;30 - l5:00
2 x 15 min Short Communications selected from submitted abstracts
D. Møthew, E. SeIIner, C. Okømura, R. Geisert, L- Anderson, and M. Lucy
Effect of Progesterone (P) Anragonist RU486 on uterine progesterone
Receptor (PGR) Expression, Embryonic Development And 'RNA
Ovarian
Function During Early Pregnancy In Pigs
F.W. Bazer, H. Gao, G.A. Johnson, G. Wu, D.W. Bailey, and R.C,
Burghardt
Select Nutrients and Glucose Transporters in Pig uteri and conceptuses
15:00 - 15:30
General Discussion
15:30 - 16:00
Break
Concluding Keynote Lecture
Chair : H e rib e rt o R o drí g u e z- M qrtín e z ( S L(), U p p s alø, S w e d e n)
Max Bell Auditorium
16:00 - 16:45
George Foxcroft ((Jniversity of Alberta, Canøda)
Keeping Pig Reproduction Research Relevant - challenges and opportunities
18:30 - 21:00
Conference Banquet and Closing Ceremony
Donald Cameron Hall Dining Room
THURSDAY, JUNE 4th
07:00 - 09:00 Marché Breakfast (Vistas Dining Room)
23

INDEX OF INVITED PAPERS
THE PHYSIOLOGICAL ROLES OF THE BOAR EJACULATE............ .......39
H. Rodríguez-Martínez, U. Kvist, F. Saravia, M. Wallgren, A. Johannisson, L. Sanz, F.J. peña, E.A.
Martínez, J. Roca, J.M. Vázquez, and J.J. Calvete
MOLECULAR KINBTICS OF PROTBINS AT THE SURFACE OF PORCINE SPERM
BEFORE AND DURING FERTLLLZAT[ON............... .....................41
P.S. Tsai and B.M. Gadella
OPTIMAL CHARACTERISTICS OF SPERMATOZOA FOR SEMEN
TECHNOLOGIES IN PIGS .......43
I. Parrilla, J.M. vazquez,r. Caballero, M.A. Gil, M. Hernandez, J. Roca, X. Lucas, and E.A.
Martínez
RECENT ADVANCES IN BOAR SEMEN CRYOPRESERVATION.................................45
D. Rath, R. Bathgate, H. Rodríguez-Martínez, J. Roca, J. Strzezek, and D. waberski
::::::i:: ::: :* :::::::: Ï: ::YÏ :Ï:T: : ::: * T:::.,
W.L. Flowers
THE ROLE OF GENE DISCOVERY, QTL ANALYSES, AND GENE EXPRESSION IN
REPRODUCTIVE TRAITS IN THE PIG...........
...........4g
S.K. Onteru, J.W. Ross, and M.F. Rothschild
PROTEOMIC ANALYSIS OF MAMMALIAN GAMETES AND SPERM.OOCYTE
INTERACTTONS......
...................s1
P. Sutovsky
NUTRITIONAL AND LACTATIONAL EFFECTS ON FOLLICULAR DEVELOPMENT
IN THE plc..........
.......................s3
H. Quesnel
APPEARANCE, FATE AND UTILIZATION OF ABNORMAL PORCINE EMBRYOS
PRODUCED BY IN VITRO MATURATION AND FERTILIZATION ...........55
K. Kikuchi, T. Somfai, M. Nakai, and T. Nagai
INTRA-FOLLICULAR REGULATORY MECHANISMS IN THE PORCINE OVARY.s7
M.G. Hunterand F. Paradis
25

TRANSCRIPTIONAL, POST.TRANSCRIPTIONAL AND EPIGENETIC CONTROL
OF PORCINE OOCYTE MATURATION AND EMBRYOGENESIS................................59
R.S. Prather, J.W. Ross, S.C. Isom, and J.A. Green
OVARIAN RESPONSES TO LACTATION MANAGEMENT STRATEGIES .................61
N.M. Soede, W. Hazeleger, R. Gerritsen, P. Langendijk, and B. Kemp
STUDIES ON FIXED-TIME OVULATION INDUCTION IN THE PIG............................63
K.-P. Brüssow, F. Schneider, W. Kanitz, J. Rátky, J. Kauffold, and M. Wãhner
PRENATAL PROGRAMMING OF POSTNATAL DEVELOPMBNT IN THE PIG........65
G.R. Foxcroft, W.T. Dixon, M.K. Dyck, S. Novak, J.C.S. Harding, and F.C.R.L. Almeida
CELLULAR AND MOLECULAR EVENTS IN EARLY AND MID-GESTATION
PORCINE IMPLANTATION SITES: A REVIEW
.......6i
B.A. Croy, J.M. Wessels, N.F. Linton, M. van den Heuvel, A.K. Edwards, and C. Tayade
FUNCTIONAL GENOMIC APPROACHES FOR THE STUDY OF
FETALiPLACENTAL DEVELOPMENT IN SWINE \ryITH SPECIAL EMPHASIS ON
IMPRINTED GENES .................69
S.R. Bischoff, S. Tsai, N. Hardison, A.A. Motsinger-Reif,,B.A. Freking, and J.A. Piedrahita
DEVBLOPMENT OF THE PIG PLACENTA.......
........7I
J.L. Vallet, J.R. Miles, and B.A. Freking
GROWTH, BODY STATE, AND BREEDING PERFORMANCE IN GILTS AND
PRTMTPAROUS SOWS................ .................73
F.P. Bortolozzo,M.L. Bernardi, R. Kummer, and I. Wentz
GENETIC SELECTION FOR LIFETIME REPRODUCTIVE PERFORMANCB ...........75
A.C. Clutter
ANTILUTEOLYTIC MECHANISMS AND THB ESTABLISHMENT OF PREGNANCY
IN THE PIG........... ......................77
A. Waclawik, A. Blitek, M.M. Kaczmarek, J. Kiewisz, and A.J. Ziecik
CONCEPTUS-UTERUS INTERACTIONS IN PIGS: ENDOMETRIAL GENE
EXPRESSION IN RESPONSE TO ESTROGENS AND INTERFERONS FROM
CONCBPTUSES........ ..................79
G.A. Johnson, F.W. Bazer, R.C. Burghardt, T.E. Spencer, G. Wu, and K.J. Bayless
26

INDEX OF SHORT ORAL PRESENTATIOI\S
CHANGES IN RESPONSIVENESS TO BICARBONATE UNDER CAPACITATING
CONDITTONS rN LIQUID PRESERVED BOAR SPERMATOZAê,^ IN V(TRO...............83
H. Henning, A.M. Petrunkina, R.A.P. Harrison, and D. Waberski
INTRAUTERINE INSEMINATION OF SOWS BY USING A TWO-CHAMBER SEMEN
BAG SYST8M........... ..................85
A.K. Olesen andC. Hansen
BINDING OF PORCINE SPERMATOZOA TO UTERINE EPITHELIAL CELLS
MODULATES THE FEMALE IMMUNE RESPONSE AND MIGHT INDICATE THE
FORMATIONOFAPRE.OVIDUCTALSPERMRESERVOIR.............. .......87
. U. Taylor,H.Zerbe, H.M. Seyfert, D. Rath, and H.J. Schuberth
SPERM SURVIVAL FOLLO\ryING COLLOID CENTRIFUGATION VARIES
ACCORDING TO THE PART OF THE SPERM-RICH FRACTION US8D.....................89
J.M. Monell, F. Saravia, M. van Wienen, H. Rodríguez-Martínez, and M. Wallgren
MAPPTNG QUANTITATTVE TRArr LOü FOR REPRODUCTTON rN PIGS...............91
S.C. Hernandez, H.A. Finlayson, C.J. Ashworth, C.S. Haley, and A.L. Archibald
GLOBAL PROTEIN PROFILING OF PORCINE CUMULUS CELLS IN RESPONSE
TO NATIVE OOCYTE SECRETED FACTORS IN VITRO ..........93
F. Paradis, H. Moore, S. Novak, M.K. Dyck, W.T. Dixon, and G.R. Foxcroft
POST.WEANING ALTRENOGEST TRBATMENT IN PRIMIPAROUS SOWS: THE
EFFECT OF DURATION AND DOSAGE ON FOLLICULAR DEVELOPMENT AND
CONSEQUENCES FOR EARLY PREGNANCY.........
...................9s
J.J.J. van Leeuwen, S.Williams, B. Kemp, and N.M. Soede
ASSESSMENT OF FOLLICLE POPULATION CHANGES IN SOWS FROM
WBANING AND AT ESTRUS USING REAL.TIME ULTRASOUND...............................97
i
R. Knox, J..Taibl, M. Altmyer, S. Breen, D. Canaday, and A. Visconti
TEMPORAL CANDIDATE GENE EXPRESSION PATTERNS IN THE SOW
PLACENTA DURING EARLY GESTATION AND THE EFFECT OF MATERNAL L.
aRGININE SUPPLEMENTATION................ ................99
S. Novak, H.S. Moore, F. Paradis, G. Murdoch, M.K. Dyck, W.T. Dixon, and G.R. Foxcroft
27

ESTROGBN AND INTERLEUKIN-IP RBGULATION OF TROPHININ,
OSTEOPONTIN, CYCLOOXYGENASE.I, CYCLOOXYGENASE.2, AND
TNTERLEUKTN-Ip SYSTBM rN THE PORCTNE UTERUS ..........:........ .......101
F. J. White, E. M. Kimball, G. Wyman, D. R. Stein, J. W. Ross, M. D. Ashworrh, and R. D. Geisert
PROTEOMIC STUDY TO IDENTIFY FACTORS IN FOLLICULAR FLUID AND/OR
::::Y i::::::: ï :: ::::: ::Y-Y* ::::*:) :: :::::Ì: :::::ff
J.Bijttebier, K.Tilleman, D. Deforce, M. Dhaenens, A.Van Soom, and D. Maes
IMPACT OF SELECTION FOR UTERINE CAPACITY ON THE PLACENTAL
TRANSCRIPTOME.. ................105
B. A. Freking, J.R.Miles, S. R. Bischoff, S. Tsai, N. Hardison, Y. Xia, D. J. Nonneman, J. L. Vallet,
and J. A. Piedrahita
ENRICHMENT OF PORCINE SPERMATOGONIA BY DIFFERENTIAL CULTURE
107
E. Behboodi, S. Mohan, J.R. Rodriguez-Sosa, Y. Li, S. Megee, and I. Dobrinski
CLONING AND EXPRESSION OF PLURIPOTBNT FACTORS AROUND THE TIME
OF GASTRULATION IN THE PORCINE CONCEPTUS............. .................109
D.R. Eborn, D.L. Davis, and D.M. Grieger
RESPONSES TO EXOGENOUS GONADOTROPHIN TREATMENT IN
CONTEMPORARY WEANED SOWS...............:.......... ..................111
J. Patterson, A. Cameron, T. Smith, A. Kummer, R. Schott, L. Greiner, J. Conner, and G. Foxcroft
BENEFITS OF SYNCHRONIZING OVULATION WITH PORCINE LUTEINIZING
HORMONE (pLH) IN A FIXED TIME INSEMINATION PROTOCOL IN WEANED
MULTIPAROUS SOWS....... ....113
L.J.Zak, J. Patterson, J. Hancock, D. Rogan, and G.R. Foxcroft
EFFECT OF PROGBSTERONE ANTAGONIST RU486 ON UTERINE
PROGESTERONE RECEPTOR MRNA EXPRESSION, EMBRYONIC
DEVELOPMENT, AND OVARIAN FUNCTION DURING EARLY PREGNANCY IN
......11s
Prcs
D. Mathew, E. Sellner, C. Okamura, R. Geisert, L. Anderson, and M. Lucy
SELECT NUTRIENTS AND GLUCOSE TRANSPORTERS IN PIG UTERI AND
CONCEPTUSES........ ................117
F.W. Bazer, H. Gao, G.A. Johnson, G. Wu, D.W. Bailey, and R.C. Burghardt
28

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INDEX OF POSTERS
P32 TYROSINE PHOSPHOPROTEINS AND THE ACROSOME
PORCINE
REACTION IN
SPERM: IMPORTANCE
cALCruM
OF EXTRACELLULAR AND INTRACELLULAR
._:.:__::_::._:___::.... .............121
J.L. Bailey, S. K. Thangavelu, and C. Lessard
THE EFFECT OF TIMING OF hCG ADMINISTRATION AFTER eCG ON FERTILITY
OF WEANED SO\ryS TIMED INSEMINATED WITH FROZEN-THAWED SEMEN..122
A. Bolarin, M. Hernande z, J.M. y árzquez,E.A. Martínez, and J. Roca
IMPORTANCE OF DIETARY OMEGA-3 FATTY ACIDS (FISH OILS) ON BOAR
SEMEN QUALITY
....................r23
C.A. Castellano, I. Audet, J.p. Laforest, y. Chouinard, and J.J. Matte
PROTECTIVE EF'FECT OF PSP-IiII SPERMADHESIN IN SORTED AND FROZEN-
THAWED BOAR SPERM.... .......:........... ....124
D. del Olmo,I. parrilla, J. Roca, E.A. Martínez, and J.M. yázquez
CRYOPRESERVATION OF SEMEN FROM NATIVE HUNGARIAN
BoARS _
MANGALICA
A prl,or sruDy.....
........__.::__::.-.:._::::_ì...............rr.
I. Egerszegi, p. Sarlós, B. Berger, and J. Rátky
RELATION BETWEEN NUMBER OF SPERM CELLS INSEMINATED AND
FERrrLrry RESULTS rN sows................ .......:.:::_.__::..:__. ..............126
-
I
H. Feitsma, J.I. Leenhouwers, and E.F. Knol
I
DOUBLE SPERM DEPOSITION TECHNIQUE (MAGAPLUS-DD@) INCREASES THE
REPRODUCTM RESULTS IN FARM CONOriloNS............. ....................127
c. Gómez-Rincón, M. García-Tomás, y. Dahmani, R. Mozo-Martín, s. Jiménez, A.N. Berges, E.
García-Bonavila, M. yeste, S. Bonet, and J. Grandía
EVALUATION OF'BOAR SEMEN FERTILIZING CAPABILITY AFTER SHORT AND
EXTRA.LONG STORAGE USING DURAGEN@ SEMEN 8XTENDER.................,.......128
c' Gómez-Rincón, M. García-Tomás, Y. Dahmani, R. Mozo-Martín, S. Jiménez, A.N. Berges, E.
García-Bonavila, M. yeste, S. Bonet, and J. Grandía
LEJA-4 COUNTING CHAMBER BXERTS NEGATIVE EI.-FECT
Morrl,rry
ON SPERM
.._.:.._*..:.:_._::-..."__:._:_ì::... ..........12s
C. Hansen
29

COMPARISON OF NUCLEOCOUNTER SP1OO AND FACSCOUNT AF SYSTEM FOR
DETERMINATION OF BOAR SPERM CONCENTRATION AND VIABILITY .......... 130
C. Hansen
SEMINAL PLASMA REGULATES PROSTAGLANDIN SYNTHESIS ENZYMES IN
THE PORCINE ENDOMETRIUM ............131
M.M. Kaczmarek, A. Blitek, J. Filant, and A.J. Ziecik
EFFECT OF BOAR SEMINAL PLASMA ON PRODUCTION OF PROSTAGLANDIN
F2o (PGF2o) AND INTERLEUKIN-6 (IL-6) FROM PORCINE AND BOVINE
ENDOMETRIAL CELLS.....
....I32
M. Madej, M.T. Madsen, M. Norrby, A. Johannisson, C. Hansen, and A. Madej
ALDOSE REDUCTASE ACCELERATES BOAR SPERM CAPACITATION. .............. 133
N. Okamura, A. Kawashima, B. Osman, K. Takebayashi, Y. Katoh, M. Takashima, S. Kohchi, K.
Kikuchi, M. Matsuda, and A. Kikuchi
CAN POST-THAW SPERM SURVIVAL BE IMPROVED BY USING A SPECIFIC
PORTION OF THE BOAR EJACULATE PACKED IN MINIFLATPACKTM...............I34
F. Saravia, M. Wallgren, A. Johannisson, and H. Rodríguez-Martínez
RELOCATION OF SWINE GENETICS USING EMBRYO TRANSFER.......................135
S.L. Terlouw, C. D. Bierman, D. L. Kohler, B.A. Didion, and J.R. Dobrinsky
ï:::*::::::::::i::T:::_"_ï::::)::T:T_:-'*)T:Tllil,.u
M. Wallgren and N. Lundeheim
STUDY OF DIFFERENT METHODS TO EVALUATE ACROSOME INTEGRITY IN
DILUTED BOAR SEMEN
.......I37
S. Williams, V. Fernández, D. Gabilondo, E. Valette, and R.L. de la Sota
BIRTH WEIGHT IMPLICATIONS FOR POSTNATAL DEVELOPMENT IN PIGS...138
F.R.C.L. Almeida, A.L.N. Alvarenga, G.G. Paneira, D.O. Fontes, G.A. Marques, P.C. Cardeal, L.
Moreira, G.R. Foxcroft, and H. Chiarini-Garcia
CHARACTERIZATION OF ITIHI,ITIH3, AND ITIH4 GENES AND THEIR
ASSOCIATION\ryITHREPRODUCTIVETRAITSINPIGS... .....................139
L Balcells, A. Castelló, R. Pena, C. Óvilo, A. Sánchez, and A. Tomás
30

DIFFERENTIAL EXPRBSSION AND LOCALTZATION OF CX43IN MALE AND
FEMALB GONADS OF NEONATAL AND IMMATURE PIGS AFTER IN ATERO
EXPOSURE TO AN ANTr-ANDROGEN, FLUTAMIDE........... .....................140
B. Bilinska,I. Kopera, M. Durlej, A. Hejmej, K. Knapczyk, M. Slomczynska, and M. Koziorowski
DELAYED PIG EMBRYO TRANSFER USING A CHEMICALLY.DEFINED
CULTURE MEDIUM: A PRACTICAL APPROACH....... ............141
P. Cherel, L. Letelu, J. Glenisson, J. pires, and p. Letelu
DIFFERENTIAL microRNA EXPRESSION AMONG NORMAL, ABNORMAL, AND
LOW MOTILITY PORCINE SPERM CELLS
...........I42
E. Curry, S.E. Ellis, T.J. Safranski, and S.L. pratt
SUCCESSFUL PRODUCTION OF PIGLETS DERIVED FROM IVF OOCYTES
MATURED IN GONADOTROPIN.FREE CHEMICALLY DEFINED MEDIA .............143
H. Funahashi, Y. Akaki, and K. yoshioka
EXPRESSION OF HOXA1O IN EARLY PREGNANT ENDOMETRIUM AND
CONCEPTUS IN PIGS WITH NATURAL AND HORMONALLY INDUCED
ovuLATroN............ .................144
M.M. Kaczmarek, D. Balcerowicz, J. Kiewisz, A.J. Ziecik and A. Blitek
INFLUENCE OF EMBRYONIC FACTORS ON LYSOPHOSPHATIDIC ACID
RECEPTOR 3 GENE EXPRESSION IN PORCINE ENDOMETRIUM DURING THE
PERIIMPLANTATION PERIOD AND CORRESPONDING DAYS OF THE ESTROUS
CYCLE..... ................145
K. Kamiúska, M. Wasielak, and M. Bogacki
BFFECT OF'INTRAUTERINE CROWDING ON FETAL DEVELOPMENT ON DAY 40
oF PREGNANCY .....................146
B. Kemp, N.M. Soede, E.H. van der Waaij, and W. Hazeleger
THE EXPRESSION OF WNT 4, WNT 5A, WNT 7A, P-CATENIN, AND E-CADHERIN
GENES IN THE ENDOMETRIUM DURING THE REPRODUCTIVE CYCLE AND
BARLY PREGNANCY IN PIGS
................I47
J. Kiewisz, M.M. Kaczmarek, A. Blitek, G. Bodek, and A.J. Ziecik
DEVELOPMENT OF NEONATAL PIGLETS FOLLOWING RECIPROCAL EMBRYO
TRANSFERS BET\ryEEN MEISHAN AND WHITE CROSSBRED GILTS....................148
J'R. Miles, J.L. Vallet, J.J. Ford, B.A. Freking, R.A. Cushman, W.T. Oliver, and R.K. Chrisrenson
31

ISOLATION OF PORCINE EMBRYONIC STEM CELLS USING SERUM-FREE
C0NDITIONS............. ...............149
M.B. Nottle, I. Vassiliev, S. Vassilieva, L.F.S. Beebe, S.J. Hanison, S.M. Mcllfatrick, and R.J.
Ashman
FACTORS INF'LUENCING IN VITRO DEVELOPMENT OF PORCINE OOCYTES
FOLLOWING TNTRACYTOPLASMTC SPERM TNJECTTON (rCSr)... .......1s0
M. Nakai, J. Noguchi, H. Kaneko, J. Ito, N. Kashiwazaki, and K. Kikuchi
SURVIVAL OF PIGLETS ACCORDING TO PHYSIOLOGICAL PARAMETERS AT
BIRTH .....................151
A.Panzardi, T. Bierhals, A.P.G. Mellagi, M.L. Bernardi, F.P. Bortolozzo, and I. Wentz
TEMPORAL AND SPATIAL CHARACTERIZATION OF HYALURONIC ACID
ASSOCIATED GENE EXPRBSSION IN THE PORCINE REPRODUCTIVE TRACT
DURING EARLY EMBRYONIC DEVELOPMENT
t52
J.A. Pasternak, F. Paradis, S. Novak, R.R.E. Uwiera, G.R. Foxcroft, and M.K. Dyck
CHARACTERIZATION OF PROGESTERONE RECEPTOR (PGR) mRNA ISOFORMS
IN THE ENDOMETRIUM OF CYCLIC AND PREGNANT PIGS..... ...........153
E. Sellner, D. Mathew, J. Ross, C. Okamura, K. Wells, R. Geisert, and M. Lucy
ESTROGEN RECEPTORS COLOCALTZATION IN THE PORCINE UTERUS
THROUGHOUT PREGNANCY
l.s4
M. Slomczynska, M. Duda, K. Knapczyk, M. Durlej, A. Tabecka-Lonczynska, and B. Bilinska
IMPACT OF A PROGESTERONE ANALOGUE TREATMENT DURING EARLY
PREGNANCY ON LITTER SIZE IN WEANED SOWS WITH AN EXTENDED
WEANING.TO.OESTRUS INTERVAL
,...........155
N.M. Soede, W. Hazeleger, and B. Kemp
INTERMITTENT SUCKLING REGIMES DURING A 4 OR 5 WEEK LACTATION
PERIOD: INDUCTION OF LACTATIONAL OVULATION AND EFFECTS ON
LITTER SIZE ............156
N.M. Soede, M. Berkeveld, R. Genitsen, B. Laurenssen, N. Kuijken, P. Langendijk, and B. Kemp
PRO-ANGIOGENIC AND ANTI-ANGIOGENIC FACTORS AND THEIR RELATION
TO PORCINE PREGNANCY SUCCESS OR FAILUR8............. ....................157
A. K. Edwards, M.J. van den Heuvel, B.A. Croy, and C. Tayade
32

EFFECT OF EMPTY UTERINE SPACE ON PLACENTAL DEVBLOPMENT,
FARROWING INTERVALS, AND STILLBIRTH.. ......................158
J.L. Vallet, J.R. Miles, T.M. Brown-Brandl. and J.A. Nienaber
PRODUCTION OF TRANSGENIC PIGS CARRYING HUMAN COAGULATION
FACTOR IX, THE FURIN PROPEPTIDE CLEAVAGE ENZYME AND SERPINA1 BY
soMATrc CELL NUCLEAR TRANSFER...............
.....................1s9
J.G.zhao, J.w. Ross, J.J. whyte, D.M. wax, L.D. Spate, M.S. Samuel, E.M. walters, A. Rieke, c.N.
Murphy, K.D. Wells, and R.S. prather
EFFECT OF SYNCHRONIZING OVULATION IN WEANED SOWS USING
OVUGELTMWITH SINGLE FIXED TIME AI oN PREGNANCY RATE AND LITTER
SIZE ......... ................160
J.N. Taibl, S.M. Breen, S.K. Webel, M.E. Swanson, and R.V. Knox
THE EFFECT OF AGE AT FIRST ESTRUS AND BREEDING ON THIRD ESTRUS ON
MATURE BODY SIZE AND LONG-TERM REPRODUCTIVE PERFORMANCE OF
sows
....161
J. Patterson, E. Beltranena, and G. Foxcroft
EFFECT OF EARLY LIFE STRBSSORS ON REPRODUCTIVE CAPABILITY..........162
c.J. Ashworth, L.E. Harker, w.c. Duncan, K. Rutherford, J.A. Rooke, and A.B. Lawrence
GENOTYPE AND FETAL SIZE DIFFERBNCES IN FETO.PLACENTAL AMINO
ACID STATUS... .....163
C.J. Ashworth and H. J.McArdle
RELEVANCE OF LOCAL PROGBSTERONE SUPPLY ON EMBRYO SURVIVAL IN
UNILATERALLY OVARIECTOMISED GILTS .......164
R.Z. Athorn, P. stott, E.G. Bouwman, R. Ashman, s. o'Leary, M. Nottle, and p. Langendijk
REPRODUCTION RESULTS AND ECONOMIC PERFORMANCE IN SOW HERDS
oFCoNSTRASTINGPRoLIFICACYLEVELS.............
S. Boulot and B. Badouard
CONJUGATED LINOLEIC ACIDS (CLA) IN GESTATING AND LACTATING SOW
DIETS AFFECT REPRODUCTIVE PBRFORMANCE AND OFFSPRING TISSUE
GAIN........
................1ó6
R. Geritsen, P. Bikker, and A.-M. pfeiffer
33

PROSTAGLANDIN ASSOCIATED WITH OXYTOCIN OR CARBETOCIN IN
INDUCTION OF PARTURITION IN SOWS ..............167
N.B. Gheller, R.F. Werlang, T.J. Mores, M. Santi, D. Gava, M.L. Bernardi, D.E.S.N Barcellos,I.
Wentz, and F.P. Bortolozzo
FACTORS ASSOCIATED \ryITH REDUCED REPRODUCTION IN SECOND PARITY
sows....... ................168
L.L. Hoving, N.M. Soede, E.A.M. Graat, H. Feitsma, and B. Kemp
EFFECTS OF REDUCED REPRODUCTION IN SECOND PARITY SOWS ON
PRODUCTION rN SUBSEQUENT pARrTrES.........
t69
L.L. Hoving, N.M. Soede, E.A.M. Graat, H. Feitsma, and B. Kemp
TIMED INSEMINATION FOLLOWING GnRH AGONIST ADMINISTRATION IN
\ryEANED SOWS....... ................170
M.E. Johnston, A.M. Gaines, M.E. Swanson, and S.K. Webel
SUMMER INFERTILITY IN SOWS IN FRANCE: DOES THE SUMMER
TEMPERATURE REALLY PLAY A PART
R. Krejcí, V. Auvigne, P. Leneveu, C. Jehannin, and E. Sallé
ULTRASONOGRAPHIC COUNTING OF SWINE EMBRYOS
....................I72
F. Martinat-Botté, S. Serriere, H. Quesnel, S. Boulot, E. Venturi, and F. Tranquart
INFLUENCE OF LESIONS DETECTED THREE WEEKS AFTER INSEMINATION
ON REPRODUCTIVE PERFORMANCE OF SOWS ...................173
A.P.G. Mellagi, T. Bierhals, A. Panzardi, N.B. Gheller, M.L. Bernardi,I. Wentz, and F.P. Bortolozzo
GENISTEIN ALTERS THE RELEASE OF CORTISOL,LH, AND PROSTAGLANDIN
F2aDURING ESTRUS AND INSEMINATION IN GILTS................ .............174
M. Norrby, M.T. Madsen, F. Saravia, E. Ekstedt, and A. Madej
ENVIRONMENTAL AND SOW RELATED FACTORS AFFECTING DURATION OF
FARROWrNG...........
...,.............17s
C. Oliviero, M. Heinonen, A. Valros, and O.A.T. Peltoniemi
DIETARY INTAKE DURING EARLY PREGNANCY DOES NOT INFLUENCE
EMBRYONIC SURVIVAL AND VARIABILITY IN GILTS...... ...............176
H. Quesnel, E. Venturi, E. Royer, F. Elleboudt, S. Boulot, S. Serriere, and F. Martinat-Botté
34

PREPUBERTAL SCORING OF SCALE ACTIVITY IN GILTS AND ITS POTENTIAL
RELATIONSHIP TO SUBSBQUENT FERTILITY AND REPRODUCTIVE
PERFORMANCE IN LANDRACE-DUROC.YORKSHIRB CROSS FEMALES ...........177
L.A. Rempel, G.A. Rohrer, and T. Brown-Brandl
y_:_T*: :::::1 T ï: :: :T: :: :* :: ::::: :* :)::y::li: Î;;
F. Rosales, v. Quintero, M. Gonzáiea A. Aguilera, M. Fernández, andM. Martens
ESTRUS SYNCHRONIZATION TO IMPROVE REPRODUCTIVE PERFORMANCE
OF PRIMIPAROUS SOWS.......
F. Rosales, V. Quintero, A. Aguilera, and M. Martens
EFFECTS OF DAY OF FARROWING INDUCTION ON SUCKLING PIGLET
PERFORMANCE.......... ............180
H.M. Smith, A.M. Williams, and T.J. Safranski
REDUCED PHOSPHORUS IN DIETS DURING GESTATION DECREASED LITTER
srzE aND LoNcBVrTy............
................18r
G. Sorensen
EFFECT OF CHRONOLOGICAL AND SEXUAL AGE AT FIRST MATING ..............182
F. Thorup
CAN PROLIFIC SOWS NURSE THEIR OWN PROGENY ........183
F. Thorup
EFFECT OF SUPPLEMENTING GILT DIETS WITH BETAINE DURING SUMMER
oN REPRODUCTTVE PERFORMANCE....... .............184
W. H. E. J. Van Wettere, P. Herde, P. E. Hughes, and S.J. pain
INVESTIGATIONS ON TNF.a MEDIATBD EFFBCTS IN PREOVULATORY
ovARrEs oF GrLTS ................185
D. waberski, M. Krüger, H.-J. Schuberth, A. Schnapper, H. Henning, K. Langner, M. Beyerbach,
and R.H.F. Hunter
REPRODUCTIVE RBSPONSES OF SOWS TO HEAT STRESS DURING DIFFERENT
PHASES OF A PRODUCTION CYCLE............... .......186
A.M. Williams, T.J. Safranski, D.E. Spiers, p.A. Eichen, E.A. Coate, and M.C. Lucy
35

ABSTRACTS OF INVITED PAPERS
37

PPl
THE PHYSIOLOGICAL ROLES OF THE BOAR EJACULATE
H. Rodríguez-Martíne1, u. Kuist', F. saravial, M. wallgrent'', A. Johannissono, L. sanrs, F.J.
Peña6, E.A. Martín ez' , J. Roca7, J.M. Vázqu ez7 , andJ.J.-Calvetes
lDiuision of Reproduction, Faculty of Veterinary Medicine & Animal Science (FVMAS),
Swedish University of Agricultural Sciences (SLU), POB 7054, SE-75007 Uppsala, Sweden.
'Center for Andrology & Sexual Medicine, Departm-ent of Medicine, Karolinska University
Hospital/Huddinge, SE-14186 Stockholm, Sweden; 3Quality Genetics, Råby 2004, SE-24292
Hörby, sweden; aDepartment of Anatomy, Physiology & Biochemistry, FVMAS, sLU, poB
7011, SE-75007 Uppsala, Sweden; slaboratory of Structural Proteomics, Institute of
Biomedicine of valencia, csIC, Jaime Roig 1 l, 46010 valencia, spain; 6section of
Reproduction & Obstetrics, Department of Medicine, Faculty of Veterinary Medicine, Avd de la
Universidad s/n, 10071 Cáceres, Spain;TDepartment of Medicine & Animal Surgery, Faculty of
Veterinary science, university of Murcia, campus de Espinardo, 30100 Murcia, Spain.
E-mail: heriberto.rodriguez@kv.slu.se
During ejaculation in the boar, sperm cohorts emitted in epididymal cauda fluid are
sequentially exposed and resuspended in different mixtures of accessory sex gland secretion.
This paper reviews the relevance of such unevenly composed fractions of seminal plasma (SP)
in vivo on sperm transport and sperm function and how this knowledge could benefit boar semen
processing for arlificial insemination (AI). The firstly ejaculated spermatozoa (first 10 mL of the
sperm-rich fraction, SRF tPll) remain mainly exposed to epididymal cauda fluid and its specific
proteins i.e. various lipocalins, including the fertility-related prostaglandin D synthase; than to
prostatic and initial vesicular gland secretions. Pl-spermatozoa are hence exposed to less
bicarbonate, zinc or fructose and mainly to PSP-I spermadhesin than if they were in the rest of
the SRF and the post-SRF (P2). Since the PI-SP is less destabilizing for sperm membrane and
chromatin, P1-spermatozoa sustain most in vitro procedures, including cryopreservaiion, the
best. Moreover, ejaculated firstly, the Pl-spermatozoa seem also those deposited by the boar as a
vanguard cohort, thus becoming overrepresented in the oviductal sperm reservoir (SR). This
vanguard SR-entry occurs before the endometrial signalling of SP components (as PSP-VPSP-I
and cytokines) and causes a massive influx of the innate defensive PMNs to cleanse the uterus
from eventual pathogens, superfluous spermatozoa, and the allogeneic SP. The SP also
conditions the mucosal immunity of the female genital tract, to tolerate the SR-spermatozoa and
the semi-allogeneic conceptus. These in vivo gathered data can be extrapolated into procedures
for handling boar spermatozoa in vitro for AI and other biotechnologies, including simplified
cryopreservation.
39

PP2
MOLECULAR KINETICS OF PROTEINS AT THE SURFACE OF PORCINE SPERM
BEFORE AND DURING FERTILIZATION
P.S. Tsai and B.M. Gadella*
Department of Biochemistry and Cell Biology, Department of Farm Animal Health, Faculty of
Veterinary Medicine, Utrecht University, Yalelaan 2 3584 CM Utrecht, The Netherlands
E-mail : B.M.Gadella @uu.nl
xPresenting author
Fertilization is a decisive moment in life and enables the combination of the DNA from two
gametes to ultimately form a new organism. The sperm surface, especially the head area, has
distinguishable subdomains that are involved in distinct fertilization pro""*i"s. It is known that
the sperm head surface undergoes constant remodelling during epididymal maturation and
migration in the male and female genital tract. But intriguingly, the identity, origin and spatial
ordering of proteins at the sperm surface that are involved in mammaú un f"rtilitution are
essentially unknown. This review deals with sperm surface protein modifications that are under
somatic cell control. As soon as the sperm is released from the seminiferous tubules it is
subjected to these modifications. These surface reorganisations continue until the sperm reside
in the fallopian tube where they meet the oocyte and may fenilize it. Most likely, a selective
process allows only functionally mature and intact sperm to optimally interact aná fertilize the
oocyte' Recent data suggest that even the perivitelline fluid is involved in sperm surface
remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize
the oocyte. In this contribution, the kinetics of proteins at the sperm surface wili be overviewed.
Better understanding of this would help to design strategies to improve male fertility or to devise
novel contraceptives.
4l

PP3
OPTIMAL CHARACTERISTICS OF SPERMATOZOA FOR SEMEN
TECHNOLOGIES IN PIGS
I. Parrillal, J.M. vázqu ezt r. cabarrero2, M.A. Gill, M. Hernandezl, J. Rocal, X. Lucas, and E.A.
Martinez
rDepartment of Animal Medicine and Surgery, Faculty óf Veterinary Medicine, University of
Murcia, E-30071, Murcia, Spain; 2 Department of Molecular Biomedical Sciences, College of
veterinary Medicine, North carolina state university, Raleigh, NC 27606,IJSA
E-mail: parrilla@ um.es
Despite the great potential of sperm technologies such as sperm cryo-preservation and sperm
sex sorting for the improvement of different aspects of swine production, artificial insemination
with fresh or stored semen is currently the only sperm technològy used at a commercial scale in
the pig industry. The lower reproductive performance associated with the use of these sperm
technologies is the reason for such limited use. Since optimal characteristics are requireã for
successful application of frozen-thawed and sex-sorted boar spermatozoa, the present paper
summarises the value of the current available methods for their functional assessment as well as
the effects ofthese technologies on boar sperm functionality. In addition, strategies developed to
reduce sperm damage and improve the yields of both sperm technologies in iwine production
are also reviewed with particular attention to the contributions of the authors.

PP4
RECENT ADVANCES IN BOAR SEMEN CRYOPRESERVATION
D. Rathl, R. Bathgate2, H. Rodríguez-Martíner3, J. Rocao, J. strzezeks, and D. waberski
llnstitute of Farm Animal Genetics, Friedrich Loeffler Institute, Federal Institute of Animal
Health, 31535 Neustadt-Mariensee, Germany; 2Faculty of Veterinary Science, The University of
Sydney, NSw 2006, Australia; 3Diuision of Reproduciion, Faculty óf v"t"rinury Medicine and
Animal Sciences, SLU, SE-75007 Uppsala, Swèden; aDepartment of Animat Medicine and
Medicine,
fÌig:ilfrlrllv^oiVererinarv
university of ruurcia, E-30071
"Department
Murcia, Spain;
of Animal Biochemistry and Biotechnology, Faculty of Animal Bioeniineering,
University of Warmia and Mazury in Olszryn, poland;õijnit for Reproductive Medicine of
Clinics for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, 30559
Hannover, Germany
E-mail: rath@tzv.fal.de
Since 35 years ago boar semen has been frozen and used for artificial insemination (AI).
However, fertility of-cryopreserved porcine sperm has consistently been low as boar sperm are
more sensitive to cellular stress imposed by changing osmotic balance, oxidative stress, lowtemperatufe
exposure' cryo-protectant intoxication etc. and are less able to "o-p"nrate
for these
deficiencies at commercially applicable dosages. Additionally, differences in sperm treezability
among individuals are well known. Here we review current-advances on tests to screen sperm
quality post-thaw, on ways of diminishing individual boar effects, on improvement of cryoprotection
by novel extender components, on packaging and freezingprotocåls and fieezing and
thawing methods, and on the handling of ^sexeJ bãar sperm. Major advances have been
registered, which have improved cryo-iurvival and the cåpacity to process boar semen for
commercialAl.
45

_-------'
PP5
SELECTION FOR BOAR FERTILITY AND SEMEN QUALITY. THE WAY AHBAD
W.L. Flowers
Department of Animal Science, North Carolina State University, Raleigh, N.C., 2i695-762I,
U.S.A
E-mail: william_flowers @ncsu.edu
Critical needs for the swine industry in terms of boar fertility evaluations are validation of
semen quality estimates with ín vivo reproductive data; estimation of the relative fertility of
boars; and elimination of sub-ferlile ejaculates. Single sire matings are the best way to validate
semôn quality estimates with reproductive performance. Sampling aboú 20Vo of the population
provides an accurate estimation of the variability among boais and should be sufficient for this
purpose. In vitro tests that measure univariate characteristics of ejaculates including motility and
morphology appear to be just as accurate as those that measure multivariate traits such as in
vitro fettilization in terms of predicting boar fertility. Reasons for this observation may be
related to how properties of sperm cells are influenced by the sow reproductive tract. Several
seminal plasma proteins show strong correlations with boai fertility *¿^ trol¿ potential for being
developed into tests that can rank the relative fertility of boars. Almost 90Vo of the variation in
boar fertility was explained when the proportion of motile and acrosome-reacted spermatozoa
was combined with relative amounts of 28 kDa, pI 6.0 and 55 kDa, p1 seminal plasma proteins.
Consequently' combining different complementaiy tests improves estimations òf boa, f'ertility.
Motility estimates routinely performed in most A.I. centres are a reasonable technique får
identification and elimination of sub-fertile ejaculates. However, the accuracy with which they
currently are conducted within the swine industry needs improvement.
47

PP6
THE ROLE OF GENE DISCOVERY, QTL ANALYSES, AND GENE BXPRESSION IN
REPRODUCTIVE TRAITS IN THE PIG
S.K. Onteru, J.W. Ross, and M.F. Rothschild*
Depaftment of Animal science and center for Integrated Animal Genomics,
Iowa State University, Ames, IA, USA
E-mail: mfrothsc @iastate.edu
*Presenting author
The reproductive performance of the sow is one of the key factors affecting production
profitability of the pig industry. Reproductive traits are in general, lowly heritable, and with
reliable markers, they can be used to enhance current selection procedures for improvement of
these traits. To find potential markers, large scale quantitative trait loci (QTL) and candidate
gene studies have been conducted for reproductive traits. The present review discusses QTL and
candidate gene discovery, large scale SNP association studies, gene expression profiling and
discovery of miRNA regulation of pig reproductive tissues. Many QTL have been found for
reproduction traits and a limited number of useful genes (e.g.: ESRI, PRLR, FSHB, EpOR and
RBP4) have been found to have significant associations with reproductive traits. Expression
studies with reproductive tissues have revealed differential expression within a few gene
networks which need further mapping and association analyses to select prospectiv" g"n"
markers. The near completion of the pig genome sequence and the development of high density
SNP chips will allow for large scale SNP association studies for pig reproductive traits in the
future' Collection of appropriate phenotypes in large numbers and in broad populations
representative of the swine industry are required if such genomic studies will ultimately be
successful.
49

PP7
PROTEOMIC ANALYSIS OF MAMMALIAN GAMETES AND SPERM-OOCYTE
INTERACTIONS
P. Sutovsky
Division of Animal Sciences, and Departments of Obstetrics, Gynecology and Women's Health,
University of Missouri-Columbia, S141 ASRC 920 East Campus Drive, Columbia, MO 65211-
5300, USA
E-mail: SutovskyP @ missouri.edu
Proteomic analysis occupies an increasingly important place in gamete and embryo biology
as an independent tool of discovery and as a means of follow-up to transcriptionai profiling.
Proteomics have been and will be increasingly helpful in many areas of repróductiv* Uiotog!,
including applied science and technology development. Areas likely to be impacted most rapiãiy
by proteomic knowledge include fertility evaluation in male farms animãls, male infertitity
diagnostics in humans, assessment and optimization of oocyte and embryo culture protocoli,
selection of fittest oocytes for assisted fertilization, and selection of most competent embryos for
embryo transfer. Oocyte proteomics will help us understand the process of oogenesis and oocyte
maturation, and to discover non-invasive markers of oocyte quality. Sperm proteomics correlate
with normal sperm structure and function and can be applied to discover novel biomarkers of
farm animal fertility and diagnostic markers of human male infertility. putative receptors
parlicipating in fertilization, as well as proteins acquired onto sperm surface from epididymal
flujd and seminal plasma, have been discovered by proteomiC analysis. An added level of
information is provided by advanced proteomic approaches, tapable of identifying
posttranslational modifications such as phosphorylation, glycosylation, anà ubiquitination which
play important functions in gametogenesis, fertilization, and embryo development. By no means
exhaustive, the present paper reviews some of the most interesting pioteomic studies of
mammalian gametes and embryos published in the last decade.

PP8
NUTRITIONAL AND LACTATIONAL EFFECTS ON FOLLICULAR DEVELOPMENT
IN THE PIG
H. Quesnel
INRA, UMR1079 Systèmes d'Elevage, Nutrition
France, Agrocampus Ouest, UMR1079 SENAH,
E-mail : helene.quesnel @ rennes.inra.fr
Animale et Humaine, F-35000 Rennes,
F-35000 Rennes, France
In sows, follicular development is inhibited during lactation, and weaning the piglets allows
recruitment and selection of follicles that will undergo preovulatory maturation and ovulate.
Lactation inhibits GnRH secretion, and in turn LH secretion, through neuroendocrine stimuli
induced by suckling. Pituitary response to GnRH and the sensitivity of the hypothalamopituitary
unit to oestradiol positive feedback are also reduced. The impact of lactation on the
reproductive axis is further complicated by the physiological and metabolic adaptations that are
developed for milk production and that depend on nutrient intake, nutrient needs, and body
reserves. A strongly catabolic state during lactation amplifies the inhibition of LH secretion,
thereby inducing a delay of oestrus and ovulation after weaning. Nevertheless, post-weaning
ovulation is less delayed nowadays than in the 1970's or 80's. Nutritional deficiency also has
deleterious effects on embryo survival, which are likely related to alterations in follicular growth
and maturation. The physiological mechanisms by which information on the metabolic changes
is transmitted to the hypothalamus-pituitary-ovary axis are not fully understood in the sow.
Glucose, insulin, and leptin are the most likely signals informing the hypothalamus of the
metabolic status, yet their roles have not been definitely established. At the ovarian level,
folliculogenesis is likely to be altered by the reduction in insulin and IGF-I concentrations
induced by nutritional deficiency. More knowledge is needed at the intrafollicular level to better
understand nutritional effects on follicular development, and also on oocyte quality and embryo
development.
53

PP9
APPEARANCE, FATE, AND UTILIZATION OF ABNORMAL PORCINE EMBRYOS
PRODUCED BY IN VITRO MATURATION AND FERTILIZATION
K. Kikuchil*, T. Somfaito, M. Nakail, and T. Nagai2
tDiuision of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki
305-8602, Japan; 2National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, 305-
0901, Japan; #Present address: National Institute of Livestock and Grassland Science, Tsukuba,
Ibaraki, 305-0901, Japan
E-mail : kiku @affrc.go jp
In vitro production (IVP) including in vitro maluration (IVM) and fertilization (IVF) is now
an important technology r for obtaining live piglets. However, there are still two significant
obstacles to the efficient production of viable porcine embryos: (1) polyspermy and (2)
fertilization of oocytes ar¡ested at the immature stage. These phenomena relate to production of
embryos with abnormal ploidy (polyploidy). To avoid these problems, careful selection of
mature oocytes for IVF, and regular monitoring of normal and abnormal fertilization
(polyspermy and/or lack of male pronucleus formation) are very important. In our recent studies,
however, we have confirmed that some oocytes with abnormal ploidy after polyspermy can
develop into diploid embryos with potentially normal developmental ability. The mechanism by
which such fertilized polyploid oocytes develop to a normal state during embryo development is
still not well understood. Attempts to clarify this mechanism would hopefully reveal data that
are very useful for not only IVP but also other technologies such as the production of transgenic
or cloned animals using IVM oocytes, including other species, also for human reproductive
manipulation. In this review, we focus on studies of normality of IVM oocytes and ploidy of
IVP embryos, and try to suggest practical ways of solving the problems mentioned above in
pigs.

I
I
I
I
I
PPlO
I
I
INTRA.FOLLICULAR REGULATORY MECHANISMS IN THE PORCINE OVARY
I
M.G. Hunterl and F. Paradis2
I
I
I
I
rSchool of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough,
United Kingdom LEl2 5RD; 2 Department of Agricultural, Food and Nutùtional Sóience, 4-10
AgricultureÆorestry Centre, University of Alberta, Edmonton, Alberta, Canada, T6G 2P5
E-mail: morag.hunter@nottingham.ac.uk
I
I
I
I
I
)
i
I
I
I
I
The mechanisms controlling the follicular growth continuum in the pig involve the
interaction between local growth factors which are expressed throughout development and extrafollicular
factors such as gonadotrophins. A large number of follicular growth factors, many
belonging to the transforming growth factor-B GGF-B) superfamily, have been identified in the
somatic cells and in the oocyte. The relative importance of these intra-follicular factors varies
with stage of development. The initiation of follicular growth and early preantral development is
controlled locally (by factors including c-kit-kit ligand, members of the bone morphogenetic
family (e.g BMP-15) and growth differentiation factor-9 (GDF-9)) and gonadorrophins are nor
thought to be involved until later. During antral follicle development, the oocyte secretes factors
that stimulate porcine granulosa cell proliferation and differentiation, modulate apoptosis and
suppress progesterone production, thereby preventing premature luteinisation. Likely candidates
for mediating these effects include BMP-6, -15 and GDF-9 that are critical for fertility and
ovulation rate in several mammals. There are also paracrine interactions between the somatic
cells, with theca derived transforming growth factor P (TGF-P) playing a key role in regulating
antral follicle maturation. Finally, during the periovulatory period, members of the EGF family
from the granulosa cells stimulate cumulus expansion and oocyte maturation. Evidence indicates
that some of these local factors may also influence oocyte developmental potential, emphasizing
further the complexity, and importance, of these intra-follicular interactions.
57

PPl1
TRANSCRIPTIONAL, POST.TRANSCRIPTIONAL, AND EPIGENETIC CONTROL
OF PORCINE OOCYTE MATURATION AND EMBRYOGENESIS
R.S. Pratherl, J.W. Ross2, S.C. Isoml, and J.A. Greenl
tDivision of Animal Sciences, university of Missouri, columbia, Mo 65211, usA,
2Depaftment of Animal science, Iowa siate university, Ames, IA 50011, usA
E-mail : PratherR @ Missouri.Edu
Embryogenesis is a complex process that is controlled at various levels. As new discoveries
are made about molecular mechanisms that control development in other species, it is apparent
that these same mechanisms regulate pig embryogenesis as well. Methylation of DNA and
modification of histones regulate transcription, and mechanisms such as ubiquiti nization,
autophagy, and microRNAs regulate development post-transcriptionally. Each of túese systems
of regulation is highly dynamic in the early embryo. A better understanding of each of these
levels of regulation can provide tools to potentially improve the reproductive process in pigs, to
improve methods of creating pig embryos and cloned embryos iniitro, and tò provide markers
for predicting developmental competence of the embryo.

PP12
OVARIAN RESPONSES TO LACTATION MANAGEMENT STRATEGIES
N.M. Soeder, W. Hazelegerl, R. Gerritsen2, p. Langendijk3, and B. Kempr
rAdaptation Physillogy Group, PO Box 338 6700 AH Wageningen University, Wageningen,
The Netherlands; 2 Current address: Schothorst Feed Research, ielystfi; tiÃlinr ñvesrock,
Roseworthy Campus, South Australia
E-mail: nicoline.soede @ wur.nl
A number of lactation management srategies can be applied to reduce negative effects of
lactation on post-weaning fertility. This paper focuses on efìåcts of lactation lenlgth, intermittent
suckling and split weaning on follicle development and subsequent oestrus. It is concluded that a
lactation length of less than 3 weeks still leads to suboptimal reproductive performance in our
modern sows. Further, both intermittent suckling and spiit weaning stimulatË lactational follicle
development and oestrus, but the variation in response between sows still limits practical
application.
61

PPI3
STUDIES ON FIXED.TIME OVULATION INDUCTION IN THE PIG
K.-P. Brüssowt, F. Schneiderl, w. Kanitzl, J. Rátky2, J. Kauffold3, and M.'Wähnera
tFBN Research Institute for the Biology of Farm Animals, D-18196 Dummerstorf, Germany;
2Research Institute for Animal Breeding and Nutrition, H-2053 Herceghalom, Hungary;'Ne*
Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square PA
19348, USA; 4Anhalt University of Applied Sciences, D-06406 Bernburg, Germany
E-mail : bruessow @ fbn-dummerstorf.de
A technology that allows for manipulating of oestrus and ovulation, and would then also
allow for fixed-time insemination, could be of great benefit for swine farms that operate using
sow batch management due, at least in part, to savings in labour and the production of large
batches of evenly developed pigs. Thanks to the current knowledge on endocrine regulation of
follicle development and ovulation, and the availability of numerous reproductively active
substances such a technology is now available. It covers procedures for synchronising oestrus
based on the use of altrenogest in gilts and of batch-wise weaning in sows, for stimulating
follicle development using eCG and for inducing of ovulation using hCG or LH as well as
GnRH analogues. While the procedures for oestrus synchronisation stand alone, other
procedures require additional treatments. If fixed-time insemination is the goal, oestrus needs to
be synchronised and follicular development and ovulation induced by the use of GnRH
analogues and hCG with ovulation occurring within 36-42 hrs. It is a general recommendation to
inseminate those animals twice, i.e. 24 and 40 hrs after ovulation induction. However, the
aforementioned technology requires healthy animals and a solid managemeht and cannot be
used to compensate for poor management.
63

PP1.4
PRENATAL PROGRAMMING OF POSTNATAL DEVELOPMENT IN THE PIG
G.R. Foxcroftl, w.T. Dixonr, M.K. Dyckl, s. Novakr, J.c.s. Harding2, and F.c.R.L. Almeida3
tSwine Reproduction-Devel-opment Program, Dept. AFNS, University of Alberta, Edmonton,
Alberta, T6G 2P5, Canada; 2Department of Largé Animal Clinical Sciences, 52 Campus Drive,
University of Saskatchewan, Saskatoon, Saskatchewan S7N 584, Canada; 3laboratory of
Structural Biology and Reproduction, Department of Morphology, Federal University of Minas
Gerais, Belo Horizonte, MG, Brazll
E-mail: george.foxcroft @ualberta.ca
Studies of low birth weight offspring (runt pigs) have a long history in pig science. Runt pigs
(< 0'8 kg at birth) can be identified as lying outside developmental norms for gestation age at a
very early stage of pregnancy. Within-litter comparisons of associations between low (- 1.0 kg)
and high (-1.9 kg) birth weight littermates and postnatal development indicate that low birth
weight pigs have reduced growth potential and poor carcass quality, linked to lower muscle fibre
numbers at birth. In contemporary commercial sows with between 10 and 15 total pigs born,
even greater between-litter differences in average birth weight appear to make the largest
contribute to variation in postnatal growth performance, independent of numbers born. At the
upper extremes of litter size, low birth weight is a predictable characteristic of the most prolific
sows, in which more than 40Vo of litters have > 15 pigs born and 4AVo of the pigs in these litters
have a birth weight < 1.2 kg.
Longitudinal studies to determine the origin of litters with low average birth weight are
limited. Cross-sectional data suggest that selection for increased numbers born has created an
imbalance between ovulation rate and uterine capacity, particularly in higher parity sows.
Increased crowding at day 30 of gestation primarily affects placental development in these sows
and persistent negative impacts on placental size affect fetal development by day 50. Although
embryonic weight and size may not be affected by crowding at day 30, there is evidence for
gender-specific effects on myogenic gene expression. The principal hypothesis driving ongoing
research is that crowding limits placental development in early gestation and is the origin of low
average birth weight litters in higher parity commercial sows.
Latent effects of metabolic state on oocyte quality and early embryonic development in the
gilt, and the lactating and weaned sow, have been reported. In contrast to effects of crowding at
day 30 of gestation, the embryo not the placenta, is primarily affected by previous catabolism.
These effects may also be gender-specific.
The large body of literature on gene imprinting, and the interactions between
metabolism, nutrition, and methylation state, suggests that classic imprinting mechanisms are
likely operating in the pig. A review of mechanisms determining prenatal programming of
postnatal outcomes based on epidemiological studies, human populations, and experimental
studies in laboratory rodents and sheep in particular, suggest avenues for further exploration in
swine. However, the potential of genomic, epigenomic, nutrigenomic, and proteomic
technologies bring new demands on experimental design and data management that present a
considerable challenge to the effectiveness of future research on prenatal programming in the
pig.
65

PP15
CELLULAR AND MOLECULAR EVENTS IN EARLY AND MID GESTATION
PORCINE IMPLANTATION SITES: A REVIEW
B.A. croyl'', J.M. wesselsl, N.F. Lintont, M. uan den Heuvert, A.K. Edwardsl, and c. Tayadel
lDepartment of Biomedical Sciences,-Ontario Veterinary College, University of Guelph,
Guelph, oN, NlG 2w1, canada and 2Department of Anatomy ãnd cell Bioiogy, eueen,s
University, Kingston, ON, K7L 3N6, Canada
E-mail: croya @ queensu.ca
commercial, North American pork breeds (Szs scrofa) experience significant loss of
genetically-normal conceptuses during the peri-implantatiån (attáchment; peîioo and at midgestation
(day 50 to,90 of the 714 day porõine geitation inrerval). Althoulh exacr causes for
these losses are not defined, asynchronous in utelo development and deficits in vascul arizalion
of the endometrium and placenta appear to be involved. understanding of normal maternal-fetal
dialogue is c¡itical to develop breølng or therapeutic strategies that improve fetal health and
overall litter size in c-ommercial pigs. The nón-invasive, epitheliochorial porcine placenta
permits investigation of maternal or fetal compartments without cross contaminating cells. we
developed and use protocols to capture single, homogenous populations of porcine cells
(endometrial lymphocytes, dendritic, ãr endothãüal cells)"from histological sections using laser
capture microdissection (LCM), a powerful tool for study of gene expression that reflects Íhe in
vivo envitonment' These data arc compared with gene á*p.Js.ion in biopsies of endometrium
and of trophoblast from the same, attachment sites. H"r" *Ë review justifications for selection of
the genes we have studied and our published and in progress work. These data provide new
insights into the roles of the endomei.iul i--un"
"nuiionirent
in the regulation of the success
and failure ofporcine conceptuses
6'7

PP16
FUNCTIONAL GENOMIC APPROACHES FOR THE STUDY OF
FETAL/PLACENTAL DEVELOPMBNT IN SWINE WITH SPECIAL EMPHASIS ON
IMPRINTBD GENES
S.R. Bischoffl'3, S. Tsair'3, N. Hardisont'0, A.A.Motsinger-Reif,g.R. Freking2, and J.A.
Piedrahita *r'3
rDepartment of Molecular Biomedical Sciences, College of Veterinary Medicine, North
Carolina State University, Raleigh, NC, USA; 2 USDA, ARS, U.S. Meat Animal Research
Center, Clay Center, NE 68933 -0|66,USA; 3 Center for Comparative Medicine and
Translational Research, North Carolina State University, Raleigh, NC, USA;a Bioinformatics
Research Center, Department of Statistics, North Carolina State University, Raleigh, NC 27695-
7566, USA
E-mail : jorge_piedrahita @ ncsu. edu
*Presenting author
This chapter describes the application of functional genomic approaches to the study of
imprinted genes in swine. While there are varied definitions of "functional genomics", in general
they focus on the application of DNA microarays, single nucleotide polymorphism (SNP)
affays, and other high coverage genomic analyses, and their combination with downsÍeam
methods of gene modification such as silencing RNA (siRNA) and viral and non-viral
transfection. Between the initial data acquisition and the actual genetic manipulation of the
system lies bioinformatics, where massive amounts of data are analyzed to extract meaningful
information. This area is in constant flux with an increased emphasis on detection of affected
pathways and processes rather than generation of simple affected gene lists. We will expand on
each of these points and describe how we have used these technologies for the study of
imprinted genes in swine. First we will introduce the biological question to provide context for
the discussion of the functional genomic approaches and the types of information they generate.
69

PP17
DEVELOPMENT OF THE PIG PLACENTA
J.L. Vallet, J.R. Miles, and B.A. Freking
USDA, ARS, U.S. Meat Animal Research Center,
P. O. Box 166, State Spur 18D, Clay Center, Nebraska 68933, USA
E-mail : j eff.vallet @ ars.usda. gov
Placental insufficiency results in fetal loss, low birth weight, stillbirth, preweaning mortality,
and poor growth. Placental development begins at conceptus elongation, which is a primaiy
factor controlling the size of the placenta. After elongation, the allantois develops outward from
the embryo to establish the allantochorion, which defines the size of the funôtional placenta.
During implantation, chorionic trophoblasts adhere to endometrial epithelial cells. placental
structures known as areolae develop at the openings of the endometrial glands and take up
endometrial gland secreted products (histotrophe). Between day 30 and 35 of gestation, thè
adhered trophoblast-endometrial epithelial bilayer undergoes microscopic folding. Fetal and
maternal capillaries develop adjacent to the bilayer and blood flows are urtangeã in a crosscountercuffent
manner. Except for nutrients secreted by the glands, nutrient exchange takes
place between these capillaries within these folds. By day 85, the folds deepen and become more
complex, increasing surface area. The epithelial bilayer thins and capillaries indent the plane of
each layer (but do not penetrate), reducing distance between capillaries. The folded bilayer is
surrounded by endometrial stroma on the maternal side and placental stroma on the fetal side.
The fetal-placental stroma is partially composed of glycosaminoglycans, the most abundant
being hyaluronan and heparan sulfate. Changes in both hyaluronoglucosaminidase and
heparanase during placental development suggest that these enzymes play a role in placental
development. In addition to structural modifications, various nutrient specific transport
mechanisms exist. These mechanisms are likely to be as important to transpofi of speóific
nutrients as placental size or structure.

PP18
GROWTH, BODY STATE, AND BREEDING PERFORMANCE IN GILTS AND
PRIMIPAROUS SOWS
F.P. Bortolotzol,M.L. Bernardi2, R. Kummer3, and I. Wentzt
I Depatlment of Animal Medicine, Veterinary Faculty, Federal University of Rio Grande do Sul,
Avenida Bento Gonçalves, 9090, CEP 91540-000 Porto Alegre-RS, Brazil; 2 Departmenr of
Animal Science, Agronomy Faculty, Federal University of Rio Grancle do Sul, Avenida Bento
Gonçalves, 7712, CF,P 91540-000 Porto Alegre-RS, Brazil; 3 Master Agropecuária Ltda, Rua
Constantino Crestani, 639, CEp 99560-000 Videira_SC, Brazll
E-mail: fpbortol@ufrgs.br; Tel. /fax: +55 51 3308 6132
Optimizing gilt management is a critical point to improve breeding herd efficiency. This
review describes the effects of growth rate (GR) and body state at onset of puberty stimulation
or at first mating on gilt puberty attainment, productivity, and sow longevity. Traditional
management practices should be re-evaluated with attention to different modern genotypes. It is
difficult to discern the real effects of age, weight, backfat depth, and estrus number at first
insemination on longevity and reproductive performance, because these variables affect one
another. GR interacts with age at boar exposure to influence age at puberty. Higher lifetime GR
gilts (>700 g/d) attain puberty earlier and have a lower anoesrrus rate. If gilis attain a target
weight (135-150 kg), are adapted to herd health status, and have at least one previously recorded
estrus, they can be inseminated. Overweight at first breeding and throughout gestation should be
avoided. There is no advantage in breeding gilts heavier than 150 kg; at first fãnowing rhe target
weight is 180-185 kg. Piglet production at first parity may be increased in gilts with a high GR
but the number of stillborn piglets can also be increased. The culling raté over 3 parities for
locomotion problems, which is one of the major risk factors for reduced herd retention rate, can
be increased in overweight gilts at first breeding (>150-170 kg).

PPl9
GENBTIC SELECTION FOR LIFETIME REPRODUCTIVE PERFORMANCB
A.C. Clutter
Newsham Choice Genetics, St Louis, MO (USA)
E-mail: archie_clutter@newsham.com
Genetic improvement of sow lifetime reproductive performance has value from both the
economic perspectives of pork producers and the pork industry, but also from the perspective of
ethical and animal welfare concerns by the general public. Genetic potential for piglets produced
from individual litters is a primary determinant of lifetime prolificacy, but femalei must be able
to sustain productivity without injury or death beyond the achievement of positive net present
value' Evidence exists for between- and within-line genetic variation in sow lifetime
performance, suggesting that improvements may be made by both line choices and genetic
selection within lines. However, some of the same barriers to accurate within-line selection that
apply to individual litter traits also present challenges to genetic selection for sow lifetime
prolificacy: generally low heritabilites, sex-limited expression, expression after the age that
animals are typically selected, and unfavorable genetic correlations with other traits in thð profit
function. In addition, there is an inherent conflict within the genetic nucleus herds where
selections take place between the goal of shofiened generation interval to accelerate genetic
progress and the expression of sow lifetime traits. A proliferation in the industry of commercial
multipliers with direct genetic ties and routine record flows to genetic nucleus Lerds provides a
framework for accurate estimates of relevant genetic variances and covariances, and estimation
of breeding values for sow lifetime traits that can be used in genetic selection.

PP2O
ANTILUTEOLYTIC MECHANISMS AND THE ESTABLISHMENT OF PREGNANCY
IN THE PIG
A. Waclawik, A. Blitek, M.M.Kaczmarek, J. Kiewisz, and A.J. Ziecik
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences,
Tuwima 10,10-747 Olsztyn, Poland
E-mail : waclawik@ pan.olsztyn.pl
Extended exposure of progesterone and conceptus estrogen influences the vascular
compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth
factors, extracellular matrix and adhesion molecules, cytokines, and transcription factors. One of
the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG
synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased
PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of
pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-
ketoreductase (CBRl). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is
involved in the process of matemal recognition of pregnancy. However, complex (direct and
indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on
day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect
another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10
and Wnt in the endometrium, to create the appropriate uterine environment for establishment of
pregnancy and implantation.
77

PP2I
CONCEPTUS.UTERUS INTERACTIONS IN PIGS: ENDOMETRIAL GENE
EXPRESSION IN RBSPONSE TO ESTROGENS AND INTERFERONS FROM
CONCEPTUSES
G'4. Johnsont, F.w. Bazer2,R.c. Burghardtl, T.E. spencer2, G. wu2, and K.J. Bayless3
lDepartment of Veterinary Integrative Biosciences, College of Veterinary Medicine and
Biomedical sciences, Texas A&M university, college station, Tx jlg43-445g, usA;
'Depattmettt of Animal Science, Texas A&M University, College Station, TX77843-Z4iI,
USA; 3Depafiment of Molecular and Cellular Medicine, Texas A&M Health Science Center,
College Station, TX 77843, USA
E-mail: gjohnson @ cvm.tamu.edu
This review highlights information on conceptus-uterus interactions in the pig with respect to
uterine gene expression in response to esffogens and interferons (IFNs) secreted from elongating
conceptuses. Pig conceptuses release estrogens for pregnancy recognition, but also secrete IFNs
that do not appear to be antiluteolytic. Estrogens and IFNs induce expression of largely nonoverlapping
sets of genes, and evidence suggests that pig conceptuses orchestrate essential
events of early pregnancy including pregnancy recognition signalling, implantation, and
secretion of histotroph by precisely controlling temporal and spatial (cell-specific) changes in
uterine gene expression through initiat secretion of estrogens, followed by cytokines including
IFNG and IFND. By Day 12 of pregnancy, esúogens increase the expression of multiple genei
in the uterine luminal epithelium including SPP1, STC1, IRF2, and STATI that likely have roles
for implantation. By Day 15 of pregnancy, IFNs upregulate a large array of IFN responsive
genes in the underlying stroma and glandular epithelium including ISG15, IRFl, STAT1, SLAs
and B2M that likely have roles in uterine remodeling to support placentation.
79

ABSTRACTS OF SHORT ORAL PRESENTATIONS
81

zsl-l
CHANGES IN RESPONSIVENESS TO BICARBONATE UNDER CAPACITATING
CONDITTONS rN LIQUID PRESERVED BOAR SPERMATOZOA rN V|TRO
H. Henningt, A.M. Petrunkinal'2, R.A.P. Harison3, and D..Waberskil
tunit for Reproductive Medicine of Clinics, University of Veterinary Medicine Hannover, Hannover,
Germany; 2Cambridge Institute for Medical Research, University of Cambridge, UK, 311 London Rd,
Great Shelford, Cambridge CB22 5DB, UK
E-mail : Heiko.Henning @tiho-hannover.de
Liquid-stored boar semen is commonly used for artificial insemination (AI) up to72h after dilution.
Insemination with semen stored for longer periods generally results in reduced fertility. Standard semen
parameters, i.e. motility and membrane integrity, usually give no indication of this reduction. Therefore,
more sensitive methods are needed for detection of storage-induced changes in sperm quality.
Capacitation has long been known to be an essential step in fertilization. In a number of studies
bicarbonate has been shown to be the key capacitating agent in boar sperm irz vitro (reviewed in Harrison
& Gadella 2005). The ability of sperm to respond to bicarbonate in vitro by undergoing capacitatory
changes can be measured as a sperm property crucial to fertilization (Petrunkina et a\.2005a; Silva &
Gadella 2006).In this study we used calcium influx as a parameter to investigate the responsiveness of
stored semen samples to bicarbonate. This parameter has been shown to be sensitive with respect to
evaluating detrimental effects of cooling during liquid storage of boar sperm (Petrunkina et a\.2005b).
Three ejaculates from each of 14 boars of proven fertility were diluted in Beltsville Thawing Solution
(BTS) extender to a concentration of 20 x 106 sperm / ml and stored at 17'C. After 12, 24, 12, 120 and
168 h of storage, motility was assessed in the diluted semen with a CASA-system, and membrane
integrity was checked with propidium iodide (PI) and FlTC-conjugated peanut agglutinin (FITC-PNA)
using a flow cytometer. Samples were then washed through Percoll, loaded with the calcium probe Fluo-
3-AM and PI, and incubated at 38"C in parallel in two variants of a Tyrode's medium. Medium A
contained 15 mM bicarbonate as well as 2 mM Ca2*; whereas bicarbonate was omitted from medium B;
incubation in medium A was performed und,er 5Vo CO2. Changes in Ca2* influx were assessed on a flow
cytometer af 3,20, 40, 60,90, I20, 150 and 180 min. The resulting kinetics of cell sub-populations were
compared between media and storage time points, based on analyses of the non-agglutinated population.
During storage, motility declined only from 89.0 -¡ 3.2 to 74.4 + l0.4%o (p

cells detected in both medium A and medium B: in medium A 10.7vo Ca2*-positive/pl-negative and
l4.7vo PI-positive after 168 h versus 3.37o and 9.Bvo respectively after 12 h;ìn medium n ísEo Curüpositive/Pl-negative
and l4.7vo Pl-positive after 168 hìersus i.rEo and7.2vo respectively after 12h.
These data could be interpreted as indicating that storage has two effects. There was on the one hand a
destabilization of the greater proportion of the populatìon such that incubation even in the absence of
bicarbonate caused membrane deterioration while incubation with bicarbonate caused such rapid
membrane destabilization that the Ca2*-positive / pl-negative state was increasingly short-lived before pI
entered- These findings are in agreement with those oi Petrunkin a et a\.2005b]rlporting that levels of
Ca2* uptake at the bèginnng Jt incubation under capacitating conditions are influenòed by storage
conditions' However, we also obtained evidence that ihe cohorts of cells that were less immediatJy
responsive to bicarbonate became refractory, reducing the overall bicarbonate response in terms of
appearance of Ca2*-positive / pl-negative plus pI-positivã cells.
As expected, motility and membrane integrity of the stored cells did not sufficiently reflect storagedependent
changes in sperm quality during prolonged storage. A recent review (petrunfona et at.,2007)
has proposed several requirements for the assessment of functional sperm parameters under capacitating
conditions. In accordanc,e with these proposals, we used a kinetic "^p"ri-"ntul
approach with welldefined
test and control media, considering equally both initial ,"rponr" ano sufsequent kinetics.
Specific spelm response to bicarbonate as measured by intracellular increases in Cazì in live celis
declined remarkably already after 72 h of liquid storage and dropped to almost zero in samples *toãd fo,
168 h' However, bicarbonate routinely caused large increases inãead cells within 20min of incubation.
lnlheir original paper on fluo-3 detection of bicarbonate-mediated changes in boar sperm, Harrison et al.
(1993) interpreted fluo-3-detectable C** entry as indicating the onset of a membrane destabilization
eventually leading to cell death. Harrison (1996) proposed ihat ¡f such destabilization were too rapid,
fertilization would be compromised. our findings *gg"rt that boar semen contains a population of cells
initially responsive to bicarbonate which becomes incieasingly intrinsically unstable åuilng storage and
responds to bicarbonate too rapidly. In addition, more stable cohorts of cells exist that during .iorug"
become less responsive to bicarbonate. We suspect that the dual opposing effects of storage, namely
destabilization and stabilizafion, may imply that ãfter normal inseminaiion the life+ime of mosr sperm in
the female tract will be insufficient to ensure satisfactory fertilization levels, while the remainder may not
respond to fertilizing conditions at all.
This work was supported by Development Associationfor Biotechnology Research (FBF e.V., Bonn)
Harrison RAP, Mairet B & Miller NG 1993 Flow cytometric studies of bicarbonate-mediated Ca2+
__ influx in boar sperm populations. Molecular Reproduction & Development 35 Ig7-20g.
Harrison RAP 1996 Capacitation mechanisms, and the role of capacih;ion as seen in eutherian
mammals. Reproducrion, Fertitity, ønd Development g 5g1_59¿.
Harrison RAP & Gadella BM 2005 Bicarbonate-induced membrane processing in sperm capacitation
Therio g e nolo gy 63 342-3 5 L
Petrunkina AM, Volker G, Brandt H, Töpfer-Petersen E & Waberski D 2005a Functional
significance ofresponsiveness to capacitating conditions in boar spermatozoa .Theriogenotogy 64
1766-1782.
Petrunkina AM, Volker G, Weitze KF, Beyerbach M, Töpfer-Petersen E & Wabersk¡ D 2005b
Detection of cooling-induced membrane changes in the rôsponse of boar sperm to capacitating
conditions. The rio g enolo gy 63 227 B-2299.
Petrunkina AM, Waberski D, Günzel-Apel AR & Töpfer-Petersen E 2007 Determinants of sperm
quality and fertiliry in domestic species Reproduction 134 3_ll.
Silva PF & Gadella BM 2006 Detection of damage in mammalian sperm cells. Theriogenology 65 95g-
918,
84

)
)
)
)
)
251-2
I
I
INTRAUTERINE INSEMINATION OF SOWS BY
BAG SYSTEM
USING A TWO-CHAMBER SEMEN
A.K. Olesenl and C. Hansenl*
rDanish Pig Production, Axeltorv 3, l1}gCopenhagen V. Denmark
E-mail : aki @ dansksvineproduktion.dk.
xPresenting author
Artificial insemination of sows is an effective method for intensive use of high breeding value boars.
In sows' intracervical insemination (ICI) using 2
!2 3 billi;;-;;"r- atozoa is an esrablished method
worldwide' resulting in a consistently higú fertility (warson ,t ot. àinoz¡.using inrraurerine inseminarion,
the number of spermatozoa per insðmiriation can be reduced even further. In intrauterine insemination
(IUI)' the tip of the catheter ìs placed in the corp_us uteri depositing the semen even closer to the site of
fertilization in the oviducf (Yazquez et aI.200Ð..If IUI can u" p"rtår-.a as easily and efficiently as ICI,
this will result in more semen doìes per boar. This will allow u it.ong". selection among boars, and lower
å|.,ji:liËitiose' rhis studv investìsarcd the
"n".,
åi"r*t,.il;* reducing the am'ount of sperm per
A total of 9272 multiparous Danish Landrace x Large white crossbred sows from seven Danish
commercial herds were randomly distributed into threeirorp, u, shown in table l. All sows were
inseminated using heterospermic i"*"n from Danish nuroJbours. Semen was collected using the gloved
hand method collecting the whole ejaculate. semen quality ru, "*luut.d
using subjective microscopic
motility score' semen concentration was measured usingîucleocounter sp100. Semen was extended
using EDTA boar semen extender. The same batch of ,"ä"n "o-prirlng
semen from 6 to l0 boars was
å:::iHTffiotto'ot
in each herd' one dose from each batch was anarysed for contenr of sperm per
Table 1: Method of insemination, no. sperm per dose, and vorume in the three groups.
TTIT
IUI-750

expected during production of semen doses and is therefore estimated not to have an influence on the
results of this trial.
The average parity in all
shown in table 2.
three groups was 4.1. Farrowing rate and total number of piglets born
Table 2: Number of sows and results achieved in the three groups.
Group Total number of
sows
ICI
IUI-750
3,Agg
3,077
rur-500 3,021
* Significantly different (p

2st-3
BINDING OF PORCINE SPBRMATOZOA TO UTERINE EPITHELIAL CELLS
MODULATES THE FEMALE IMMUNB RESPONSE AND MIGHT INDICATE THE
FORMATION OF A PRE-OVIDUCTAL SPERM RESERVOIR
U. Taylorl, H.Zerbe2,H.M. Seyfeft3, D.Rathl, and H.J. Schubertha
I Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Federal Research Institute for Animal
Health, 31535 Neustadt, Germany; 'Clini" for Ruminants, LMU Munich, 85164 Oberschleissheim,
Germany; 3 Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany;
a
Institute of Immunology, University of Veterinary Medicine ,30173 Hanover, Germany
E-mail : taylor@ tzv.fal.de
Inseminations in pigs are characterized by the tremendous amount of spermatozoa needed for
successful fertilisation. If, in contrast, spermatozoa are deposited at the tip of the uterine horn, a fraction
of the usual porcine insemination dose suffices (Johnson 1991, Y azqtez et al. 2005). It thus seems to be
the uterine passage where the need for such high sperm numbers arises. In the past the provision of
sufficient sperm numbers to reach acceptable fertility rates did not pose a problem due to the abundance
of spermatozoa in one single boar ejaculate. However, modern biotechnological procedures, such as sex
sorting of spermatozoa, require insemination of sperm portions containing no more then 50 x 106
spermatozoa. To facilitate insemination with such small sperm doses also for conventional AItechniques,
more knowledge has to be gathered about fundamental sperm transport and selection
mechanisms within the uterus. Previous studies on the pig uterus (Lovell & Getty 1968), the utero-tubal
junction (Rodriguez-Mafünez et al. 1990), and the oviduct (Wagner et al. 2002) suggested that
spermatozoa are indeed subject to close interaction and even binding with the epithelial structures of the
female genital tract. The present study aimed to further our understanding of such interactions
specifically in the porcine uterus.
For this purpose an ex vivo model was developed using uterine segments of 10 cm derived from 50
freshly slaughteredperi-ovulatory German Landrace gilts. In each segment 100 x 10ó spermatozoawere
incubated for 60 min at 38"C. The sperm cells originated from ejaculates provided by 4 boars of the same
breed and of proven fertility. Previous to incubation spermatozoa were either washed and diluted in the
semen extender AndroheprM or diluted with autologous seminal plasma without further washing. The
spermatozoa were subsequently flushed out of the segments, counted and their viability parameters were
established flow cytometrically using the stains PI and JCi for membrane integrity and mitochondrial
membrane potential respectively.
The results indicated a retention of viable spermatozoa within the uterine cavity since only 55+7Vo of
the intact spermatozoa (PIJJCI+) were rediscovered in the flushing, while the damaged sperm
population (PI+/JCl-) was flushed out almost in its entity (93x.127o; p

7.1-fold), TNF-u (AH: 1.9-fold) and COX-2 (AH:7-fold). Interestingly, despite their majorly different
composition AndroheprM and seminal plasma elicited a significantly different response only concerning
the arachidonic acid metabolite COX-2. Most surprisingly, however, was the result that the presence of
spermatozoa led to a significant down-regulation back to baseline levels for every mediator tested.
In conclusion, based on the described results we propose the hypothesis that the immense amounts of
spermatozoa needed for insemination in pigs is at least partially due to sperm binding sites along the
endometrium, which need to be saturated before unbound sperm can proceed to the oviduct.
Interestingly, the majority of bound spermatozoa are membrane intact and possess functional
mitochondria, i.e. are potential candidates for fertilisation. The exact binding mechanism and the
biological consequences remain, however, elusive. The sperm binding sites might serve several purposes.
For one, the bound spermatozoa might serve as a secondary reservoir to feed the oviductal reservoir in
case of a delayed ovulation. The fact that bound sperm are viable supports this presumption. On the other
hand, they could also function as part of a negative selection mechanism, which prevents boars with
oligozoospermia to mate successfully, a fact that would have to be overcome for low dose insemination,
for instance, by changing the site of application close to the tip of the uterine horn as described by
Yázqtez et al. (2005). Furthermore, the observed sperm-associated modulation of uterine gene
expression strongly suggests that sperm binding to endometrial cells has a considerable influence on the
female immune response. The down-regulation of inflammation-relevant cytokines and arachidonic acid
metabolites might serve to tightly control the issuing post-mating inflammatory reaction described in pigs
(Matthijs et al. 2003). As a long-term perspective it could also be part of the tolerance induction process
against paternal antigens or otherwise set off the preparation of the uterus for the reception of the
conceptus.
We gratefully acknowledge the financial support of the Hans Wilhelm Schawnann Foundation and the
G e nnan R e s e arch F oundation.
Johnson, LA 1991 Sex preselection in swine: Altered sex ratios in offspring following surgical
insemination of Flow Sorted X- and Y-bearing sperm. Reproduction in Domestic Animals 26 309.
3r4'.
Lovell JE & Getty R 1968 Fate of semen in the uterus of the sow: histological study of endometrium
during the 27 hours after natural services. American Journal of Veterinary. Research 29 609-625.
Matthijs A, Engel B & Woelders H 2003 Neutrophil recruitment and phagocytosis of boar spermatozoa
after artificial insemination of sows, and the effects of inseminate volume, sperm dose and specific
additives in the extender. Reproduction 125 351-361.
Rodriguez-Martinez H, Nicander L, Viring S, Einarsson S & Larsson K 1990 Ultrastructure of the
uterotubal junction in preovulâtory pigs. Anatomy Histology Embryology 19 76 - 36.
Yazqaez JM, MartinezB{, Roca J, Gil MA, Parrilla I, Cuello C, Carvajal G, Lucas X, & Vazquez
JL 2005 Improving the efficiency of sperm technologies in pigs: the value of deep intrauterine
insemination . Theriogenology 63 536-541 .
Wagner A, Ekhlasi-Hundrieser M, Hettel C, Petrunkina A, Waberski D, Nimtz M, & Toepfer-
Petersen E 2002 Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in
the pig. Molecular Reproduction Development 61 249-25.
88

251-4
SPBRM SURVIVAL FOLLOWING COLLOID CENTRIFUGATION VARIBS
ACCORDING TO THE PART OF THE SPERM.RICH FRACTION USED
J.M. Morrelll, F. saravial, M. van'wienent, H. Rodríguez-Martínez1, andM. wallgren2
lDepaflment of Reproduction, Division of Clinical Sciences, SLU, Uppsala, Sweden;
2Quality
Genetics, Hörby, Sweden
E-mail: Jane.morrell @ kv. slu. se.
Spermatozoa present in the first 10 ml (portion 1, Pl) of the sperm-rich fraction (SRF) of boar
ejaculates show increased resilience to cooling and cryopreservation compared to those in the rest of the
ejaculate (Saravia et al. 2008) presumably because of differences in exposure to seminal plasma. A new
technique for selecting the most robust animal spermatozoa, Single Layer Centrifugation (SLC), has
recently been developed at SLU (Morrell & Rodriguez-Martinez 2009).It was shown that SLC of SRF
using Androcoll-PrM followed by storage at room temperature (22"C) in extender without antibiotics
resulted in sperm preparations of high motility which survive longer than unselected sperm samples
(Wallgren et al. 2008). However, it is not known how SLC using Pl instead of SRF would affect boar
sperm survival. Furthermore, the technique requires scaling-up to enable larger volumes of ejaculate to
be processed. Scaling up has been shown to be possible for stallion spermatozoa (Monell et al.
unpublished data).
In experiment 1, Pl from 12 ejaculates (4 boars, 3 ejaculates per boar) and 12 SRF from the same
boars (again 3 ejaculates from each boar), collected on different occasions, were extended in Beltsville
Thawing Solution (BTS) without antibiotics to achieve a sperm concenrration of 100x106 mL. Aliquots
(1.5 ml) of these sperm samples were prepared by SLC on Androcoll-PrM by centrifugation at 300xg for
20 min. The resulting sperm pellets were washed in 5 ml BTS + bSA (5 mg/ml) by centrifugatioì at
500xg for l0 min. before resuspending in 1.5 ml of the same extender. The sperm suspensions were
stored in a styrofoam box at room temperature (22"C) for up to 6 days. Sperm motility was assessed
subjectively on a daily basis using phase contrast microscopy, after incubating the sperm suspensions at
38'C for 30 min. Differences between means were tested for statistical significance by ãnalysis of
variance, where P < 0.05 was considered significant.
In the second experiment ("scale-up"), larger volumes of extended semen were used, e.g. 3 ml or 4.5
ml were pipetted on top of 4 ml Androcoll-PrM and 10 ml, I2.5 ml or 15 ml extended semen were
pipetted on top of 15 ml Androcoll-P Large, an optimized formulation for larger tubes. After
centrifugation, the sperm pellet was resuspended in 3 ml or 10 ml BTS with bSA (1.25 mg/ml)
respectively. Sperm motility was analysed by computer assisted sperm analysis (CASA) on 5pl aliquots
of sperm samples placed in a pre-warmed Makler chamber (Sefi Medical Instruments, Haifa, Israel),
depth 10 pm, using a Mika Cell Motion Analyzer (MTM Medical Technologies Montreux, Switzerland)
and a microscope equipped with a warm stage and phase contrast optics l20x objective, Optiphot-2,
Nikon, Japan). Two hundred spermatozoa per sample were examined.
In experiment l, the mean differences in Eô sperm motility between control (non-SlC-selected) and
SlC-selected samples were as follows: time 0 h,Pl +13.3Vq SRF +16.6Vo; at24 h, Pl +23.7%, SRF
+23.47o; at 48 h, Pl +70vo, SP.F +24.6vo; ar 72 h, Pt +9.6vo, SRF +39.2vo; at 96 h, p1 +9.4%, SRF
+39.2Vo; at 120h,Pl +6.1%o, SRF +25.87o. The non-SlC-selected samples showed bacterial growrh afrer
the first 24 h, which may have contributed to the subsequent deterioration in sperm motility. SLCselected
sperm samples from SRF had a higher motility than non-selected sperm samples and retained
their motility for longer (P

colloids respectively, there was no difference between the "small" and "large" preparations (sperm
motility: "small" = 81.7 +7.J,"lar1e" =84.9 x.6.1 Vo; yield: "small" =43.J + I0.l Vo, "large" =42.4 +
23 7o). The motility of the unselected spermatozoa in these experiments was 77.4 + I9Vo. It was
interesting to note that the yield of spermatozoa was lower than in the previous experiment (performed 15
months earlier) which may be due to deteriorating quality in the semen of boars as they age.
The findings indicate that SLC selected the most motile spermatozoa from both Pl and SRF.
Moreover, SlC-selection removed bacterial contamination occurring during semen collection, enabling
the sperm suspensions to be kept without antibiotics in the semen extender or requiring a reduced
temperature. SlC-selection of SRF resulted in better sperm motility than SLC of P1 suggesting a
potentially critical relationship between initial sperm numbers and accessory gland secretions. These
observations are of interest to the swine Al-industry for the following reasons: (i) reducing the use of
antibiotics in semen extenders; (ii) simplifying sperm storâge requirements; (iii) providing additional
information about the effect of seminal plasma on boar sperm survival. Further studies on all these issues
are warranted. Finally, the results of the second experiment showed that it was possible to scale-up the
volumes of semen used from the 1.5 ml used in the first experiment to 15 ml. This experiment is
continuing, to scale-up to even larger volumes and to assess other parameters of sperm quality.
Funded by the Swedish Farmers' Foundation for Agricultural Reseqrch and FORMAS, Sweden.
Saravia F, Wallgren M, Johannisson A, Calvete JJ, Sanz L, Pena F, Roca J & Rodriguez-Martinez
H 2008 Exposure to the seminal plasma of different portions of the boar ejaculate modulates the
survival of spermatozoa cryopreserved in MiniFlatPacks. Theriogenology 71662-675.
Morrell JM & Rodriguez-Martinez H 2009 Biomimetic techniques for improving sperm quality in
animal breeding: a review. The Open Andrology Journal I l-9.
Waflgren M, Saravia F, Rodriguez-Martinez H & Morrell JM 2008 Effect of density gradient and
single layer centrifugation on motility and survival of boar spermatozoa. Reproduction in Domestic
Animals 43 53,241.
90

252-l
MAPPING QUANTITATIVE TRAIT LOCI FOR REPRODUCTION IN PIGS
S.C. Hernandez, H.A. Finlayson, C.J. Ashworth, C.S. Haley, and A.L. Archibald
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin,
Midlothian EH25 9PS, Scotland, UK
E-mail: silvia.hernandez@roslin.ed.ac.uk
Reproductive performance is a critical component of sustainable animal production systems. The low
heritability of reproductive performance traits such as litter size, ovulation rate, and prenatal survival and
their expression only in females limits improvement of these traits through traditional selective breeding
programs. However, there is abundant evidence of genetic variation in these traits between pig breeds,
which could be exploited to improve reproductive performance through selective breeding. The Chinese
Meishan breed is one of the most prolific pig breeds known, displaying grearer litter size than
commercial rffestern breeds, such as Large White, through higher levels of prenatal survival for a given
ovulation rate. But Meishan pigs have poor growth rates and high carcass fat content. However,
increasing the number of viable and productive offspring per reproductive female reduces financial and
environmental costs and improves the sustainability of the system. Thus, the superior Meishan alleles for
reproduction traits are potentially commercially valuable. As only a fraction of the genes / loci that
underpin the Meishan's superior reproductive performance have been identified to date, it is evident that
the genetics of reproductive performance merits further investigation. In an earlier study we mapped a
QTL (quantitative trait loci) with effects on embryo survival and litter size to the distal end of pig
chromosome 8 (King et a|.2003). The objective of this study is to identify QTL affecting ovularion rate,
teat number, litter size, number born alive and embryo survival, and characterize candidate gene(s)
underlying such QTL.
Our strategy to identify genetic markers for reproduction traits combines identifying QTL (regions of
the genome linked ,to the phenotypes) through genome scans using interval mapping and testing genes
recognized as candidates on both positional and physiological grounds. The QTL analyses involve
testing for associations between variation in the trait(s) of interest and the inheritance of chromosomal
segments from the parental animals. The inheritance of chromosomal segments through the QTL
mapping population is tracked by genotyping the population for polymorphic genetic markers -
microsatellites and single nucleotide polymorphisms (SNPs). The three-generation Roslin Institute
Meishan x Large White F2 QTL mapping population was genotyped for ten additional markers across the
QTL found previously on chromosome 8 and for I27 markers evenly spaced across the rest of the
genome. The marker genotypes and trait data were lodged in the resSpecies database
(www.resSpecies.org). Linkage maps were constructed using Multimap and Crimap (Green et at. 1990)
and the resulting maps checked for anomalous double recombinants with the chrompic function.
Anomalous genotypes were checked and corrected or omitted from the analysis. The marker orders in
the linkage map exhibited good agreement with international reference linkage maps. QTL analyses
were performed using the "fixed QTL allele" model on the GridQTL portal (www.gridqtl.org.uk/)
(Seaton et a|.2002).
A genome-wide scan was conducted for QTL with effects on the following traits: embryo survival,
total ovulation rate, total teat number, litter size, and number born alive. This scan revealed QTLs for
litter size (total number born) on SSC6 (102 cM), SSC8 (114 cM), SSClO (111 cM) and SSC18 (37 cM);
QTL for number born alive on SSCS (l14 cM); QTL for embryo survival on SSC8 (135 cM), SSCS
(29cM) and SSC10 (111cM) and QTL for total ovulation rate on SSCT (55cM), SSC13 (39cM),
SSC15 (8cM), and SSC18 (37cM), and for total teat number on SSC5 (57cM), SSC6 (20cM) and
SSCl8 (0 cM). These results were used as background effects in further analyses to confirm the presence
or absence of these QTL and to potentially reveal additional QTL. This new scan revealed QTL for litter
size (total number born) on SSC13 (33 cM), not confirming any of the previous ones; QTL for number
born alive SSC13 (41 cM); QTL for embryo survival on SSC8 (135 cM), SSC! (29 cM) and SSC10
(23 cM) and QTL for total ovulation rate on SSCT (9-12 cM), SSCl3 (39 cM), and SSC14 (21 cM), and
91

for total teat number on SSC5 (57 cM), SSC6 (79cM), SSC11 (0cM), and SSC12 (3 cM). These QTL
require further studies to confirm the effect on the different traits. As noted above, QTL with effects on
litter size and embryo survival had been discovered colocated at the distal end of chromosome 8 in an
earlier study (King et al. 2003). The fine mapping of these QTL enabled by genotyping the population
for additional markers revealed two QTL locations - a QTL for embryo survival on chromosome 8
(SSC8) at I34 cM, a QTL for litter size at position 114cM, together with a QTL with effects on the
number born alive. The emerging pig genome sequence and the homologous regions of the human and
mouse genome sequences will be inspected for potential positional and comparative positional candidate
genes. The SPP1 (Secreted phosphoprotein 1) gene, which is known for its role in communication
between the developing embryo and the sow, with a key role in conceptus implantation and maintenance
of pregnancy, is a strong candidate gene as it is located under the peak of the SSC8 QTL with effects on
embryo survival.
The resolution for the QTL found previously has been improved by the addition of a further ten
markers across the region of interest. The genome scan revealed twelve QTLs of which only two are
annotated in the pigQTldb (www.animalgenome.org/QTldb/pig.html). Future studies will involve fine
mapping the QTL revealed by the genome scan and prioritisation of positional candidate genes
underlying the QTL for functional analyses. For example, candidate genes for embryo survival QTL will
be examined for their expression at the RNA and protein levels in relevant tissue samples (feto-placental
tissue) from animals of different breeds.
{
I
a
d
We thank the British Pig Executive (SCH) and BBSRC (ALA, CJA, CSH) for fínanciøl support and
Birrel-Gray Travelling Scholarship (SCH) lor the award to attend the conference.
Green P, Falls K, & Crooks S 1990 Documentation for CRI-MAP, Version 2.4. St.Louis, Mo.,
Washington Univesity School of Medicine.
King AH, Jiang Z, Gibson JP, Haley CS, & Archibald AL 2003 Mapping quantitative trait loci
affecting female reproductive traits on porcine chromosome 8. Biology of Reproduction 68 2172-
2179.
Seaton G, Haley CS, Knott SA, Kearsey M, & Visscher PM 2002. QTLExpress: mapping quantitative
trait loci in simple and complex pedigrees. Bioinformatics. 18 339-340.
92

252-2
GLOBAL PROTEIN PROFILING OF PORCINE CUMULUS CELLS IN RESPONSE
TO NATIVE OOCYTE SECRETED FACTORS IN VITRO
F. Paradisl, H. Moorel, s. Novakl, M.K. Dyck1, w.T. Dixonl, and G.R. Foxcroftl
tswine Reproduction-Development Program, Dept. AFNS, University of Alberta, Edmonton, AB, T6G
2P5, CANADA
E-mail : fparadis @ ualberta.ca
Until recently, the traditional view was that the oocyte played a passive role in folliculogenesis,
relying on paracrine signalling from surrounding somatic cells to acquire developmental competence.
However, recent evidence suggests that the oocyte plays an active role in the proceis of folliculågenesis
by secreting soluble factors that act on the neighboring somatic cells. Studies using rodent and ruminant
animal models have shown the importance of oocyte-secreted factors for cill proliferation and
differentiation, steroidogenesis, metabolism, and, in certain species, in determining ovulation rate.
However, little information is available in the pig species. Therefore, the objective of the cunent study
was to determine the effects of native oocyte-secreted factors on cumulus cell protein expression in the
pig follicle.
Three groups of four sows were euthanized on day 19 + 1 after the l't post-weaning oestrus at a time
point when the pre-ovulatory follicle population was established and the oocytes stroul¿ have acquired
full developmental competence. Follicle size and follicular fluid oestradiol concentration were used to
confirm that oocytes were derived from highly oestrogenic follicles that had not been exposed to the
preovulatory-LH surge. Cumulus-oocyte complexes (n = 19 + 2 per sow) were aspirated from these large
pre-ovulatory follicles, washed three times in PVA TL-HEPES, and denuded inO; trom their cumulus
cells' Concomitantly, gilt ovaries were obtained from a local slaughterhouse and oocytectomized
cumulus complexes (OOX) were prepared by microsurgically removing the oocytes from their
surrounding cumulus cells. Groups of 16 OOX were then cultured for 22h with or without the DOs
obtained from individual sows (n = 4 groups of OOX per treatment per replicate) in 36 pl droplets of
modified M199 medium under mineral oil at 38.5'C in a humidified atmospñere of 5Vo COz. For each of
the three replicate cultures, 3 groups of 16 OOX incubated with or without DOs were pooled and
subjected to 2-dimensional gel electrophoresis on 7cm lPGstrips pH 3-10 in the first dimension and 10Vo
SDS-PAGE slab gels in the second dimension. The SYPRO Ruby. (BioRad, CA) stained gels were
imaged on a Typhoon Trio (GE Healthcare). Due to incomplete isoelectric focussing for one replicate,
only the gel images from the remaining two replicates were finally analysed using Pro-genesis SameSpots
software (Nonlinear Dynamics, UK). Individual spot intensities were normalized with the total spot
volume from the gel of origin. A preparative gel containing the protein from the cumulus cells of 500
cumulus-oocyte complexes was also run under the same conditions but using an 18 cm IpGstrip pH 3-10.
The preparative gel was matched with the analytical gels and protein spots of interest were manually
excised and sent to a mass spectrometry facility for identification by LC MS/MS (Centre Genomique du
Quebec, Sainte-Foy, Canada). To confirm proper matching between the analytical and preparative gel, 6
abundant protein spots ofinterest from the analytical gel were also sent for identification.
than
]More
600 spots were matched across the 4 gels of which 14 proteins were found to be
differentially expressed when the OOX were incubated alone or with DOs. Interestingly, 9 of the 14
differentially expressed proteins were up-regulated (1.2- to 2.7-fold change) in the absence of oocytes.
Conversely, 5 of the 14 differentially expressed proteins were down-regulated in the OOX incubated
without DOs; however, the magnitude of the down-regulation was lower, with only a l.l- to I.2-fold
change, representing aI}-2OVo decrease in protein abundance.
These results suggest that removing the oocyte from the cumulus-oocyte complex leads to specific,
and predominantly suppressive, effects on cumulus cell protein expression. Amongst the l3 proteins that
were identified, 5 up-regulated proteins and I protein that was down-regulated in ObX incubated without
DOs appear to be of particular interest (Table 1). First, 14-3-3r1is a member of the 14-3-3 protein family
that modulates the interactions between proteins. The 14-3-3 proteins aie involved in signaì ffansduction
93

and interact with various receptors including the glucocorticoid receptor, androgen receptor, insulin and
IGF-I receptor, as well as FSHR. In addition, they are also involved in the regulation of cell cycle
progression, apoptosis, and metabolism. NPM1 is a nuclear chaperone capable of acting as a
transcription coactivator and modulates cell proliferation and apoptosis. P4HB is a key enzyme in
collagen biosynthesis that appears to be essential in the peri-ovulatory period after the LH surge.
Collagen synthesis is considered to be essential for repairing the ruptured follicle after ovulation but may
also be necessary for the follicle-luteal transition that occurs at ovulation. TCTP is involved in
modulating apoptosis but also alters cell morphology and positively regulates cell proliferation. The Rho
GDP-dissociation inhibitor I prevents the activity of Rho GTPase which is involved in the organization
of the actin cytoskeleton and in cell proliferation. Finally, PGAM1 is a glycolytic enzyme that catalyzes
the interconversion of 2- and 3-phosphoglycerate. Changes in the activity of this enzyme modify the
quantity of glucose 6-phosphate available for the pentose-phosphate pathway which ultimately modulates
the amount of NADPH available for downstream biosynthetic pathways such as steroid biosynthesis.
Altogether, these observations suggest that the porcine oocyte potentially modulates cumulus cells with
regards to metabolism, apoptosis, proliferation, and steroidogenesis. These findings are, therefore,
consistent with the concept that oocytes secrete soluble factors that act on the cumulus cells to modulate
their functions and phenotype.
Table 1: Identity of 6 proteins whose expression was differentially regulated when oocytectomized
cumulus complexes (OOX) were cultured with or without the denuded oocytes (DOs) harvested
from large, oestrogenic porcine follicles that had not been exposed to the pre-ovulatory LH surge iz
vivo.
Jl in
oox
slot w/o DOs
' (fotd
change)
2 1Q.7)
3 1Q.t)
4 1 (1.8)
s 1(1.7)
20 10.2)
Protein name
(symbol)
14-3-3 protein eta
(YWHAH)
Nucleophosmin
(NPM1)
Prolyl 4-hydroxylase,
beta subunit (P4HB)
Translationallycontrolled
tumor
protein (TCTP)
Rho GDP-dissociation
inhibitor 1
(ARHGDTA)
Accession
#
Theoretical # of
MW
(kDa)/pl peptides (Vo)
uPr0000880531 28 t 4.8 21
P06148
P05307
P73693
P19803
33 / 4.6
57 / 4.1
20 t4.8
23 /5.0
:3 r (1 ls) äï:ii','|å"iXi,, P18669 29 t 6.7 20
Protein
unique coverage
F. Paradis is supported by a Natural Science and Engineering Research Council of Canada (NSERC) and
an Alberta Ingenuity Doctoral Scholarship. Thís research was supported by the Natural Science and
Engineering Research Council of Canada (NSERC).
l0
l5
60
39
32
3l
43
78
-ì
(
I
(
I(III
(
I (
I I
(
II
(
I
(
a
I
i I
a
(I
a
(
a
(
I II
a
(
q

2s3-l
POST-WEANING ALTRENOGEST TREATMENT IN PRIMIPAROUS SowS: THE
EFFECT OF DURATION AND DOSAGE ON FOLLICULAR DEVELOPMENT AND
CONSEQUENCES FOR EARLY PREGNANCY
J.J.J. van Leeuwenl3, S.Williams2, B. Kempl, and N.M. Soedel
rAdaptation Physiology Group, Department of Animal sciences, wageningen university, wageningen,
l1:It,.t|jii{,r: 2Facultad de cienåias veterinarias,
-racurtacr
universidad Nacional de La plara, Argenrina;
cfe clencias veterinarias, universidad Nacional de La pampa, Argentina
E-mail : Jessika.vanleeuwen @ wur.nl
Many first litter sows suffer from a suboptimal reproductive performance in the second cycle, shown by
l iryreased weaning-to-oestrus interval, a low farowing rate, ånd
"
rt"¿rliii"r;i;;. ir,ut"' & Bilkei
2005)' This so-called' 'second litter syndrome' may bJ related to the suppresrive èffãcts of a negative
energy balance on lactational follicle development, since several authors have shown that pre-follicular
phase feeding levels (either during lactation oi during the luteal phase of the oestrus cycle) affect the antral
follicle pool and subsequent follicle development (Quesnel "tàt.2000¡,
oocyte oevltopment (zak et at.
1997), ovulation rate (Hazeleger et al.200s), embryo development (Algriany et al.200+¡,and embryo
mortality levels (Almeida et al. 2000). Postponing oestrus by administration of a progesterone analogue
(altrenogest) from weaning onwards positively affeãts subsequent reproductive performance (e.g. Martinat-
Botte et al' 1994, Patterson et al. 2a08). ir"""r* of altrånogest rreatment is probably related to the
improvement of follicle development during treatment. Therefore, the objective of this study was to
investigate follicle development at weaning una drring and after difierent altrenogest treatments, and relate
this to subsequent ovulation rate and early ãmbryonic ãevelopment..
. *For this study, 48 primiparous sows were weaned from tieir (9.4¡r.l)piglets at Day 2r+3 oflactation
(=Day 0) and were randomly assigned to the following treatmentì: control (no altrenogest, n=l1), RUg-15
(15 mg of altrenogest, n=l1, Day -1 till Day 7), RU8-t0 (20 mg of alrrenoges L, n=r2,Day -l till Day 7) or
RU15-15 (15 mg of altrenogest, n=12, Day -l till Day i4)IFrom weaning onwards, trans-abdominal
ultrasound was performed daily and the five' largest follitles ãn on. ovary were measu¡ed until ovulation
occumed' oestrus detection was performed twiðe daily and sows were inseminated 12 hours after first
detected oestrus on 12 hours intervals during standing o"rt*r. sows were slaughtered 5 days after ovulation
to determine ovulation rate and to reco verimbryoslAt slaughter a blood sample was coílected fiom each
sow to determine progesterone concentrations. Èmbryo mori'hology and quality was assessed after which
embryos were treated to spread and count cell nuclei and number oiä.."rrory sperm cells, and to determine
the number of cell cycles (2log of the nuclei count) and homogeneity of the litter (expressed as the range in
number of cell cycles for embryos with more than 90vo of the iitter uu"rug" in cell cycles).
Follicle size at weaning was 2.6+l'1 mm. Follicle size increased during treatment to reach a plateau of
4'6tr'6 mm around Day 6, irrespective of treatment. This increase resurted in larger follicles at the onset of
thefollicularphasefortreatedanimals(4.8+1.8,4.8+7.4,and4.g+o.gmmforRUg-l5,RUg,20,andRU15-
15) compared with controls (2.910.8; P=0.0002). Follicles of treated animals remained signifìcantly larger
tlltlit lne
fourth day of the follicular phase. Pre-ovulatory follicle size tended to be larger for treated animals
(7'9t2'4mm' 7'9ú.7mm, and 8.6i1.3mm for RU8-is, nus-zo, and RU15-15, respecrively) rhan for
controls (6.9+0.9mm; p=0.07).
The interval to onset of oestrus (from weaning for controls and from 24h after last treatment for treated
animals) was shorter for t¡eated animals (4.6x.1.4, 4.J+0.g, and 5.2+1.6 days for RUg-15, RUg-20, and
RU15-15) compared with controls (6.5+1.4 days; P=0.0001). Duration of oestrus was not afîected by
treatment, neither was ovulation rate (18.9t4 .O vs.2l.2+5.4 vs. 18.814.0 vs.l9.5+3.1 for RUg-15, RUg-20,
RU15-15 and controls, respectively (P>0.1)), .luteal
weight, or progesterone levels at slaughter. Average
fertilization rate of the recovered embryos and oocytes l,,tasgo.6E"lwhich was not affecteà'by treatment.
The majority of the recovered embryoi consisted of compact morulas (26vo) and blastocysts (53%). No
treatment effect was found on embryo quality (visual appråisal), number of accessory sperå cers, number
of cell cycles, or homogeneity of the litter. Futher, none^of these parameters were related with follicle size
95

at weaning, follicle size at the start of the follicular phase, or pre-ovulatory follicle size. For treated animals,
the increase in follicle size during treatment was significantly related with ovulation rate (p=0.05); every
mm increase in size during treatments resulted in an increase in ovulation rate of 1 .1.
The fact that altrenogest treatment after weaning did not affect ovulation rate and embryonic
development may be related with the low lactational burden of these sows (on average 9.4 piglets ãnd a
relative weight loss of 47o).However, even under these circumstances treated animals had larger follicles
than controls at the onset and first four days of the follicular phase, tended to have larger pie-ovulatory
follicles, and the follicle growth during treatments was positivèly related with subsequeit ovulation rate.
Further studies need to evaluate subsequent reproductive performance of different altrenogest treatments.
Algriany O, Bevers M, Schoevers E, Colenbrander B & Dieleman S 2004 Follicle size-dependent
effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blãstocyst
formation of sow cumulus oocytes complexes. Theriogenology 62(s)t4g3-14g7.
Almeida FRCL, Kirkwood RN, Aherne FX & Foxcroft GR 2000 Consequences of different patterns
of feed intake during oestrous cycle in gilts on subsequent fertility. Joimat of Animat Science 78
I 556-1563.
Hazeleger W, Soede NM & Kemp B 2005 The effect of feeding strategy during the pre-follicular phase
on subsequent follicular development in the pig. Domestic Animal Endocrinology)l ZeZ-ZlO.
Martinat-Botte F, Bariteau F, Forgerit Y, Macar C, Poirier P & Terqui fvl ßg+ Control of
reproduction with a progestagen-Altrenogest (Regumate) in gilts and at weaning in primiparous sows:
effect on fertility and litter size. Reproduction in Domestic Animals 29(5) 362-365.'
Patterson J, Wellen A, Hahn M, Pasternak A, Lowe J, DeHaas S, Kraus D, Williams N & Foxcroft
GR 2008 Responses to delayed estrus after weaning in sows using oral progestagen treatment.
Journal of Animal Science 861996-2004.
Quesnel H, Pasquier A, Mounier A & Prunier A 2000 Feed restriction in cyclic gilts: gonadorropinindependent
effects on follicular growth. Reproduction Nutrition Development 40(4) +Oi-+t+.
Thaker M & Bilkei G 2005 Lactation weight loss influences subsequent reproductive performance of
sows. Animal Reproduction Science 88 309-318.
ZakLJ, Xu X, Hardin RT & Foxcroft GR 1997 Impact of different pattems of feed intake during
lactation in the primiparous sow on follicular development and oocyte maturation. Journal of
Reproduction and Fertility ll0 99-106
96

'r4 mm are LH responsive (Dufour & Mariana 1993;Lucy et at.2}}r).Counts and size
measures for ovulatory follicles may differ by whether physical diameter or ultrasound assessmenr was
used and the follicle classification limits (Soede et al.199d; rnox et a\.2002;Bracken et at.2006).we
hypothesized that the numbers of follicles classified as ovulatory at estrus may not reflect expected
ovulation rate or litter size' We petformed two experiments to characteri ze tîe changes in follicle
populations from 'onset of estrus (Experiment 1) and frãm dme of weaning to ovulation (Experim ent 2).
In experiment 1, our objectives were to measure proportions of wean-ed sows having large, medium,
and small follicles on two days before ovulation. A lotal of 21 sows that had exprãssed estrus and
ov-ulated between day 2 and 3 and which had real-time ultrasound digital video recoidings for both the
left and right ovaries on the first (period 1) and second day (period 2) of estrus were included in this
study' The images of the ovaries were obtained transrectally urìng un Aloka 500v ultrasound with a 7.5
MHz linear transducer' The images were digitally recoided u"nd folli"l., individually counted and
measured using a digital display system that was calibrated to the measures of the ultrasound. The
follicles were classified as small (S,

day
1d of weaning (d 0). Sow had no impact on small or Ml follicles but did impact M2 and larger
follicles. Day post weaning had a significant effect on most follicle parameters assessed. The numbers
of small follicles differed by day (P847o) on all days but numbers differed by day and ranged from 3-9
follicles' The number of M2 follicles/sow (n=6) did not differ by day but peróentáges of sows with M2
follicles wete 50Vo on day 1 and >85Vo on days 2-6. The number of L I follicles differed by sow and day.
Sows had < 3 Ll follicles on d 1-3, and on d 5 and 6, increased their numbers to 5-6 Li foilicles. tfre
percentage of sows with L1 follicles was low on d 1, and reaching 46Vo on d2, ancJ then increasing to
>857o of sows on d 3-6. The L2 class was not present on d 1-2 but was present in -50Vo of sows on dãys
3-4, and, in >807o of sows on d 5-6. The L3 class was represented in ònly 10Vo of sows and estimates
were < 1 follicle on average. The total numbers of follicles >6.49 mm on day 1 of estrus was 8.3 with the
total number of large follicles reduced on the second day of estrus due to ovulation in process in half of
the sows. Since total numbers of large follicles was lower than CL counts (11.3) in sóws that could be
scanned for CL counts, and was also lower than total born pigs, we analyzed all follicles >5.0 mm. The
result was 14.6 follicles at estrus and suggests that ultrasound -easutes should include 5.0 mm follicles
at onset of estrus for assessment of ovulation rate. In summary, it would appear that for experiments 1
and 2, heterogeneity occurs in follicle sizes that ovulate, and that follicles 5.ô mm or greater at onset of
estrus in weaned sows will ovulate and contribute oocytes that will eventually affect litær size.
Bracken CJ, Radcliff RP, McCormack BL, Keisler DH & Lucy MC 2006 Decreased follicular size
during late lactation caused by treatment with charcoal-treated follicular fluid delays onset of estrus
and ovulation after weaning in sows. Journal of Animal science g42110-211l..
Dufour JJ, & Mariana JC 1993 Comparative follicular development in Meishan and Large white gilts
during prepubertal periods and its relation to hormonal stimulation. Biolog of Reproduction 4g 1020-
1025.
Ilunter MG, Grant SA & Foxcroft GR 1989 Histological evidence for heterogeneity in the
development of preovulatory pig follicl es. Journal of Reproduction and Fertility 86 táS-nO.
Knox RV, Miller GM, Willenburg K.& Rodriguez-Zas SL zooz Effect of frequËncy of boar exposure
and adjusted mating times on measures of reproductive performance in weaned sows. Journal of
Animal Science 80 892-899.
Lucy MC, Liu J, Boyd CK & Bracken CJ 2001 Ovarian follicular growrh in sows. Reproduction
Supplement 58 31-45.
Soede N, Hazeleger W & Kemp B 1998 Follicle size and the process of ovulation in sows as studied
with ultrasound. Reproduction in Domestic Animals 33 Z3g-244.
98

)

Table 1: Reproductive Characteristics of the experimental sows at day 30 of gestation.
Item Control sows (n=6) L-arginine sows (n=6) P-value
Parity
4.0 t 0.35
3.8 t 0.37
0.698
Ovulation Rate
25.8 x.1.12
23.2 + 1.80
0.305
Live Embryos
15.9 x.l.l4
13.8 t 1.19 0.2t6
Embryo Survival (7o)
64.1 + 4.6
60.6 t 4.8
0.603
Embryo Weight at Day 30 1.98 r 0.1 1
2.26 t0.12
0.105
Placental Weight at Day 30 30.60 x.3.66
34.94 x.3.43
0.402
Temporal gene expression profiling in the Control samples revealed that angiogenin (ANGI) and
VEGFA mRNA expression did not change (Þ0.05) from d 17 through 49 of gestation. However,mRNA
expression increased (P

2s2-4
ESTROGEN AND IITTILEUKIN.IF REGULATION OF TROPHININ,
osrEoPoNTIN, CYCLOOXYGENASE- 1, cycLooxyGENASE-2, AND
INTERLEUKIN.Ib SYSTEM IN THE PORCINE UTERUS
F. J. whitet, E. M. Kimbaill, G. wymanl, D. R. stein2, J. w. Ross3, M. D. Ashworth2, and R. D.
Geiserta
tcameron universitv, Lawton, oK; 'okrahoma s_tate university, Stillwater, oK; 3lowa State university,
Ames,IA;aUniversity of Missouri, Columbia, MO, USA
E-mail: fwhite@cameron.edu
Embryonic loss during early gestation limits litter size in swine production. Failure of the conceptus
to attach properly to the uterine surface may contribute to the high rate of embryonic loss observed in
swine' Attachment to the uterine surfaóe is a highly syncfrronized event that requires precise
communication between the expanding conceptus and endomãtrial tissue. conceptus aiiarh-ent to the
uterine surface includes upregulation o1 adhesion molecules at the matem alJreÂlinterface for attachment
as well as a pregnancy-specific inflammatory response. Trophinin and osteopontin u* cell adhesion
molecules that may function in initial attachment 6etween conieptus trophectoderm and uterine luminal
epithelium of the pig and human- Leukocytes infiltrate the endãmerium during implantation, and the
pro-inflammatory cytokines cyclooxyg"nur" (cox)-l and coX-2 u.. "*pr"rJ"d
in human and pig
endometrium during pTgn1lty, wrreróirrey are proposed to regulate conceptus implantation and uterine
angiogenesis' Interleukin-l0 (IL-IB) incrðases àurìng implaniation in the mouse, hr-un, and pig and
may regulate uterine inflammatory cytokines. Estrogen also controls uterine events necessary for
attachment and implantation of the mouse and pig co"nceptus and may act in synergy with IL-1B to
prepare the uterus for the implanting embryo. FurtiJrmore, irophinin expression wãs inãúce¿ by IL-18 in
human endometrial cells, and uterine osteopontin expression is regulated by estrogen in the pig and
mouse. The objective of the current study was to evaluate the hypotheses that estrogen regulates the
uterine-inflammatory response induced uy i-rB during rtr. "r,"ilir'r,ment
of pregnancy.
cyclic gilts were treated with corn oil or estradiollypionate (5 mg) on Day 1l of the estrous cycle.
on Day 12, gilts were subjected to mid-ventral laparotoÁy una utèrlne honrs were randomly infused with
either saline or porcine IL-18 (ls ¡rg). uterinê horns íere ."-;J;;'l;;jiäöåst-infusion (4
giltvtrlsampling periods) and endometrial mRNA was quanrifi;ã ùf quantttarive RT-pcR.
Estrogen did not influence (P> 0.1) concenrrationi or en¿ometrial coX-l and coX-2 nRNA;
however, IL-lp increased (P = 0-01) endomerrial cox-2 mRNA by 3.5 fold and rended (p = 0.06) to
increase cox-l mRNA \v.1fold
4h post-infusion. cyclooxygenase-t and coX-2 regulare urerine
prostaglandin secretion, which is essentiai to normal implántation ãnd pregnancy in pigs (Kraeling et al-
1985)' cyclooxygenase-2 null mice are infertile un¿ fuil to irnprunt; however, implantation is not
impeded in the cox-l null mouse (Lim et at. 1997). attnougtritre conceptus induces uterine cox-2
expression at implantaúon sites, estrogen did not increase coi-zmRNA in ovariectomized mice which
is true in our pig study (Chakraborty et al. 1996). Furthermore, IL-18 regulates ovulation in mice
through cox-2 and prostaglandin production (Davis et al. 1999). we hypothãs ize thatconceprus IL-IB
regulates uterine prostaglandin secretion by increasing endometri al cox-2. prostaglandins regulate
angiogenesis and the conceptus may increase uterine vascularity through IL-lB induce d cox-z.
The elongating porcìne conceptus secretes IL-1p on Day 12 of pregnancy during conceptus
attachment and maternal recognition of pregnancy, and this córrelates with endometrial Interleukin-l
receptor type 1 and interleukin receptor accessory protein gene expression suggesting conceptus
interleukin upregulates its own endomãtrial receprors (Ross er
-at. 2a0i). In the cürent study, IL-lp
increased (P = 0'05) its own expression by 3.5 fold and tended (P = .08) to increase Interleukin-l receptor
type I by 2'5 fold; however, estrogen not IL-IB increased þ < o.os¡ interleukin receptor accessory
protein (2'5 fold increase)' similarly' IL-lB increased Interleukin-l receptor type I in human endometrial
101

stromal and glandular cells (Soloff et al. 2004). Interleukin-lp and estrogen differentially regulated
uterine interleukin receptors; therefore their combined effects may alter the endometrial response to IL-
1B during implantation.
Endometrial osteopontin and trophinin were increased (P < 0.05) at 36 h but not 4h after estrogen
treatment. Estrogen treated gilts had 11.3 fold greater OPN and 3.6 fold greater trophinin endometrial
nRNA than gilts treated with corn oil. Trophinin mRNA and protein are located at the maternallfetal
interface during implantation of pigs, mice, and humans (Suzuki et at. 2000, Nakano et at. 2003,
Sugihara et a\.2008). Uterine trophinin is increased by estrogen in mice but IL-1p in human endometrial
cells (Sugihara et a|.2008), suggesting expression is pigs and mice are controlled by similar mechanisms
which differs from humans. The fact that OPN and trophinin receptors are located on conceptus
trophectoderm suggests a role in attachment and communication at the maternal/fetal interface. We
hypothesize that IL-18 and estrogen secretion by the pig conceptus differentially modulates uterine
expression of COX-I, COX-2, trophinin, osteopontin, and the interleukin-lp system providing an
inflammatory environment that is essential to establish pregnancy.
Chakraborty I, Das SK, Wang J, & Dey SK 1996 Developmental expression of the cyclo-oxygenase-l
and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation
by the blastocyst and ovarian steroids. Journal of Molecular Endocrinology 16 107 -122.
Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC & Langenbach R 1999
Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-l8.
Endocrinolo gy 140 2685 -269 5.
Kraeling RR, Rampacek GB & Fiorello NA 1985 Inhibition of pregnancy with indomethacin in mature
gilts and prepubertal gilts induced to ovulate. Biology of Reproduction 32 105-1 10.
Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM & Dey SK 1997 Multiple
female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91 I91-208.
Nakano S' Kishi H, Ogawa H, Yasue H, Okano A & Okuda K 2003 Trophinin is expressed in the
porcine endometrium during the estrous cycle. Journal of Reproducrion and Development 49 I27-
134.
Ross JW, Malayer JR, Ritchey JW & Geisert RD 2003 Charactenzation of the interleukin-lbeta
system during porcine trophoblastic elongation and early placental attachment. Biology of
. Reproduction 69 1257-7259.
Soloff MS, Cook DL, Jr, Jeng Y-J & Anderson GD 2004 In situ analysis of interleukin-l-induced
transcription of cox-2 and il-B in cultured human myometrial cells. Endocrinology 145 1248-1254.
Sugihara K, Kabir-Salmani M, Byrne J, Wolf DP, Lessey B, Iwashita M, Aoki D, Nakayama J &
Fukuda MN 2008 Induction of trophinin in human endometrial surface epithelia by CGbeta and ILlbeta.
FEBS Letters 582197-202.
r02

)30 kDa) versus l0zo pFF (>30
kDa) obtained from sows in the preoìulatory stage of the estrous cycle.
shotgun proteomics analysis of the pFF and serum fractions tâotou from 3 sows was perf'ormed by
application of the 'isobaric Tag for Rélative and Absolure euantitation'(iTRAe) rechnology (Applied
Biosystems) followed by ,2D-LC
ESI-Q-TOF MSMS. of each sample, 100¡rg prorein marerial was
loaded and runs were performed in dupúcate. The processed data, obiained froà mascot deamon, was
searched against the pig EST databaie for protein identification (http;/þigest.ku.dk). protein ratios
resulting from duplicate runs were averaged, log-transformed, and analyzed by Student,s t-test. In
a{{tion, 600 prepubertal gilt oocytes were inature d in vitro for 26hin NCsÛ23 supplemente d, with l¡vo
pFF (>30kDa) or l07o serum (>30kDa) of each of the 3 sows. After IVM, thï expanded cumulus
matrices were collected and subjected to proteomic analysis. Proteins in the matrix extracts were
separated using 2D-PAGE' Two spots that were absent in -matrices matured in pFF were excised and
submitted to mass spectrometric analysis using ESI-Q-TOF MSMS. The processed data, obtained from
mascot deamon' was searched against the pig nSr ¿atà¡ase for protein identification.
First of all, serum and.pFF were not ãeptetea for high abundant proteins like albumin, because the
depleted sample did not show the same biological effeci on the IVM of porcine oocytes. Therefore an
exclusion list was used based on the first run to exclude abundant peptides derived from albumin.
Proteomic analysis of serum and pFF revealed 63 unique proteins present in both fluids of which 13
showed significantly (P30 kDa).
In-conclusion,2 proteins that are upregulated in autologous serum were also solely retrieved in the
cumulus matrices of oocytes matured in l}vo serum (>30 kDa). one of them, azna, miint be involved in
the observed differences-in cumulus expansion. Alpha2- macioglobulin, as a major proteinase inhibitor,
might degrade ADAMTS-1, which has been shown to be essen"tial for gonadotåpin-r.guiated cumulus
expansion of porcine cumulus-oocyte-complexes (Shimada et al. 20041. Howevå, furtlher experiments
should be performed to elucidate the mechânisms responsible for the possible inhibiting effects of A2M
on cumulus expansion.
103

TABLE 1: Differentially expressed proteins (p

252-6
IMPACT OF SELECTION FOR UTERINE CAPACITY ON THE PLACENTAL
TRANSCRIPTOME
B. A. Frekingr, J.R.Milesl, s.^R. Bischoff, s. Tsai2, N. Hardison2, y. xia3, D. J. Nonneman,, J.
L. Valletl, and J. A. piedrahita2
tUSoA, ARS, U.S. Meat Animal Research Center, Clay Center, NE USA;
2Department of Molecular
Biomerclical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC
USA; 3Center for Biotechnoìogy, Universiiy of Nebraska, Lincoln, NE USA
E-mail: Brad.Freking @ars.usda. gov
Direct single trait selection for 11 generations resulted in a 1.6 pig advantage for uterine capacity
(UC) while average birth and placental weights at term remained ùninangea. Uterine capacity was
defined as the total number of fully-formed pigs produced to term when ovulation rate *us nãt limiting,
using a unilateral hysterectomy-ovariectomy model. A serial slaughter experiment conducted throughout
gestation determined the critical time period for the line difference in litter size was already estabúshed
between d 25 and 45 of gestation and generated direct evidence of differential relative growth rates for
placental tissues at these times. Timing of line differences in fetal survival as well u, un"-.dotul evidence
of tissue structural differences pointed to the developing placental tissue as a target ofparticular interest.
Our objective was to gain insight into placental transcriptional changes duriig this critical stage of
gestation and identify genetic loci impacted by quantitative ielection for uterine calacity.
Thirty gilts each from the UC and control (Co) lines were subjecred ro u;ilateial hysterectomyovariectomy
at approximately 160 d of age and mated within line ar approximately 280 d. Gilts were
slaughtered at d 25,30, or 40 of gestation. Fetal and placental tissues were obtãined from each live
embryo' Fetal liver samples were used to extract DNA and determine sex of each fetus by pCR. Two
male and two female embryos closest to the litter mean for placental weight were chosen to represent
each litter sampled (n = 3 litters per line and time point combination). Placental tissues were pooled
within litter and total RNA was extracted. Samples were labeled and hybridized to Affymetrix porcine
array chips (n = 18) using the manufacturers suggested protocols. Signal intensities were normalized
using GC content Robust Multi-array Average (GCRMA) õn tne probe lãvel data. Filtering was based on
perfect match intensities as implemented for Affymetrix Human arrays. Two-way ANOVA (two lines
and three stages) was performed. Threshold values were set at a minimum of l.S fôl¿ difference and the
false discovery rate was set to P < 0.05 (Benjamini and Hochberg algorithm). Less stringent two-way
comparisons (t-tests) were also conducted between lines within eaõh gestation stage. GeneSifter@
software web tools were utilized to conduct the analyses and generate the glne lists.
An additional analysis was conducted to examine the potential for a bioinformatics method of
identifying single feature polymorphism (SNP) targets beiween the two lines associated with rhe
behavior of the expression data. A gene by gene linear mixed model of probe intensity variation from I 1
probes per gene target was investigated to identify line x probe interactions. Log2-transformed perfectmatch
intensities for all observations were fit to a linear mixed model that broadl/corrected for effects of
breed and array. A gene-specific mixed model was fit to the normalized intensiiies (residuals from fjrst
model) accounting for fixed breed, probe, and breed-by-probe interaction effects, and a random arïay
effect. .Probes
rhat produced sratistical evidence (qvalue i O.OS to account for multiple tesring and lfolá
changel > 2) for the breed-by-probe interaction were identified as candidates coitaining SNp. The
interaction was declared significant when Prob t < 0.0064 and would indicate a putative äoing region
polymorphism associated with line within that probe location. Validarion õf *ris approach was
conducted in a previous experiment comparing Meishan and occidental breed placental tissues with an
877o success rate using the same threshold values of q < 0.05 for false discovåry rate and with greater
than 2-fold difference in estimated corrected probe-by-6¡sed interaction effects. The great majoiity of
these single feature polymorphisms are discovered in the middle portions of the 25-mer froue rargets.
A total of 4l7l targets on the array exceeded P value and rhrêshold limits for the måin effecåf stage
(2230 tp-tegulated and 1839 down-regulated from d 25 to d 40). Two targers, LIM domain and acrin-
105

inding protein 1 (LIMAI) and a hypothetical protein LOC5L524 (transmembrane protein 138)
approached highly stringent thresholds for significance of the main effect of line (P < 0.08) in the twoway
ANOVA with both targets up-regulated in the UC line and increasing during these stages of
gestation. Expression data for both loci were validated as differentially expressed by QPCR (P < 0.05)
and also increased with gestation age. The function of LIMAI is to bind to actin monomers and
filaments, increase the number and size of actin stress fibers, and inhibit membrane ruffling. It inhibits
actin filament depolymerization, promotes bundling of actin filaments, delays filament nucleation, and
reduces formation of branched filaments. Both genes could be predicted to play roles in cell migration,
suggesting participation in placental folded bilayer formation.
Less stringent analyses of two-way comparisons within each time point indicated increased expression
occurred more frequently than decreased expression in the UC line. The number of up-regulated targets
relative to the CO line identified totaled 219, 407, and 234 for gestation srages d 25,30, and 40,
respectively. In contrast, the number of down-regulated targets relative to the CO line identified totaled
36, 71, and 4'l for gestation stages d 25, 30, and 40, respectively. This overall expression profile is
consistent with observations reached previously, that these lines differed in relative growth patterns of
fetal and placental tissues. Genes of interest identified in these lists revealed the importance of pathways
such as integrin signaling pathway (calpains, collagen, fibronectin) and lipid metabolism (adiponectin
receptors).
Analysis of probe x line data yielded a large number (5617) of putative transcribed SNP between the
lines. Merging these data with the expression information indicated an overlap of 762 targets where day
effects were also significantly different in the two-way ANOVA, and an overlap of 179 targets from the
pair-wise contrasts within each stage. Therefore this overlapping list of targets offers evidence of both
expression differences in the placenta as well as genetic variation between the lines that could be
contributing to genetic variation for uterine capacity. Both lines originated from the same four-breed
composite genetic base prior to the 11 generations of selection applied. Due to the small sampling of
data generated from the microarrays, it is also likely a large number of these putative line-specific
polymorphisms are associated with effects of genetic drift. However, these targets offer an intriguing
start to directly untangling the impact of genetic selection for uterine capacity. It is clear that a more
thorough investigation by cluster and pathway or network analysis will also require better annotation of
targets on the array. The experimental and analytical approaches in this study identified both expression
and polymorphism differences to pursue for identification of genetic markers contributing to genetic
variation in uterine capacity from a unique germplasm resource.
106

'r\)-1
ENRICHMENT OF PORCINE SPBRMATOGONIA BY DIFFERENTIAL CULTURB
E. Behboodi, S. Mohan, J.R. Rodriguez-Sosa, Y. Li, S. Megee, and L Dobrinski*
Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of
Pennsylvania, Kennett Square, PA 19348, USA; * current address: Department of Comparative Biology
& Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta T2N
4Nl, Canada
E-mail: idobrins @ucalgary.ca
Undifferentiated spermatogonia are a potential source of pluripotent cells and could be used for
targeted genetic alteration in pigs. Our understanding of mechanisms maintaining porcine spermatogonial
stem cells (SSCs) in vivo and of conditions to propagate SSCs in vitro remains limited. This is largely
due to the small number of SSCs present in the testis and the lack of specific morphological and cellsurface
markers to isolate a purified population. The goal of this study was to establish a modified
differential culture system to effectively enrich SSCs from prepubertal porcine testes for subsequent
culture.
Germ cell enrichment was quantified by immunocytochemistry and RT-PCR analysis of proteins and
genes known to be specifically expressed in spermatogonia (PGP 9.5, VASA, and DBA). Testes were
collected from 10 week-old pigs and washed with PBS and transported on ice within 24 h to the
laboratory in PBS that was supplemented with antibiotics. Testes were pooled for the isolation of germ
cells (2-4 testes per trial). Cells were isolated by a two step enzymatic digestion (Honaramooz et al,
2002). The cells were incubated in DMEM containing 0.1 mM l-mercaptoethanol, 0.1mM MEM nonessential
amino-acids, 200mM L- Glutamine and 5Vo FCS supplemented with 100 IU penicillin
strepto_mycin, in tissue culture dishes coated with 0.0IVo gelatin 12 h prior to use, at a concentration of
50x106 cells per dish (60x 15 mm) for t h at 3l oC in 5Vo COz in air. By counting cells in the supernarant
it was determined that 5OVo of the total cells attached to the culture plates after t h, most of which were
somatic cells. Germ cells largely remained in suspension and were transferred to new culture dishes.
After an additional 14 h of incubation, unattached cells were collected, concentrated by centrifugation for
5 min and counted before use for long term culture. Enrichment of germ cells at each time point (0, 14 h)
was determined by immunocytochemistry for alkaline phosphatase activity, and expression of DBA, PGP
9.5, and VASA. Counterstaining for vimentin was employed to identify somatic cells. mRNA was
isolated for RT-PCR analysis to confirm expression of PGP 9.5 and VASA. Isolated cells were seeded at
a density of 5 x 105 cells in 6-well plates in DMEM medium as above, supplemented with glial cellderived
neurotrophic factor (GDNF, 20 ng/ml), epidermal growth factor (EGF; 200 ng/ml), and basic
fibroblast growth factor (bFGF; 200 ng/ml). Comparison between groups was by Srudent's t-test.
The two-step differential culture increased the concentration of germ cells from5.4+3Vo in the initial
cell suspension to 46.6+22Vo in the non-adherent population at 14 h of culture (Table 1). Enriched germ
cells formed more (21-t-7 versus I0+3.4; P

Table 1: Germ cell enrichment by differential culture
Testis cells (0h) Germ cells Enriched testis
cells (14h)
Cell no. (x 106) (vo\ Cell no. (x 10ó)
290
300
300
300
200
218144
8.2
5.3
4.1
5.3
4.0
5.4x.1.7
12
13
8
6
6
5.4x.3.3
Germ cells
(Vo)
74.0
4r.3
62.5
35.0
r7.5
46+22
Table 2: Colony formation after 7-10 daysin vitro
Replicates per
experiment (0h)
2
2
2
2
2
Colony No. per
dish
(0h)
6
12
l5
t2
9
lO+3.4
0h: Cells plated without enrichment
14 h: Cells plated after enrichment
Student's T-test P

252-8
CLONING AND EXPRESSION OF PLURIPOTBNT FACTORS AROUND THE TIME
OF GASTRULATION IN THE PORCINE CONCEPTUS
D.R. Eborn, D.L. Davis, and D.M. Grieger
Animal Sciences and Industry, Kansas State University, Manhattan, KS, 66506 USA
E-mail: dgrieger@ksu.edu
The expression of the transcription factors Nanog, Sox-2, and Oct-4 is required for maintaining the
inner cell mass and ensuing epiblast of the developing mouse embryo as well as pluripotency of
embryonic stem cells in culture. Nanog and Oct-4 are down regulated about the time of gastrulation
(Rosner et aI. 1990, Chambers et al. 2003) whereas Sox-2 expression is observed in other tissues
including the developing nervous system (Avilion et al.2003). In embryonic stem cells, these factors
suppress differentiation and promote self-renewal by forming an autoregulatory and feedforward
network. The expression pattern of these markers in farm animal species is not well characterized and
may differ from that of the mouse (Degrelle et a|.2005). Therefore, we have partially cloned the porcine
Oct- , Nanog, and Sox-2 transcripts and characterized their expression in day-10, -12, -15, and -Ll
embryonic and extraembryonic tissues as well as endometrium, myometrium, placenta, and fetal liver at
day 40 of pregnancy (day 0 is the onser of estrus).
Embryos were flushed from sows 10, 12, 15, and 17 days post-insemination. Day-10 and -I2
embryos were processed as whole conceptuses. Day-15 and -I1 embryonic tissue (embryonic disk) was
separated by closely trimming the adjacent extraembryonic tissue (proximal extraembryonic) with a
scalpel using a stereo-microscope (5 to 50X). Additional extraembryonic tissue (distal extraembryonic)
was collected after removal of the embryonic disks. Total RNA was isolated using RNeasy Mini or
RNeasy Micro Kits (Qiagen; Valencia, CA) according to manufacture's instructions. Sequence for each
transcription factor was obtained by full-length RNA ligase-mediated rapid amplification with either the
RLM-Race (Ambion; Austin TX) or GeneRacer (Invitrogen; Carlsbad CA) kits according to
manufacture's instructions. Total RNA was reverse transcribed and real-time PCR was peformed using
TaqMan probe-based assays (Applied Biosystems; Fqster City CA). ,Threshold values were normalized
using 18s ribosomal RNA as the endogenous control. Using the adjusted threshold values, tissue means
were compared by the GLM procedure of SAS (SAS Institute Inc.; Cary NC) and pair-wise comparisons
were made between tissues. For each gene, the tissue with the lowest adjusted threshold value was
designated as the reference tissue. Relative expression differences were calculated by taking the
difference in threshold values with the reference tissue and raising itby 2".
The coding sequence for porcine Nanog (Genbank: DQ44120I) including 452basepairs of the Nanog
promoter, and partial coding sequences of Oct-4 and Sox-2 were obtained. The homeodomain and c-
terminal tryptophan repeats are highly conserved in porcine Nanog compared to the mouse, human, and
bovine. In the promoter, the highly conserved Octamer and Sox binding sequences are also present.
Oct-4 and Sox-2 expression (see Table l) was lowest in day-40 tissues except for fetal liver which was
2O and lI fold, respectively, higher than endometrium. The pattern of Nanog RNA expression differed
from Oct-4 and Sox-2. Day-40 tissues demonstrated the highest expression of Nanog, including
endometrium (7 fold), fetal liver (27 Told), placenta (40 fold), and myometrium (72 fold) when compared
to day-15 distal extraembryonic tissue. Expression of these transcription factors in fetal liver may
indicate the presence of a stem cell population in the developing liver. This could coincide with
hematopoiesis in the developing fetus. Expression in the endometrium, placenta, and myometrium was
unexpected. Expression of Nanog in adult mouse tissues has been reported (Hart et aI. 2004) and the
relatively high expression in these gravid tissues may be associated with rapid growth or other
physiological responses in pregnancy.
Oct-4 expression levels were similar for day-10, -72 and -15 conceptuses and disk but dropped 3 fold
in d.ay-17 disk. On the other hand, Sox-2 was up regulated 1000 fold in the day-15 disk and 2000 fold in
the day-17 disk when compared to the day-12 conceptus. The up regulation of Sox-2 occurs when the
initial neural structures are appearing in the disk.
109

Overall, the expression of Nanog, Oct-4, and Sox-2 in pig pregnancies reveals similarities to that in
the mouse but expression in the early fetal period may indicate functions beyond early embryogenesis.
Further study of the regulatory circuits at these stages is needed.
Tabfe l: Relative Expression of Nanog, Oct-4, and Sox-2 in the Conceptus and Gravid Uterus
Tissue
D10 Conceptus
D12 Conceptus
D15 Embryonic Disk
D15 Proximal Extraembryonic
D15 Distal Extraembryonic
D17 Embryonic Disk
D17 Proximal Extraembryonic
D17 Distal Extraembryonic
D40 Endometrium
D40 Myometrium
D40 Liver
D40 Placenta
4
5
5
7.9'
6.9'
123"d
3.9b"
Oct-4
61.
52.gd
Sox-2
29.
2l.gd"
Lß4.6f
5
3
1.0u
lr.2"d
3.zub"
l7.7b"d
34.gb"d
7.4ub
2022.6f
1.4ub
J
J
J
J
l.2ub
7.0"
72.6"
27.4d"
g.gb
l.3u
1.0u
1.5u
3.gub"
1.0u
1.80"
71.5"
J 39.gd"
26.'lh"d
5.zub"d
uo"o"tExpression values having different superscripts within column are different (P < 0.05)
65.7d
51.3d
g.5b
J.4h"d
Avilion AA, Nicolis SK, Pevny L[,Perez,Vivian N & Lovell-Badge R 2003 Multipotent cell lineages
in early mouse development depend on SOX2 function. Genes & Development l7 126-140.
Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S & Smith A 2003 Functional
expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell ll3 643-
655.
Degrelle SA, Campion E, Cabau C, Piumi F, Reinaud P, Richard C, Renard JP & Hue I 2005
Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts.
D ev elopment al B iol o gy 288 448-460.
Hart AH, Hartley L, Ibrahim M & Robb L 2004Identification, cloning and expression analysis of the
pluripotency promoting Nanog genes in mouse and human. Developmental Dynamics 230 I87-198.
Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW & Staudt LM 1990 A POUdomain
transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345
686-692.
110

253-4
RESPONSES TO EXOGENOUS GONADOTROPHIN TREATMENT IN
CONTEMPORARY WEANED SOWS
J. Pattersont, A. cameront, T. smithl, A. Kummer', R. s"hottt, L. Greiner', J. conn"rt, and G.
Foxcroftl
tswine Reproduction-Development Program, Swine Research & Technology Centre, University of
Alberta, Edmonton, AB, T6G 2P5 Canada; 2PIC North America, 100 Bluegrass Commons Boulevard,
Suite 2200, Hendersonville, TN 37075;3lnnovative Swine Solutions, LLC, PO Box 220,Carthage,IL
62321,U5A
E-mail : jennifer.patterson @ualberta.ca
The principal goal of commercial breeding herds is to consistently meet weekly breeding targets.
Weaned sows failing to return to estrus within 7 d after weaning contribute to missed breeding targets
and increased non-productive sow days. Treatment with low doses of exogenous gonadotrophins (GT)
has traditionally been used to advance and synchronize estrus in weaned primiparous sows. However, in
well-managed contemporary commercial sow farms, more than 907o of sows may return to estrus within
7 d aftet weaning, posing questions about the likely efficacy of exogenous GT treatment. Therefore, the
primary objective of the present study was to determine the response to GT treatment at weaning in
contemporary parity I commercial sows with lactation lengths typical of the North American swine
industry.
Primiparous crossbred sows (PIC C22, n = 275; and PIC C29, n = 131) from a 5,000 sow commercial
farrow-to-wean facility (lMildcat, Carthage Veterinary Service, Carthage, IL) were blocked by estimated
farrowing weight and genetic line and then randomly allocated to either receive a combination dose of
400IU eCG and 200IU hCG (PG600, Intervet, USA, De Soto, KS) I.M. in the neck on rhe morning of
weaning (PG group; n = 189), or to be untreated controls (CON group; n = 218). From rhe day after
weaning, all sows were provided twice daily fence-line contact with mature boars for stimulation and
detection of estrus. Sows were bred according to estâblished herd protocols with doses of
3.0 x 10e sperm cells/insemination. Reproductive paiameters analyzed were éstrus synchronization rate
(ESR), determined as the number of sows with observed standing heat within 7 d after weaning,
weaning-to-estrus interval (WEI), proportion of sows bred over a 3-d period (BRD3), proportion of sows
bred that farrowed (FR), total litter size (TB), and total live born piglets (BA) at farowing. Based on
WEI, sows were retrospectively grouped into 4 categories (WCAT); 1) Target Breed Week: sows bred
within 7 d post-weaning,2) "Tail-enders": sows with an extended 8-19 d WEI ,3) "Missed heats" sows
detected in heat > 20 dpost weaning, or 4) No detected heat by d 30.
Estimated farrowing (193.6 + 1.5 vs 192.2 x.1.6) and weaning (1S9.4 + 1.3 vs 188.0 + 1.4) weights
state units were similar in CON and PG sows, respectively, and marginal weight loss in lactation was
recorded across all sows (4.2 kg). Considering data from all sows assigned to treatment (Table 1),
treatment did not affect the proportion of sows bred within 7 d after weaning (ESR), or within a 3-d
breeding window (BRD3). However, the timing of this 3-d breeding window (d 4, 5 and 6 for CON vs d
3,4 and' 5 for PG sows) reflected a shofier (P

GT treatment may reflect the very synchronous return to estrus seen in the Control sows and good
lactational management as reflected in the marginal overall loss in estimated sow body weight at
weaning.
Table 1. Effects of treatment (T; exogenous GT at \üeaning (PG) or untreated controls (CON)),
genotype (G)' and their interaction on the distribution of observed estrus in all sows on experiment,
and productivity of those sows bred within 7 days after weaning.
Treatment CON PG
P-Value of effect
T
Sows assigned (n)
ESR (7o)
wEr (d)
BRD3 (7o)
Sows farrowed (n)
FR (7o)
TB
BA
Fertility indexr
Sows per WEI
r/2/3/4
218
88.1
4.5 x..01
19.2
166
86.5
72.9 x..3
12.7 x..3
983
CON
88.t/2.8/2.3/6.9
189
92.1
4.0 x. .07
85.2
144
82.8
12.2 x..3
11.6 t .3
930
PG
92.1/0.5/3.7t4.2
0.22
0.001
0.27
0.7 t
0.11
0.20
G
0.07
0.42
0.34
0.28
0.45
0.38
TxG
0.38
0.88
0.13
0.32
0.24
0.22
T
0.34 0.001 0.t4
WCAT TxWCAT
= sows bred in < 7 days after weaning and farrowed per 100 sows weaned x TB
The absence of sows classified as "tail enders" in the PG group may be of practical significance.
Assuming that the incidence of "tail enders" will increase at more adverse times of year due to seasonal
effects on lactation feed intake and overall reproductive performance, the apparent ability of GT
treatment to induce an early ovulation in these sows is of interest. Those GT treated sows showing a
silent first heat and then detected in estrus at l9 or more days after weaning will have active corpora lutea
at days 77-19 after weaning. These sows should therefore respond to luteolytic prostaglandin treatment
and could then either be bred if they show a clear standing estrus or be reliabty culled as either showing
persistent silent heats or as being totally anovulatory. The retum on investment of being able to impose a
standardized protocol for reaching a culling decision based on reliable indicators of reproductive
performance after weaning, and thus reduce unwarranted non-productive sow days seen in many herds,
may be substantial.
Bates R0, Kelpinski J, Hines B & Ricker D 2000 Hormonal therapy for sows weaned during fall and
winter. Journøl of Animal Science. 78 2068-207 l.
Kirkwood RN 1999 Pharmacological intervention in swine reproduction. Swine Health Production 7 29-
35.
Knox, RV, Rodriguez-Zas SL, Miler GM, Willenburg KL & Robb JA.200l Administration of P.G.
600 to sows at weaning and the time of ovulation as determined by transrectal ultrasound. Journal of
Animal Science 79 796-802.
Vargas AJ, Bernardi ML, Wentz I, Borchardt Neto G & Bortol ozzo F. 2006 Time of ovulation and
reproductive performance over three parities after treatment of primiparous sows with PG600.
Theriogenology 66 2017 -2023.
112

253-6
BENEFITS OF SYNCHRONIZING OVULATION \ryITH
HORMONE (pLH) IN A FIXED TIME INSEMINATION
MULTIPAROUS SOWS
L.J Zakl,J. Patterson2, J. Hancock3, D. Roganl, and G.R. Foxcroft2.
PORCINE LUTEINIZING
PROTOCOL IN WEANEI)
rBioniche Animal Health, P.O. Box 1570, Belleville, ON, K8N 5J2, Canada; 'Swine Reproduction-
Development Program, Swine Research & Technology Centre, University of Alberta, Edmonton, AB,
T6G 2P5. canada;3Picron Animal Hospital,lg Mcsre;en Drive, picron, KOK 2To, canada
E-mail: Louisa.Zak @Bioniche.com
Insemination protocols are usually based on the onset of estrus, with a view to inseminating within a
24 hour window before or up to 16 hours after ovulation (Kemp & Soede 1996). However, use of
transrectal ultrasonography has shown that the time of ovulation relative to onset of estrus is highly
variable, and dependent on weaning-to-estrus interval (WEÐ, duration of estrus, and frequency of estrui
detection (Weitze et al- 1994). Variation in the timing of the pre-ovulatory LH surge relative to onset of
estrus is assumed to cause these differences in the time of ovulation during estrus (iilton et al. l9g2) and
injection of porcine luteinizing hormone (pLH) induces ovulatioi approìimately 3g h after
administration (Cassar et al. 2005). Using pLH at the onset of esrrus, timeã insemination 24 or more
hours after pLH then ensures optimal fertility outcomes. The efficacy of combining pLH administration
at first detection of standing heat in weaned multiparous sows in a commercial opeiatìon, followed with
fixed- time double insemination, was initially compared to results from sows óonventionally managed
and inseminated multiple times until no longer in estrus.
Sows (parity 2-9) wete assigned to treatment at weaning on d. 21 of lactation. From the day of
weaning, twice-daily boar exposure at 8am and 2pm facilitaied estrus detection. Onset of behavioural
estrus was defined as the time that sows first showed a standing response to back pressure in the presence
of a boar. In accordance with normal farm practice, untrãated- control ro*i ICON; n=116) were
inseminated at 6-, 18- and24-h intervals based on their WEI and the time (am or pm) they were first
recorded in estrus (See Table 1). After the second insemination, additional inseminations were employed
at 24-h intervals until sows were no longer in standing heat. Sows assigned to LUT (n=163j were
administered 5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, ON; t.l¿. in 4 ml of vehicle)
concomitant with the fi¡st detection of standing heat. If sows were first detected in estrus in the morning
(8am) they were inseminated at 8am and 2pm the next day (24 and 30 h after pLH injection). If estrus
was first detected in the afternoon (2pm), sows were inseminated at 2pm the next äay and 8am the
following day (24 and 42 h after injection; see Table 2). Extended semen was < 3 days olã and contained
a minimum of 3x I 0e live spermat ozoa per dose. Data from sows that had a weaning to last insemination
interval (!u-Ð 10 d were removed from the analysis. Parities 7, 8 and 9 were analyzed, as one
group (Parity 7+). The effect of treatment, parity, and their interaction on number of inseminations, age
of semen, WLII, litter size and weight born, and individual piglet weight were analyzed, using a linear
mixed effect model, with lactation length as a covariate. Propórtìons of sows bred thai were pregnant and
farrowed were analyzed using a chi-squared test.
There were significant effects of treatment and parity but no treatment by parity interaction for any
variable measured. For the purposes of this presentãdon only the main effects of treatment are reported.
The last insemination occurred sooner after weaning (5.3 t 0.07 vs. 5.8 t 0.08d; p < 0.001), the number
of inseminations was fewer (2.0 + 0.02 vs.2.2 + 0.02; P < 0.001) and farrowing rare tendeá to be higher
(87'4 vs' 82.37o; p = 0.1) in LUT compared to CoN sows, respectively. Totaipigs born and born alive
was also greater (P < 0.01) in LUT (12.9 ¡0.3 and I 1.7 * 0.3, reipectiveiy) "o-purla
to coN (11.g + 0.3
and 10.6 + 0.3, respectively) sows. Treatment had no effect on uuìrug" pigtet wËignt or on piglet removal
rate due to low viability before cross fostering at d 3 after farrowing.

Table 1: Untreated control sow insemination schedule
wEI (d) Estrus onset 1"'insemination time /
Estrus to AI interval (h)
< 4 days
> 4 days
am
pm
am
pm
Same day pm (6 h)
Next day am (18 h)
Same day am (0 h)
Same day pm (0 h)
x then inseminated at 24-h intervals until no longer in standing estrus
2nd insemination* /
Estrus to AI interval (h)
Next day am (24h)
Following day am(42h)
Next day am (24h)
Next day am (18 h)
Table 2: Lutropin treated sow insemination schedule
Estrus onset / pLH
administration
am
pm
l"t insemination time /
pLH to AI interval (h)
Next day am(24h)
Next day pm (24 h)
2"d insemination time /
pLH to AI interval (h)
Next day pm (30 h)
Following day am(42h)
These results indicate that administration of pLH at onset of behavioural estrus to control time of
ovulation enables a double fixed-time insemination protocol to be employed, which resulted in reduced
semen usage and labour devoted to estrus detection, and improved sow productiviry. These data also
provide preliminary information suggesting that administration of pLH concomitant with onset of estrus
could be used to facilitate a fixed-time insemination protocol in which a single insemination is performed
24 or 30 hours after pLH administration.
Cassar G, Kirkwood RN, Poljak Z, Bennett-Steward K & Friendship RM 2005 Effect of single or
double insemination on fertility of sows bred at an induced estrus and ovulation. Journal of Swine
Health and Production 13 254-258.
Kemp B & Soede NM 1996 Relationship of weaning to estrus interval to timing of ovulation and
fertilization in sows Journal of Animal Science 74 944-949.
Tilton JE, Foxcroft GR, Ziecik AJ, Coombs SL & Williams GL 1982 Time of the preovulatory LH
surge in the gilt and sow relative to the onset ofbehavioural estrus Theriogenology 18277-236.
Weitze KF, Wagner-Rietschel H, Waberski D, Richter L & Krieter J 1994 The onset of heat after
weaning, heat duration and ovulation as major factors in AI timing in sows Reproduction in
Domestic Animals 29 433-443.
r14

252-9
EFFECT OF PROGESTERONE ANTAGONIST RU486 ON UTERINE
PROGESTERONE RECEPTOR
'RNA
EXPRESSION, EMBRYONIC
DEVELOPMENT AND OVARIAN FUNCTION NUNTÑC EARLY PREGNANCY IN
PIGS
D' Mathewt, E. sellnert, c. okamurat, R. Geisertt, L. Anderso n2, andM. Lucyl
rDivision of Animal Science, university of Missouri, Columbia, uSA;
2Department of Animal Science,
Iowa State University, Ames, USA
E-mail: ems7Oe @mizzou.edu
Porcine peri-implantation development and matemal recognition of pregnancy is temporally
associated with down-regulation of progesterone receptor (PGR) in the endometrial epithelium on days
70 ro 72 (Geisert et at.^20a6). one trreory for down-regulaiion of uterine epithelial pGR is progesterone
stimulates epithelial P9lj" induce exprðssion of RAN:KL [receptor activator for nuclear factor-kappa B
(NF-KB) ligand or TNFSFIll' RANKL binds to is receproi, nÀN¡i (TNFRSFIlA) ro activare NF-KB.
NF-KB and PGR are mutually antagonistic to one another. Activation of NF-rcB, túerefore, may inhibit
PGR expression and induce the increase in endometrial prostaglandin-endoperoxide synthase 2 (pTGS2
or CoX2) expression'that occurs in the endometrium of ryclic ind pregnant pigs on days 10 to 12.
The PGR antagonist, RU486, could be used to deteimine ir utoctcing Þdn ¿o,"n-regulation in the
uterine epithelium prey:lts RANKL expression and NF-rcB activation. To-test this hypothesis, gilts were
inseminated at estrus (d 0) and assigneã to one of three treatments: RU4g6 (400 mg/d) on d 3, 4, and 5
(T1;n = 10);RU4B6 on d 6 and7 (T2; n = 9); or control (n = 9). Blood was colleJteá daily forplasma
progesterone analysis, and the uterus and ovaries were harvested aftei slaughter on ¿ s or d 12.
Endometrial total RNA was isolated and, analyzed with specific primers for RANKL, PTGS2, pGR
isoform B (PGR-B), or the region coûrmon to PGR isoforms A and B (PGR-AB) by real-time reverse
transcriptase PCR (RTPCR). NF-KB activation was measured by immunohistochernistry and scored
objectively by three independent individuals.
Gilts treated with RU486 (T1 and 12) had heavier ovaries (17.g,1g.g,and 16.1 g tsEM = 1.11; -tr,T2
and control; P < 0'05), greater average follicular diameters rs.ø,
q.g,and 3.6 mm [sEM = 0.5]; p < 0.01),
a tendency for a greater number of corpora lutea (16.8, 15.0, and 13.7 tsEM = t.0j; p < 0.07) and greater
mid-cycle plasma progesterone concentration (25.2,28.0, and 20.6 ng/mL;p < 0.05; d 9 to l l). uterine
weight (g) was reduced (P < 0.05) for T1 (608 t 46) compared with T2 (7go + 4g) orconrrol (7g5 + 44).
Treatment of gilts with RU486 affected early embryänic development. The proportion of gilts with
normal early embryonic development was lowest ror gitts in Tl (chi-sqüâre=l1.2;p

Table 1: Number and percent of gilts with normal embryonic development, relative nRNA amount
in endometrium and luminal epithelium NF-KB activation (0 = cytoplasmic localization to l0 =
nuclear localization) for pigs (d 0 = estrus) treated with RU486 on d 3, 4, and 5 (Tl), RU486 on d 6
andT (T2), or untreated (control) with tissue collected on d 8 or d 12.
Normal development
(vo)
RANKL mRNA
PGR-B mRNA
PGR-AB mRNA
NF-KB activation
PTGS2 mRNA
Day 8 Day 12
T1 T2 Control T1 T2 Control
3t5
(60)
17.8+9.8
11.0-¡1.3
10.4x.7.3
2.4+0.6
1.0t0.6
4/4
(100)
3.7x.10.9
4.9+l.5
6.1x.1.4
2.6¡0.1
0.5t0.6
5/5
(100)
1.8t9.8
1.9x.1.3
2.8x.1.3
2.2+0.6
0.3t0.6
0t5
(0)
26.6+70.9
7.3x.1.5
10.9x.7.1
2.5t0.6
0.3t0.6
3/5
(60)
25.2¡9.8
6.9x.1.3
8.0t1.4
4.0+0.6
4.4+0.6
4t4
(100)
2.8x]0.9
4.3+1.5
4.2x.1.4
4.8t0.6
3.4+0.6
RU486 treatment stimulated ovarian follicular and luteal development perhaps by removing the
inhibitory effect of progesterone on the ovary or hypothalamus. Early embryonic development was
compromised for gilts treated with RU486 on d 3, 4 and 5 (Tl). Inhibition of progesterone action shortly
after insemination, therefore, is incompatible with conceptus development and survival. There was a
slight numeric reduction in the percentage of normal embryos on d 12 for gilts treated with RU486 on d 6
and 1 (T2). Blocking progesterone action with RU486 during early pregnancy increased PGR-B and
PGR-AB mRNA expression on d 8 and 12. This supports the general concept that progesterone is
responsible for PGR down-regulation in the uterine epithelium. Our results do not support the hypothesis
that RANKL mediates NF-KB inhibition of PGR. Both RANKL and PGR expression were clearly
elevated in T1 gilts on d 8; a time when activated NF-rB was low. Endometrial RANKL expression does
not appear to be responsible for NF-KB activation in the uterus and it is possible that another pathway for
NF-KB activation such as Toll-like receptor 4 could be involved. Activated NF-KB was only detected on
d12in treatments that were conducive to early conceptus development (T2 and control). Activation of
NF-KB, therefore, was temporally associated with PGR down-regulation and the secretion of IL-lp by the
elongating porcine conceptuses on d 12 (Ross er a\.2003). The activation ofNF-rcB on d 12 coincided
with greater PTGS2 expression; a response observed previously in the pig (Ashworth et al. 2005).
Thís project was supported by National Research Initiative Grant no. 2007-35203-17836 from the USDA
Cooperative State Research, Education and Extension Service.
Ashworth MD, White FJ, Ross JW, Hu J, DeSilva U, Johnson GA & Geisert RD 2005 Expression of
porcine endometrial prostaglandin synthase during the estrous cycle, early pregnancy and following
endocrine disruption of pregnancy . B i ol o gy of Rep ro duction 7 4 I 007- 1 0 1 5.
Geisert RI), Ross JW, Ashworth MD, White FJ, Johnson GA & DeSilva U 2006 Maternal
recognition of pregnancy signal or endocrine disruptor: The two faces of oestrogen during
establishment of pregnancy in the pig. In: Control of Pig Reproduction VII. pp 131-145 Eds R
Kraeling & C Ashworth. Nottingham University Press.
Ross JW, Malayer JR, Ritchey JW & Geisert RD 2003 Involvement of the interleukin-lb system in
porcine trophoblastic elongation and at the fetal-maternal interface during peri-implantation
development . Biolo gy of Reproduction 69 7251 -1259.
116

2s2-10
SELECT NUTRIENTS AND GLUCOSE TRANSPORTERS IN PIG UTERI AND
CONCEPTUSES
F.w. Bazer', H. Gaot, G.A. Johnson2, G. wu1, D.w. Bailey2, andR.c. Burghardt2
Departments of Animal Science¡ and Veterinary Integrative Biosciences', Texas A&M University,
College Starion, T exas j j 843-247 l, U S A
E-mail : fbazer @ cvm.tamu.edu
Glucose present in the intrauterine environment can be metabolized, activate cell signaling pathways,
or be converted to a "storage" form. Total recoverable glucose in uterine fluid ofpregnãnt, Uut not cyciic
pigs increases from Day 12 after onset of estrus in concert with conceptus elongation (Bazer et at. l99l).
Transport of glucose into the ovine uterus and its uptake by conceptuses involves sodium-dependent and
facilitative glucose transporters (Gao et al. 2009f. Glucóse can activate the FRApI/mToR ,,nutrient
sgnsing" pathway in which protein kinases activate p70S6 through phosphorylation to increase
translation of 5'ToP mRNAs (terminal oligopyrimidine trãct) (wen et at. ioosj. Aciivate¿ FRApI also
regulates differentiation of trophectoderm (ir) via Ras transformation by phosphorylating eukaryotic
initiation factor 4E binding protein 1 (eIF4EBPI), a translational repressor or cal,-aependent translation
(De Benedetti & Rhoads 1990). Select nutrients that stimulate mapr activity in 'ir include glucose,
arginine (Arg), leucine (Leu), and glutamine (Gln) which may increase of IGF2, ODC, and
NoS mRNAs (Nielsen et al. 1995; Kimball et al. I999:Martin & Surherland ""pr"riion 2001) which are required
for conceptus development, differentiation, and implantation through effects on production of NO
(Kaliman et al. 1999) and polyamines (Van Winkle & Campione 1983). FRAPI nrll -i"" die shorrly
after implantation due-to impaired cell proliferation and trypertroptry in both the embryonic disc and Tr
(Murakami et al' 2004)- There are 14 isoforms of faclútativ" glu.o*" transporterl and 6 sodiumdependent
glucose transporters. of these, 9LC2A\, sLCSAi, an¿ sicsul mRNAs are most abundant in
endometria and SLC2A3 is uniquely expressed by ovine conceptus Tr and endoderm (Gao et at.2009).
The objective of this study with sexually mature gilts was to ìdentify effecrs of pregìancy, long-term
treatment of ovariectomized gilts with progesterone (P4) and estradiol-induced pr"uaoi'r"gnancy (pp) on
changes in amounts of select nutrients (glucose, Arg, Leu, and Gln) in uterinè fluid-anãexpression of
glucose transporters in endometria and conceptuses.
Experiment 1 determined effects of day of the estrous cycle and pregnancy on total recoverable
glucose, Arg, Leu, and Gln in uterine flushings from gilts on ôuy, s, g, tù an¿ i s of the estrous cycle
(Cy) and Days 9, 10, 12, 13, 14, and 15 of pregnancy (Px). Total-recoverable glucose, Arg, Leu and Gln
increased (P

SLC2A2 mRNA was most abundant in conceptuses from Days 12 to 40 of Px, decreased to Day 50 and
then increased and was maintained specifically in placental areolae and apical regions of interdigitating
endometrial folds to Day 80 of Px. SL2CA2 was expressed in uterine LE of Px and PP gilts and LE and
GE of OVX-P4 gilts.
Results indicated that: 1) glucose and Arg in particular, but.also Leu and Gln, increase in uterine
fluids of Cy and Px gilts; 2) these select nutrients are abundant in uterine flushings of PP and OVX-P4
gilts; and 3) temporal and cell specific changes occur in expression of specific glucose transporters in the
uterus and conceptus. These select nutrients likely stimulate FRAPI cell signaling in trophectoderm cells
of conceptuses to influence proliferation, migration, attachment and gene expression necessary for
conceptus development and survival in pigs.
ßazer FW, Thatcher WW, Martinat-Botte F, Terqui M, Lacroix MC, Bernard S, Ravault M &
Dubois DH 1991 Composition of uterine flushings from Large White and prolific Chinese Meishan
gilts. Reproduction, Fertility and Development 2 5l-59
De Benedetti A & Rhoads RE 1990 Overexpression of eukaryotic protein synthesis initiation factor 4E
in HeLa cells results in aberrant growth and morphology. Proceedings National Academy of Science,
usA87 8212-8216
Gao H, lVu G, Spencer TE, Johnson GA, Li X & Bazer FW 2009 Select nutrients in the ovine uterine
lumen: I. Amino acids, glucose and ions in uterine lumenal fluid of cyclic and pregnant ewes.
Biology of ReproductionS0 86-93
Gao H, Wu G, Spencer TE, Johnson GA & Bazer FW 2009 Select nutrients in the ovine uterine
lumen: II. Glucose transporters in the uterus and peri-implantation conceptuses. Biology of
Reproductíon 80 94-104
Kaliman P, Canicio J, Testar X, Palacin M & Zorzano A, 1999Insulin-like growth factor-Il,
phosphatidylinositol 3-kinase, nuclear factor-B and inducible nitric-oxide synthase define a common
myogenic signaling pathway. Journal of Biological Chemistry 274 17437-11444
Kimball SR, Shantz LM, Horetsky RL & Jefferson LS 1999 Leucine regulates translation of specific
mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and
phosphorylation of ribosomal protein 56. Journal of Biological Chemistry 274 11647-17652
Martin PM & Sutherland AE 2001 Exogenous amino acids regulate trophectoderm differentiation in
the mouse blastocyst through an mTOR-dependent pathway. Developmental Biology 240 182-193
Murakami M, Ichisaka T, Maeda M, Oshiro N, Hâra K, Edenhofer F, Kiyama H, Yonezawa K &
Yamanaka S 2004 mTOR is essential for growth and proliferation in early mouse embryos and
embryonic stem cells. Molecular Cell Biology 24 6710-67L8.
Nielsen FC, Ostergaard L, Nielsen J & Christiansen J 1995 Growth-dependent translation of IGF-II
mRNA by a rapamycin-sensitive pathway. Nature 377 358-362
Van Winkte LJ & Campione AL 1983 Effect of inhibitors of polyamine synthesis on activation of
diapausing mouse blastocysts in vitro. Journal of Reproduction and Fertility 68 437-4M
Wen HY, Abbasi S, Kellems RE & Xia Y 2005 mTOR:A placental growth signaling sensor. Placentq
26 S63-569.
118

251-5
P32 TYROSINE PHOSPHOPROTEINS AND THE ACROSOME REACTION IN
PORCINE SPERM: IMPORTANCE OF EXTRACELLULAR AND INTRACELLULAR
CALCIUM
J.L. Bailey, S.K. Thangavelu, and C. Lessard
Centre de Recherche en Biologie de la Reproduction, Département des sciences animales, Université
Laval, Québec City, Canada
E-mail: janice.bailey @fsaa.ulaval.ca
Capacitation is a signal transduction-mediated event that involves protein phosphorylation at tyrosine
residues and primes spermatozoa for the acrosome reaction. In the pig, capacitation is associated with
the calcium-dependent appearance of p32 tyrosine phosphoproteins. The exact role or regulation of p32
tyrosine phosphorylation is still not established, although it has been suggested that it is involved in the
acrosome reaction. Indeed, sp32, a proacrosin binding protein, is a major component of the group of
proteins that make ry p32, and is tyrosine phosphorylated during capacitation. We hypothesised that p32
appearance and sp32 activity are coupled to the release of sperm calcium stores and related to the
acrosome reaction.
To test these hypotheses, fresh pig sperm were subjected to capacitation in the presence of modulators
of sarcoplasmic and endoplasmic reticulum Ca2*-ATPases (SERCA) or a calcium chelator. P32 and sp32
appearance, (pro)acrosin size, and spontaneous acrosome reactions were assessed as indicators of sperm
functional state. The presence of SERCA in pig sperm was investigated by Western blotting of extracted
proteins and immunolocalisation using a pólyclõnal anti-SERCA 2 (N-19) goat antibody. Cytosolic
calcium levels of sperm loaded with the fluorescent calcium indicator indo-l were assessed by flow
cytometry.
Thapsigargin, a SERCA inhibitor that should release calcium stores, markedly accelerated the
appearance of p32, confirming the dependence of this group of tyrosine phosphoproteins on calcium. In
contrast, gingerol, a SERCA activator, did not alter p32 appearance. However, both p32 appearance and
the elevation of cytosolic calcium during capacitation were prevented by the inclusion of a calcium
chelator, BAPTA-K+, in the medium. Furlhermore, thapsigargin could not override the effect of
BAPTA-K+ on p32 appearance. During incubation in capacitating conditions ,1 + 4 % of control sperm
underwent spontaneous acrosome reactions, whereas thapsigargin induced 29 + 12 7o spontaneous
acrosome reactions, or l0 + 5 7o and I + 3 Vo with gingerol and BAPTA-K*, respectively. Thapsigargin
hastened the formation of acrosin from its proacrosin precursor and sp32 size decreased with the
conversion of proacrosin to acrosin, likely indicative of sp32 hydrolysis. These data indicate that the
release of calcium from SERCA-mediated intemal stores by thapsigargin accelerates p32 appearance and
other signalling events leading to the acrosome reaction. Nonetheless, the release of calcium stores in the
absence of extracellular calcium is insufficient for these events because even with thapsigargin,
extracellular calcium remains essential. Finally, Western blotting with anti-SERCA 2 antibody revealed
a band at Mr-100,000, similar to the molecular weight (-110 to 115 kDa) of SERCA 2 isoforms reported
in the literature. Indirect immunofluorescence of fixed, permeablised sperm revealed SERCA 2 localised
at the acrosome.
These results suggest that store-operated Ca2* entry regulates p32 appearance, sp32-mediated acrosin
maturation, and the spontaneous acrosome reaction. Moreover, a SERCA-like pump is present on the
porcine acrosome that may play a role in regulating capacitation and/or the acrosome reaction.
Semen was generously donated by the Centre d'insémination porcine du Québec and NSERC of Canada
funded the research.
I2t

2st-6
THE EFFECT OF TIMING OF hCG ADMINISTRATION AFTER eCG ON FERTILITY
OF WEANED SOWS TIMED INSEMINATED WITH FROZEN.THAWED SEMEN
A' Bolarin, M. Hernandez,J.M.yâzqaez,E.A. Martínez, andJ. Roca*.
Department of Medicine and Animal surgery, university of Murcia, 30100 Murcia, spain
E-mail: roca@um.es
xPresenting auLhor
The short life span of thawed spermatozoa is a major factor limiting the reproductive performance of
frozen-thawed G-r) boar semen. Therefore, inseminaiion in a "safe" 4-8 h interval before ovulation is a
priority to achieve high fertility. The control of ovulation by gonadotropin treatment (eCG + hCG) allows
for a single insemination within the "safe" interval before éxpected timã of ovulation. The timing of hCG
administration post-eCG has not clearly been established, suggesting a range between 54 and 96 h as
appropriate' The aim of this study was evaluate 3 different ti*ingr of hCG administration in order to
define the most suitable for the efficient use of FT semen in pig commercial farms.
Multiparous sows (n-254) receiving 1250 IU of eCG 24 i after weaning were randomly allocated to
receive 750 IU of hCG at 60 h (n=81), 72 h (n=85) or 84 h (n=88) afrer eCG. Timed arrificial
inse.mination (TAI) was performed 38 h after hCG by means of a deep intrauterine deposition of 1,500
x10" FT-sperm. ovaries were monitored by transrectal ultrasonography to determine the ovarian status at
the TAI time (pre-, peri- and post-ovulatory TAIs). Differences in ti" p"r""ntage of pre-, peri- and postovulatory
TAIs were compared using a chi-square tesr. Ferrility (fanôwing .ut", enl, pión¡"u"y 1ìitt".
size, LS) and fecundity index (fanowing rate iimes average number of piglets born pei litter times 100,
FI) were analyzed by ANOVA using a mixed model with effect of TAI day as a random variable.
_- M9t" pre- and less post-ovulatory TAIs were performed in sows with an eCG-hCG interval of 60 and
l2hthan 84h(P

251-7
IMPORTANCE OF DIETARY OMEGA-3 FATTY ACIDS (FISH OILS) ON BOAR
SEMEN QUALITY
C.A. Castellanor, I. Audetl, J.P. Laforest2, Y. Chouinard2, and J.J. Mattel
tDairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College
Street, P.O. Box 90, Lennoxville STN, Sherbrooke, QC, Canada JIM lZ3
2Faculty of Agriculture and Agri-Food Sciences, Laval University, Quebec, QC, Canada GlK 7P4
Boar semen has a particular fatty acid profile, especially for long chain polyunsaturated fatty acids
(PUFAs), with high concentrations of docosahexanoic acid (DHA, n-3) and docosapentanoic acid (DPA,
n-6) and zero content of eicosapentaenoic acid (EPA, n-3). The present experiment was conducted to
evaluate the influence of dietary incorporation of fish oils (rich in long chain PUFAs) vs hydrogenated
animal fat (saturated fatty acids) on semen production and quality, fatty acid composition, and
preservation properties in boars.
Forty boars, 6-7 months old, were fed 2.5kg/day of a basal diet supplemented with 0.3 kg/day of a
specific top dressing for each different treatment mixtures during 7 months. Boars were randomly
assigned to one of the four treatments. The first group was supplemented with hydrogenated animal fat
(C, n=10); the others groups received 2.I7o menhaden oil with a DHA contenÍ of lTEo (M,n=10),2.77o
tuna oil with a DHA content of 30 Vo (T, n=I}) or 2.IVo menhaden oil with 2 ppm of biotin (MB, n=10).
Biotin is a critical factor in the elongation of PUFAs (EPA to DHA). All diets were isoenergetic and
provided daily 989 mg of vitamin E. After a training period of 12 weeks, the semen collection was
divided in 3 phases: l-regular (twice a week during 4 weeks); 2-intensive (daily during 2 weeks); 3-
recovery (twice a week during 10 weeks). Classical measurements on sperm quality and quantity were
taken throughout those phases. A semen preservation test was also performed during 9 days at 1'7"C at
the end of the experiment.
In C boars, similar proportions of both DHA and DPA (22.27o and 25.67o of all fatty acids,
respectively) were measured in spermatozoa. In the three fish oils treatments, the DHA/DPA ratio was
substantially higher: DHA increased (P

251-8
PROTECTIVE EFFECT OF PSP.VII SPERMADHESIN IN SORTED AND FROZEN-
THAWED BOAR SPERM
D. del Olmo,I. Parrilla, J. Roca, E.A. MartÍnez, andJ.M.Yâzqtez
Department of Animal Medicine and Surgery, University of Murcia, 30100 Murcia, Spain
E-mail ; Parrilla@um.es
The improvement of freezing-thawing procedures for sexed boar spermatozoa is an essential step for
the practical application of this sperm biotechnology in swine. The aim of the present study was to
evaluate the effect of the presence of heterodimer PSP-VI in the collection medium, on motility and
viability of sex-sorted and frozen-thawed boar spermatozoa.
Hoechst 33342-stained spermatozoa from 5 boars were flow cytometrically sorted for the X
chromosome using a MoFlo SX (Dako Colorado Inc., Fort Collins, CO, USA) collected in two different
media, Medium A: TES-Tris-Glucosa (TTG) + 27o egg yolk + 107o whole seminal plasma (v/v) or
Medium B: TTG + 2Vo egg yolk + 0.15 mg/ml of PSP-VII, and cryopreserved according to a standardized
procedure in our lab. Sperm motility and viability parameters were measured at four different steps
during the cryopreservation process (after centrifugation for sperm concentration prior to freezing, during
cooling at 5oC, and either 15 min and 45 min after thawing). All viability analyses were performed by
analytical flow cytometry (EPICS XL; Coulter Corporation, Inc., Miami, FL, USA) using triple
fluorescent staining (SYBR-14/PI/PE-PNA). Percentage of progressive motile spermatozoa was analyzed
by a computer-assisted sperm analysis (CASA) system (ISAS@, Proiser SL, Valencia, Spain).
Analyses of variance (ANOVA) were carried out and were compared with a Tukey test, using the SPSS
11.5/PC statistical package (SPSS, Inc., Chicago, IL, USA). All data are expressed as mean + SEM.
Mean values for percentage of viable spermatozoa in Medium A and Medium B during the four different
timepointswereasfollows: aftercentrifugationS3.4+2.37ovs90.6+2.0Vo,at5"C,80.5-r3.25vs89
+ 1.3 7o,15 min after thawing 70.1 + 5 7o vs75.3 x.2.7 7o, and 45 min after thawing 68.1-1 4 7o vs72.2 +
4 Vo for Medium A and Medium B, respectively. The percentage of progressively motile spermatozoa for
Medium A and Medium B, respectively were as follows: after centrifugation 83 + 4.2 7o vs 88.2 + 2 7o, at
5'C 79.8 + 3.5 Vo vs 86.8 + 1.8 Vo, 15 min after thawing 40.6 x.6;7 7o vs 61.8 t 5.9 Vo, and 45 min after
thawing 40.2 + 6.2 Vo vs 50.2 + 2.9 7o. These data indicate as expected, that the freezing-thawing
procedure induces a significant decrease in sex-sorted boar sperm quality, regardless of the medium used
(P

251-9
CRYOPRESERVATION OF SEMEN FROM NATIVE HUNGARIAN MANGALICA
BOARS-APILOTSTUDY
I. Egerszegir, P. Sarlósl, B. Berger2, and J. Rátkyt
rResearch Group of Reproductive Biology, Animal Breeding Division, Research Institute for Animal
Breeding and Nutrition, Herceghalom, Hungary; 2lnstitute for Organic Farming and Biodiversity of Farm
Animals, Thalheim, Austria
E-mail: istvan.egerszegi @ atk.hu
Cryopreservation of semen is a useful method for conservation of farm animal biodiversity and
nowadays there is an increased demand of commercial use of frozen semen in the pig sector. However,
post-thawing survival of frozen boar semen does not reach the levels achieved in ruminant species. Thus,
improvement and adaptation of currently used freezing protocols are needed for cryopreservation of pig
semen to become a viable option. The aim of the study was to adapt a current semen freezingprotocoi in
pig and to test two different extenders in cryopreservation of semen from a native Hungarian pig breed.
Five mature Mangalica boars were used in this experiment. Three of them were pie-sel ,òtea çao uo
fresh semen motility, >10 Vo motility 24 h,

251-10
RELATION BETWEEN NUMBER OF SPERM CELLS INSEMINATED AND
FERTILITY RESULTS IN SOWS
H. Feitsmal'2, J.I. Leenhouwers', and E.F. Knol2
tnig AI Netherlands, Deventer, The Netherlands;
2lnstitute for Pig Genetics, Beuningen, The Netherlands
E-mail : hanneke.feitsma @ ipg.nl
The use of Artificial Insemination (AI) on sow farms in The Netherlands is over 987o. ,\I centres
therefore play a key role, both in fertility results and in genetic progress. Large numbers of insemination
doses produced per ejaculate and thus per AI boar ensure that the genetics of this boar will be spread
extensively throughout the population. Additionally, AI centres can produce insemination doses more
efficiently. However, at a certain point it is expected that lowering sperm numbers per dose will affect
fertility results. In this study data analysis was performed on semen quality data from the AI centres of
Pig AI Netherlands combined with fertility data available from the breeding database present at the
Institute for Pig Genetics. The aim of this study was to investigate the relationship between the number of
sperm cells per insemination dose and sow fertility results.
The AI and breeding datasets, containing records from January 1998 until April 2006, were checked
for incorrect data / outliers. The breeding data were standardised for farm, sow line, line and/or breed of
the litter, year, month and parity, double or re-insemination. The datasets were merged based on boar
name and ejaculate number. In the case no ejaculate identification was found in the sow record, the
ejaculate of the respective boar that was produced within 3 days prior to the first insemination was
assigned to the insemination. Statistical analysis was performed with SAS@ 9.1 using the General Linear
Models procedure, thereby adjusting for: boar line, boar age (day of collection), month, year, days
between ejaculation, AI centre, and number of sperm cells per dose. Number of sperm cells per
insemination dose was calculated using the formula: ejaculate volume x ejaculate concentration / number
of doses produced from ejaculate.
After the merge of the AI and breeding datasets 110,161 records remained. The number of cells per
dose (80 ml) ranged from 1.5 billion cells up to 6 billion cells.In total8.67o of the doses had less than2
billion cells, 50.7Vo contained between 2 and 3 billion cells, and 40.6Vo had over 3 billion cells per dose.
For the statistical analysis the doses with more than 3 billion cells were ignored, because in practice 3
billion cells is the upper limit for number of cells per dose. Approximately 66,000 records remained for
statistical analysis. Results showed that 6.7Vo of the total variation in litter size was explained by the
model. The following variables contributed significantly (P

25r-lt
DOUBLE SPERM DEPOSITION TECHNIQUE (MAGAPLUS.DD@) INCREASES THE
REPRODUCTIVE RESULTS IN FARM CONDITIONS
C. Gómez-Rincónl, M. García-Tomásl, Y. Dahmanil, R. Mozo-Martínl, S. Jiménez3, A.N.
Berges3, E. García-Bonavila 2, M. Yeste2, S. Bonet2, and J. Grandía3
t R&D Department Magapor S.L Zaragoza Spain;
2 TechnoSper, Scienlific and Technologic Park of the
University of Girona, Spain; 3 Agrotest-Control S.L Zangoza, Spain
The satisfactory reproductive results in the current pig production system are highly dependent on AI
with elevated numbers of spermatozoa (3 billion). In recent years, intrauterine insemination (IU) and
deep intrauterine insemination (DIU) methods have been developed in order to reduce the number of
spermatozoa to 1/3 and 1/20, respectively. In practice, both methods can potentially approach normal
fertility rates. However, DIU significantly decreases litter size, mainly due to the unilateral fertilization
phenomenon. The present study was performed in order to evaluate the reproductive efficiency of a new
AI device (Magaplus DD@) that combines both IU and DIU. The Magaplus DD@ allows the deposition
of 807o of the seminal dose at the uterine level, and 20Vo of Íhe dose inside the uterine horn.
Ejaculates from two adult boars were collected. Each ejaculate (n=6) was extended and seminal doses
were prepared as follows: 3/90; 1.5/50; L5/30; 0.751501' 0.75/30 billion spermatozoa/ml. Landrace x
Large White multiparous crossbred sows (n=118) were randomly divided into six groups. Five groups
were inseminated with Magaplus DD@ according to semen processing. The sixth group was traditionally
inseminated (Cervical 3/90). In all treatment groups, insemination was performed at 0 and 24 h after
external oestrus signs were observed. Pregnancy rate (PR) was evaluated 21 days after insemination by
ultrasound scan. The sows were slaughtered at 35 days of pregnancy, their genital tracts were removed
and total embryos (TE) were recorded. Logistic regression and analysis of variance were used to study
the effect of treatment on PR and TE, respectively.
Pregnancy rate values were compatible with farm production. However, significant differences
associated with dose volume were found in PR between treatments. Animals inseminated with 30 ml
volume dose showed PR lower fhan 85Vo, whereas the other treatments reached values around 95Vo.
When all experimental sows were considered, no significant differences were observed on TE.
Nevertheless, when analyses data were limited to low potential prolific females (TE

25t-12
EVALUATION OF BOAR SEMEN FERTILIZING CAPABILITY AFTER SHORT AND
EXTRA-LONG STORAGE USING DURAGEN@ SEMEN EXTENDER
C. Gómez-Rincónl, M. García-Tomásl, Y. Dahmanil, R. Mozo-Martínl, S. Jiménez3, A.N.
Berges3, E. García-Bonavila2, M. Yeste2, S. Bonet2, and J. Grandía3
t R&D Department Magapor S.L Zaragoza Spain;
2 TechnoSper, Scientific and Technologic Park of the
University of Girona, Spain; 3 Agrotest-Control S.L Zaragoza, Spain
The productivity of the swine industry is highly dependent on artificial insemination and consequently
on the production and transport of boar semen. For this reason, developing an extender that preserves
sernen for a long time without a decrease in sperm quality is a major goal in swine reproductive
technologies. The present study was performed to investigate the potential of extra-long term extender
Duragen@ to keep the fertilization capability of semen after 1, 12, and 15 days of preservation.
The sperm rich fraction of ejaculates from two fertile boars were collected, evaluated and pooled.
Seminal heterospermic doses of 3 billion spermatozoa in 90 ml were prepared and stored at 7J"C before
being used for l, 12, and 15 days for Duragen@ and I day for BTS extended semen. 84 multiparous
crossbred sows (Landrace x Large'White) were randomly divided in four groups according to semen
processing: BTSI (control group); Duragen for 7, 12, or 15 days. Cervical insemination (Foam catheter,
Magapor) was performed at 0 and 24 hours after the observance of external estrus signs. Pregnancy rate
(PR) was evaluated 21 days after insemination by ultrasound scan. The sows were slaughtered at 35 days
of pregnancy, their genital tracts were removed, and total embryos (TE) were counted.
A logistic regression and analyse of variance were used to study the effect of treatment on PR and TE,
respectively.
Pregnancy rate values were clearly compatible with farm production in BTSr; D¡ and Dr2 groups
(100Vo;1007o and 75.67o respectively). Even sows inseminated with 15 days preserved doses (D15)
showed PR values over 50 7o. No significant difference was found in TE between BTS1, D1 and Dr2 (13.3
+ l.I7; 15.4 + 0.88; 14.4 x. I.14) but semen storage for 15 days clearly decreases this variable (p

251-13
LEJA.4 COUNTING CHAMBER EXERTS NEGATIVE EFFECT ON SPERM
MOTILITY
C. Hansenl
rDanish Pig Production, Department for Nutrition and Reproduction, Axeltorv 3, DK-1609 Copenhagen,
Denmark
E-mail : cha @ dansksvineproduktion.dk
The most objective motility estimate is performed using computer assisted semen analysis (CASA).
Various instruments are available for this purpose, often used in combination with the Leja-4 counting
chamber. This chamber is considered appropriate for both motility and estimation of sperm concentration
for CASA instruments. The aim of this study was to estimate the effect of sperm motility depending on
position in the Leja-4 counting chamber.
Semen from 11 DanBred Duroc boars was included in this experiment. Semen was diluted to a
concenftation of approximately 30-35 million sperm per mL using EDTA boar semen extender. The
semen doses were stored for 3 days at I7"C before analysis. From each dose, approximately l0 mL of
semen was transferred to each of four 10 mL Nunc tubes and heat-reactivated for 50 minutes at 38 'C.
Semen was analysed with four different batches of 20 ¡rm deep Leja-4 counting chambers. The semen in
each tube was analysed four times with only one of the four batches of counting chambers. Analysis of
sperm motility was performed using a SpermVision CASA system. Analysis of motility comprised 15
separate fields evenly distributed in the "sample inlet - sample outlet" axis of the chamber. An estimate
of-sperm's straight line velocity (VSL) (¡rm/sec), curved line velocity (VCL) (¡rm/sec) and sperm average
path velocity (VAP) (pm/sec) for each field was analysed using mixed model procedure in SAS
including field and batch of counting chamber as fixed effect and ejaculate as random ãffe"t.
The results clearly demonstrated a negative effect on sperm motility parameters. The effect was linked
to position in the chamber. Thus, motility declined when moving from sample inlet to sample outlet. The
decline in motility was evident for VSL, VCL, VAP as well as percent motile sperm. The reduction in
motility was between 10 and 50 Vo andwas statistically significant for VSL, VCL and VAP (p

25t-14
COMPARISON OF NUCLEOCOUNTER SP1OO AND FACSCOUNT AF SYSTEM FOR
DETERMINATION OF BOAR SPERM CONCENTRATION AND VIABILITY
C. Hansenl
tDanish Pig Production, Department for Nutrition and Reproduction, Axeltorv 3, DK-l609 Copenhagen,
Denmark
E-mail : cha @ dansksvineproduktion.dk
Quality of semen is of great importance in the production of semen doses for artificial insemnation.
Quality of semen can be assessed using different assays of which sperm viability is one of the available
assays. The aim of this study was to compare NucleoCounter SP100 (SP100) with FACSCount AF
System (FACS) for determination of sperm viability.
A total of 238 commercial semen doses from DanBred AI stations were included in this study. The
semen originated from Landrace, Large White and Duroc boars and some doses consisted of
heterospermic semen and others of homospermic semen. All semen doses we¡e stored at 17 C' until
analysis. All semen doses were analysed in duplicates with FACS as well as SPl00. Each analysis
included a determination of sperm concentration as well as sperm viability. Results for sperm
concentration and sperm viability were analysed with linear regression using SAS (version 9.1).
The results showed high repeatability for determination of sperm concentration. Sperm concentration
for the majority of the semen doses varied from approximately 23 to 40 million sperm per mL., and
represented normal commercial semen doses. It was found that SP100 consistently underestimated sperm
concentrationby 6.77o.The957o confidence interval for the regression for sperm concentration was 3.47.
The results for determination of sperm viability also showed a high correlation between FACS and
SPl00. The resulting regression equation was: Viability G.ecs) = 12.6 + 0.905 x viability (sproo). (r2 = 0.89).
The results demonstrated a high correlation between FACS and SPl00. These regression results will only
be valid for analysis of sperm concentrations between 25 and 40 million sperm per mL. Previous
analyses of raw semen showed even higher correlation between FACS and SP100 for determination of
sperm concentration.
This study demonstrated the relation between FACS and SP100 for determination of sperm
concentration and sperm viability measured on semen from semen doses.
This work was paftly funded by Danish DanBred Al-stations.
130

zst-15
SEMINAL PLASMA REGULATES PROSTAGLANDIN SYNTHESIS ENZYMES IN
THE PORCINE ENDOMETRIUM
M.M. Kaczmarek, A. Blitek, J. Filant, and A.J. Ziecik
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, poland
E-mail: moka @pan.olsztyn.pl
Seminal plasma (SP) has been viewed as a transport and survival medium for mammalian sperm but it
seems that its role.extends beyond this to target female reproductive tissues. Several studies have
indicated that the introduction of semen into the female reproductive tract may induce molecular and
cellular changes that facilitate conception and pregnancy. Recently, SP-regulated expression of
prostaglandin GIH synthase 2 (PGHS-2) gene has been demonstrated in pigs (O'Leary ei at., 2004).
Since prostaglandins are crucial for pregnancy success, we investigated the effect of Sp on the expression
of prostaglandin synthesis enzymes in the porcine endometrium. Moreover, prostaglandin concentrations
in the uterine lumen after SP-treatment were determined.
Fifty six pre-pubertal crossbred gilts of similar age (approx. 5-6 mo) and weight (104.5t6.5 kg) were
synchronized (750 IU of PMSG and 500 IU of hCG 72 h later). One day aiter hCG injection gilts
received 100 ml intrauterine infusion of either SP or phosphate buffered saline (pBS; control). Luminal
fluids and endometrial tissues were collected on days 1, 3, 5, 10 after SP- or PBS-treatment (n=5-
8ldayltreatment). The levels of PGE2, PGF2,, and its merabolite (PGFM) in the uterine flushings were
measured by ELISA. Gene expression of PGHS-2, PGF synthase (PGFS), PGE synthase (mpGES-1), pG
9-ketoreductase (9KR), and PGI synthase (PGIS) relative to p-actin (ACTB) was srudied by the real-time
PCR. Results were analyzed using two-way ANOVA followed by Bonferroni's post hoc tests.
The levels of PGE2, PGF2', and PGFM in the luminal fluids were not significantly affected by the
treatment. However, 24 h after infusion into the uterine horns, mean concentrations of pGE2 in the
uterine lumen were 2-fold lower in SP-treated animals than in controls (0.36t0.06 and 0.74+0.20 ng/ml,
respectively). In addition, the PGE2:PGF2' ratio was lower 24 h afær SP infusion when compared to
PBS-treated pigs (P

251-16
EFFECT OF BOAR SEMINAL PLASMA ON PRODUCTION OF PROSTAGLANDIN
F2a (PGF2a) AND INTERLEUKIN-6 (IL-6) FROM THE PORCINE AND BOVINE
ENDOMETRIAL CELLS
M. Madejl, M.T. Madsen2, M.Norrbyl, A. Johannissont, C. Hansen2, and A. Madejl
iDepaftment of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences,
P.O Box 7011, SE-750 07 Uppsala, Sweden. 2Danish Pig Production, Axeltorv 3, DK-1609 Copenhagen,
Denmark
A better understanding of seminal plasma signaling in the female reproductive tract would contribute
to improving reproductive efficiency in the pig. Human seminal plasma is activating the expression of
pro-inflammatory and immune-regulating cytokines in human cervical cells. Thus, the objective of this
study was to investigate the effect of seminal plasma on production of PGF2a, its metabolite PGFM and
IL-6 from porclne and bovine endometrial cells.
Porcine endometrial epithelial cells were isolated from uteri of 4 gilts based on methods described by
Blitek and Zíeclk (2004). Bovine endometrial epithelial cells were purchased from Cell Applications,
Inc., USA. The viability of the cells was around 90Vo and all tests were performed when the cells were 80
to 90Vo confluent. Porcine and bovine epithelial cells were cultured with 0.2 ml seminal plasma, pooled
from 3 Danish Duroc boars, in Medium 199 for 3 and 24 h in triplicate. Control epithelial cells were
incubated with or without arachidonic acid (AA) (20 þglml) in Medium 199, also in triplicates and rhe
tests were repeated 3-4 times. The collected media was analyzed by EIA for the content of PGF2a,
PGFM, porcine and bovine IL-6. IL-6 was also analyzed by the xMAPTM technology. The cells were
lysed with 0.1 N NaOH for determination of the total cellular proteins.
The basal concentrations of PGF2u and PGFM in the collected media from the porcine epithelial cells
after 3 h incubation were 1.49 + 0.2I and 0.19 t 0.07 pg/¡rg protein, respectively. Addition of AA to the
medium significantly increased the production of PGF2c and even PGFM to 3.37 + 0.7 and I.28 ¡ 0.2
pglpg protein, respectively. Addition of 0.2 ml seminal plasma to the medium with or without AA totally
inhibited production of PGF2o to values around the detection limit. Concentrations of PGFM in the
medium were significantly reduced up to 4 Vo of control values after incubation with 0.2 ml seminal
plasma and were 0.03 t 0.01 pg/¡rg protein. The inhibition of PGF2u and PGFM was also seen after 24 h.
The basal concentrations of PGF2u and PGFM in the collected media from the bovine epithelial cells
after 3 h incubation were 2.8 + 0.31 and 0.46 + 0.06 pg/¡rg protein, respectively. Addition of AA to the
medium did not increase production of PGF2aafter 3 h but did increase it after 24h.'lhere was no effect
of AA on concentrations of PGFM. Addition of 0.2 ml seminal plasma to medium with or without AA
totally inhibited production of PGF2a to values around the detection limit. Concentrations of PGFM in
medium were significantly reduced up to 17 7o of control values after incubation with 0.2 ml seminal
plasma and were 0.08 t 0.01 pg/¡rg protein. Concentrations of IL-6 in control media were around the
detection limit. Incubation of porcine and bovine uterine epithelial cells with 0.2 ml of seminal plasma
resulted in an increased production of IL-6 expressed per ml of medium as well as per protein content.
In conclusion, the boar seminal plasma has an inhibitory effect on production of PGF2o and its
metabolite PGFM and possibly stimulatory effect on IL-6 from porcine as well as bovine endometrial
epithelial cells after 3 and24 h of incubation.
This study was supported by Danish Pig Production.
Blitek' A & Ziecik, ÃJ.2004 Prostaglandins F and E secretion by porcine epithelial and stromal
endometrial cells on different days of the oestrous cycle. Reproduction in Domestic Animals 39 340-
346.
t32

251-Lt
ALDOSE REDUCTASE ACCELERATES BOAR SPERM CAPACITATION
N. okamurat'', A. Ku*ushima2, B._osmanl, K. Takebayashil, r y. Katohl, M. Takashimat, s.
Kohchir, K. Kikuchi3, M. Matsudar, and A. Kikuchir - -- ' '
rGraduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba,
lbqati 305-8575,.Japan. 2Graduate School of Sciences, The University of Tokyo, Hongo 3-7-i, Tokyo
113-0033, Japan. 3Division of Animal Sciences, National Institute of Ágrobiolågical Sc"iences, Tsukuba,
Ibaraki 305-8602, Japan
E-mail : naooka@md.tsukuba. ac jp
Capacitation in the female reproductive tract is the final process through which mammalian sperm
acquire the ability to ferlilize an oocyte. Although the molecular basis of cipacitation has nor beenlully
elucidated, tyrosine phosphorylated proteins are considered to play central roles in its completion. In the
present study, the novel proteins which are tyrosine-phosphorylated and involved in the aðcomplishment
of capacitation have been identified.
Porcine cauda epididymal sperm were incubated in the capacitation buffer containing BSA, CaCl2,
and sodium bicarbonate. Capacitation-induced tyrosine-phosphorylated proteins were" identified by
proteomic analysis in combination with two-dimentional gel èlectrophoreìis and mass spectrometry.
Among those proteins, the involvement of aldose reducáse in capacitation was analyzed using a
membrane permeable specific inhibitor of aldose reductase, alrestatin, in connection with the effects of
reactive oxygen species on capacitation.
It was found that aldose reductase was tyrosine-phosphorylated during capacitation induced in vitro,
which resulted, in 10ovo stimulation of its enzymaìic activiiies. Alrestatinlnhibited the capacitationinduced
tyrosine-phosphorylation of sperm proteins, the production of reactive oxygen species, and the
induction of hyperactivated motility by g\vo,35vo, and 90%, respectively.
These results strongly suggest that aldose reductase is activaied by the phosphorylation of its tyrosine
residue(s) and stimulates the production of reactive oxygen species, *tri.t - in turn accelerates
capacitation and induces hyperactivation of sperm motility.
133

251-18
CAN POST.THAW SPERM SURVIVAL BE IMPROVED BY USING A SPECIFIC
PORTION OF THE BOAR EJACULATE PACKED IN MINIFLATPACKTM
F. Saravial, M. Wallgrenl'3, A. Johannisson2, andH. Rodrígu ez-Martínezl
tDivision of Reproduction;
2Dept of Anatomy, Physiology and Biochemistry, Faculty of Veterinary
Medicine and Animal Sciences, sLU, uppsala, sweden; 3euality Genetics, Hörby, Sweden
E-mail: fernando.saravia @kv.slu.se
Cryopreservation of boar semen is, as yet, sub-optimal; with sperm survival usually below 507o when
either whole ejaculates or their sperm-rich fraction (SRF) are used, often packed in medium straws. Here
we present post-thaw survival results when using a specific portion of the boar ejaculate, packed in
MiniFlatPacksrM (MFP), and cryopreserved either with a conventional or with a simplified handling
procedure, aimed at deep-intrauterine AL
Semen from 19 sires (103 ejaculates) was collected as the SRF or as Portion 1 (P1, the first 10 mL
from SRF). Semen from SRF or P1 was frozen either conventionally (Saravia et a1.,2005) (SRF-CF, pl-
CF), or using a simplified protocol (SRF-SF and PI-SF; Saravia et al. Reprod Domest Anim 2008 43:64)
and packed in MFPTM. After thawing (35'C for 2O sec), sperm motility was evaluated by CASA while
plasma membrane integrity (PMI, SYBR-L4/PI), stability (Annexin-V/Pl) and chromatin structure
(Acridine Orange) were checked by flow cytometry. Data were examined by ANOVA using GLM or
PROC MIXED procedures.
Post-thaw motility was significantly (P

2s1-19
RELOCATION OF SWINE GENETICS USING EMBRYO TRANSFER
s.L. Terlouwr, c.D. Bierman',D.L.Kohlerr2, B.A. Didionr, and J.R. Dobrinskyr
tMinitube International Center for Biotechnology, Mt. Horeb, wI, USA and zBabcock Genetics Inc.,
Rochester, MN, USA
Consistent swine production requires stable health status. Stable health status is defined by
economically viable production in the presence of known and unknown pathogen(s). Genetic change or
introduction of replacement stock can expose farms to health risks that can potentiaily disrupt consistent
production. Therefore, genetic advancement may be limited by the health status of the genetic supply
herd due to known or unknown pathogen(s). Our objectiv" *ur to use embryo transfer to avoid
transmission of unknown pathogens during genetic relocation of healthy animals.
Forty genotype specific (GS) donor females were scheduled for thrle sessions of embryo recovery in
six week intervals using Altrenogest (Matrix@, Intervet, Millsboro, DE), 1250 IU pregnant mare serum
gonadotropin (Sigma, St' Louis, MO) and 750IU þCG
(Chorulon@, Inrerver, Millsboro-, DE). Single-sire
GS matings were made 34 hours after Chorulon@ injection. To accomplish single-sire transf-ers, color
specific (CS) supplemental embryos were used to assist in maintenance of recipient pregnancy. CS
embryo donors and GS embryo recipients were synchronized with Matrix@, p.G. 60õt 1zo'o ru hCG, 400
IU PMSG, Intervet, Millsboro, DE) and Chorulon@. Embryos from GS donors were suigically recovered
on day 5 post-insemination, washed per IETS requirements using a zwitterion buffered culture medium
(PorcPro E-Blast, Minitube of America, Verona, WI) and transpãrted in a portable incubator (Minitube
of America, Verona' wÐ 2.5 hours to the recipient herd. Embryãs from CS donors were recovered at the
recipient site as described above. Embryos, GS and CS, were surgically transferred into minus 24 hour
asynchronous recipients within 6-14 hours after recovery.
A total of 620 (10.3t4.9) embryos were recovered from 65.27o (60192) of GS matings and shipped to
the recipient site. A total of 587 (9.3¡4.1, 59.2vo) GS and 402 (6.4t3.0, 40.gvo) c-S embryos were
transferred into 63 recipients for an average of 75.1t2.0/recipient. A total of 33 GS embryos were
discarded prior to transfer. To achieve ararget of 17 embryor pãt Íansfer, 59 (93.6Vo) embryo transf-ers
required CS embryos (range, 2-13) in addition to GS embryos (range, 2-I5),4 embryo transfers were GS
only embryos (range, 15-23). Fifty three (84.lvo) recipienti were cãnfirmed pregnani by ultrasound at 35
days of gestation. of the 40 GS donors, 1 was cullãd for genetic ,"uronr, o ãi¿ noi give transferable
embryos, and 1 gave transferable embryos but the recipient returned to "orr"rponding
ã.t*, for a total
genetic transfer rate of 80Eo (32/40). After three sessions oÌ emuryo transfer, 32/33 (g7vo) GS donors that
produced embryos for transfer were represented by a minimum of I pregnant recipient at 35 days of
gestation; 1l/32 GS donors are represented by single pregnancy and l5/i2 Ëy multiplè pregnancies. Þifty
two (82'5vo) recipients fqowed
429 live pigs (8.25t2.5) consisting of 23I cs 6.+i*2.s, 53.BVo) and
198 (3'81t2.4,46.2Vo) CS. Gestation length, interval from hCG to fànowing, was 118-r1.6 days. Serum
from GS donors was evaluated for porcine Reproductive and RespiratoÐ, syndrome (pRRSV) and
circovirus
fot:tl:
type 2 (PCV2) prior to each embryo recovery sessi,on. Seiology resulrs iere negative
for PRRS (0/98)) and27 -Svo (27/98) were positive roi pcvz. Embryo wash medium from pCV2 positive
GS donors delivering embryos for transfer was pooled from the lasi 2 washes and individually evaluated
for PCV2 after transfer, all samples tested negãtive (0/18). Day 5 zona pellucida intact embryos were
successfully used to relocate swine genetics from the donor herd into a récipient herd with no observed
health status change in the recipient herd-
135

251-20
INTRA.CERVICAL PIG AI WITH TWO DIFFERENT SPERM CONCENTRATIONS
M. Wallgrenl'2 and N. Lundeheim3
rQuality Genetics, Råby 2003, 262 94 Hörby, 'Division of Reproduction, Dept of Clinical Sciences, Box
7054, 3Division for Pig Breeding, Dept of Animal Breeding and Genetics, Box 7023, SLU, 150 07
Uppsala, Sweden
E-mail : Margareta.Wall gren @ kv. slu.se
Crises (epidemics, fire etc) at an AI stud can result in limited semen supplies, leading to the
following question: can the concentration of spermatozoa (spz) in Al-doses for standard (intra-cervical)
insemination, be reduced without severely impairing fertility Today, the standard AI dose in Sweden is
comprised of a total of 2.3 x lOe spz, derived from 3-6 boars (mixed ejaculates), suspended in 80 mL
extender. To answer the question given, fertility results following AI using standard doses (2.3 x 10e spz;
C= Control) were compared to those after using doses containing 1.8 x 10e spz; X= Experiment).
For each produced X-batch, a corresponding C-batj:h, including semen from the same boars, was
produced on a weekly basis using two extenders (X-celltM or BTS+@, IMV, LÁigle Cedex, France) at a
iommercial AI stud using establiihed AI boars. X-doses were prepared on Fridays, extended in X-cellrM
and kept in the dark at 18-20' C until delivery to the farm. The next Monday morning, C-doses extended
in BTS, were prepared. The X- and the C- doses were considered of equal quality regarding sperm
viability at the time of AI (DeAmbrogi et aI. 2006, Haugan et al. 2007). Doses were coded and sent
simultaneously to the farm (a sow pool; 1,600 sows). Sperm concentration in all semen batches was
verified, using a Bürker haemocytometer. Data related to that Al-dose was discarded when results
deviated >I07o from what was expected. The Al-doses.were used within three days after delivery to the
farm, where inseminations were performed solely on sows (i.e. no gilts) by the herd staff as praxis. The
resulting data was retrieved and analyzed using the SAS statistical package. Data covering 6l weeks of
inseminations, a total of 3,534 AIs and resulting in 3,269 litters, were analysed by ANOVA (PROC
MIXED; total born, born alive) and logistic regression (GLIMMIX; return to oestrus within the interval
18-48 days after AI). The statistical model included fixed effects of parity number, AI group (C or X),
interactions between parity number and AI group, and the random effect of insemination week.
AI doses with the reduced number of spz resulted in a significantly lower litter size (-0.3 total piglets
per litter, P < 0.01, -0.3; born alive piglets per litter, P

251-2t
STUDY OF DIFFERENT METHODS TO EVALUATE ACROSOME INTEGRITY IN
DILUTED BOAR SEMEN
s. williamst'',v. Fernándezr, D. Gabilondo2, E. valetter, and R.L. de la sotar
tcátedra y Servicio de Reproducción Animal ,tcártedru d,e Zootecnial, Facultad de Ciencias Veterinarias,
Universidad Nacional de La Plata, La plata, Argentina
E-mail: swilliams @fcv.unlp.edu.ar
To evaluate the potential fertility of a boar, the minimum necessary analysis concer-ns semen quality
(concentration, motility, and morphology). As these parameters have limitations, other analyses includL
the evaluation of the sperm membrane and the integrity of the acrosome, that is essential to the
occulrence of the oocyte fertilization. The objective of.this study was to determine capacity of two
different methods to evaluate acrosome integrity in diluted boar semen.
Sperm-rich fractions of semen were collected from commercial genotype boars that were greater than
2 years of age. Boars were housed in 6m2 individual pens with solidfloor and fed a commercial balanced
diet. Semen was collected by gloved-hand technique and filtered with gauze during collection. For each
ejaculate, semen quality was evaluated by percentage of progressively motile spermatozoa and
percentage with normal head and tail morphology. Semen concentration was calculated by a manual
count of sperm cells on a hemacytometer Bürker chamber. Semen was then diluted using the commercial
extender MR-A@ (Kubus, SA, Spain). Viable and non-viable spermatozoa were identified by eosin
staining. Samples were prepared by mixing an aliquot of spermatozoa suspended in saline medium with
eosin for 30s before preparing a smear and drying on a warm plate at 37'C. E¡a.ulates that showed
progressive motility >75Vo, normal spermatozoa > 80Vo and viable cells > 70Vo were used. Acrosomal
integrity was evaluated by phase-contrast microscopy on samples previously diluted with an g Vo
gluteraldehyde solution and by fluorescence stain using pisum saiivum- agglutinin (pSA; Sigma L O:.70).
An aliquot of each ejaculate was treated with cycles of freezing to -20"C and thawìng to room
temperature to deliberately increase the number of abnormal acrosomes, and these samples were used as
control to test the sensitivity of the protocols. Analysis of sensitivity and predictiv" uulu" for a positive
result was performed on phase-contrast and PSA techniques. Values *.r"
"ãrnpured
using ¡r
The sensitivity of acrosomal integrity analysis by phase-contrast was glVo and 89Vo when using the
PSA fluorescence staining. Meanwhile the predictive value of a positive result (PpR) was 50Vo and,53Vo
when using phase-contrast or PSA staining, respectively. No statistically different differences were found
between the two procedures (P

252-tt
BIRTH WEIGHT IMPLICATIONS FOR POSTNATAL DEVBLOPMENT IN PIGS
F.R.C.r. Almeidal,4.L.N.Alvareng-ar, G.G. parreirat, D.o. Fontes2, G.A. Marquest, p.C.
Cardeal1, L. Moreirar, G.R. Foxcrofi3, and H. Chiarini-Garcial
I Laboratory of Structural Biology and Repr-oduction, Department of Morphology, Federal University of
Minas Gerais, Belo Horizonte, MG, Brazil;2 Faculty of Veterinary Medicine, Department of Animal
Science, Federal University of Minas Gerais, Belo Horizonte, MG, Brazll; ' Swine Reproduction-
Development Program, Swine Research and Technology Centre, University of Alberta, Edmonton, AB,
Canada
E-mail : falmeida @ icb.ufmg.br
Selection for prolificacy appears to have created an imbalance between the number of conceptuses
surviving to the post-implantation period and uterine capacity. Limited placental development, due to
intra-uterine crowding in early gestation, results in entire litters with characteristics of Intra-Uterine
Growth Restriction (IUGR) at birth. The present study confirmed and extended evidence for IUGR in
light-weight offspring.
New-born male pigs (n = 80; DanBred X PIC terminal line), born to 4'h- 6th parity sows and in litters
of 10 to 15 pigs, were identified as falling into two birth weight (birth-wt) groups: high (HW: range 1800
¡o 2200 g) and low (LW: range 800 to 1200 g) birth-wt littemates. One sub-set of 20 animals from each
group was necropsied at birth and the heart, pancreas, liver, spleen, small intestine, large intestine,
kidneys, testes, semitendinosus (semi-ten.) muscle, and brain were weighed. Evidence of IUGR was
analyzed by comparing brain:organ wt ratios. Data were analyzed as a fully randomized design and the
comparison between means was performed by t-test.
All organs of H'W pigs were heavier than in LW counterparts (P

252-12
CHARACTERIZATION OF ITIHI,ITIH3 AND ITIH4 GENES AND THEIR
ASSOCIATION WITH REPRODUCTIVE TRAITS IN PIGS
I' Balcellsl, A. castellór, R. pena2, c. óvilo3, A. sánchezr, and A. Tomásr
rDepartament de Ciència Animal i dels Aliments, Facultat de Veterinària, UAB, Bellaterra, Spain
2Genètica i Millora Animal,IRTA, Lleida, Spain
3Deparramento de Mejora Genética Animal, bclf-Wn, Madrid, Spain.
Reproductive traits, such as prolificacy, are complex traits of economical interest for swine breeders.
v/ith the aim of studying the genes related wittr prótificacy in pigs, an F2 intercross was created from 3
Iberian boars and 18 Meishan sows. These two parental breeds shãw highþ divergent phenotypic records
for this trait, with mean numbers of piglets born alive of 7 and 14 for the Iberian und M"irhun breeds,
respectively' The inter-alpha-trypsin inhibitor heavy chains I (ITIH\),3 (|TIH3), and 4 (ITIH4) genes
have been related with the stabilization of the cumulus extracellular matrix and with the maintenance of
the uterine glycocalyx.surface during placental attachment. For these reasons, these genes were selected
as physiological candidate genes for prolificacy traits.
cDNA sequencing and quantitative PCR were performed to search for polymorphisms or variable
gene expression that could explain the differences in prolificacy of the F2 iows. soïs were classified
into 3 groups according to the number of embryos NÊ) at the iacrifice dãy ß0-32 days of gesration):
low (NE

2s2-13
DIFFERENTIAL EXPRESSION AND LOCALIZATION OF CX43IN MALE AND
FEMALB GONADS OF NEONATAL AND IMMATURE PIGS AFTER IN UTERO
EXPOSURE TO AN ANTI-ANDROGEN, FLUTAMIDE
B. Bilinskal, L Koperar, M. Durlejr, A. He¡me¡t, K. Knapczykl, M. Slomczynskar, and M.
Koziorowski2
iDepartment of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow;
2Department of
Physiology and Animal Reproduction, University of Rzeszow, Poland
E-mail: bbili @ zuk.iz.uj.edu.pl
Connexin 43 (Cx43) is one of the predominant gap junction proteins in the gonads. The physiological
significance of the gap junctional coupling in testes is attributed to the coordination of the secretory
activities of Leydig cells in the interstitium and the capacity for interactions between Sertoli and germ
cells, whereas in the ovary junctional coupling is important for proper interactions between granulosa
cells and the oocyte. The objective of this study was to show the role of androgen receptors (AR) in
mediating the action of androgens on gap junctional communication in testes and ovaries of neonatal and
immature piglets exposed in utero to an anti-androgen, flutamide.
To test the influence of flutamide on Cx43 gene expression in porcine gonads, nine-month-old pigs
were injected starting at 20 or 80 day of pregnancy with a dose of 50 mgikg body weight or vehicle (flax
oil), five times, every second day. Thereafter, testes and ovaries were isolated from two-day-old and
three-month-old boars and gilts. Immunohistochemistry (IHC) was performed on tissue sections using a
polyclonal rabbit antibody against Cx43 followed by an anti-rabbit IgG and the standard AB-HRP
complex. Bound antibody was visualized by diaminobenzidine. Tissue homogenates were used to assess
the presence of Cx43 by Western blot analysis. For this, proteins (20 pg/lane) were subjected to
electrophoresis on 127o SDS-PAGE under reducing conditions and transferred to nitrocellulose
membranes. To show Cx43 mRNA in the male and female gonads RT-PCR was performed. Total
cellular RNA from porcine gonads was isolated using.Nucleo Spin RNA II. Complementary DNA was
synthesized using MMLV reverse transcriptase. Next, PCR amplification was performed. All PCR
products were electrophoresed on agarose gels and visualized with UV light, following ethidium
bromide staining.
Immunohistochemical analysis revealed Cx43 protein to be distributed ubiquitously in the interstitial
area between Leydig cells of the testes and in granulosa cells of primary ovarian follicles in both
control- and flutamide-in utero-treated piglets. Strong staining was confined to testicular Leydig cells
and to the ovarian interstitial cells surrounding clusters of oogonia in the cortex. In Western blot
analysis, Cx43 appeared as several bands including a band of 43 kDa. Screening for Cx43 expression
revealed the presence of a transcript in all the tissues examined. Electrophoresis revealed PCR products
of the predicted sizes; 232 bp for Cx43 and 220 bp for GAPDH. However, due to the semi-quantitative
nature of the PCR method applied, we were not able to determine the exact amount of the transcript.
Since we observed the presence of the Cx43 protein and its mRNA in the male and female gonads of
both control and flutamide-in utero- exposed pigs it seems possible that androgens acting through the
AR receptors are not involved in the control of Cx43 gene expression in neonates, whereas in immature
pigs differential expression of Cx43 likely indicates androgen-dependent alterations.
Supported by the Ministry of Science and Higher Education - Grant N N303 339835 and by The
Foundation for Polish Science - Acqdemic Grants for Professors 2008 (to B.B).
140

252-14
DELAYED PIG EMBRYO TRANSFER USING A CHEMICALLY.DEFINED
CULTURE MEDIUM: A PRACTICAL APPROACH
P. Cherell, L. Letelul, J. Glenissonl, J. pirest, and p. Letelul
1FRANCE HYBRIDES, F 4580g Sr Jean de Braye, France
E-mail : pierre. cherel @ hendrix-genetics. com
Practical applications of embryo transfer procedures in pig breeding are hampered by transfer
procedure inefficiency, low embryo survival during freezing protocols, or difficult workload logistics
when transferring fresh embryo over short times (0-1 days). As a low-end alternative to a long sought
robust protocol of embryo freezing, we investigated here the potential of a delayed embryo transfei(4
days delay) allowing more flexibiliry in transfer schedules, using a surgical transfer procedure and a
chemically-defined culture medium.
Two groups of donor gilts, pre-pubertal cross-bred (FH100, FRANCE HYBRIDES: LW-DU
background genotype) or cyclic purebred (FH012, FRANCE HYBRIDES: Landrace type) were used to
collect Z-cellsl4-cells embryos. This followed 18 days of synchronization with altrenogest for cyclic
females, gonadotrophin superovulation treatment (PMSG 850 UI, 36 hours after lãst altrenógest
treatmen| hCG 500 UI, 48 hours following PMSG injection) and two artificial inseminations. Embryos
recovered by oviduct flushing 2 ro 3 days following hCG treatment, were cultured 96 hours in a
chemically defined culture medium (PZM4, as in Yoshioka et a1.,2002), under hypoxic conditions (5Zo
02, 57o C02,90Vo N2). Groups of 14 to 27 developing embryos (morula to early blastocyst stages) were
surgically transferred to altrenogest synchronized cross-bred recipients (FH300, FRANCE HyBRIDES :
LR x LW type recipients).
A total of 6 litters from 12 transfers were obtained from transfer of embryos of pre-pubertal gilts,
while embryos of cyclic gilts allowed production of 5 litters from 7 transfers. Reduceã litier sizes were
observed in both groups, with averages of 6.2 born alive (7.0 total born) in litters produced from prepubertal
embryos and 6.6 born alive (8.0 total born) in litters from cyclic gilts embryos. Altogether, these
results, produced in the context of a running nucleus herd, clearly point to workable solutions, likely to
comply with a range of health control regulatory standards, as using zona pellucida intact embryor und u
chemically-defined culture medium. A trend for lower competence of embryos produced fro- pr"-
pubertal gilts stems from the two groups comparison. However, in both cases, substantially reduced litter
size also suggests that the development potential of transferred (although morphologically normally
developing) embryos is clearly compromised by the culture conditions and/or collection and transfer
procedures. This calls for further improvement of basic IVC conditions of pig embryos, as a much
needed foundation for a whole range of embryo centered applications, including IVM and IVF
procedures.
141

252-15
DIFFERENTIAL micToRNA EXPRESSION AMONG NORMAL, ABNORMAL, AND
LOW MOTILITY PORCINE SPERM CELLS
E. Curryr, S.E. Ellisr, T.J. Safranski2, and S.L. prattr
rDepartment of Animal and Veterinary Sciences, Clemson University, Clemson, SC, USA
2Division of Animal Sciences, University of Missouri, Columbia, MO, USA
MicroRNAs (miRNA) are short (20-24 nt), non-coding, single-stranded, ribonucleic acids which alter
messenger RNA translation. miRNAs affect diverse biological processes such as insulin secretion,
adipocyte differentiation, B-cell development, and tumorigenesis, but little is known as to the identity of
miRNA in porcine gametes or their potential involvement in gametogenesis or reproductive processes.
Recent investigations have demonstrated that the miRNA synthesis pathway is necessary for
differentiation of mature sperm in mice; however, the specific miRNAs responsible for spermatogenesis
have not been identified. The objective of this study was to determine the identities and compare the
expression of miRNAs in normal porcine sperm cells, sperm cells with abnormal morphology, and sperm
cells with low motility.
Total RNA enriched for small RNAs was isolated from three types of porcine sperm cells using the
mirYanarM miRNA Isolation Kit (Ambion Inc., Austin, TX). The control iample (C) consisted of RNA
from sperm cells of normal motility and morphology, the abnormal sample (AB) included RNA from
morphologically abnormal sperm cells with cytoplasmic droplets, and the low motility sample (LM)
exhibited a low percentage of forward moving sperm cells. Each sample was hybridized to commercially
available anays (LC Sciences, LLC; Houston, TX) consisting of 1097 known miRNA sequences from 8
different species and, because many miRNAs are highly conserved among species, allowed for efficient
hybridization. Tag detection was determined using fluorescence labeling with tag-specific dyes. Data
were analyzed by first subtracting the background and then normalizing the signals using a LOWESS
filter (Locally-weighted Regression) to compensate for the intensity difference between Cy3 and Cy5.
The ratio of the two sets of detected signals (log2 transformed, balanced) and p-values of the t-test were
calculated. Differentially detected signals were those ryith p-values less than 0.01.
Of the 1097 miRNAs probed, results showed that 62 miRNAs were differentially expressed between
control and AB (5.77o), with 38 upregulated in AB and 24 downregulated. There were 66 miRNAs
differentially expressed between control and LM (6.0Vo), with 33 upregulated in LM and 33
downregulated. Forty-eight miRNAs were differentially expressed in both the AB and LM samples, with
3 miRNAs (miRs-25, -92, -92a) upregulated in the AB sample but downregulated in the LM sample.
Interestingly, numerous miRNAs that were differentially expressed (i.e. ler7i, miRs-l5b, -150, -34b, -
182) are predicted to target messenger RNA that code for human proteins involved in sperm structure
and/or motility (carbonic anhydrase, isocitrate dehydrogenase, glycogen synthase kinase, motile sperm
protein domain 1, and Rho GDP-dissociation inhibitor 1).
Altered miRNA expression was associated with, and may result in, aberrant spermatogenesis in boars.
Although there is no single cause for male infertility, the identification of specific miRNAs that are
associated with sperm motility, morphology, or structural integrity could lead to the development of a
microarray-based diagnostic assay to provide an assessment of male fertility status. Future studies are
being directed at validating miRNA expression levels from different boars in each group via qRT-PCR
and confirming messenger RNA targets.
The authors would like to thank Premium Standard Fanns (Princeton, MO) for providing the semen
samples.This project was supported ín part by National Research Initiative Competítive Grant no. 2007-
35203-18275 fromthe USDA Cooperative State Research, Educarion, and Extension Service.
t42

252-16
SUCCESSFUL PRODUCTION OF PIGLETS DERIVED FROM IVF OOCYTES
MATURED IN GONADOTROPIN.FREE CHEMICALLY DEFINED MEDIA
H. Funahashil, Y. Akakir, and K. yoshioka2
rDepertment of Animal Science, okayama University, okayama, Japan;
2National Institute of Animal
Health, Tsukuba, Japan
To induce meiotic resumption, it is thought to be necessary to expose porcine oocytes-cumulus
complexes (OCC) to gonadotropins during maturation. Howeïer, thé Oetaited mechanism is still
unknown and- any piglet production has not been reported by using IVF oocytes matured in
gonadotropin-free media.-The p-resent study was undertakeito obtain piglets"from thoseiocytes.
oCC were aspirated from 3-6mm diameter follicles of prepuberal ovaries and used in the current
study. Basic culture medium was a chemically defined -"ålu-, porcine Oocyte Medium (pOM;
Research Institute for the-Functional Peptides,_Yamagata, Japan). oCC were "*por"á
to EGF (10 ng/ml),
amphiregulin(1^000ng/ml) and dbcAMÞ (1 mM) duãng tnó tist 20 h of tvvt in ân armosphere of 5vo
COz in air at 39"C and then continued the culture in thJabsence of EGF-like factors and dbcAMp in the
same atmosphere and temperature. Control oocytes were cultured using a standard IVM protocol that
replaced EGF and amphiregulin with 10 iu/d èCG and 10 iu/ml hCGl The oocyres were co-cultured
with fresh spermatozoa for 8 h in a chemically defined IVF medium, porcine Gamete Medium (pGMtac4;
Research Institute fo¡ lhe
Functional Peptides, Yamagata, Japan) in an atmosphere of 5Vo CO2in air
at 39'C and then cultured in a chemically defined culturã rnedium, Porcine zyþote Medium (qZM-S;
Research Institute for the_Fu¡ctional Peptides, Yamagata, Japan) in an atmosphãre of 5Vo 02,5Vo COz
and 90vo N2 at 39"C for 5 days. Blastoõysts were su-rgically transferred into the uterine horn of three
recipients (18-19 blastocysts/recipients) whose estrous cycles were synchronized at 5 days after human
chorionic go-nadotropin (hçG) injection. Pregnancy diagnosis was óarried our by ultraÉonography 21
days after.hCG injection. Statistical analyses was perforãred by ANovA with Bónfe¡oni/Dñnn"s post
hoc test (significance, P

252-17
EXPRESSION OF HOXA1O IN BARLY PREGNANT ENDOMETRIUM AND
CONCEPTUS IN PIGS WITH NATURAL AND HORMONALLY INDUCED
OVULATION
M.M. Kaczmarek, D. Balcerowicz, J. Kiewisz, A.J. Zieclk, and A. Blitek
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland.
E-mail: agas @pan.olsztyn.pl
Proper development of endometrial receptivity, controlled by ovarian steroids, is required for
successful embryo implantation. Homeobox (Hox) 410, a member of abdominal subclass of homeobox
genes functioning as transcription factors, is responsible for uterine homeosis during development.
Interestingly, HOXAI0 has been demonstrated in adult human endometrium and regulated by estradiol
and progesterone. Peak expression of HOXA1O during the window of implantation suggests important
role for this protein as a potential marker of uterine receptivity (Daftary and Taylor 2006). However, the
role of HoxAlO in adult porcine endometrium is not known. Therefore, the present studies were
undertaken to examine the expression of HoxAlO in endometrium and conceptus of early pregnant pigs
with natural and hormonally stimulated ovulation.
Pre-pubertal crossbred gilts (5-6 months of age) of similar weight were divided into two experimental
groups - with natural (n=22) and hormonally induced (750 IU of PMSG and 500 IU of hCG 72 h later,
n=19) ovulation. Endometrial tissue samples were collected on Days 10, 17, 12, and 15 after
insemination. Conceptuses were flushed from uteri with sterile phosphate buffered saline and based on
their morphology classified into following days of pregnancy: Days 10-11 (spherical and tubular), Day
12 (filamentous), and D 15 (elongated). Expression of HoxAlO mRNA, and protein was determined
using Real-Time PCR and Western blot techniques, respectively. Additionally, blood samples were
collected for EIA of progesterone. Results were analyzed using two-way ANOVA followed by
Bonferroni's post hoc tests.
Serum progesterone content did not differ through the studied period of early pregnancy, however
decreased level of this hormone was detected on Day 12 in gonadotropin stimulated gilts in comparison
to non-treated animals (P

252-18
INFLUENCE OF EMBRYONIC FACTORS ON LYSOPHOSPHATIDIC ACID
RECEPTOR 3 GENE EXPRESSION IN PORCINE ENDOMETRIUM DURING THE
PERIIMPLANTATION PERIOD AND CORRESPONDING DAYS OF THE ESTROUS
CYCLE
K. Kamiúska, M. Wasielak, and M. Bogacki.
Institute of Animal Reproduction and Food Research of Polish Academy of Science, Tuwima 10, l0-.,4j
Olsztyn, Poland
E-mail: katka@pan.olszfyn.pl
our previous sfudies indicated that like in rodents, lysophosphatidic acid recepror 3 (LpA3), plays an
important role during early pregnancy in pigs and embryonlc signáts may influence receptor LpA3 expression
in the porcine endometrium. ciltt suuiectãa to surgical ligatún of the one uterine horn were used in the
present study' In the unilaterally d14 pregnant (n4) ãnd cyãic gilts (n=4) the correlations between LpA3 and
E2 synthase (PGES) prostaglandin rzì sptlase (PGFS) ;RNÀ revels in the endomerrium were invesrigated
as well as the concentrations of prosøglan din9-2 (PGE2), prostaglandin F2u (pGF2o) and estradiol (E2)
endometrium in the
and uterine flushings. Àdditionally, tt e of p'otential embryo products "tr*ct
such as pGE2, interferon y (IFNy) and Fo,
interleukin-6 (IL6) on LPA3 g"n" was examined
endometrium ""pr"rrion
in the porcine
in an in urTro system.
The mRNA expression levels of LPA3, PGES, and PGFS were determined using real
contents
time
of
RT-pcR,
E2 were determined using RIA and PGE2, PGF2ü using ELISA. To
influence
úamine the porential
of embryonic factors on ,J""pto, LPA3 mRNA expression in the endometrium,
explants
endometrial
recovered from pregnant (l4d) ánd cyclic (14d) gilts, were incubate d. in vitro
Preliminary with various factors.
studies were carried out to determine the optiLal dose of the investigated
incubation
factors and
time
length
for the
of
proper experiment. In the proper experiment, 6 hours incubation
significantly
and doses which
changed the level of rpa: werË 'RNA
app[àa. Thus, tissue explanrs
rocking
were incubated
and
with
rreared separarery with pGE2 (10nM), a ii0oMl, IFNy (5¡rg/rnr), and IL6 (rpg/rnl)
atmosphere in an
of 5vo carbon dioxide in air at 37oc for 6 hours. Tíie RNA was extracred and
transcript
levels
were
of LpA3
determined using e_pCR.
In unilaterally pregnant gilts, a negative correlation (R2=0.52 p

2s2-19
EFFECT OF INTRAUTERINE CRO\ryDING ON FETAL DEVELOPMENT ON DAY 40
OF PREGNANCY
B. Kempl, N.M. Soeclet, E.H. van der Waaijl, and W. Hazelegerl
lAdaptation Physiology Group, wageningen university, wageningen, The Netherlands
E-mail: Bas.Kemp @wur.nl
Due to selection for increased litter size in pigs, uteri have become more crowded. There are
indications this has a negative effect on fetal development. Sensitive periods with respect to embryo
mortality are the period immediately after fertllization of the oocytes (quality of fertilization), the period
around Day 12-14 of pregnancy (success of implantation in the uterus) and the remainder of pregnancy
(consequences of uterine crowding and placental quality). The objective of this study was to investigate
the effect of intrauterine crowding on fetal development atday 40 of pregnancy.
Fifty-five commercial crossbred cyclic gilts were subjected to hormonal treatment to synchronizethe
estrous cycle and to achieve crowded uteri. The oral progesterone analogue altrenogest was given for 18
days, followed by eCG (1000 i.u. Folligonan) 24h after last altrenogest and hCG (500 i.u. Chorulon) 3
days later. Gilts were inseminated during oestrus, at 24h and 40h after hCG and slaughtered at days 39,
40, or 4I of pregnancy. At slaughter, the number of corpora lutea (CL) and the number of vital and dead
fetuses was counted, the length and width of the implantation sites and distance between implantation
sites were determined, and for each fetal-placental unit, fetal weight, placental weight, and placental
length were determined. Data were analysed at the fetal level, with gilt as a random effect in the model.
Number of CL ranged from 22 to 16, total number of implantations ranged from 12 to 49 and the
number of vital fetuses ranged from 10 to 24. An increase in number of CL resulte d in a 0.29 times lower
increase in total number of implantations, but not in an increase ìn number of vital fetuses. In other
words, the total number of implantations present had no effect on the number of vital fetuses to day 40 of
pregnancy. On average, vital fetuses weighed 11+ 2 grams, their placenta weighed 32 x. 15 grams and
was 26 t 10 cm long, their implantation site was 12 +- 5 cm long, and their implantation site was at a
distance of 9 + 5 cm from neighbouring implantation sites (e.g. 2 times 4.5 cm). Vital fetuses in a uterus
with more than 24 implantations in the uterus on average had a 4 cm shorter implantation site, a 6 g
lighter and 7 cm shorter placenta, and a 0.4 g lighter weight than vital fetuses in a uterus with fewer than
24 implantations. Fetuses at the beginning or end of the uterine horns on average had an up to 2.5 times
higher probability to survive until day 40 of pregnancy, a 2 cm longer implantation site, 8 g heavier and
10 cm longer placenta than fetuses with two neighbors, especially if those two neighbors were dead. Vital
fetuses in a uterus with a high rate of vital fetuses on average had a 7 cm larger and 1 6 g heavier placenta
than vital fetuses in a uterus with a higher rate of non-vital implantations.
In conclusion, the late embryonic mortality that occurs in crowded uteri (i.e. non-vital implantations
or fetuses) negatively affects the surviving fetal-placental units at Day 40 of pregnancy.
t46

252-20
THE EXPRESSTON oF \ryNT 4, wNT sA, \ryNT 74, Þ-CATENIN, AND B-caDHERrN
GENES IN THE ENDOMETRIUM DURING TTTN ÉiiPNODUCTIVE CYCLE AND
EARLY PREGNANCY IN PIGS
J. Kiewisz, M.M. Kaczmarek, A. Blitek, G. Bodek, and A.J. Ziecik
Institute of Animal Reproduction and Food Research Polish Academy of Sciences, olsztyn, poland
E-mail : kiewisz @pan.olsztyn.pl
-The Wnt are morphogens that play an important role in cell fate, growth, differentiation, and cell-tocell
contacts' They can activate transãucdon iignaling pathway which leads to accumulation of B-catenin
(CNNTBl) in cells' The intracytoplasmic B-catenin binds E-ðadherin (cDHl) rhat is atached to the E-
cadherin in an adjacent cell. All oi these molecules are thought tá be involveá in endometrial-conceptus
interactions in animals characterized by invasive o, non-inouäu" fru""ntution. Thus, in thl present study,
we compared the expression of wnt 4, w1t 5a, wnt 7a, B-catenin, and E-cadherin genes in the
endometrium during the reproductive cycle and early p."gnun"y in the pig.
Thirty crossbred gilts at the third "rtrou,
cycle were divi¿eã into a pregnant (artificially inseminated)
and-a non-pregnant group,(non-inseminated). on day(s) 9 (n=5),72(n=5), and l5-16 (n=5) ofthe estrous
cycle or pregnancy' samples of endometrial tissue r"ré "oìt".táã.
rn" profile of wnt 4, wnt 5a, wnt 7a,
B-catenin, and E-cadherin gene expression was established using the Real-Time pCR method. Results
were analyzed using rwo-way ANovA folrowed by Bonferron|s lost hoc tests.
Expression of wnt 4, p-catenin, and E-cadherin was maintåined at constant levels that were not
affected by day or by reproductive status. The expression of wnt 5a gene was notably influenced by
reproductive status (P

252-21
DEVELOPMENT OF NEONATAL PIGLETS FOLLOWING RECIPROCAL EMBRYO
TRANSFERS BETWEEN MEISHAN AND WHITE CROSSBRED GILTS
J.R. Miles, J.L. vallet, J.J. Ford, B.A. Freking, R.A. cushman, w.T. oliver, and R.K.
Christenson
USDA-ARS, U.S. Meat Animal Research Center, Clay Center, NE 6g933
Sow productivity has a significant economic impact on the swine industry and is influenced by a
number factors including preweaning piglet mortality. In Western breeds, low birth weight piglets extri¡it
the greatest susceptibility to prewean mortality. However, despite their overall decreãsed weights,
Meishan (MS) piglets have lower preweaning mortality rates. The objective of the cur¡ent study rã* to
determine the contributions of the piglet and maternal genotypes and their interactions on the
development of neonatal piglets pertaining to preweaning survival using reciprocal embryo transfer
between MS and White crossbred (WC) gilts.
Twenty-five successful pregnancies were produced by embryo transfer in two farrowing seasons. All
piglet and maternal genotype combinations were represented; MS x MS (n=4 litters), WÕ x WC (n=7
litters), MS x WC (n=7 litters), and WC x MS (n=7 litters). At approximately 24 h of age (day l), piglets
were weighed and a blood sample was taken. Hematocrit, hemoglobin, glucose, nitrogen, albumin, free
fatty acids, and cortisol were measured in all blood samples. In addition, representativepiglets from each
litter were sacrificed and body measurements (i.e. organ weights, tissue glycogen .õnt"nt, and body
composition) were determined. All data were analyzed for analysis of variance using MIXED model
procedures.
Both WC piglet (P=Q.Ql) and maternal (P=0.004) genotypes had significanr effects on piglet weighrs
demonstrating that not only were WC piglets heavier than MS piglets, but piglets from WC gilts were
heavier than piglets from MS gilts. In addition, WC piglets had increased (p=0.04) eviscerated body
weight compared with the MS piglets. However, total visceral weight prior to emptying the stomach was
increased (P=0.01) in the MS piglets despite no significant differences in organ .èights between WC and
MS piglets. Interestingly, stomach content weight was greater (P=0.02) in the IVIS piglets, suggesting
greater milk intake in MS piglets. Furthermore, the percentage of fat and nitrogen within tf,ð UoOy
composition were greater (P

,

)

252-24
SURVIVAL OF PIGLETS ACCORDING TO PHYSIOLOGICAL PARAMETERS AT
BIRTH
A. Panzardil, T. Bierhalsr, A.P.G. Mellagil, M.L. Bernardi2, F.P. Bortolozzol, and L Wentzr
rFaculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre,
RS/Brazil;2Faculdade Agronomia - UFRGS, Porto Alegre, RSÆrazil
E-mail : fpbortol @ ufrgs.br
Neonatal mortality makes a substantial contribution to swine production losses. Adaptation to extrauterine
life is a considerable challenge for the neonatal piglet, as evidenced by the majority of preweaning
deaths occurring within the first 72h post-partum. Newborn, and particularly low birth weight,
pigs are very susceptible to cold because of their low birth energy reserves and poor thermoregulation,
thereby affecting their survival rate. The aim of this study was to verify the impact of physiological
characteristics at birth on the survival ofpigs during the first week of life.
Pigs evaluated in this study were born from 56 Agroceres PIC sows of parity 3 to 5 in which
farrowing was induced. Stillborn and mummified piglets were recorded but they were not included in the
analysis. At birth, the following parameters were measured: heart rate (HR), blood oxygen saturation
(pSatO2), and blood glucose (BG) concentration. Rectal temperature was measured at 0h (RTO) and 24h
(RT24) after birth. Birth order (BO) was recorded and piglets also had their individual weight measured.
For each parameter, the 612 piglets were classified into three groups representing low (75Vo) quartiles. Mortality rates until 3 and I days after birth were
compared among these groups using Chi-Square test. Correlation between the parameters evaluated was
investigated with CORR procedure (SAS).
Mean values observed were 40 (t 16) for BG, 158bpm (t 56) for HP.,167o (t 12) for pSatO2, 37.6C
(t 1.4) for RT0, 38.4'C (t 0.8) for RT24 and 1518 g (t 3659) for birth weight (BW). Significant
correlations (P

)

252-26
CHARACTERIZATION OF PROGESTERONE RECEPTOR (PGR) mRNA ISOFORMS
IN THE ENDOMETRIUM OF CYCLIC AND PREGNANT PIGS
E. Sellnerl, D. Mathewl, J. Ross2, C. okamural, K. wellsl, R. Geisertl, and M. Lucyl
tDivision of Animal Science, University of Missouri, Columbia, MO;
2Department of Animal Science,
Iowa State University, Ames, IA
The down-regulation of the PGR within the luminal epithelium enables conceptus attachment and
signaling within the porcine uterus. In humans, three PGR mRNA isoforms (PGR-A, PGR-B, and PGR-
C) arise from alternative transcription start sites. These mRNA encode proteins with different N-termini
that may confer distinct biological functions. The objective was to identify PGR isoforms in the pig and
to characterize their mRNA expression in endometrium during the estrous cycle and pregnancy.
Primer sets for quantitative RT-PCR were developed from porcine genomic and mRNA sequences
and used to amplify porcine PGR fragments from cDNA. RT-PCR primers for PGR-B (5'-
TCAGACTGAAGTCGGGGAAC-3' AND 5'-GGGTGAAATCTCCACCTCCT-3') and PGR-AB
(region common to both PGR-A and PGR-B; 5'-GCTCCATGGTTCCACTTCTG-3' and 5'-
GATGGGCACGTGGATAAAAT-3') were developed to study PGR regulation in endometrial tissue
from cyclic (d 0, 5, 7.5, rc, n, ß, 15, 17) and pregnanr (d 10, 12, 13, 15, Ii) pigs (n = 53 samples;
minimum of 4 pigs per status and day). Statistical analysis was performed by using the Proc GLM
function of SAS v 7.1 with day, status and day by status interaction included as independent variables in
the model. Homology of mRNA and protein was determined by using the NCBI BLAST program
against Refseq RNA or Refseq protein databases, respectively.
Based on cDNA sequencing and porcine genomic sequence, the porcine PGR mRNA is 4.3 kb and
84Vo identical to human PGR mRNA. The porcine PGR protein is 938 amino acids in length and 84Vo
identical to human PGR protein. The porcine PGR protein was 75Vo identical to human PGR protein
within the AÆ (variable) region and was 97Vo identical to human PGR protein within the C, D and E
regions. There was a tendency for an effect of day on PGR-B expression (P < .10) because PGR-B
mRNA was more abundant on day 0 (0.52 t 0.07) and day 5 (0.51 t 0.07) compared with day 7.5 (0.31 +
0.07) and day 15 (0.30 -r 0.05). The remaining days were intermediate. Abundance of PGR-AB mRNA
remained low through day 13 (0.13 + 0.01; d 0 to 13; cyclic and pregnant) and increased on day 15 in
both pregnant (0.90 t 0.07) and cyclic (0.41 + 0.07) pigs (P < .001). The PGR-AB mRNA remained
elevated in pregnantpigs on d 17 (0.33 t 0.06).
We conclude that PGR isoform mRNA abundance may fluctuate during the estrous cycle and
pregnancy, leading to functional differences in PGR action.
This project was pørtially supported by National Research Initiative Grant no. 2007-35203-17836 from
the USDA Cooperative State Research, Education and Extension Service.
1s3

252-27
ESTROGEN RECEPTORS COLOCALIZATION IN THE PORCINE UTERUS
THROUGHOUT PREGNANCY
M. Slomczynskal, M. Dudal, K. Knapczykt, M. Durlejl, A. Tabecka-Lonczynska2, and B.
Bilinskal
rDepartment of Endocrinology & Tissue Culture, Institute of Zoology,Jagiellonian University, Kraków,
Poland. 2 Department of Physiology and Reproduction of Animals, University of Rzeszow, Rzeszow,
Poland
E-mail: slom @zuk.iz.uj.edu.pl
The uterus is a well-known target of endocrine, paracrine, and autocrine acting molecules among
which steroid hormones are of special importance. The objective of our work was to localize estrogen
receptors (ERc and ERp) in the pig uterus throughout pregnancy.
Porcine uteri were obtained from pregnant pigs (3 or 4 animals per each day) on days 10, 18, 32, 50,
71, and 90 post coitum (p.c.). Uterine horns were washed in PBS and cut into small fragments. They were
immediately frozen in liquid nitrogen (for RNA isolation and Western blot analysis) or fixed for
immunohistochemical localization of both ER types. Mouse monoclonal anti-human ERu antibody
(Dako A/S, Glostrup, Denmark) and mouse monoclonal anti-human ERB1 isoform antibody (Serotec,
Kidlington, Oxford, UK) were used. The intensity of immunoreaction in the luminal epithelium,
glandular epithelium, and myometrium was measured and expressed as an overall mean (+SEM).
Statistical difference was assessed by analysis of variance (ANOVA) with Tukey's post hoc comparison
test (p

252-28
IMPACT OF A PROGESTERONE ANALOGUE TREATMENT DURING EARLY
PREGNANCY ON LITTBR SIZE IN \ryEANED SO\ryS \ryITH AN EXTENDED
WEANING.TO.OESTRUS INTERVAL
N.M. Soede, W. Hazeleger, and B. Kemp
Department of Animal "Sciences,
Wageningen University, Wageningen, The Netherlands
E-mail: nicoline.soede @ wur.nl
Suboptimal plasma progesterone in early pregnancy is related with increased embryo mortality.
(Pharazyn et aL(199I)CJAS71:9491. Jindal et a/.(1996)JAS74:620; van den Brand et aL
(2000)JAS:78:405). Injections with progesterone in the critical phase resulted in increased plasma
progesterone and increased embryo survival in gilts (Jindal et al.(1997)IAS75:1063) bur nor in weaned
primiparous sows (Mao et al.(1998)JAS16:I922). The question is whether treatment with an oral
progesterone analogue would also increase embryo survival. However, in an earlier study, oral
progestagen treatment starting between 0 to 2 days after ovulation lowered embryo survival at Day 35 of
pregnancy (Soede et al.(2004)IPVS:492). In the current study, therefore, we examined effects of
progesterone analogue treatment starting at a later stage of pregnancy (day 4 to 6 after onset of postweaning
oestrus).
At a Dutch 1600-sow farm, 98 Dutch Landrace sows (parity 2.9 x.1.9; lactation length2T + 2 days
and 17.2 + 2.0 weaned pigs) were fed altrenogest, an oral progesterone analogue, as a top dressing on the
morning feed at Day 4 and at Day 6 after onset of the first post-weaning oestrus, at a dosage of 20mg
(ALT20; n=49) or 10mg (ALT10; n=49); Control sows (n = 51) were not treated (CTR). Sows with
similar weaning-to-oestrus intervals (WOI) of 6 days, 7 days, and 8 to 14 days were equally divided
âmong the treatments. This allocation accounted for previous farm records indicating that sows with
extended WOIs had a lower litter size (of 1.0 pig for WOIs of 6 and7d, and of 1.8 pigs for V/OIs of 8 to
14d) than sows with a WOI of 5 days (data not shown). Statistical analyses were done using the
GLIMMIX-procedure (chance of farrowing) and the GlM-procedure (litter size) of SAS9.1, taking into
account relevant factors. Parity (1, 2,3+), number of piglets weaned (10, 11, l2+) and lactation length
(27) of the preceding lactation were analysed as class variables. Means are presented as
mean+sD.
Treatment did not affect farrowing rate (90Vo for ALT20, 8l7o for ALT1O and 90Vo for CTR); neither
was farrowing rate influenced by WOI. Average litter size was ll.7 + 4.1 for ALT20, I2.3 -r2.9 for
ALT1O and 13.3 + 3.1 for CTR and the difference between ALT20 and CTR approached significance (P
= 0.10; corrected for parity). Litter size was not affected by WOI (from 12.3 + 3.1 for Day 6 to 12.4 ¡-
3.6 for Day 7 and 12.5 + 3.8 for Days 8 to 14; P = 0.90). Effects of treatment on litter size were not
affected by number of piglets weaned or lactation length of the preceding lactation
In contrast to expectations, CTR sows with a weaning-to-oestrus interval of 8 to 14 days did not show
a lower litter size, as seen on this farm previously. However, the experiment clearly indicates that
supplementation with a progesterone analogue at day 4 to 6 after onset of oestrus did not increase, but
even seems to decrease, fertility of weaned sows independent of their WOI; a dosage of 20mg tended to
decrease litter size by 1.7 piglets and the 10mg dosage showed intermediate results. The results,
therefore, support our previous negative responses to altrenogest supplementation (20mg) at days 1 to 4
or 2 to 4 after onset of oestrus (Soede et a1.,2004). Thus, further experimental studies are needed to
clarify timing and dosage of altrenogest treatment on ovarian, uterine, and embryo functioning before
progesterone analogue treatment can be considered for subfertile sows in practice.
155

)0.05). These results were not significantly affected by the timing of oestrus (lactational or not) or by
the duration oflactation (26 vs 33 d).
In conclusion, a total of 54 to 62Vo of IS sows showed lactational oestrus, with a low incidence in first
parity sows and no improvement by later onset of IS. Pregnancy rates and litter size were similar for IS
and control sows, regardless of the timing of oestrus. In contrast to expectations, ongoing lactation during
early pregnancy (IS19-14) did not negatively affect fertility. Despite promising results for IS sows thus
far, the lack in IS sows of consistent timing of lactational oestrus might limit practical use of IS to benefit
piglet performance after (postponed) weaning in this line of sows.
156

252-30
PRO-ANGIOGENIC AND ANTI-ANGIOGENIC FACTORS AND THEIR RELATION
TO PORCINE PREGNANCY SUCCESS OR FAILURE
A. K. Edwardsl, M.J. van den Heuvelr, B.A. Croyr'2, andC.Tayader*
rDepartment of Biomedical Science, University of Guelph, Guelph, ON, Canada, NlG 2Wl
2Department
of Anatomy and Cell Biology, Queen's University, Kingston, ON, Canada, K7L 3N6
-Presenting Author
Two waves of spontaneous fetal loss occur in North American pork breeds (Sus scrofa); one during the
peri-implantation period (-gestation day (gd) 20), and another at mid-gestation (-gd50-70). Insufficient
blood supply to the developing conceptus is thought to be a participating cause. Previous studies from
our laboratory have shown that a decrease in vascular endothelial growth factor (VEGF) family members
(primary pro-angiogenic family) is associated with conceptus growth arrest. We propose that a balance
between pro-angiogenic and anti-angiogenic factors are needed for successful conceptus growth. Proangiogenic
factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor
(PDGFB), and their receptors FGFRI, FGFR2, and PDGFRBB, along with anti-angiogenic factors
Thrombospondin-l, 2 (TSP-I, TSP-2), TSP receptor CD36, and endostatin were investigated. Expression
of these factors in endometrial and trophoblast samples was analyzed by quantitative real time PCR
during peri-implantation (gd20) and mid-gestational porcine pregnancy (gd50). Statistical analysis was
done using ANOVA on Ranks, with Dunn's post test. At gd20, endometrial samples from arresting
conceptuses had elevated transcripts for bFGF, and PDGtrB, while at gd50, bFGF, FGFR2, and CD36
were found to be elevated at arresting conceptus attachment sites compared to their healthy counterparts
(p

252-31
EFFECT OF EMPTY UTERINE SPACE ON PLACENTAL DEVELOPMENT,
FARROWING INTERVALS, AND STILLBIRTH
J.L. Vallet, J.R. Miles, T.M. Brown-Brandl, and J.A. Nienaber
USDA, ARS, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center NE
Prolonged farrowing intervals (FI) are associated with stillbirth and decrease as litter size increases,
but the reason is unclear. Our primary objective was to determine whether unoccupied uterine space
associated with small litters could present a barrier to delivery of piglets and increase FI. A secondary
objective was to determine whether fetal loss on day 35 of pregnancy influenced placental and fetal
development of the remaining fetuses during later gestation.
Empty uterine space was created in gilts by crushing I or 2 fetuses on either the ovarian (O, no
impediment to farrowing) or cervical (C, possible impediment to farrowing) end of uterine horns on day
35 ofpregnancy. This also provided a test of the effects of adjacent empty uterine space on placental and
fetal development. A subset (n = 27) of gilts were slaughtered at 105 days of gestation to determine
whether the treatment generated the required empty uterine space and to measure effects on the placenta
and fetus. The lengths of empty regions of the uterus were measured. Each fetus was removed and
weighed and the lengths of the ovarian- and cervical-facing halves of each placenta were measured from
the umbilicus; then, each placenta was weighed. The remainder of the gilts (n = 75) were farrowed and
monitored with24-h video. Each piglet's status (live, stillborn, mummy) and FI were recorded..Empty
uterine lengths, placental and fetal weights, and placental half-lengths were analyzed using PROC
MIXED. Farrowing intervals were analyzed using PROC MIXED and stillbirth rates were analyzed using
PROC GLIMMIX.
At slaughter, treatment successfully (P < 0.01) increased the length (cm) of empty uterine horn (O
fteatment,Oend=25.8+2.1 vs.Cend=4.6+2.0;Ctreatment,Oend='7.2+2.8vs.Cend=22.0+
2.7). This resulted in increased (P < 0.01) placental-half lengths (cm) adjacent to the empty spaces (O
treatment,Ohalf=21.4+1.2vs.Chalf=12.5+-1.2;Ctreatment,Ohalf=11.3+1.2vs.Chalf=19.2+
1.2). Placental and fetal weights were greater e f 0.05) for empty space adjacent compared with
nonadjacent fetuses. There were no effects of treatment on piglet FI or stillbirth.
These data clearly indicate that (1) placental potential is not fixed by day 35 ofgestation, (2) adjacent
empty uterine space is advantageous to developing fetuses after day 35 of pregnancy, and (3) empty
uterine space does not influence FI or stillbirth rates and therefore does not explain the effect of litter size
on Fl.
158

7

253-3
EFFECT OF SYNCHRONIZING OVULATION IN WEANED SOWS USING
OVUGELT'\ryITH SINGLE FIxED TIME AI oN PREGNANCY RATE AND LITTER
SIZE
J.N. Taibll, S.M. Breen', S.K.Webel2, M.E. Swanson2, and R.V. Knoxl*
lDepartment of Animal Sciences, University of lllinois, Champaign-Urbana, IL 61801-3939, USA;
2
Pennatek, LLC, Sheridan IN,46069, USA
E-mail : rknox @ illinois.edu
*Presenting author
Controlling time of ovulation could eliminate breeding based on estrus, and allow sows in weaning
groups to receive one AI for optimal fertility. A unique method for delivering the GnRH agonist
triptorelin (OvuGeFu; intravaginally has been developed for use in weaned sows (Pennatek, Sheridan,
IN). The treatment synchronizes ovulation in sows but the optimal time for AI has not been determined.
The objective of this study was to determine the effect of a single, fixed-time AI at different times
following OvuGel on pregnancy rates and litter size.
This study was performed in 3 replicates at an 800 sow research farm during the summer of 2007. All
replicates included estrus and ovulation data and two replicates included AI, pregnancy and litter results.
At weaning, mixed parity sows (n=32/tealment) received either vehicle carrier at 96 h post-weaning and
a single AI24h later (Placebo), OvuGel at96h post-weaning and a single AI 18 h later (OG18), OvuGel
at 96 h post-weaning and a single AI 24 h later (OG24), or OvuGel at 96 h post-weaning and a single AI
30 h later (OG30). OvuGel was administered -2 cm posterior to the cervix using the deposition catheter.
Estrus detection was performed once daily from d 3 after weaning until d 7. Ultrasound was performed
every 8 h from d 5 through d 6. On d 30 of gestation, the sows were sacrificed and reproductive tracts
examined. There was no effect of treatment on sows expressing estrus within 7 days of weaning (79Vo) or
on the wean-to-estrus interval (4.7 days). The percentage of sows that ovulated by 48 h after treatment
was greater (P = 0.005) in OGl8, OG24, and OG30 than in the Placebo treatment (Table 1). The interval
from AI to ovulation was different among treatment groups (P

253-5
THE EFFECT OF AGE AT FIRST ESTRUS AND BREEDING ON THIRD ESTRUS ON
MATURE BODY SIZE AND LONG-TERM REPRODUCTIVE PERFORMANCE OF
SOWS
J. Pattersont, E. Beltranena2, and G. Foxcroftl
tswine Reproduction-Development Program, Swine Research & Technology Centre, University of
Alberta, Edmonton, AB, T6G 2P5 2#204 J.G. O'Donoghue Building,7000 - 113 Street, Edmonron,
Alberta, T6H 5T6
One of the most critical factors driving the reproductive performance of the sow herd is gilt
development and management. Large variation exists with respect to the successful introduction and
retention of high value replacement gilts into the herd. Developing management practices that produce
gilts with the greatest potential lifetime performance is crucial to the productivity of conventional
production systems. Even minor improvements in gilt management can lead to large increases in
breeding herd efficiency by meeting replacement targets from smaller pools of "select" gilts with
improved lifetime performance. Therefore, the primary objective of this present study was to determine
the effect of age at first estrus and breeding at third estrus on mature body size and long-term
reproductive performance of sows.
The objective of this trial was to determine the effect of age at first estrus on mature body size and
long-term reproductive performance of sows. At approximately 100 d of age, prepubertal C22 gilts
(n=431) were originally allocated to trial, and at a pen average of 140 d of age, gilts began daily direct
contact with mature boars to stimulate onset of puberty. "Select" gilts (n=275,757o)were recorded as
cyclic by 180 days of age and were further classified on the basis of age at puberty into 3 groups: l)
Early Puberty (EP) (< 153 d of age) n=85; 2) Inrermediare Puberry (IP) (154 - 161 d of age) n = 140; or
3) Late Puberty (LP) (168 - < 180 d of age;n= 90). Gilts not exhibiting the standing reflex by 180 d of
age were considered Non-responders (NR) n = 81.
Mean d to puberty and age at pubeÍy attainment in each of the Select group classifications were: EP:
9.6 t 0.5 d and 147.4 + 0.5 d; IP: 19.3 + 0.5 d and 159.9 +- 0.3 d; LP: 33.8 + 0.7 and 175.1 + 0.6 d,
respectively. NR gilts that did not reach puberty within the allotted time were removed from the
intensive portion of the trial ar l':.9.5 {- 0.7 d. Body weight increased over the productive life of the sow,
and EP gilts were lighter than LP gilts at every measured event (P < 0.05). Plasma IGF-1 only differed
between classification groups at d 100 (EP: 166.8 + 4.0, IP: 154.6 ¡3.2 andLP: 142.6 t a.0) (P < 0.05).
Fewer NR gilts (73.0 7o) originally on inventory were bred than were EP (97.'7 7o),IP (93.2 Vo) orLP
(93.0 7o) gilts (P < 0.05). Numbers born were not different between treatment classifications and
increased over successive parities in EP, IP, and NR gilts. For gilts initially served, there was no
classification effect on retention in the herd until farrowing a third litter (P = 0.12), but the rate of fallout
per parity tended to be highest for NR gilts (17.6 Vo) and lowesr for EP (12.4 Vo) and LP (14.2 Vo) gilts,
with IP (J5.5 Eo) gilts again being intermediate (P < 0.09). Percenr lifetime NPD decreased with
increasing parity and the slope of the regression lines were statistically parallel but not the intercepts (P <
0.05), indicating that EP and IP gilts accumulating lower 7o NPD than LP and NR gilts.
Taken together, these data suggest that the response to a standardized protocol ofboar stimulation can
identify the top '75 7o of gilts that are most fertile over their productive lifetime in the breeding herd. An
advantage was seen in the increase in percentage of animals served, pigs born lifetime, retention rate,
decreased accumulated NPD, and physical body size at mating, in animals known to have reached
puberty within 40 d of good boar contact compared to those that were non-pubertal.
161

253-7
EFFECT OF EARLY LIFE STRESSORS ON REPRODUCTIVE CAPABILITY
C.J. Ashwoftht'2,L.8. Harker2'3, V/.C. Duncan3, K. Rutherford2, J.A. Rooke2, and A.B.
Lawrence2
rThe Roslin Institute, University of Edinburgh, UK;
2SAC, West Mains Rd, Edinburgh, UK;
3eMRI,
University of Edinburgh, UK
E-mail: cheryl.ashworth @roslin.ed.ac.uk
In rodents, stresses imposed during pregnancy compromise the reproductive function of offspring.
This experiment determined whether stresses imposed during mid-pregnancy and/or soon after birth
affected reproductive function in the pig.
In a 2x2 factorial design, Large White x Landrace gilts were stressed by mixing with unfamiliar older
sows for two one-week periods during mid-pregnancy (M), whereas control (C) gilts were not mixed.
Their piglets either had their tails docked on days 2-4 of life (D) or remained intact (I). A blood sample
and the gonads from 2'7 male and 25 female 8- to lO-week old pigs were collected at slaughter. Plasma
concentrations of testosterone in males and oestradiol in males and females were determined. Numbers of
oocytes and proportions of ovarian follicles at different stages of development were assessed following
Haematoxylin and Eosin staining. Immunohistochemical staining for the germ cell marker VASA was
used to count spermatogonia in testes. Immunohistochemistry or PCR was used to measure indices of cell
proliferation (Ki67) and steroidogenic potential (StAR,3p-hydroxysteroid dehydrogenase, l7u
hydroxylase, aromatase and LH receptors) in ovaries and testes. Data were analysed in a2x2 ANOVA,
[itting age at slaughLer as a covariate.
Neither prenatal stress nor early post-natal pain consistently affected gonad weight, numbers of
proliferating cells in the gonads, the number of oocytes, or the proportion of interstitial cells in the testes.
There was an indication (P=0.08) that testes from tail-docked animals had fewer, smaller seminiferous
tubules. Expression of l7a hydroxylase/GADPH in the testis was higher in pigs born to mothers that
were sffessed during pregnancy (C=0.884 versus M=1.048, s.e.d. = 0.0844; P=0.016), but lower in taildocked
pigs (I=1.036 versus D=0.914, s.e.d. = 0.078; P=0.006). No other markers of steroidogenic
potential, including circulating oestradiol and testosterone concentrations, were affected by stress.
These studies provide preliminary evidence that early life stress may affect components of the
reproductive axis in pigs and suggest that the developing testis may be more susceptible than the ovary.
Fun.ded by the sconísh Government and the BBSRC Animal welfure Initiative.
162

253-8
GENOTYPE AND FETAL SIZE DIFFERENCES IN FETO.PLACENTAL AMINO
ACID STATUS
C. J.Ashworthr and H. J.McArdle2
lThe Roslin Institute, University of Edinburgh, UK;
2The Rowett Research Institute, University of
Aberdeen, UK
E-mail: cheryl.ashworth @roslin.ed.ac.uk
The regulation of fetal and placental growth differs between indigenous genotypes and prolific
Meishan pigs. The biochemical correlates of these differences are poorly understood. This study
compared placental protein synthesis and fetal amino acid concentrations between normally-sized fetuses
and their placentas with the smallest fetus and its placenta in Large White x Landrace (LWxL) and
Meishan (MS) gilts.
Plasma concentrations of amino acids in the smallest and a normally-sized fetus of 9 LWxL and 8 MS
gilts on day 100 of pregnancy were estimated by ion exchange chromatography. Sections of the
chorioallantoic membrane of the placentas supplying these fetuses were cultured in the presence of 3Hleucine
for 24 h to estimate de novo protein synthesis and secretion. Effects of breed, fetal size, and their
interaction were analysed by ANOVA ina2x2 design.
Placental tissue from MS gilts secreted more protein in vitro than placental tissue from LWxL gilts
(11.19 ng/mg vs 6.81 ng/mg, s.e.d. = 1.35; P

2s3-9
RELEVANCE OF LOCAL PROGESTERONE SUPPLY ON EMBRYO SURVIVAL IN
UNILATERALLY OVARIECTOMISED GILTS
R.Z. Athornt, P. Stottt, E.G. Bouwman', R. Ashmanl, S. o'Learyl, M. Nottlel, and P.
Langendijk2
rUniversity of Adelaide, Adelaide, South Australia;
2South Australian Research and Development
Institute, Roseworthy Campus, South Australia
Embryo mortality in pigs is estimated to range from 10 to 40Vo of the originally fertilized oocytes.
Progesterone has been identified as an important driver of endometrial function, and as such is important
for early embryo survival. High feeding levels have been shown to reduce peripheral progesterone levels
during early pregnancy, but effects of feeding level on embryo survival have been equivocal (e.g. Jindal
et a1.,7996; Virolainen et a1.,2004). This may be due to an underestimated contribution of local
progesterone supply from the ovary to the uterus, which may be enhanced at a higher feeding level. In
addition to progesterone supply from the systemic circulation, a local supply ofprogesterone occurs
(Stefanczyk-Krzyzomowski et al., 1998), and variations in the proportions of the supply from these two
sources may explain the inconsistent response to feeding levels. The objective of this study was to
identiff if there was any difference in embryo development between ipsi - and contralateral horns in
unilaterally ovariectomised gilts.
Large Whitellandrace crossbred gilts were induced into puberty at 24 w of age by an injection of
PG600. ULO (n=15) gilts were unilaterally ovariectomised during the luteal stage of their first oestrous
cycle. CTR (n=10) gilts were not ovariectomised. All animals were mated by artificial insemination (AI)
at their second oestrous cycle. CTR gilts were fed a 1.8M feeding level, whilst gilts in the ULO treatment
received a2.4M feeding level in order to exaggerate the difference in progesterone supply between ipsiand
contralateral horns. The gilts were then slaughtered at approximately day 35 of pregnancy and
embryo and uterine parameters were assessed.
Table 1: Macroscopic meâsurements (Means t SE) for unilaterally ovariectomised gilts (n=15).
Number of
lmnlantations
Ovulation ipsilateral contralateral  ipsirate
contralateral
15.1 ¡.0.6 6.5 + 0.4 5.3 x.0.4 1.1 -r Q.5xx
Numtler of Embryos
ipsilateral contralateral  ipsicontralateral
6.I + 0.4 5.0 t 0.4 1.13 + 0.4x*
x*Significantly different (P < 0.05) from 0, tested within gilts.
ULO gilts had an ovulation rate (15.7 + 0.6 vs. 15.9 + 0.8) and total embryo number (11.1 -¡ 0.6 vs
11.5 + 0.7) similar to CTR gilts indicating compensatory follicle development and no adverse effects of
the surgery in ULO gilts. However, in ULO gilts, the number of implantation sites and number of
embryos were lower (P

253-10
REPRODUCTION RESULTS AND ECONOMIC PERFORMANCE IN SOW HERDS
OF CONSTRASTING PROLIFICACY LEVELS
S. Boulot and B. Badouard
IFIP Institut du Porc, La Motte au Vicomte, BP 35104, 35651 Le Rheu Cedex, France
E-mail : Brigitte.badouard@ifip.asso.fr
The development of hyper prolific sows has been associated with a dramatic increase in perinatal
mortality. The objective of the present study was to re-evaluate possible other side effects of high
prolificacy on reproduction, subsequent weaning-to-sale performance and global economic results in
French pig farms.
This study was performed on data from the French National Pig Management database (GTTT-GTE):
average 2007 annual reproduction (2 435 herds) or economic results (1 340 farms). Analysis was
restricted to indoor production farms (selection or multiplication unit excluded). Herds were split into
four groups according to their average annual litter size: 13 total born or less (T813-, n=422), 13-14
(T814, n=951), 14-15 (T815, n=883), over 15 (T815+, n=779). They were compared using variance
analysis procedures with herd size as covariate. Largest litters were associated with the highest numbers
of stillborn piglets (average * sd values for the TB13-, T814, T815, and TB15+ respectively: 0.8 t 0.4,
1.0 t 0.3, I.2 + 0.3, 1.4 + 6.3;. Despite high total pre-weaning mortaliry (17.5 + 5.0, 20.5 + 4.2,22;7 +
4.I,25.4 ! 4.37o), the size of weaned litters increased with total born (10.2 + 0.6, 10.7 I 0.6, 11.1 + 0.6,
11.5 t 0.7). No detremental effect of high prolificacy was seen on reproduction performance nor on sow
longevity: weaning-to-first service interval 6.6 + 2.2 days, weaning-to-effective service interval 9.6 + 3.4
days, conception rate at first service 88.3 + 6.7 Vo, and weaned litters at culling 5.2 t 1.1 (average * sd
values n=2435 herds). Global annual productivity peaked in "T815+" (27.9 + 1.8 piglets
weaned/productive sow, 22.6 t 1.9 pigs sold/sow) and was minimum in "T813-"(24.4 t 2.1 piglets
weaned/productive sow, 19.8 t 2.3 pigs sold/sow). Performance during the subsequent weaning-to-sale
periodsuchas pigmortality (6.4+2.6,6.3+2.5,6.1+2.1,6.011.87o),technicalfeedconversionratio
(2.67 + 0.11 ,2.63 + 0.16, 2.61 t 0.15,2.601 0.13), and daily weight gain (664 + 53,678 + 43, 682 + 41,
598 + 35 g/day) were not impaired in prolific herds. The annual gross margin/sow differed from 200
euros between the extreme groups. Despite higher veterinarian and medecine expenses, "TBl5+" herds
have better economic results mainly because of higher productivity and better feed efficiency.
165

253-11
CONJUGATED LINOLEIC ACIDS (CLA) IN GESTATING AND LACTATING SOW
DIETS AFFECT REPRODUCTIVE PERFORMANCE AND OFFSPRING TISSUE
GAIN
R. Gerritsent, P. Bikk"tl, and A.-M. Pfeiffer2
lschothorst Feed Research, Lelystad, The Netherlands;
2
BASF, Berlin, Germany
E-mail : rgerritsen @ schothorst.nl
Conjugated linoleic acids (CLA) are isomers of linoleic acid with many unique biological properties
compared to the common unsaturated fatty acids, e.g. linoleic acid. Effects of CLA have mainly been
studied in dairy cattle and growing pigs, but only a small number of studies have been performed in
sows. The aim of this study was to determine the effects of incremental levels of CLA in diets of
gestating and lactating sows on their reproductive performance and offspring performance.
The experiment was comprised of four treatments; a control diet and three experimental diets only
differinginthelevelof Luta-CLA (l7o,2Voor3%o).Dietswerefedduringthelast6weeksof gestation,
throughout lactation, and the weaning to estrus interval. In total 80 sows (GY x SL) were blocked based
on parity. Sows from each block were randomly allocated to the treatments. Observations included
repioductive parameters of the sows and performance of the offspring. Data were analyzed by ANOVA
foilowed by a least significant difference-test using GenStat software. In addition, CLA effects were
tested for linearity by regression analysis.
Results of the study are shown in Table 1. Litter size (total pigs born) differed between dietary
treatments, which must have been present before the start of the experiment since the CI-A treatments
were only supplied from day 72 onwards. Also no effect of parity was found for this parameter. CLA
inclusion level did not significantly affect the number of stillborn piglets. Dietary CLA linearly increased
birth weight (P=0.02), most likely as a result of the amelioration in the sows' negative energy balance
with increasing CLA level (P=0.02). Birth weight of the subsequent litter was also higher with increasing
levels of CLA suggesting a long-term effect of CLA on reproductive performance. Dietary CLA reduced
piglet body fat content at weaning (P

2s3-12
PROSTAGLANDIN ASSOCIATBD WITH OXYTOCIN OR CARBETOCIN IN
INDUCTION OF PARTURITION IN SOWS
N.B. Ghellert, R.F. werlangr, T.J. Moresl, M. santil, D. Gaval, M.L. Bernardi2, D.E.S.N
Barcellosl, I.'Wentzl*, anO p.p. Bortolozzol
rFaculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), porto Alegre,
RS/Brazil; 2Faculdade de Agronomia-UFRGS, porto Alegre ,Brazil
E-mail : fpbortol @ ufrgs.br
xPresenting author
Induced parturition allows efficient use of labor and facilities and increases the opportunities for early
cross-fostering and supervision of colostrum intake. Lower dosages of prostaglandin analogues may be
used if injected into the vulvomucosa rather than given intramuicularly. The aim of this study was to
compare the efficiency of farrowing induction through the application of a prostaglandin F2a ãnalogue
(cloprostenol) associated with carbetocin or oxytocin.
Agroceres PIC sows (n=276) were uniformly allocated according toparity into fourtreatments: Tl-
0.175 mg of cloprostenol via the vulvomucosal (VM) route; T2- 0.1t5 m; of cloprostenol VM and 0.10
mg of carbetocin intramuscularly (IM) 24 hours later; T3- 0.175 mg of cloprostenãl VM, and 0.05 mg of
carbetocin IM 24 hours later;T4- 0.175 mg of cloprostenol VM anã t0IUif oxytocinlM24hours later.
The application of cloprostenol was performed at 113 days after first insemination in all treatments. Litter
size, farrowing duration, and induction to farrowing interval were analyzed by GLM procedure of SAS
and treatments were compared by Tukey-Kramer test. Stillbirth percentages were compared by Kruskal-
Wallis test. The percentage of manual obstetrical interventionJ and peicentages of sows farrowing at
different intervals (26,28 and 30 h) after induction were compared by Chi-Squaie test.
Treatment did not affect total born piglets, live born piglãts, percentage ãf stillborns, and percentage
of farrowings with obstetrical intervention (Table 1). Farrowing duration was shorter in carbetocin
groups (T2 and T3). Although the mean interval from cloprostenol injection to farrowing was similar
among the groups, the efficacy of induction within 26 and 28 h was higher inT2,T3, an¿ i¿. Therefore,
although farrowing duration is shorter when cloprostenol is used in association with carbetocin, good
synchronization of farrowing is obtained when parturition is induced with cloprostenol associated with
either oxytocin or carbetocin treatment.
Table 1: Efficacy of different treatments for induction of parturition in swine females.
T1 T2 T3 T4
Farrowi n gs with obstetric al intervent i on (Vo) 1 I .6" ll .4u 15.9" 77 .4u
1g6" 144b I lgb 1g0^
13.0" 12.6^ 12.9^ r2.4^
t2.2^ 12.0^ r2.0^ ll.7^
Farrowing duration (minutes)
Total born piglets
Live born piglets
Percentage of stillborns
Percentage of intrapartum stillborns
Induction to farrowing interval (hours)
Vo of fanowings within 26 hours after induction
7o of fanowings within 28 hours after induction
7o of fanowings within 30 hours after induction
2.9"
2.3^
2.gu 5.20
2.lu 4.1"
3.0"
2.lu
23.7^ 24.9^ 24.7^ 24.9"
63.gb gl.3^ 94.2^
75.4b 9g.6" 9g.6" g1.3^
95.6^
gg.gb 100" 100" g7.7ub
MeanswithdifferentletterSinthesameIowweredifferent(@
cloprostenol + 0.10 mg of carbetocin; T3 - cloprostenol + 0.05 mg of carbetocin; T4 -
cloprostenol + i0 IU ofoxytocin.
167

253-13
FACTORS ASSOCIATED WITH REDUCED REPRODUCTION IN SECOND PARITY
SOWS
L.L. Hovingt'', N.M.Soedet, E.A.M. Graa(,H. Feitsma3, and B. Kempl
I Adaptation Physiology Group and
2
Quantitative Veterinary Epidemiology Group, Wageningen
Institute of Animal Science (WIAS), Wageningen University, Wageningen, The Netherlands; 3 Varkens
KI Nederland 8.V., Deventer, The Netherlands
Email: Lia.Hoving @wur.nl
Reproductive problems, such as reduced farrowing rates and reduced litter sizes, in second parity
sows are often referred to as the Second Litter Syndrome (SLS). SLS decreases reproductive efficìency
of sows and can lead to early culling of sows. Occurrence of the SLS is believed to be related to sow
development and body condition in the first reproductive cycle. In a retrospective study we aimed to
quantify SLS at the sow level and to identify and quantify factors associated with SLS.
Data on measures of sow development and reproduction were obtained from two experimental farms
in The Netherlands between August 1999 and June 2005. Two binary outcome variables were defined:
non-pregnant (NP: no/yes) and 'at least one piglet less in second litter compared to first litter' (RTB,
reduced total born: no/yes). Univariate logistic regression was performed. Only variables with significant
effects (p

II
253-14
EFFECTS OF REDUCED REPRODUCTION IN SECOND PARITY SOWS ON
PRODUCTION IN SUBSEQUENT PARITIES
L.L. Hovingt'', N.M.Soedel, E.A.M. Graa(,H. Feitsma3, and B. Kempl
1
Adaptation Physiology Group and 2 Quantitative Veterinary Epidemiology Group, Wageningen
Institute of Animal Science (WIAS), Wageningen University, Wageningen, The Netherlands; 3 Varkens
KI Nederland 8.V., Deventer, The Netherlands
Email: Lia.Hoving@wur.nl
Reproductive problems, such as reduced farrowing rates and reduced litter sizes, in second parity
sows are often referred to as the Second Litter Syndrome (SLS). SLS might decrease reproductive
efficiency of sows and might lead to early culling. The occurrence of SLS has been related to sow body
condition parameters in the first cycle. Effects of SLS on subsequent production, however, are not clear.
In a retrospective study we aimed to describe effects of the SLS on farrowing rate, litter size, and sow
culling in subsequent parities.
Data were obtained from 87 Dutch farms using the sow management program Farm (Agrovision
B.V.). In total, information of about 160,000 cycles from 47,000 sows was available for analysis. SLS
was defined as a) having at least one piglet less in second litter compared to first litter (RTB; reduced
total born) or b) returning to heat in second parity (RTH). Generalized linear regression analyses were
performed (proc Mixed and Glimmix for continuous and binary dependent variables, respectively; SAS@,
2004). A random farm effect was included in the models. Results are presented as mean+std.
Farrowing rate from first insemination for second parity sows was 80.07o, with a litter size of 12.0 t
3.4 pigs and 16 7o of sows being re-bred. Second parity litter size was 9.7 + 3.0 piglets for sows with
RTB (387o of the sows) and I3.4 * 2.8 pigs for sows without P.TB (62Vo of the sows). Sows with RTB
produced fewer (0.25
- 0.35, p

253-15
TIMED INSEMINATION FOLLOWING GnRH AGONIST ADMINISTRATION IN
WEANED SOWS
M.E. Johnstonr, A.M. Gaines2, M.E. Swansonr, and S.K. Webell
rPennatek, LLC, Sheridan,fn46O69, USA;2The Maschhoffs, Carlyle,ll-62231,UïA
Swine producers spend a large part of each day observing sows for behavioral estrus. However, the
signs of estrus do not identify the precise time of ovulation and optimal time for a single insemination.
The common practice among producers is to inseminate sows two or three times during their observed
estrus period in an effort to ensure a successful mating. Intravaginal treatment with the GnRH agonist,
triptorelin, at a fixed time after weaning has the potential to synchronize ovulation and thus eliminate the
need for heat detection and multiple inseminations. The objective of this study was to determine the
effect of a single, fixed timed insemination following GnRH administration on subsequent farowing rate
and litter size.
Three hundred weaned sows (PIC) were blocked by parity (parities 1 through 6; average parity 2.8),
previous lactation length (average length 21 d), and body condition score (range 2.5 to 3.5; average score
2.8) and allocated to one of two treatments. Control sows were observed for 7 d after weaning for
behavioral estrus and bred according to normal farm procedures, with Control sows being inseminated
the day they were observed in estrus and at24-h intervals for the duration of standing heat. Sows in the
GnRH treatment group were also observed for 7 d after weaning for signs of behavioral estrus. However,
GnRH sows were treated intravaginally with a proprietary GnRH agonist preparation (OvuGelrM,
Pennentek, USA) on d 4 after weaning and then inseminated once 24 + 2 h post-OvuGelrM treatment,
regardless of whether or not they showed signs of standing estrus. Normal farm management practices,
including housing, lighting, feeding, heating/cooling were followed during the breeding, gestation, and
farrowing periods. Data were analyzed using the PROC MIXED procedure of SAS (ver. 9.1) to test the
main effect of OvuGelrM on farrowing rate and litter size.
Responses to treatment (means + SEM) and comparisons between Control and GnRH sows are
presented in the table below.
Sows Allocated to Treatments
Sows Inseminated in 7-d period after weaning
Sows In Estrus at AI (Vo)
Weaning-to-Estrus Interval (d)
No. of Inseminations /Sow
No. Sows Farrowed
Sows Farrowed of Sows Allocated (7o)
Total Pigs Born/Semen Dose
Total Pigs Born Alive (all litters)
Pigs Born Alive/Litter
Control GnRH
150
t26
100
4.7
2.3
109
72.7
5.3
I 191
10.9
SEM Contrast,
P<
150**
150**
84.0 2.34 0.0001
4.4 0.11 0.05
1.0 0.04 0.0001
115 * x
76.7 3.56 0.43
9.6 0.43 0.0001
1300 * *
11.3 0.29 0.37
These data indicate that treating sows with the proprietary GnRH agonist, OvuGelrM and inseminating
once with respect to the time of GnRH treatment results in farrowing rates and litter sizes comparable to
sows receiving multiple inseminations during behavioral estrus. The major improvement in pigs born per
semen dose used for insemination indicates the potential for maximizingrhe genetic value of a smaller
number of high index boars, as part of a strategy to improve overall pork production efficiency.
170

253-16
SUMMER INFERTILITY IN SO\ryS IN FRANCE: DOES THE SUMMER
TEMPERATURE REALLY PLAY A PART
R. KrejcíI, V. Auvigne', P. Leneveu', C. J"hunnina, and E. Sallél
I Ceva Santé Animale, ZILa&allastière, BP 126,3350I Libourne Cedex France ;
2
Ekipaj, C/ Valencia 3,
28223 Pozuelo de Alarcón, Madrid, Spain; 3 Ispaia, Zoopole les Croix, BP 7 ,22440 Plóuiragan, France ;
o Gènes Diffusion, 3595 route de Tournai, BP 7A023,59S0t Oouai Cedex, France
E-mail : vincent.auvigne@ekipaj.com
Summer infertility is described in many countries, from Australia to Finland. The relative importance
of parameters such as high temperatures, lactational catabolism, or photoperiod is widely discussed.
The results of five years (2003 to 2007) of ultrasound pregnancy diagnosis carried out in 266 indoor
farms were analyzed. For all farms the data cover the entire study period. Farms were situated in 4 areas,
with a marked difference in average summer temperatures, in the west and north of France. The data of
22,713 batches and 610,117 sows were included. Summer infertility was defined as the difference
between the fertility rate in "summer" (inseminations during weeks 25 to 42) and "winter" (inseminations
during weeks 1 to 18 of the same year). For each farm, baseline fertility was defined as the average
winter fertility over the past five years. In each area, two meteorological variables were defined on a
weekly basis, using data from a reference weather station: the number of warm days (maximum
temperature > 25 'C) and the number of tropical days (maximum temperature > 32 oC and minimum
temperature > 18 "C). Statistical unit was "farm year" (n=1330) using Mann-Whitney/Wilcoxon test
(Epilnfo 3.4.3 - November 2007).
The mean fertility was 85Vo. The median summer infertility was 2.8Vo and more than 1.l%o for a
quarter of farms. Summer infertility was observed every year in every area. Summer infertility did not
vary between areas and was independent of the baseline fertility of the farms (Table 1, p=0.38). In the
four areas, the summer of 2003 was exceptionally hot and the summer of 2007 exceptionally cold.
Summer infertility during 2003 was significantly higher (p< 0.01) than the other four years, which did
not differ among each other. Thus infertility had not improved during the summer of 2001 even if this
summer was exceptionally cold. Lastly, the correlation between surrìmer infertility of two subsequent
years on the same farm is significant but weak.
It can be concluded that peaks of high temperatures may exacerbate summer infertility. However,
other yearly recurrent factors (such as photoperiod) probably play an essential role in this phenomenon.
Table 1: Summer infertility Ís not linked to baseline fertility (p=0.38)
Su mmer inf ertilitv (7o)
Baseline fertility n lsr quart. Median 3rd quart.
82.6 - 86.2 Vo 345
>86 - 89.4 7o
>89.4 Vo
315
33s
-8.8
-6.9
-5.8
-3.5
-2.3
-J
-2.5
1.6
t.3
0.1
0.3
t7t

2s3-17
ULTRASONOGRAPHIC COUNTING OF SWINE EMBRYOS
F. Martinat-BottéI, S. Serriere2, H., Quesnel3, S. Boulot4, E. Venturis, and F. Tranquart2
t INRA, UMR 85 PRC, 37380 Nouzilly, France ;'Inserm U619, CHRU Tours, Hôpital Breronneau,
37000 Tours, France ; 'INRA, UMR1079 SENAH, 35590 Saint-Gilles, France ; 4IFIP Insrirut du Porc, La
Morre au Vicomre, BP 35104,35651 Le Rheu Cedex, France ;'INRA, UPEAO, 31380 Nouzilly, France
E-mail : Francoise.B otte @ tours.inra.fr
Detection of large litters and the progession of normal gestation may improve economic efficiency in
pig farms. This implies early detection of the number of viable fetuses. Despite great technical
improvement in echography, literature on this topic is scarce, with no practical conclusions. The
objective of the present study was to assess the accuracy of in vivo embryo counting using 2 different
devices at 2 different stages ofpregnancy.
The trial involved 5 groups of six Large White cyclic gilts from the INRA-PRC herd (Nouzilly,
France). They were synchronized with Regumate@ (Janssen, France) and artificially inseminated twice, 6
to 12 hours after the onset of estrus and 24 hours later. During ultrasonographic examinations of the
embryos, gilts were restrained in a crate and received small amounts of feed to reduce agitation.
Scannings were performed by trained operators at two stages (23-25 and 26-28 days after insemination),
using successively two different devices (ESAOTE TECHNOS and ALOKA SSD-900, with 5MHz
convex transducers) providing different picture qualities. The probe was located on the abdominal flank
and was moved slowly and linearly from the back to the front, between the bladder and the ribs. Embryos
were counted on the right and on the left sides. After the second control, gilts were slaughtered in the
INRA-PRC slaughterhouse and their genital tracts were collected immediately to record living, dead, and
resorbed embryos (empty allantoic sacs) in both uterine horns. Ultrasonographic (US) embryo counts
were compared to true embryo numbers at slaughter, using either raw values or accuracy ratios (US
counts / true counts in Vo). Correlations were calculated and variance analyses for repeated measurements
were performed using device, pregnancy stage, replicates, and interactions as main factors.
Data were recorded for 29 pregnant gilts (1 empty). At27 days, the average number of living embryos
was 17.3 x.3.2(lOto25). Total numbers were respectively 11.5 ¡.3.2 and 17.8 + 3.1 when dead and dead
+ resorbed embryos were included. The two different devices provided highly correlated counts
(P

253-18
INFLUENCE OF LESIONS DETECTED THREE WEEKS AFTER INSEMINATION
ON REPRODUCTIVE PERFORMANCE OF SOWS
A.P.G. Mellagir, T. Bierhalsr; A. Panzardil, N.B. Ghellerr, M.L. Bernardi2,I.wenfzr, and F.P.
Bortolozzol
lFaculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre,
RS/Brazil;2Faculdade Agronomia - UFRGS, Porto Alegre, RS/Brazil
E-mail : fpbortol @ ufrgs.br
Service groups must be formed by sows in a healthy condition to achieve satisfactory herd
productivity. The presence of lesions may impair reproductive performance and compromise the
longevity of sows. The aim of this study was to evaluate the impact of the presence and severity of
lesions observed at 20 to 23 days after AI on reproductive performance.
The study was conduced with 1,087 females of parity order 1 to 9. Detection and scoring of lesions
were performed at 20 to 23 days after AI. Abscesses, shoulder ulcers and locomotor disorders were
scored according to their severity as degree 1 (ligh$, 2 (moderate), and 3 (high). Abscesses were scored
as follows: degree I (small abscess of 1 to 3 cm in diameter), degree 2 (abscesses with 5 to 10cm in
diameter), and degree 3 (opened abscess associated with discomfort). For shoulder ulcers, degree I was
scarring of skin over the tuber of the scapula, degree 2 was broken skin up to 3 cm in diameter, and
degree 3 represented broken skin over the tuber with more than 3 cm in diameter. Finally, locomotor
disorders of degree 1 were injuries without sensitivity on the legs or claws, degree 2 was attributed to
lesions with sensitivity, and degree 3 represented an inability to remain standing. For the analysis three
classes were considered: class 1 (without lesions or degree 1), class 2 (degree 2) and class 3 (degree 3).
Farrowing rate (FR), return to estrus rate (RER) and abortion rate (AR) were compared among classes
using a chi-square test. Total born (TB) was analyzed using the GLM procedure of SAS and TB in
previous parturition was used as a covariate.
Shoulder ulcers did not affect RER, AR, and FR (data not shown). When all kind of lesions were
grouped, an increase in RER was observed when lesions had a high severity (Table 1). Sows of class I
achieved higher FR and more TB compared to classes 2 ancl 3. Abscesses of higher severity resulted in
higher RER and lower FR. There was a severe reduction in FR in sows with locomotor disorders of
degree 2 or 3.
The reproductive performance of sows is negatively affected by the presence of lesions at 20 to 23
days after AL The greater contribution to reproductive failure is observed when sows have abscesses of
high severity or locomotor disorders of moderate and high severity.
Table 1: Reproductive performance according to lesions observed at20-23 days after AI
Lesion score classes
t2 3 P value
All RER, 7o 1.2a(13/1020) l2.5ab(4132) l7.lb(6135) 0.041
lesions AF-, Vo 2.4a(2411020) 9.4b(3132) 2.9ab(1135) 0.046
FP.,7o 89.0a(908/1020) 68.1b(22/32) tt.4b(25/35)

253-19
GENISTEIN ALTBR THE RELEASE OF CORTISOL,LH, AND PROSTAGLANDIN
F2aDURING ESTRUS AND INSEMINATION IN GILTS
M. Norrbyl, M.T. Madsen2, F. Saravia3, E. Ekstedtl, and A. Madejt
1Dept. of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala,
Sweden; 2Danish Pig Production, Axeltorv 3, DK-1609 Copenhagen, Denmark; 3Dept. of Clinical
Science, Div. of Reproduction, Swedish University of Agricultural Sciences
Phytoestrogens are a group of chemicals produced naturally by a number of edible plants including
soy. Soy and soy products are commonly used as an important source of protein to feed pigs. Genistein is
one of the phytoestrogens, which can activate both estrogen receptor o (ER u) and B @R B). The aim of
this work was to study the effects of pure genistein in gilts fed a phytoestrogen-free diet, during estrus
and insemination.
Seven gilts (Swedish purebred Yorkshire) were given a phytoestrogen-free diet from the first estrus.
Three days before expected second estrus, four (4) gilts were given 2 mg/kg BW per day of pure
genistein orally (GEN-group), and the other three (3) were controls (C-group). All gilts were fitted with a
catheter in one of the ears, vena jugularis extema through vena auricularis. Blood samples were taken 10
and 5 minutes before, during, and for 80 minutes after insemination (AI). In total, 21 samples per gilt
were collected. All animals were inseminated, after high human stimulation in front of a boar, with 80 ml
EDTA-extended semen. The blood was collected in EDTA tubes containing 0.7 mg aprotinin with and
without indomethacin. Cortisol analyses were done using a radioimmunoassay kit. LH was analyzed by
an EIA sandwich assay and PGF2u analyses were performed by a competitive immunoassay kit. All
analyses were done according to the manufacturer's protocols. Statistical analyses were executed with
SAS program package, all values were first log transformed and then repeated measures analysis of
variance was performed.
All results are given in mean + SEM. Cortisol concentrations did not change significantly in GENgroup
but had a significant rise in C-group, from 136.3 + 19.4 nmol/l during AIto23I.4 + 15.2 nmol/l
six minutes after AI. Afterwards, cortisol concentrations were elevated for another 4 minutes and then
slowly decreased to levels around 130 nmol/I. LH concentrations decreased significantly in GEN-group
from 0.52 + 0.04 ng/ml during AI to 0.32 t 0.06 ng/ml20 minutes after AI. The concentrations of LH
varied around this level for another 40 minutes and then increased again to 0.52 x.0.07 nglml. At 40 and
50 minutes after AI, concentrations of LH in GEN-group were significantly lower then in C-group. The
C-group displayed no significant changes in concentrations of LH during the experimental period. The
PGF2cr concentration in GEN-group was 0.57 + 0.19 ngiml at 5 min before AI and then displayed 4
significantpeaks,2.4l x.0.12,3.49 t 1.55, 4.51-t- 1.30, and 3.56+1.37 nglml attime of AI,25,35-40
and 50 minutes after AI, respectively. The C-group showed a single significant peak 15 minutes after
insemination, from 0.20 t 0.07 ng/ml at AI to 3.55 + 2.45 nglml. The duration of this peak was 10 min
and afterwards the concentrations of PGF2' slowly decreased to 0.53 t 0.35 ng/ml. At 35 and 40 minutes
after AI, PGF2a concentrations were significantly higher in the GEN-group then in C-group.
This study suggests that ingestion of genistein has an impact on hormonal patterns during estrus and
insemination in gilts. Previous observations made that genistein decreases corlisol production from
porcine adrenals, inhibits secretion of LH in ovariectomized ewes, and significantly increases plasma
levels of PGF2o, in heifers fed soy may give support to our results. Further studies are needed to
elucidate effects of genistein on endocrine consequences in pigs.
This workwas supported by Danish Pig Production.
t74

2$-2A
ENVIRONMENTAL AND SOW RELATED FACTORS AFFECTING DURATION OF
FARROWING
C. Oliviero r, M. Heinonen 1, A. Valros r, and O.A.T. peltoniemir*
lDepartment of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki,
Paroninkuja 20, 04920 Saarentaus, Finland
Email: Olli.Peltoniemi @Helsinki.fi
xPresenting author
Prolonged farrowing is associated with complications and a higher number of stillborn piglets.
Therefore, the aim of the present field trial was to explore factors affecting the duration of farrowing on
two high health status farms with 440 crossbred Yorkshire x Landrace sows each. Factors initially
included were parity, breed, herd, group, back fat, design of the farrowing pen (open pen / crate), litter
size, number of stillborn piglets, pregnancy length, and constipation. Each of the farrowing pens (n=16)
were equipped with video recording cameras and beginning / end of parturition were verified by
reviewing the recordings. A stepwise multivariate linear regression model was built in order to find
significant predictors of duration of farrowing. Sows (n=170) included had an average parity of 4.4 x.I.B
and the average number of piglets bom alive was 11.9 + 2.9.The average back fat 3 weeks prior to
farrowing was 14.5 + 3.6 mm and the average constipation score was 2.0 + 0.6. Overall, the mean
duration of fanowing was 268.8 + 154 minutes. Constipated sows (score below 2.0) and sows with a
back fat score higher tban l7 mm had a prolonged duration of farrowing (P

253-21
DIETARY INTAKE DT]RING EARLY PREGNANCY DOES NOT INFLUENCE
EMBRYONIC SURVIVAL AND VARIABILITY IN GILTS
H. Quesnell, E. Venturi', E. Roy"r', F. Elleboudt2, S. Boulot3, S. Serrierea, and F' Martinat-
Botté5
tINRA, UMR 1079 SENAH, 35590 Saint-Gilles, France;'INRA, UPEAO, 37380 Nouzilly, France;
3IFIP
Institur du Porc, BP 35104, 35651 Le Rheu Cedex, France; olnserm U619, CHRU Tours, Hôpital
Bretonneau, 37000 Tours, France; tINRA, UMR 85 PRC, 37380 Nouzilly, France
E-mail : Helene.Quesnel @ rennes.inra.fr
A high level of feeding during early pregnancy has been associated with a reduction in embryonic
survival in some studies, *nl"n ¡¡uy r"rult in reduced litter size in moderately prolific sows. In highly
prolific sows, embryonic survival is generally not a limiting factor for litter size. Furthermore, high
ãmbryonic survival ieported in hyperprolific sows results in uterine crowding and an increased risk of
intraúterine growth retardation. Moreover, a good embryonic survival rate has been associated with
greater variability in embryo development. In hyperprolific sows, thus, a reduced embryo survival in the
period could have beneficial effects on litter characteristìcs at birth through an early
freimplantation
iedu"tìon in embryo heterogeneity and uterine crowding. Our objective was to determine if a high level
of feeding during early pregnancy influences embryo survival and weight in prolific gilts.
Thirty Large Wnit" gittr *"r" arlificially inseminated twice at 227 + ldays of age and 733 x 2 kg live
weight. buring the 7 days after the first AI, they received 4 or 2 kg daily of a gestation diet (groups High
andtontrol, réspectively, n = 15/group). Before and after this period, all gilts received 2 kg of a gestation
diet. Gilts were slaughtåred at Zl.O x.0.1 days of pregnancy. The genital tract was collected and living,
dead, and resorbed embryos were recorded. Embryos were considered dead when allantoic and amniotic
fluids were dark and not iimpid. Resorptions were characterized by the absence of both embryo and fluid.
Each living embryo was weighed and measured. For each gilt, calculated criteria were embryo survival
(number oi liuing embryos ai slaughter/number of corpora lutea); "post-implantation" embryo survival
(number of living embryos at slaughter/total number of embryos); within-litter mean embryo weight and
iength, and coefficients of variation; and in utero density (number of living embryos per meter of uterine
horn;. Variance analyses were performed using dietary treatment, replicate and the interaction as the
main factors. Correlations between criteria were calculated. The level of significance was P < 0.05.
At slaughter, rwenty-eight gilts were pregnant (15 and 13 in groups High and Control, respectively)'
Ovulation rate averaged 21.0 + 0.5 and ranged from 14 to 27. None of the criteria was significantly
influenced by the level of feeding during the first week of pregnancy. The number of living embryos
averaged tl.S x.0.6 (10-25), embiyo survival 85.47o (55-l00%o), and post-implantation embryo survival
96.1% (71-IO}Vo). Given the great number of ovulations for such young gilts, embryo survival was good,
which is consistent with previous findings in highly prolific sows. Across treatments, embryonic survival
was not correlated with ovulation rate or uterine length. Within-litter variability in embryo weight
averaged 107o, with 6 to lSVo extreme values. Embryo heterogeneity was not correlated with ovulation
rate, number of total or living embryos, or embryo densiry. Similar results were obtained for embryo
length and its variation.
Ás a conclusion, for prolific gilts, a high level of feeding during early pregnancy did not reduce
embryo survival and had no beneficial nor detrimental effects on embryo size and variability at 27 days
of gestation.
176

253-22
PREPUBERTAL SCORING OF SCALE ACTIVITY IN GILTS AND ITS POTENTIAL
RELATIONSHIP TO SUBSEQUENT FERTILITY AND REPRODUCTIVE
PERFORMANCE IN LANDRACE.DUROC.YORKSHIRE CROSS FEMALES
L.A. Rempel, G.A. Rohrer, and T. Brown-Brandl
USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE, USA;
E-mail : lea.rempel @ ars.usda. gov
A majority of animals culled due to reproductive failure are skewed towards younger parities (l or
less), decreasing the economic potential of the herd, thereby leading to an overall reduction in the herd's
sow lifetime productivity. The ability to identify young females with superior reproducrive potential
would have a large economic impact on commercial swine production. Therefore it was our objective to
monitor temperament in prepubertal females and determine if a relationship existed between behavior
and reproductive and production performances.
One thousand two hundred thirty-two Landrace-Duroc-Yorkshire females were scored for behavioral
tendencies at approximately 154 days of age during a scheduled weighing. Animals were scored from 1
to 5 at 0.5 increments while being weighed. A description of rated scale activity is as follows: 1) remains
calm with little or no movement; 2) walks forward and backward at a slow pace; 3) continuously moves
forward or backward at a rapid pace; 4) continuously moves forward or backward at a rapid pace with
vocalization; and 5) continuously moves forward or backward at a rapid pace with vocalization and
attempts to escape. Subsets of scored animals were also monitored for age at puberty (AP) measured as
first detected estrus; day 35 post-breeding pregnancy diagnosis; farrowing rate; and production
parameters including total number born (TNB), number born alive (NBA), and number weaned by dam
(WND-DAM); ovulation rate (OR); and weaning-to-estrus interval (U¡EÐ. Data were collected on
animals through panty 2.
Animals that were scored at 3 or greater had a decreased (230.0 + 2.78 d; P = Q.9948) AP in conrrast
to animals that were scored between I and.2.5 (236.9 + l.l3 d). Scale activity scores were related to
pregnáncy diagnosis at 35 days post-breeding as well as farrowing rate where animals with greater
activity scores were more often diagnosed pregnant (P = 0.0264) and farrowed (P = 0.0278), respectively.
Regression analysis indicated that a one-degree increase in scale activity score increased pregnancy rate
by approximately 3.3Vo (P = 0.0173) and farrowing rate by 37o (P = 0.0345). However no significant
correlations were detected between AP and early pregnancy diagnosis or farrowing rate (P = 0.1537 and
P = 0.2063, respectively). None of the measured production traits (TNB, NBA, WND_DAM) were
affected by the early activity score (P > 0.05). Likewise, V/EI and OR were not impacted by the scale
activity score (P > 0.05).
These results indicate that prepubertal behavior may be useful as an indicator for females that have an
increased fertility as measured by AP, pregnancy rate, and farrowing rate. Furthermore, since animals
with greater scale âctivity scores at 154 days of age had no negative impacts on litter production
performance, it may be plausible that earlier selection for these active animals will not deirimentally
affect herd production, but rather increase it as a function ofimproved fertility.
177

2s3-23
IMPROVING FERTILITY AND SAVING LABOUR BY FIXED TIME INSEMINATION
F. Rosalesl, V. Quintero2,M. Gonzátlez3, A. Aguilera.l, M. Fernánd ez3, andM. Martensa
rlnterveyschering Plough Animal Health - Mexico; 2Universidad Nacional Autónoma de México;
3Hacienda alntervelschering de la Flor farm; Plough Animal Health - The Nederlands
E-mail: jose.andres.francisco.rosales.espinosa@sp.intervet.com, patologiavictor@yahoo.com.rlìx
In the modern pork industry, efficient management and effective cost-control are of the highest
importance. Reproáuctive efficiency can usually be improved, in pafiicular the non-productive period'
which tends to be overlooked. The Weaning to Service Interval (WSI) is a major contributor to the
number of non-productive days, and relies heavily on the estrus detection skills of the farm staff. The aim
of this trial was to reduce the WSI by using hormone treatment to allow insemination at a fixed time after
weaning, and to measure the effect of the treatments on reproductive parameters such as farrowing rate
(FR) and litter size (LS).
' i*o strategies (Phase I and Phase II) were applied on a single-site breeding farm in the central part of
Mexico, comprised of 300 sows with a 30Vo replacement rate. From the several procedures described in
the literature, the ones chosen for this farm were tailored to the fertility regime already in place. Sows of
similar age and parity were randomly allocated to treatment groups (TG; N=40) or their respective
untreared control groups (CG; N=38). Sows (n=14) in the Phase I TG were injected with eCG(400IU) +
hCG(200IU) (PG-600o, Intervet/Schering Plough) at weaning, another 14 sows were remain as CG.
Those in the Phase II TG (n = 26) received the same injection and, in addition, 0.01mg of the
Gonadotrophin-Releasing Hormone analogue containing the active 0.004 mg Buserelin
lReceptal@TConcepral@, Intervet/Schering Plough) 78 hours later. For the Phase II TG the insemination
14,u, out aistrictly 24 and 42 hours after the last treatment, for the CG (n = 24) the inseminations
were "ãoi"d performed in accordance with the farm common procedures; the sows were AI the next morning (9
AM) ãr the next afternoon (6 PM) depending on the detection of estrus and the observation of standing
reflex. Otherwise, the entire program accommodated the farm's existing routines. In total, there were
three repetitions for the Phase I treatment and six for the Phase II-
In the Phase I there was a significant reduction in WSI for the TG compared to CG (T student test,
p

2s3-24
ESTRUS SYNCHRONIZATION TO IMPROVE RBPRODUCTIVE PERFORMANCE
OF PRIMIPAROUS SOWS
F. Rosalesl, V. Quintero2, A. Aguileral, and M. Martens3
llntervet/Schering Plough Animal Health - Mexico; 2Universidad Nacional Autónoma de México;
3lntervet/Schering Plough Animal Health - The Nederlands
E-mail: jose.andres.francisco.rosales.espinosa@sp.intervet.com, patologiavictor@yahoo.com.mx
One option for increasing a pig farm's reproductive turnover is by improving the performance of
primiparous sows (Pls) between their first and second farrowing, which is often not ideal. The present
trial was carried out with the aim of improving the reproductive parameters of Pls by reducing the nonproductive
days (attributed to the length of the Weaning to Service Interval; WSI), Fertility Percentage
(FP) and Litter Size (LS).
A single-site breeding farm in central Mexico, with 350 sows, practicing 3-week lactations, and with a
30Vo relacement rate was used. A total of 40 Pls were rreared with eCG (400IU) + hCG (200IU)
GG600@ - Intervet/Schering Plough) at weaning, between February and Septemb er 2007. The WSI and
LS were recorded for each sow and the FP for the whole group. The data from the Treated Pls were
compared with equivalent data from 59 Un-treated Pls from the previous year (February to September
2006), and analyzed by a student's T test.
Table 1. General data: Treated and Un-treated Pls.
TREATED
UN.TREATED
GROUP SOWS WSI (days)
mean+SE
No.
Vo
No.
Vo
40
100
59
100
5.575u
+0.228
9.415b
t0.831
RETURNS
TO
ESTRUS
3
1.57o
6
r0.2vo
OTHER FERTILITY
FAILURES
I (abortion)
2.5Vo
1
1.77o
JI
92.5To
52
88.l%o
GROUP
TOTAL LS (piglets)
BORN Mean+SE
TREATED
UN-TREATED
No.
7o
No.
7o
36
100
52
100
376
459
10.44
+0.641
g.g3b
+O.447
Different superscripts indicate statistically different values; Student's T test, P value

2s3-2s
EFFECTS OF DAY OF FARROWING INDUCTION ON SUCKLING PIGLET
PERFORMANCE
H.M. Smith, A.M. Williams, and T.J. S.afranski
University of Missouri, Department of Animal Science, Columbia, MO, U.S.A
Today's commercial farrowing induction protocols were developed mostly in the 1970s and 80s, and
the average non-induced gestation length was found to be 114 d. No detrimental effects of prostaglandin
protocols were found when inducing no more than two days early, and addition of oxytocin could more
tightly synchronize farrowing and facilitate supervision. "Normal" gestation length today has been
reported to be 115 to ll7 d, and some have suggested that induction is associated with increased
morbidity or mortality. The objective of this study was to compare the effects of inducing sows on d 113,
114, or 116, with the expectation that most d 116 sows would farrow spontaneously.
The genetic lines for females and males were Genetiporc F20 and G Performer, respectively. Records
on 472 first and second-parity sows and 5,493 piglets born alive were collected on a 3,000 sow
commercial farm in central Missouri. Inductions stafted July 14 and ended August 5, 2008. Sows were
allocated to ffeatment group when they entered the farrowing rooms. Parturition induction consisted of
intramuscular injections of 1.5 ml LutalyserM at 0300 and 0600 on d 113, lI4, or 116 of gestation to
sows not having farrowed or in labor; first day of mating was d 0. One-half ml oxytocin was given
intramuscularly at 0530 the next day to treated sows that had not farrowed. Supervision of farrowing was
only available from 0600 to 1400. Piglets were cross-fostered within 24 h only within treatment groups
and processed on d 2. One way ANOVA (two-by-three factorial) was used to analyze the data, with
mean differences determined by Fisher's LSD. The frequency of treatment was analyzed using row by
column chi-square.
Averagegestationlengthwas 114.00+0.61, 114.65 +0.89, and115.26+I.32 dfor113,Il4,and 116
groups, respectively; spontaneous farrowing occurred in 8.33, 16.88, and 79.497o of sows. Litters were
weighed at processing and again the day before weaning at d 22.12, 21.44, and 2I.01 of lactation,
respectively for groups ll3, I74, and 116. There was no difference among heatment groups in number
born alive, mummies, or litter weaning weight (Table 1). Sows induced on d 116 tended (P=0.07) to have
more stillborns (0.80 t 0.71) compared to sows induced on d 113 (0.56 -r 1.11). Percentage of litters
receiving medical intervention was 3.21 (dl13), 3.80 (dl14), and 5.73%o (dl16) and not different among
treatments.
Farrowing induction is still a valuable tool when average non-induced gestation length of the sow
population is known. Further investigation in herds with higher morbidity and older parity sows are
necessary to confirm broad applicability of these results.
Table 1: Mean production parameters for select traits without significant treatment differences.
Born Alive
Mummies
Litter Wean Weieht, ke
d 113 d 11,4 d 116
11.56-t- 3.20
11.88 + 3.02
17.99 ¡2.84
61.60 -r 10.66 60.28 + IL34 6030 + 12.30
0.21 + 0.53
0.29 x.0.61
0.23 + 0.53
180

2s3-26
REDUCED PHOSPHORUS IN DIETS DURING GESTATION DECREASED LITTER
SIZE AND LONGEVITY
G. Sorensenl
lDanish Pig Production, Axeltorv 3,1609 Copenhagen V. Denmark
E-mai I : gs @dansksvi neprodu ktion.dk
The theoretical phosphorus requirement of gestating sows can be calculated at 1.0-1.9 g digestible
phosphorus per FUgp. The requirement is lowest for old sows. Correspondingly, the theoretical
requirement of lactating sows can be calculated at 2.4-2.6 g digestible pnospfrorui per FUgp. The
standard for Danish sows is 10-15 per cent higher (1), (2) than the theoreticãl ."qìir"-"nt, which means
that it should be possible to make a further reduction compared with the current stãndard.
The trial was carried out in four herds: two herds used purchased feed and two herds used homegrown
feed' Different types of housing were represented in the herds. The study was based on approx.
5'000 litters from 1600 gilts that were monitored for a period of three years. In ihe gestation periõã, the
sows in the control group were fed a diet containing 2.2 g digestible phosphoro. p"t FUgp ana rhe trial
group was given a diet containing 1.8 g digestible phosphorus per FUgp. Both grõups were tested at the
same time in each herd. The sows were fed with the same diet in the lactation period containing 2.7 g
digestible phosphorus per FUgp and 7.0 g calcium per FUgp. The prorein and enìrgy levels + the CA:p
ratio were held constant for all diets.
Farrowing rate, litter size, weaning weight and culling rate were recorded for all sows. Strength of
bone, collected at slaughter, was determined with DEXA according to Nielsen et al. (3). Datã was
analysed with proc GLM and Mixed in SAS.
The trial concluded that litter size (first and second parities) and longevity - expressed as born litters
before culling - of the young sows were negatively affected when the phósphórus content of the gestation
feed was reduced firom2.2 to 1.8 g digestible phosphorus per FUgp (p

253-27
EFFECT OF CHRONOLOGICAL AND SEXUAL AGE AT FIRST MATING
F. Thorupl
lDanish Pig Production, Axeltorv 3,1609 Copenhagen V. Denmark
E-mail: ft@dansksvineproduktion.dk
In gilts, various recommendations for optimal chronological and sexual age and weight are given. As
few investigations look into more that one of these factors, conclusive results are sparse. Also, it is
questionable if the same recommendations are valid for gilts from prolific breeds. The aim of this study
was to investigate the effect ofchronological and sexual age on fertility, productivity, and longevity.
A total of 646 Danbred Landrace cross Large White gilts delivered to two herds were included. At an
age of 4 to 6 months gilts were blood sampled to test if a high level of progesterone could indicate
cyclicity. The gilts were randomised into three groups to be mated at first, second, or third observed
oestms. The gilts underwent oestrus detection performed with a boar outside the pen every day. Gilts
were mated according to their designated breeding group. The gilts were offered the same amount of feed
per day before mating at first, second and third oestrus. Piglet growth and survival was followed in 20
litters born to females in each of the original breeding groups in each herd. Pigs in these litters were
weighed after litter size was adjusted and at weaning. Runt pigs could be removed from these lit¡ers, but
new pigs from non-experimental sows could not enter. Gilt longevity will be calculated as the number of
gilts farrowing their second litter.
Only 1.2 percent of the gilts had a progesterone level above 7 gllitre. This indicates that only a few
gilts were cyclic when entering the trial between approximately 120 and 180 d of age. Gilts mated at first
observed oestrus had an average of 13.2 total born piglets at first farrowing. Gilts mated at second
observed oestrus farrowed 1.7 piglets more. This significant effect was present in both herds, and in gilts
that were either 6, 7, or 8 months old when first oestrus was observed. Waiting to mate the gilts until
third oestrous did not increase litter size significantly compared with gilts mated at second oestrous. No
effort was made to increase feed intake before mating at second or third oestrus (flushing), which could
have explained the increased litter size following mating at second and third oestrus. Litter growth and
survival did not differ significantly between the three groups. After weaning the first litter, the same
percentage of gilts was mated and the farrowing percentage did not differ between breeding groups.
Postponing mating until second or third oestrus increased the cost of feeding and housing for three or
six weeks extra. This did not alter productivity in the form of pig growth rate in the nursing period. Of
the gilts mated, 78, 81, and 85 per cent did farrow their second litter. Postponing mating until second
oestrus gave a significant increase in piglets born per litter. This effect was seen in prolific gilts, also
achieving a high litter size when mated at first oestrus. Mating gilts at second observed oestrus seems to
be cost efficient under most commercial conditions.
r82

253-28
CAN PROLIFIC SOWS NURSE THEIR OWN PROGENY
F. Thorupr
lDanish Pig Production, Axeltorv 3,1609 Copenhagen V. Denmark
E-mail: ft@dansksvineproduktion.dk
Modem breeding has resulted in highly prolific sow lines. This calls for new management practices to
rear this high number of live born pigs. A high number of pigs nursing the sow will increase the risk of
malnutrition of some pigs, and may also reduce daily weight gain in all pigs. The aim of this study was to
investigate the effects on pig mortality and average weight gain in sows nursing litters of 11, 13 or 15
pigs.
In this study 132 prolific Danbred Large White and Landrace crosses were used. All sows had at least
15 functional milk glands at farrowing. Sows included in this trial had an average of 15.1 live born
piglets per litter. As the groups of three sows farrowed, they were assigned randomly to nurse 11, 13, or
15 pigs, and 39 pigs from their litters were weighed and randomly assigned to the three groups. Surplus
pigs from these litters were transferred to nurse sows and were not included in this experiment. During
the trial period runt piglets could be transferred from the trial sows to nurse sows, but transfer of new
pigs to the trial sows was not allowed. Runt piglets transferred to nurse sows were followed to estimate
the total mortality and weaning weight resulting from the three litter size groups.
Table 1: Performance of experimental litters of 11, 13, or 15 pigs per litter.
Pigs per litter 11 pies 13 pies 15 niss
660
12
Total pigs allocated per group (n = 44 sows) 484 5j2
Runt piglets taken away (per cent) 8
10
Mortality, including mortality in transferred runt
5
piglets (percent)
6
Mean pig weight at weaning (kg) 1.9
1.5
Table 2 gives the number of nurse sows needed to handle surplus and runt piglets, when sows are
allowed to have different numbers of piglets after fanowing. Runt piglets were transfer¡ed to a nurse sow
after an average of 6.5 days of nursing. In a system where all sows nurse 11 piglets, nurse sows receiving
surplus pigs can only accept 11 nurse piglets, to allow all transferred piglets to have an active gland.
Surplus or runt piglets in a system where sows nurse 15 piglets can be transferred to a nurse sow with 15
active glands. The number of litters produced per sow and year was calculated by the number of days
pregnant (116), number of lactation days (24), number of empty days per litter (16), and number of nurse
days per litter born (see table 2). The number of piglets weaned per farrowing is based on the mortality
achieved in this trial.
Table 2: Pigs weaned per so\ry per year.
10
7.2
Piglets per litter 11 pies 13 niss 15 niss
Nurse days needed per litter born
Nurse days needed for runt pigs
Weaned pigs per farrowing
Litters per sow per year
weaned per sow
7.6 days
1.1 days
14.3
2.20
31.5
3.2 days
1.4 days
14.1
2.26
31.9
t.zãays
13.5
2.30
31.1
In conclusion, in sows farrowing 15 live born piglets per litter, the highest number of pigs weaned per
sow per year was achieved when 13 piglets were nursed by each sow.

253-29
EFFECT OF SUPPLEMENTING GILT DIETS WITH BETAINE DURING SUMMER
ON REPRODUCTIVE PERFORMANCE
W. H. E. J. Van Wettere*I, P. Herde2, P. E. Hughes2, and S.J. Pain3
lDiscipline of Animal Science, University of Adelaide, SA 5371, Australia
2Pig and Poultry Production Institute, SA 5371, Australia
3lnstitute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New
Zealand
*Email : william.vanwettere @ adelaide.edu. au
Prolonged exposure to high ambient temperatures and the resultant heat stress can impair ovarian
function and embryo development (Wolfenson et a1.,200I), and may be at least partially responsible for
the depression in fertility commonly observed in domestic sows. The current study determined the effects
of supplementing gilt diets with betaine, a potent organic osmolyte and methyl donor, during summer on
the timing of the pubertal response to boar stimulation and potential litter size.
A total of 84 purebred maternal (Large White) / terminal (Duroc) line gilts were used in this study.
Commencing at 21 weeks of age, gilts received either a standard finisher diet (CONTROL) or a betaine
supplemented (2 elkÐ finisher diet (BETAINE) (n = 42 gilts / treatment). Diets were fed until puberty
attainment, with all gilts received 3 kg of feed per day (13.0 MJ DE/kg, 15.57o protein, 0.69 avail
lysine/ìvIJ). Boar contact commenced at 25 weeks of age, and consisted of 20 minutes/day of full contact
with a vasectomized boar in a detection-mating area. Gilts were artificially inseminated at their first
observed oestrus. Reproductive tracts were collected post slaughter at 30.6 t 0.14 days after first mating,
and numbers of corpora lutea (CL) and viable embryos were recorded. A two-way analysis of variance
was used to examine treatment effects on all parameters measured. Data is expressed as mean + S.E.M.
Table 1: Days-to-puberty and potential litter size of BETAINE and CONTROL gilts maied in
summer at their pubertal oestrus (Mean r SEM)
Dietary Days-to-
Ovulation rate Number of Embryo survival
treatment puberty (davs) (Number of CL) Embryos (7o\
CONTROL Il.3 + 1.23* 13.5 -r 0.37" 10.9 r 0.50 0.80 + 0.03*
BETAINE 8.0 + 1.18x 14.6 + 0.38b 10.3 + 0.51 0.71 t 0.04*
oÐifferent superscripts, within column, indicate significant differences (P

253-30
INVESTIGATIONS ON TNF-o MEDIATED EFFECTS IN PREOVULATORY
OVARIES OF GILTS
D. waberskil, M. Krtigetr'2'3,H.-J. schuberth2, A. schnapper3,s, H. Henningl, K.Langner2, M.
Beyerbacha, and R.H.F. Hunterr
tunit for Reproductive Medicine / Clinic for Pigs and Small Ruminants,
2lmmunology Unit,
3Department
of Anatomy, aDepartment of Biometry, Epidemiology and Processing Information, Ùniversity of
Veterinary Medicine, Hannover, Germany; sDepafiment of Veterinary Anatomy, Vetsuisse Faculty,
University of Ztnch, Switzerland
E-mail : dagmar.waberski @ tiho-hannover.de
The process of ovulation has been linked to an inflammatory reaction, which includes the infiltration
of different leukocyte populations into the tissue of mature Graafian follicles and the local production of
cytokines. Seminal plasma has been demonstrated to advance ovulation in gilts. The natureìf this signal
transfer is essentially unknown. Signal pathways could involve locally induced cytokines such as TNF-o
and GM-CSF by epithelial cells or other cells present the uterus. In the rat model, TNF-u has been shown
to trigger events leading to ovulation. In the present study, we tested the hypothesis that TNF-a reaching
the porcine ovary via the arteria ovarica triggers resident mast cells and thereby induces degranulation
and mediator secretion which eventually promote preovulatory events.
A bilateral surgical approach in the pig using 14 spontaneous cycling gilts under general anaesthesia
was used. Recombinant porcine TNF-a (2 or 20 ng in I .0 ml of PBS) was injected into rhe ovarian arrery
of one side and 1.0 ml of PBS alone into the contralateral side up to 10 h after the detection of oestrus.
The ovaries were removed 45 min later. Follicular fluid was analysed by radio-immunoassay for
histamine concentrations. Ovarian tissue was dissected into four regions to be analysed by histoiogy,
immunohistochemistry, and quantitative RT-PCR. The Pappenheim stain was used for the quantitative
analysis of mast cells. Immunohistochemistry was performed using specific anti-TNF- s and anti-TNF.- a
receptor I antibodies. The mRNA expression of eight chemokines, cytokines, and enzymes (TNF- a,
GM-csF, TGF-ß, L-10, cxcl-8, rL-6, cox-2, and,Lipoxygenase 5) was analysed by qRT-pcR.
Mast cells were only found in connective tissue in the zona vasculosa; they expressed neither TNF_ u
nor TNF- ü, receptor I. The numbers of mast cells did not differ between treated and control ovaries
(Þ0'05). Moreover, histamine concentrations in follicular fluids did not differ significantly between
treated and control sides. TNF-ü-positive cells in connective tissue were identified as macrophages and
detected in the zona parenchymatosa as well as in the zona vasculosa. TNF-g receptor I was expressed in
functionally heterogeneous cell populations: follicular epithelial cells in primordial and primary follicles,
granulosa cells in secondary and tertiary follicles, theca cells, and fibroblasts. Interestingly, the
application of 20 ng TNF- o resulted in a reduction of mRNA copy numbers of CXCLS (IL-8, induction
fold 0.43) and TNF- o (induction fold 0.38), whereas the expression of lipoxygenase-S, IL-10 GM-CSF,
IL-6, and TGF-ß remained unaffected. The only upregulated gene appeared to be COX-Z (induction fold
2.6)
The results indicate that a putative role of TNF-c in the ovulatory process is not mediated by a
primary activation or degranulation of resident mast cells in the pig. However, a variety of other cells
expressing TNF- cr receptor I are present in preovulatory porcine ovaries which synergistically may be
involved in the ovulatory cascade triggered by TNF-u The selective upregulation of the pro-inflammatory
COX-Z and the selective down-regulation of other pro-inflarrunatory mediators indicatei that this cascade
is not a classical inflammatory response.
Supported by Germøn Research Foundation (DFG Ha 433/6-1, 6-2)
185

253-31
REPRODUCTIVE RESPONSES OF SOWS TO HEAT STRESS DURING DIFFERENT
PHASES OF A PRODUCTION CYCLE
A.M. Williams, T.J. Safranski, D.E. Spiers, P.A. Eichen, E.A. Coate, and M.C. Lucy
Division of Animal Science, University of Missouri, Columbia, USA
Seasonal infertility is prevalent in sows during summer months and is problematic for producers
striving to increase production and maximize reproductive efficiency. While not all aspects of the
condition are understood, it is universally recognized that the increase in weaning to estrus intervals
(UIED and anestrus are largely a result of increased ambient temperatures. The objective was to
determine when sows are most sensitive to heat stress during a production cycle and examine production
and reproductive responses.
First parity Landrace and Landrace/I-arge White Fl sows (n=58) were rotated through chambers in the
Brody Environmental Center for 58 d beginning in late gestation [gest], continuing through lactation
flact] and ending at breeding. The ambient temperature sequences (treatment; trt) included either
thermoneutral (TN; 18 to 20'C) or heat stress (HS; 24 to 30'C) for each production phase (TN-TN-TN
[n=15], TN-HS-TN [n=14], HS-TN-HS [n=14] or HS-HS-HS [n=15] for gestJact-breeding [20,24, and
14 d, respectivelyl). Rectal temperature, body weight (BW), backfat (BF), loin eye area (LEA), feed
intake (FI), piglet weights, WEI, ovarian follicular growth and subsequent breeding performance were
measured. Data were analyzed for the effects of trt and day by using the General Linear Models
Procedure (Proc GLM) of SAS v. 9.1.
Rectal temperature differed across phases and conditions (38.3 and 38.2, 39.5 and 39.2, 39.0 and
38.9'C ISEM < .04] for HS and TN during gest, lact, breeding, respectively; P

INDEX BY AUTHOR
A
Aguilera, A.,178, 179
Akaki, Y.,743
Almeida, F'.,65, 138
Altmyer, M.,97
Alvarengø, A.,738
Anderson, L., ll5
Archibald, A.,97
Ashman, R.,149,164
Ashw orth, C., 91, 162, 163
Ashworth, M.,101
Athorn, R.,164
Audet, L,123
Auvigne, V.,177
B
Badouard, 8,,165
Bailey, D.,117
Bailey, J.,l2l
Balcells,1.,139
Balcerowic4 D.,744
Barcellos, D.,167
Bathgate, R.,45
Bayless, K.,79
Bazer, F.,79,117
Beebe, L.,149
Behboodi,8.,107
Beltranena, 8.,761
Berger, 8.,125
Berges, A.,127,128
Berkeveld, M.,156
Bernardi, M., 7 3, 151, 167, 173
Beyerbach, M.,185
Bierhals, 7., 151, 173
Bierman, C.,135
Bijltebier, J.,103
Bikker, P.,166
Bilinska,8.,140,754
Bischoff, S.,69,105
Blitek, A.,77 , 131, 144, 147
Bodek, G.,7M
Bogacki, M., 145
Bolarin, A.,122
Bonet, 5.,127,128
Bortolozao, F,, 7 3, 75 1, 167, 17 3
Boulot, 5., 165, l'12, 17 6
Bouwman, 8.,164
Breen, 5.,97,160
Brown-Brandl, 7., 158, 17'7
Brüssow, K.,63
Burghardt, R.,79, 117
c
Caballero, 1.,43
Calvete, I.,39
Cameron, A,,117
Canøday, D.,97
Cardeal, P,,138
Castellano, C,,123
Castelló', A.,139
Cherel, P.,l4l
C hiarini- Garcia, H., 138
Chouínard, Y.,123
Christenson, R,,748
Clutter, A.,75
Coate,8.,186
Conner, f.,717
Croy, 8,,67,157
Curry,8.,142
Cushman, R.,148
D
Dahmanì, Y., 12'7,128
Davis, D.,709
ile In Sota, R.,737
Deþrce, D., 103
del Olmo, D.,124
Dhaenens, M.,103
Díilíon, 8.,135
Dixon, W.,65,93,99
Dobrinski, 1., 107
Dobrínslcy, J., 135
Duda, M.,754
Duncan, W.,162
Durlej, M.,140, 154
Dyck, M.,65,93,99,152
E
Eborn, D.,109
Edwards, A., 67 ,157
Egerszegi, 1.,125
Eichen, P.,186
Ekstedt, 8., 174
Elleboudt, F,,176
Ellß, s.,142
F
Feitsma, H., 126, 168, 169
Fernándeç M.,178
Fernánilez, V.,137
Fiúønt, J.,131
Finlayson, H.,91
Flowers, W.,47
Fontes, D.,138
Ford, J.,148
Foxcroft, G., 65, 93, 99, 111, I 1 3, 138, 152, 161
Freking, 8., 69, 7 1, 105, 148
Funahashi, H.,143
r87

G
Gabilondo, D.,137
Gadella, 8.,47
Gaines,4,, 170
Gao, H.,717
García-Bonavila, 8., 127, 128
García-Tomós, M., 127, 128
Gøva, D.,167
Geísert, R., l0l, 115, 153
Gerritsen, R., 61, 156, 166
Gheller, N.,167,173
Gil, M.,43
Glenisson, J.,141
Gómez-Rincón, C., 127 , 128
Gonzólez, M.,178
Graat, 8.,168, 169
Grandía, 1.,127,128
Green, J.,59
Greiner, L.,717
Grieger, D.,109
H
Haley, C,9l
Hancock, J.,113
Hansen, C.,85, 129,130, 132
Harding, J.,65
Hardison, N.,69, 105
Harker, L.,762
Harrison, R.,83
Harrison, 5.,149
Holeleger, W.,61 , 146, 155
Heinonen, M.,175
Hejmej,4.,140
Henning,1L, 83, 185
Herde, P.,184
Hernandez, M.,43,122
Hernandez,5.,91
Hoving, L.,168,169
Hughes, P.,784
Hunter, M.,57
Hunter, R.,185
I
Isont, 5.,59
Ito, I.,150
J
Jehannin, C.,171
Jimênez, 5.,127,128
Johannßson, A., 39, 132, 134
Johnson, G.,79,117
Johnston, M.,170
K
Kaczrnarek, M., 7 7, l3l, 144, 147
Kamiúska, K.,145
Kaneko, H.,150
Kanitz, W,63
Kashíwazaki, N.,750
Katoh, Y.,133
Kuulfod, J.,63
Kawashima,4.,133
Kemp, 8., 61, 95, 146, 155, 156, 168, 169
Kiewßç J.,77,144,147
Kikuchi, A.,133
Kikuchi, K.,55, 133, 150
Kimball,8.,701
Knapczyk, K.,140,154
Knol,8.,726
Knox, R.,97,160
Kohchi,5.,133
Kohler, D.,135
Koperq 1.,740
Koziorowski, M.,140
Krejcí, R.,l7l
Kräger, M.,185
Kuijken, N., 156
Kumnter,4.,177
Kummer, R,,73
Kvist, U.,39
L
LaÍorest, J,, 723
I-angenilijk, P.,61, 156, 164
Inngner, K,,185
Iturenssen, 8,,756
kwrence, A., 162
I*enhouwers, J.,126
I*neveu, P.,771
I*ssard, C.,127
I*telu, L.,141
I*telu, P.,141
Li, Y.,101
Linton, N.,67
Lucas, X.,43
Lucy,M.,1l5, 153, 186
Lundeheim, N., 136
M
Madej, A., 132, 174
Madej, M.,132
Madsen, M.,132,174
Maes, D.,lO3
Marques, G.,138
Marlens, M,,178, 179
Martinat-B otté, F,, l7 2, 1'1 6
Mørtínez, 8., 39, 43, 122, 124
Mathew, D.,115,153
Matsuda, M.,133
Matte, J., 123
McArdle, H.,163
Mclfutick, 5.,149
Megee,5.,107
Mellagi, A., 151 , 173
Miles, 1.,71, 105, 148, 158
Mohan,5.,107
Moore, H.,93,99
Moreira, L.,138
Mores, T.,167
Morrell, J.,89
Motsinger-Reif, A.,69
Mozo-Martín, R., 127, 128
Murdoch, G.,99
Murplry, C.,159
188

N
Nagøi, T.,55
Nakai, M.,55, 150
Nienaber, J.,758
Noguchí, J.,750
Nonneman, D,,105
Norrby, M.,132,174
Nottle, M.,149,164
Novak, 5., 65, 93, 99, 152
o
O'I*ary,5.,164
Okamura, C.,115,153
Okamura, N.,133
Olesen, A.,85
Oliver, W.,148
Oliviero, C.,175
Anturu, 5.,49
Osman, 8.,133
Ovito, C., 139
P
Paín, 5.,184
Panzardi, A,, l5l, 173
Paradß, F., 57 ,93,99, 152
Parreírø, G.,138
Panilla, 1.,43, 124
Pasternak, J,,152
Patterson, J., lll,1 1 3, 161
Peltoniemi, O., 175
Peña, F.,39
Pena, R,139
Petrunkina, A.,83
Pfeiffer, A.,166
Piedrahita,,/., 69, 105
Pires, J.,741
Prather, R.,59, 159
Pratt, 5.,742
o
Quesnel, H., 53, 172, 176
Quintero, V.,178,179
R
Rath, D.,45,87
Rátkt, J.,63,125
Rempel, L.,177
Rieke, A.,759
Roca, J., 39, 43, 45, 122, 124
Rodrtguez-Martínez, H., 39 , 45, 89, 134
Ro drigue z- S o sa, J., 107
Rogan, D.,113
Rohrer, G.,777
Rooke, I.,162
Rosales, F.,178,179
Ross, ,/., 49,59,101,153, 159
Rothschild, M.,49
Royer, 8.,176
Rutherford, K.,762
S
Safranskí, T., 142, 180, I 86
Sallê, 8.,171
Samuel, M.,159
Sánchez, A.,139
Santi, M.,767
Sanz, L,,39
Saravia, F., 39, 89, 134, 1'7 4
Sarlós, P.,725
Schnapper, A.,785
Schneider, F.,63
Schott, R.,ll1
Schuberth, H., 87 , 185
Sellner, ¿'.,115,153
Serriere, 5., 172,176
Seyfert, H.,87
Slomc4t nskø, M., I 40, lS4
Smith, H.,180
Smíth, T.,177
Soede, N.,61,95,146,155, 156, 168, 169
Somfai,7.,55
Sorensen, G,,l8l
Spate, L.,159
Spencer,7.,79
Spiers, D.,186
Stein, D.,101
Stott, P.,764
Strzezek, I.,45
Sutovsþ, P,,51
Swanson, M.,160, 170
T
Tabecka- Lonczy nska, A., 7 54
Taibl, J.,9'7,160
Takashima, M.,133
Takebayashi, K.,733
Tayade, C.,67,157
Taylor, U.,87
Terlouw,5.,135
Thangavelu, 5,,727
Thorup, F.,182,183
Tilleman, K.,703
Tomóq A,,139
Trønqaart, F,,172
Tsai, P.,41
Isal, S.,69, 105
U
Uwiera, R., 152
l
I
189

V
Valette,8.,737
Vøllet, J., 71, 105, 148, f58
Valros, 4.,775
vøn den Heuvel, M.,67,157
yan der'Waaij, 8,,146
van lteuwen, J.,95
Van Soom, A.,103
Vøn Weltere, W.,184
van Wienen, M.,89
Vassiliev, 1.,149
Vøssilieva, 5.,749
Vázquez, 1., 39, 43, 122, 124
Venturi, 8.,1,'12,176
Visconti,4.,97
W
Waberski, D.,45, 83, 185
Wøclawik, A.,77
Wähner, M.,63
.Wallgren, M.,39, 89, 134, 136
Walters, 8.,159
Wøsielak, M.,145
Wax, D,,159
Webel, 5.,160, 170
Wells, K.,153, 159
Went6 1.,73, 157, l6't, 173
Werlnng, R.,767
Wessels, J.,6"1
White, F.,l0L
Whyte, J.,159
Williøms, A.,180, 186
Willíams, S.,95, 137
Wu, G.,79,711
Wyman, G.,107
x
Xíß, Y.,105
Y
Yeste, M., 127 , 128
Yoshioka, K.,743
z
Zak, L,,ll3
Zerbe, H.,87
Zhuo, J.,159
Zíecik, A., 7'7, 131, 144, 147
190