2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A255 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” ULTR TRASONOGR ASONOGRAPHIC APHIC PROFILE OF EMBRYONIC/FET ONIC/FETAL GROWTH OF TRANSGENIC GOATS FOR HG-CSF Dárcio Ítalo Alv lves Teix eixeir eira, Car arla Rozilene Guimarães Silv ilva Oliv liveir eira, Car arlos Henr enrique Sousa de Melo elo, Antônio Car arlos de Albuquer lbuquerque Teles Filho ilho, Ribr ibrio io Ivan Tavar ares Per ereir eira Batista, Joanna Mar aria Gonçalv onçalves de Souza, Ale lexsandr sandra a Fer ernandes Per ereir eira, Luciana Magalhães Melo & Vic icen ente e José de Figueirêdo Freitas UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL. Transgenic goats should be evaluated by their embryonic/fetal growth, what could be done by real time ultrasonography. The aim of the study was to evaluate the morphological and morphometric development of transgenic goat embryos and fetuses for human Granulocyte Colony Stimulating Factor (hG-CSF). The project was approved by the ethics committee and biosafety of UECE. Four non-transgenic goats pregnant after mated by the transgenic Canindé male were used. Ultrasonographic exams (Falco 100 ® , Pie Medical, Maastricht, Netherlands) were performed at 30 and 40 days (transrectal via, linear array transducer, 6.0/8.0 MHz) and at 50, 60, 90 and 120 days of pregnancy (transabdominal via, convex transducer, 3.5/5.0 MHz). Images were recorded for posterior evaluation of gestational development and embryonary and fetal parameters such as Heart Rate (HR), conceptus movement, Embryonic Vesicle Diameter (EVD), Crown-Rump Length (CRL), Diameter of Thorax (DT), Abdome (DA), Umbilical Cord (DUC) and Placentomes (DP). Images were measured by the Image J software (National Institutes of Health, Millersville, EUA), with prior calibration for each frequency. Morphological aspects such as formation of skeleton and heart were also assessed. Four transgenic goats (two males and two females) besides two non-transgenic males were obtained after PCR analysis. After this moment, transgenic and non-transgenic sonograms were separated. In all conceptus the HR decreased during pregnancy. The movements of fetus were present in all exams, being less intense in the beginning and more evident after 50 days of pregnancy. EVD and CRL were evaluated at 30, 40, 50 and 60 days. It was observed that conceptus gradually grew, and the evaluation of other parameters was necessary to better follow the growth in the final stage. After the 50 initial days it was also possible to evaluate the DT, DA, DUC and DP. During the pregnancy, all fetuses grew as expected, with a higher development on the final third of pregnancy. Bone structures development occurred at 60 days, with hiperechoic areas forming the skull, thorax, spine and long bones. At 30 and 40 days of pregnancy, the heart appeared as a small anechoic area and with intense movements. After 50 days it was already possible to evaluate the cardiac chambers, consisting in four anechoic well defined areas. Transgenic goats embryo and fetuses showed a morphologic development similar to the non-transgenic ones and remained viable during all period evaluated. Keywords: transgenic, goat, hG-CSF. A256 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” PROTEOME-B TEOME-BASED INSIGHTS S ON OOCYTE COMPETENCE FROM A RETROSPECTIVE ANALYSIS OF BOVINE FOLLICULAR FLUID Felipe Perecin 1 , Lilian Jesus Oliveira 2 , Juliano Rodrigues Sangalli 3 , Tiago Henrique Câmara de Bem 4 , Gustavo Henrique Martins Ferreira Souza 5 , Jerusa Simone Garcia 6 , Ricardo Pimenta Bertolla 7 , Christina Ramires Ferreira 8 , Marcos Nogueira Eberlin 9 & Flávio Vieira Meirelles 10 1,2,3,4,6,10 DEPTO. CIÊNCIAS BÁSICAS-FZEA-USP, PIRASSUNUNGA, SP, BRAZIL. 5 WATERS CORPORATION, LABORATÓRIO DE DESENVOLVIMENTO DE APLICAÇÕES EM ESPECTROMETRIA DE MASSAS, SÃO PAULO, SP, BRAZIL. 6 INSTITUTO DE CIÊNCIAS EXATAS, UNIVERSIDADE FEDERAL DE ALFENAS, ALFENAS, MG, BRAZIL. 7 UNIVERSIDADE FEDERAL DE SÃO PAULO (UNIFESP/EPM), SÃO PAULO, SP, BRAZIL. 8,9 LABORATÓRIO THOMSON DE ESPECTROMETRIA DE MASSAS, INSTITUTO DE QUÍMICA-UNICAMP, CAMPINAS, SP, BRAZIL. The proteome composition of follicular fluid produced during folliculogenesis reflects the metabolism of the ovarian follicle and the competence of the oocyte to finish maturation and to support embryo development. In order to study the proteome of bovine follicular fluid (bFF), 46 ovarian follicles (3.14+0.56 mm diameter) were dissected so that the oocyte and follicular fluid could be individually recovered. The bFF samples were stored at -80°C while oocytes were individually submitted to in vitro maturation and parthenogenetic activation. Oocyte maturation and embryo development were assessed through first polar body (1.PB) extrusion and development until blastocyst stage, respectively, generating three groups: (1) Non-matured (NM) – oocytes that did not extruded 1.PB; (2) Matured (M) – oocytes that extruded 1.PB but did not reached blastocyst stage and (3) Blastocyst (BL) – oocytes that reached blastocyst stage. Bovine FF of these groups were pooled and retrospectively used for label-free comparative shotgun proteomics analysis using MudPit (Multidimensional Protein Identification Technology) tandem MSE acquisition to investigate differential protein expression. Functional and pathway analysis using FatiGo (www.babelomics.org); Pathway Express (http://vortex.cs.wayne.edu/ontoexpress) and Ingenuity Pathway Analysis (www.ingenuity.com) were used to identify relevant ontologies and canonical or noncanonical pathways represented by the expressed proteins in the bFF. A total of 105 proteins or randomic sequences were identified corresponding to 59 unique proteins present in the bFF. Out of 59 proteins, 11 were exclusively expressed in the bFF from NM oocytes, 13 in the bFF from M oocytes and 10 in the bFF of BL oocytes whereas were 18 proteins commonly expressed among groups. There was a co-expression of 2 proteins in the bFF from NM and M, 3 proteins in the bFF from NM and BL groups and 2 proteins in the bFF from M and BL groups. The ontology and pathway analysis showed the overrepresentation of proteins from extracellular space, acute inflammatory response, vitamin D and single-stranded RNA binding process in all groups. Among the unique proteins present in the BL group we found Apolipoprotein A1 (APOA1), involved in the metabolism of cholesterol and steroid, and the Secretogranin-1 (CHGB) involved in vesicle sorting and exocytosis. Our results bring new insights about the proteins present in the bovine follicular environment and involved in the establishment of oocyte competence. Keywords: follicular fluid, oocyte competence, proteomic. s464
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A257 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” RELATIVE ABUNDANCE OF MATERNAL TRANSCRIPT ANSCRIPTS S IN OOCYTES WITH AND WITHOUT POLAR BODY AFTER IN VITRO MATUR TURATION TION Lilian Tam amy Iguma 1 , Michele Munk Per ereir eira 2 , Sabine Wohlr ohlres-V es-Viana 3 , José Nelio S. Sales 4 , Car arolina Cap apobiango R. Quin uintão 5 , Isab sabella Silv ilvestr estre e B. Pin into 6 , Raquel Varella Serapião 7 ; Savana Giacomini Brito 8 , Bruno Campos Caravalho 9 , João Henrique Moreira Viana 10 , Luiz Sérgio Almeida Camargo 11 & Marta Fonseca M. Guimarães 12 1,5,6,9,10,11,12 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2,3,6 UNIVERSIDADE FEDERAL DE JUIZ DE FORA, JUIZ DE FORA, MG, BRAZIL. 4 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 7 PESAGRO-RIO, NITERÓI, RJ, BRAZIL. 8 UNIVERSIDADE PRESIDENTE ANTÔNIO CARLOS, JUIZ DE FORA, MG, BRAZIL. The oocyte ability to undergo maturation and early cleavages during the maternal–zygotic transition (MZT) is associated to transcript levels stored in its cytoplasm. The extrusion of the polar body is an indicative of oocyte ability to undergo maturation. However, to our knowledge, no data comparing mRNA levels among bovine oocytes maturated in vitro presenting polar body (PB) and oocytes maturated in vitro without the presence of polar body (NPB) are available. The aim of the present study was to compare the relative abundance of maternal transcripts of Maternal Antigen That Embryo Requires (MATER), Zygote Arrest (ZAR1), TEA domain family member 2 (TEAD2) and High Mobility Group N (HMGN1) genes between oocytes with or without PB (PB and NPB groups, respectively) following in vitro maturation. Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 199 (Gibco Life Technologies, New York, USA) containing 10% estrus cow serum and 20 µg/mL FSH (Pluset, Serono, Italy), for 24 h under 5% CO2 in air at 38.5°C. Oocytes separated according to the presence or absence of PB and then they were denuded and frozen in liquid nitrogen. Three pools of ten oocytes for each group were subject to RNA extraction. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (WTA2 – Sigma Aldrich) according to the manufacturer’s instructions. Relative abundance of the target transcripts was performed by real-time PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, EUA) using the betaactin gene as the endogenous reference. The relative expression of MATER (0.81±0.11), ZAR1 (0.82±0.09), TEAD2 (0.83±0.07) and HMGN1 (0.90±0.08) was dowregulated (P < 0.05) for oocytes NPB after in vitro maturation. We conclude that the presence of polar body may be related to abundance of maternal mRNA and can contribute to predict quality of in vitro maturation conditions.[Financial support: Embrapa – Animal Genomics Network Project (01.06.9.01.01.00) and Innovations in Animal Reproduction Network Project (01.07.01.002), CNPq, CAPES and Fapemig]. Keywords: gene expression, oocytes, in vitro maturation. A258 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” RELATIONSHIP BETWEEN ACR CROSSOME REACTION, POST-THAW SPERM EVAL ALUATION AND TOTAL PROTEIN IN MOXOTÓ GOAT SEMEN R ober erta V ianna do Valle alle, Ângela Mar aria Xavier Elo loy, Nadiana Mar aria Mendes Silv ilva, Fr ancisco W ilson Venâncio Silv ilva & João Ric icar ardo Furtado UVA/EMBRAPA CAPRINOS E OVINOS, SOBRAL, CE, BRAZIL. Even been able to fertilize, sperm cells from epididymis can present alterations when in contact with the seminal plasma during the ejaculation, losing its capacity of suffering the acromosse reaction (AR) and to penetrate the oocyte. This study aimed to evaluate the relation between (AR) with the post-thaw semen evaluation and the total proteins (TP) in the seminal plasma of Moxotó bucks in Brazilian Northeast. The semen was collected from five males from January to March/2011, totalizing three samples per animal, being one week of the month used for semen freezing and the other for total protein analysis. For semen freezing, Tris-glycerol at 2% was used, being the sperm stored in 0.25 mL straws , using the portable system Tetakon, TK 3000. The post-thawed spermatic motility (%) was evaluated and the TP analysis followed the Bradford method (1976, M Analyt Biochem 72:248-254). In the AR evaluation, the Naftol yellow/erythrosine B was used aiming to identify the percent of post-frozen/thawed sperm reaction. The Pearson correlation was run between post-thawed spermatic motility and total plasma protein. The mean spermatic motility and the post-thawed AR were 20% and 35%, respectively. It was verified a positive and high intensity correlation (r= 0.40) between AR and progressive post-thaw motility, according to studies that are in agreement with these results (Correa et al., 1997, Theriogenology 48:721-731). It was not observed a correlation (P > 0.05) between AR and seminal TP. It is concluded that probably Moxotó animals that showed higher spermatic motility can also be able to suffer good rate of AR and, in consequence, can probably show higher fertility. However, it is recommended more studies on this subject. Keywords: cryopreservation, fertilization, proteomics. N s465
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