Fecundação in vitro de ovócitos bovinos com sêmen submetido a diferentes diluidores
Carregando...
Data
2000-04-01
Autores
Coelho, Lia de Alencar
Esper, Cesar Roberto [UNESP]
Garcia, Joaquim Mansano [UNESP]
Vantini, Roberta [UNESP]
Almeida Junior, Ivo Luis [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Sociedade Brasileira de Zootecnia
Resumo
O objetivo deste trabalho foi avaliar a influência do diluidor do sêmen no desenvolvimento in vitro de ovócitos bovinos após a maturação e fecundação in vitro. Ejaculado de um reprodutor foi fracionado e submetido a três diluidores: Lactose/gema de ovo (LG), Citrato/gema de ovo (CG) e Tris/gema de ovo (TG). Amostras deste material foram envasadas, congeladas e estocadas em N² e, posteriormente, descongeladas; a fração móvel foi separada por gradiente descontínuo de Percoll. A concentração espermática foi ajustada para 10 x 10(6)/mL e a capacitação espermática, induzida com 10 µg/mL de heparina. Após 24 horas de cultura para maturação in vitro, os ovócitos, aspirados de folículos ovarianos, foram inseminados com sêmen diluído em meio TALP e, após 48 horas de cultura, os zigotos foram transferidos para gotas de meio TCM 199, com 5% de soro fetal bovino, 5% de soro de vaca em estro e suspensão de células epiteliais do oviduto bovino, cobertas com óleo de silicone, e mantidos em cultura por nove dias. Todas as culturas foram realizadas a 38,5ºC em atmosfera com 5% de CO2. Os dados foram analisados pelo teste do qui-quadrado e houve diferença com relação à taxa de clivagem (TC), sendo as médias de 66,0; 69,3; e 54,4% para LG, CG e TG, respectivamente. Não houve diferença entre tratamentos com relação às taxas de mórulas/blastocistos ou de eclosão. O diluidor do sêmen não teve efeito sobre o desenvolvimento in vitro de embriões bovinos, embora a TC tenha sido afetada.
This study investigated the effect of semen extender on in vitro development of bovine oocytes after in vitro maturation and fertilization. The ejaculate from one bull was divided in three parts which were diluted with three different extenders: Egg yolk/Lactose (EYL), Egg yolk/Citrate (EYC) and Egg yolk/Tris (EYT). Samples of semen diluted with these extenders were frozen in liquid nitrogen and, after thawing, were filtered by discontinuous Percoll gradient. The sperm concentration was adjusted to 10 x 106 cells/mL and the sperm capacitation was induced with 10 µg/mL of heparin. After 24 hours of culture for in vitro maturation, the oocytes, aspirated from ovarian follicles, were inseminated with semen diluted in TALP medium and 48 h after the zigotes were transferred into droplets prepared with TCM-199 medium containing 5% of bovine fetal serum, 5% of estrous cow serum and bovine oviduct epithelial cells in suspension under silicon oil and were further cultured of 9 days. All the cultures were performed at 38.5ºC in 5% CO2 in air. Data were analyzed by chi-square analysis. There was a significant difference in terms of cleavage rate (CR) with means of 66.0, 69.3 and 54.4% for EYL, EYC and EYT, respectively. There were no differences in terms of the morulae/blastocysts and hatching rates. These results suggest that the semen extender had no effect on in vitro development of bovine, even the CR have been affected.
This study investigated the effect of semen extender on in vitro development of bovine oocytes after in vitro maturation and fertilization. The ejaculate from one bull was divided in three parts which were diluted with three different extenders: Egg yolk/Lactose (EYL), Egg yolk/Citrate (EYC) and Egg yolk/Tris (EYT). Samples of semen diluted with these extenders were frozen in liquid nitrogen and, after thawing, were filtered by discontinuous Percoll gradient. The sperm concentration was adjusted to 10 x 106 cells/mL and the sperm capacitation was induced with 10 µg/mL of heparin. After 24 hours of culture for in vitro maturation, the oocytes, aspirated from ovarian follicles, were inseminated with semen diluted in TALP medium and 48 h after the zigotes were transferred into droplets prepared with TCM-199 medium containing 5% of bovine fetal serum, 5% of estrous cow serum and bovine oviduct epithelial cells in suspension under silicon oil and were further cultured of 9 days. All the cultures were performed at 38.5ºC in 5% CO2 in air. Data were analyzed by chi-square analysis. There was a significant difference in terms of cleavage rate (CR) with means of 66.0, 69.3 and 54.4% for EYL, EYC and EYT, respectively. There were no differences in terms of the morulae/blastocysts and hatching rates. These results suggest that the semen extender had no effect on in vitro development of bovine, even the CR have been affected.
Descrição
Palavras-chave
Bovine, cleavage rate, Embryo, In vitro fertilization, semen extender, Bovino, diluidor do sêmen, Embrião, Fecundação in vitro, taxa de clivagem
Como citar
Revista Brasileira de Zootecnia. Sociedade Brasileira de Zootecnia, v. 29, n. 2, p. 397-402, 2000.