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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A151 OPU-IVP AND ET IN VITRO PRODUCTION OF OVINE EMBRYOS OF DORPER AND SANTA A INES BREED ASPIRATED BY VIDEOLAP APAR AROSC OSCOPY OPY AND TRANSPOR ANSPORTED OVER LONG DISTANCES Marcos Chalhoub 1 , Andrea Cristina Basso 2 , Janaína Silva Chacon 3 , Antônio de Lisboa Ribeiro Filho 4 , Marcus Vinícius Galvão Loiola 5 , Leandro Moureira de Castro Feitosa 6 , Bruno Henrique de Araújo Andrade 7 , Alexandra Soares Rodrigues 8 , Priscila Assis Ferraz 9 , Endrigo Adonis Braga de Araujo 10 , Sidnei Nunes Oliv liveir eira 11 & Ale lexandr xandre Far aria Tab abet 12 1,4,5,6,7,8,9,10,11 UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR, BA, BRAZIL. 2 IN VITRO BRASIL SA, MOGI MIRIM, SP, BRAZIL. 3 AUTÔNOMO, SÃO PAULO, SP, BRAZIL. 12 AUTÔNOMO, SÃO PAULO, SP, BRAZIL. The sheep industry in Brazil is in a time of expansion, increasing thus the demand for biotechnologies of reproduction in this specie, amongst which stands out in vitro production of embryos (IVP), which allows the intensive exploration of the genetic and reproductive potential of females. However, this technology still has not been adequately explored in small ruminants, and several factors may influence results, for example, the breed of the donors. Aimed to compare the efficiency of IVP in Dorper and Santa Ines breed sheep after videolaparoscopic aspiration of oocytes and transport over long distances. We analyzed the number of oocytes retrieved and the rates of cleavage. We used 59 donors ewes, single, with three years of age on average, created in an extensive way, being 34 Dorper and 25 Santa Ines ewes. All animals were previously synchronized and superstimulated according to Baldassare et al. (2004, Animal Reproduction Science 82-83, 255-266). At the beginning, follicular aspiration was performed by videolaparoscopy in Dorper and Santa Ines donors created in the microregion of the Chapada Diamantina in Bahia. The aspirated follicular fluid was evaluated under stereomicroscope and cumulus-oocyte complexes visualized were counted, sorted and transferred to cryogenic tubes containing maturation medium on temperature, humidity and CO2 concentration suitable. Later sent to a commercial laboratory in the state of São Paulo, which followed the steps of in vitro maturation, fertilization and culture, according to Basso et al. (2008, O embrião 38, 9-13). In order to compare the number of oocytes, embryo produced and cleavage rate in different breeds, was employed the statistical package SAS, version 6.0 (Procedure MEANS and GLM with P < 0.05). The average of total oocytes retrieved and embryo produced of Dorper ewes of 11.90±7.18 and 8.83±5.37 was higher (P < 0.05) to that obtained in Santa Ines ewes of 9.00±4.47 and 4.62±2.48, respectively. The cleavage rate (embryos produced/oocytes recovered) in the Dorper ewes was of 76.09%, significantly higher compared to the Santa Ines ewes of 57.47%. These results suggest that exist a superiority in the production of oocytes by videolaparoscopy and in the cleavage rate of in vitro produced embryos and transported over long distances, in the Dorper in relation to Santa Ines breed. Evidenced the feasibility of transporting long distance oocytes in sheep. Keywords: ivep, videolaparoscopy, cleavage rate. A152 OPU-IVP AND ET IN VIVO EMBRYO PRODUCTION IN COWS SUPEROVUL OVULATED 1 OR 2 DAYS AFTER OPU Ricardo Silva Surjus 1 , Alexandre Barbieri Prata 1 , Marta Borsato 1 , Fernada C.S.Z. Mattos 2 , Mariana Curci Martins da Silveira 1 , Gerson Barreto Mourão 1 , Alexandre Vaz Pires 1 & Roberto Sartori 1 1 ESALQ-USP, PIRACICABA, SP, BRAZIL. 2 UNESP, BOTUCATU, SP, BRAZIL. The aim of this study was to evaluate the superstimulatory and superovulatory responses and in vivo embryo production in cows treated with FSH starting at 1 or 2 days after OPU. Thirty two nonlactating Nelore cows, weighing 489.5±11.3 kg, handled in a two cows per stall system, were fed a diet with corn, sugarcane bagasse, urea and mineral salt, according to the NRC (2000). All cows were subjected to aspiration of follicles >2 mm for OPU. Recovered oocytes were used in another experiment. After OPU, the cows were randomly divided into two groups: D1 and D2, in which the follicle superstimulatory treatments with FSH started 1 or 2 days after OPU, respectively. The embryo donors received an intravaginal device (IVD) of progesterone release (Sincrogest ® , Ouro Fino, Ribeirão Preto, Brazil), soon after OPU. The cows received eight decreasing doses of FSH (100 mg, i.m., Folltropin-V ® , Bioniche, Ontario, Canada), and concomitant with the fifth and sixth treatments of FSH, PGF2α (150 µg each, i.m., d-cloprostenol, Sincrocio ® , Ouro Fino) was administered. The IVD was removed at the time of the last FSH injection. Twelve hours after IVD removal ovulation was induced with GnRH (0.01 mg, i.m., Buserelin acetate, Sincroforte ® , Ouro Fino). Twelve and 24 h later, cows were inseminated. Embryo collection was performed 8 days after GnRH injection. Data were analyzed using the GLIMMIX procedure of SAS and the results are presented as least squares means ± SE. On the fourth day of FSH treatment, cows in Group G1 had a greater superstimulatory (follicles =6 mm in diameter) response (21.1±3.0 follicles) than those in Group G2 (13.7±2.4 follicles, P = 0.08). In addition, there was a much greater proportion (P < 0.001) of follicles =8 mm compared to follicles =6 mm and 0.10). Nevertheless, the cows in Group D2 had more viable (3.3±1.2 vs.. 1.8±0.7, P = 0.09) and freezable (3.2±1.1 vs.. 1.3±0.5, P < 0.05) embryos than those in Group D1. In conclusion, to obtain a better superovulatory response and higher embryo production in superovulated cows after OPU, it is recommended to wait at least 2 days to start treatment with FSH. [Acknowledgements: We thank FAPESP, CNPq, and InVitro Brazil for financial and technical support]. Keywords: embryos, superovatory, opu. s412
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A153 OPU-IVP AND ET HORMONAL PROTOC OCOLS OLS FOR OOCYTE RETRIEVAL AND IN VITRO PRODUCTION OF EMBRYOS IN BOVINE DONORS Tatimar timara a Mar aria Miy iyauchi 1 , Tochimar chimara a Apar parecida Miy iyauchi 1 , Miller Per ereir eira Palhão 1 , Aline Sousa Camar amargos 2 , Car arlos Antônio de Car arvalho alho Fer ernandes 1 & Mar arilu Martins Gioso 1 1 UNIFENAS, ALFENAS, MG, BRAZIL. 2 FMVZ-UNESP, BOTUCATU, SP, BRAZIL. The in vitro embryo production (IEVP) can improve the genetic outcome of the zootechnical features in bovine specie. However, the efficiency of this technique is directly related to the number and the quality of the oocytes recovered after follicular aspiration (OPU). The control of the follicle development can improve the results of the IEVP. The aim of this study was to evaluate different protocols for synchronization follicle development in B. taurus and B. indicus donors. The animals were randomized in one of the four experimental groups: TI - Control (N = 81): OPU regardless the day of the estrous cycle with no previous hormone treatment; TII - (N = 97): synchronization of the follicular wave (2 mg BE1) five days before OPU; TIII - (N = 97): synchronization of the follicular wave and no corpus luteum (CL). Five days before the OPU, the follicular wave was synchronized with 2 mg of EB, an ear implant (Norgestomet) was placed and 0.150 mg of D- cloprostenol was given; TIV - (N = 69): the same protocol of the TIII plus stimulation of the follicular development. Two injections of FSH (each injection of the 50 UI for indicus and 75 UI for taurus) were performed 12 h apart four days after starting protocol. The OPU section was held 12 to 16 h after last FSH injection. The oocytes were sent to PIV laboratory for a period until 6 h. Procedures were the same performed in routine of the commercial laboratory. The oocyte retrieval (total and viable) and embryo production were higher in indicus than in taurine (17.7 ± 0.9 vs. 11.9 ± 0.6, 14.2 ± 0.7 vs.. 8.8 ± 0.5, 4.3 ± 0.3 vs. 2.1 ± 0.2, respectively, P < 0.05). There was an increase in number and quality of oocytes recovered from donors from TIII and TIV (16.8 ± 1.2 and 17.8 ± 1.4 vs. 10.7 ± 1.0, 13.2 ± 1.0 and 13.8 ± 1.1 vs.. 8.1 ± 0.8, respectively, P < 0.05). The total embryo production was higher in all treatments when compared to control (P < 0.05). The FSH injections (TIV) to promote the ultimate follicle development and increased the average of the embryo production when compared to the other groups (4.6± 0.6 and 2.8± 0.3). In taurine, the OPU section from treatments III and IV provided more oocytes (total and viable) (12.7 ± 1.5, 14.3 ± 1.3 and 9.8 ± 1.2, 10.9 ± 1.1, P < 0 05). In Zebu, the embryo production was higher in treated groups when compared to control (5.1 ± 0.5 vs. 2.0 ± 0.4, P < 0.05). The synchronization treatments were efficient for oocyte retrieval in taurine and embryo production and zebu donors. [Support: FAPEMIG]. Keywords: opu, gyr, follicle wave. A154 OPU-IVP AND ET TECHNICAL REPORT: OVUN PICK UP BY SINGLE FOR RECOVER OVERY OF OOCYTES IN MARES IN TRANSITION PERIOD André Gomier omiero o Rigo 1 , Bruno Valen alente e Sanches anches, José Henr enrique For ortes Pon ontes es, Luc ucas Lop opes Moino oino, Rodr drigo Mendes Untur tura, Per erla Dagher Cassoli Fleur leury, Mar ario Martinez-Dias , Janaina Ferreira Nagao & Andrea Cristina Basso IN VITRO BRAZIL S.A., MOGI MIRIM, SP, BRAZIL. The most difficulty founded for practicing reproductive biotechnologies in horses as in vitro fertilization, intracytoplasmic sperm injection, oocyte transfer, gamete intrafallopian transfer and nuclear transfer is the difficulty of aspiration and oocyte retrieval in large quantities. For the present research we used 26 crossbreed mares, 4 to 16 years old, body scores from 3.0 to 3.5, average weight of 400 kg, daily subjected to gynecological examination. The mares presented multiple follicles with size between 5 and 22 mm and complete absence of uterine edema or diestrus featuring transitional period between March to April 2011. For follicle aspiration the animals were treated with 0.5 mL of acepromazine 2% EV. The aspiration was preceded by rectal cleaning using carboxy-methyl cellulose additioned of 10% lidocaine hydrochloride 2%. Epidural anesthesia was not performed. After cleaning the perineal area with water and 70% alcohol, the procedure was guided with ultrasound MINDRAY 2200, 5 MHz sector transducer. The transducer was connected to a single-way needle and positioned transvaginal, beside to the cervix and the ovary where the follicles were aspirated. The ovary was placed by transrectal manipulation to the follicular wall being juxtaposed to the transducer and the cannula guide. The needle was inserted through the cannula guide to perform the follicular puncture. The follicular fluid was aspirated without washing the follicle, using TERUMO 16 gauge needle (0.9 X 50 mm) and vacuum pump (Cook Veterinary Products, New Buffalo, MI, USA) using 200 mm Hg pressure. After follicular aspiration proceeded up the search and evaluation of oocytes in a petri dish with the aid of a binocular dissecting microscope with a magnification between 15 and 60 times. After completion of the total scan of the ovaries, 298 follicles were aspirated, 84 oocytes were recovered (recovery rate 28%), yielding an average of 3.23 of total oocytes and 2.75 viable oocytes per mare. After complete aspiration of the ovaries, these mares were monitored daily for 21 days by ultrasound, all returned to a growth of new follicular wave 6-8 days after the first aspiration and there were no sequelae from the procedure. This research puts the follicular aspiration in mares receiving transitional or diestrus phase as an alternative to obtaining oocytes needed for the development and study of new biotechnologies, without affecting the commercial routine of an equine recipient center. Keywords: ovum pick up, mares, oocytes. N s413
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