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. A159 OPU-IVP AND ET USE OF RETINOIDS TO INHIBIT THE IN VITRO APOPTOSIS OSIS OF CAPRINE OOCYTES AND EMBRYOUS Juliana Costa Conceição 1 , Ricardo de Macedo Chaves 2 , Madson Atila Vidal Silva 3 , Edivaldo Rosas dos Santos Júnior 4 , Leopoldo Mayer Freitas Neto 5 , Fabíola Freitas de Paula-Lopes 6 , Paulo Bayard Dias Gonçalves 7 , Paulo Fernandes Lima 8 & Marcos Antonio Lemos de Oliveira 9 1,4,8,9 UNIVERSIDADE FEDERAL RURAL DE PERNAMBUCO, RECIFE, PE, BRAZIL. 2,3 UNIVERSIDADE ESTADUAL DO MARANHÃO, SAO LUIS, MA, BRAZIL. 5 FACULDADE PIO DÉCIMO, ARACAJU, SE, BRAZIL. 6 UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 7 UNIVERSIDADE FEDERAL DE SANTA MARIA, SANTA MARIA, RS, BRAZIL. The objective was to evaluate the apoptosis of goat oocytes and embryos through the activity of enzymes of group II caspases and DNA fragmentation by TUNEL assay, which underwent in vitro maturation (IVM) in medium with or without retinol (TR) and retinoic acid (RA). Ovaries were obtained from a slaughterhouse and transported to the laboratory in warm saline at 30°C. The oocytes were collected from follicles measuring 2-6 mm in diameter by aspiration with a syringe needle attached. The oocytes, tested in 10 replicates, were initially washed five times on medium itself, then screened and distributed in droplets of TCM 199 (Control Group - CG) and this medium supplemented with retinol (retinol group - GRT) or Retinoic Acid (Group Retinoic Acid - OHR), to finally being incubated at 39°C in a humidified atmosphere containing 5% CO2 for 24 h. After this period, a proportion of oocytes was processed through mDM and exposed to sperm for 18 h in the same atmospheric condition. Subsequently, presumptive zygotes were transferred to drops of KSOM medium containing the monolayer of cells in the oviduct, and finally incubated at 39°C in humid atmosphere with 5% CO2. After 48 h, the structures that were not cleaved and removed 30% of the medium was partially renovated and remained so for eight days. The other part of the oocytes and blastocysts in each experimental group were prepared to locate the changes characteristic of apoptosis, using the reagent PhiPhiLux-G1D2 and solutions of 4% paraformaldehyde and Phosphate-Buffered Saline + 1 mg/mL polyvinylpyrrolidone. For statistical analysis, variance analysis was performed by the method of least squares. The activity of caspase enzymes did not differ (P > 0.05) between oocytes in the CG (10.32%), GRT (8.08%) and the OHR (8.45%) and between GRT and the OHR. The oocytes and blastocysts positive for TUNEL assay were higher (P < 0.05), respectively, in the CG (11.46%, 9.22%) than in GRT (6.86%, 5.45%) and the OHR (7.41%, 6.12%), no difference (P > 0.05) between the GRT and the OHR. Zygotes GC had lower (P < 0.05) capacity development to the blastocyst stage (5.32 ± 0.81) than those of the GRT (7.94 ± 0.93) and GAR (7.36 ± 1.02), no difference (P > 0.05) between the GRT and the OHR. This result indicates that the addition of retinoids in the middle of oocyte maturation reduces cell apoptosis, and increases in vitro production of goat embryos. Keywords: retinoids, apoptosis, blastocyst. A160 OPU-IVP AND ET FSH IN PROTOC OCOLS OLS FOR FOLLICULAR WAVE SYNCHRONIZA ONIZATION AND EMBRYO PRODUCTION IN TAURIN AND ZEBU DONNORS Carlos Antônio de Carvalho Fernandes 1 , Ana Cristina Silva de Figueiredo 1 , Bruno Fernandes Ludgero Alves 2 , Eduardo Ramos de Oliveira 2 , Thais Camargo Rossi 2 , Adriana Agostini 1 & Ana Cristina Silva de Figueiredo 1 1 UNIV. DE ALFENAS, ALFENAS, MG, BRAZIL. 2 BIOTRAN LTDA, ALFENAS, MG, BRAZIL. The current knowledge in physiology of reproduction and hormone therapies can improve the results of the follicle aspiration (OPU) and the quality of the oocytes recovered. Including the FSH in protocols available reducing the atresia and the inconsistency of the oocytes retrieval after OPU, mainly for Taurus breeds. This study was designed to evaluate the inclusion of FSH in protocols for follicle synchronization and to compare the IVPE results between Taurus (Holstein, Jersey and Angus) and Zebu (Gir, Nelore and Guzerá) breeds. Twenty-one Taurus and 23 Zebu donators were included for this purpose. All animal were randomized in one of the three treatments: T1) Control - OPU without follicle synchronization; T2) Day 0 (D0) – implant of progesterone, 2 mg of Estradiol Benzoate (EB) and 0,530 mg of cloprostenol. Five days later (D5), two injections of FSH - 60UI intramuscularly (IM) during the morning and 40 UI IM 12 h later; T3) D0 - the same for T2, D4 afternoon – two injections of FSH - 25 UI (IM) and 75 UI subcutaneously (SC) - given at the same time. The interval from previous aspiration was always greater than 30 days and all animals underwent all treatments. The cows were submitted to OPU six days after the beginning of the treatment. The procedures for IVEP were performed in the same laboratory with X sorted semen. Means among treatments for oocyte recovered and produced embryos were compared with Tukey test at 5% of probability. Zebu cows provided more oocytes and produced more embryos than Taurus (P < 0.05) in all treatments. Within subspecies (Zebu and Taurus), there were no statistical differences for oocytes (total and viable) recovered from different breeds. In Zebu donators, the total oocytes (19,9+8,3, 22,6+9,7 and 21,4+9,8) and those classified as viable (11,3+7,5, 14,1+7,6 and 13,7+8,8) were similar for T1, T2 and T3, respectively. The same was observed in Taurus, 22,8+6,4, 15,6+5,2 and 14,4+6,2 oocytes, and 7,6+6,6, 9,2+7,1 and 8,7+6,8 viable for T1, T2 and T3, respectively. In Zebu, the embryo production by donator was 4,3+2,6b, 6,6+2,7a and 6,6+2,9a, and for Taurus it was 2,5+2,0b, 3,8+1,8a and 3,5+2,2a, respectively, for T1, T2 and T3. The treatments did not change the number and the quality of the recovered oocytes; however, the embryo production was improved. It seems that the inclusion of the FSH in protocols for follicle synchronization improved the intrinsic quality of the oocytes. Additionally, treatments 2 and 3 were similar and the injection of FSH given by SC could shorten the animal manipulation. [Support: FAPEMIG]. Keywords: bovine, opu, Bos taurus. s416
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A161 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION USE OF β-MER -MERCAPT APTOETHANOL FOR IN VITRO MATUR TURATION TION AND CULTURE OF OVINE EMBRYOS Jorgea Pradieé 1 , Liziane Lemos Vianna 1 , Alexander Oliveira Gonçalves 1 , Elisa Caroline da Silva Santos 1 , Rafael Gianela Mondadori 1 , Thomaz Lucia Jr. 1 , Arnaldo Diniz Vieira 1 & Ligia Margareth Cantarelli Pegoraro 2 1 UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS, RS, BRAZIL. 2 EMBRAPA, PELOTAS, RS, BRAZIL. One of the factors limiting the success of ovine IVP is the formation of reactive oxygen species (ROS) resulting from cell metabolism. The action of the ROS can be physiologically inhibited by antioxidants such as glutathione (GSH). As GSH’s concentration is reduced during most steps of the IVP, the media used for IVP are commonly supplemented with antioxidant substances. This study evaluated the development of ovine embryos after supplementation of the media for IVM and IVC with 20 µM β-mercaptoethanol (βME), a GSH precursor. Ovaries from pubertal sheep were collected in slaughterhouse and transported to the laboratory in a saline antibiotic solution at 30ºC. Cumulus-oocytes complexes (COC) were recovered and selected for IVM, which was conducted during 22-24 h, using the 199 TCM medium including estradiol, FSH, LH, pyruvate, estrus sheep serum and antibiotics. For this experiment 6 replicates were performed. Further comparisons were done between the IVM medium with (n = 328) or without (n = 320) βME. Sperm selection was conducted though swim-up method using fresh sperm and tris-citric acid-glucose medium. The IVF was done with sperm concentration of 1 x 106/mL in SOF medium with 2% estrus sheep serum. Thereafter, presumptive zygotes were denuded and cultured for 8 days in SOFaa medium and 2.5% FCS with or without âME. The IVM, IVF and IVC were conducted in an incubator with 5% CO2 and saturated humidity, at 39ºC. The rates of embryo development at D2 (cleavage) and D8 (blastocyst) were compared between treatments through the chi-square test. Although cleavage rates were the same (70%) for medium with or without âME, the blastocyst formation rate was greater (P < 0.001) for the control (17%) than for the βME-supplemented medium (5%). Therefore, supplementation of IVM and IVC medium with âME at the tested concentration did not benefit cleavage rates and was associated with reduced blastocyst rate. Further studies are still needed to investigate potential benefits of supplementation with âME at distinct concentration for the development of ovine embryos. Keywords: sheep, embryo, antioxidant. A162 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION ADDITION OF NERVE GROW TH FACT CTOR ON MATUR TURATION TION AND DEVEL VELOPMENT MEDIA FOR IN VITRO PRODUCTION OF SHEEP EMBRYOS M. Vilar ilariño iño 1 , M. Crisp ispo 2 , P.C. dos San antos-N os-Net eto 1 , R. Wijma 1 , N. Bar arrer era 1 , A. de León 2 , L. Barb arbeit eito 2 & A. Menchac enchaca 1 1 FUNDACIÓN IRAUY, MONTEVIDEO, URUGUAY. 2 INSTITUTO PASTEUR DE MONTEVIDEO, MONTEVIDEO, URUGUAY. Nerve Growth Factor (NGF) is a neurotrophic factor that promotes survival and proliferation in different types of non-neuronal cells. In the present study we assessed whether NGF could improve the in vitro embryo production in sheep. A total of 1342 cumulus oocyte complexes (COCs) covered by at least two layers of granulosa cells and homogeneous cytoplasm were selected for in vitro maturation (IVM) after aspiration from ovine ovaries obtained in a local slaughterhouse. The selected COCs were incubated in groups of 25-30 in 100 µL drops of TCM199 supplemented with 10% estrous ovine serum, 10 µg/mL FSH, 10 µg/mL LH, 100 ìM cysteamine and antibiotics; covered with embryo tested mineral oil for 24 h, at 39°C and 5% CO2 in air. For in vitro fertilization (IVF), matured oocytes (25-30/100 µL drop) were incubated for 22 h in fertilization medium with 1x106 frozen-thawed spermatozoa selected by swim-up method. The fertilization medium consisted of synthetic oviductal fluid (SOF) supplemented with 2% estrous ovine serum, 10 ìg/mL heparin and 10 ìg/mL hypotaurine. In vitro development (IVD) was performed in groups of 25-30 zygotes in 100 ìL drops of SOFaa BSA covered with mineral oil at 39°C and 5% O2, 5%CO2, 90% N2. NGF was evaluated using 0, 100 or 1000 ng/mL during IVM (0 ng/mL, n = 226; 100 ng/mL, n = 264; and 1000 ng/mL, n = 286), or IVD (0 ng/mL, n = 191; 100 ng/mL, n = 189 and 1000 ng/mL, n = 186). Cleavage rate (2 cell embryos/oocytes) and development/ rate (morula and blastocysts/oocytes) were recorded after 48 h and 6 d in culture after fertilization, respectively. Statistical analysis was performed by logistic regression. For IVM, no differences were found in the cleavage rate (46%, 104/226; 54.5%, 144/264; and 50%, 143/286), the development rate (12.4%, 28/226; 17.8%, 47/264; and 13.6%, 39/286), and the developed/cleaved rate (26.9%, 28/104; 32.6%, 47/144; and 27.3%, 39/143) for 0, 100 and 1000 ng/mL NGF, respectively. When NGF was used during IVD, no differences were found in the development rate (41.4%, 79/191; 45.5%, 86/189; and 46.8%, 87/186) and developed/cleaved rate (53.7%, 79/147; 57%, 86/151; and 56.5%, 87/154) for 0, 100 and 1000 ng/mL NGF, respectively. These results suggest that the addition of NGF in the maturation or development media does not affect the number of in vitro produced sheep embryos. Keywords: ngf, in vitro production, embryos sheep. N s417
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