Bovine embryo development produced by in vitro fertilization cultured with oviductal cell or conditioned medium and transfer to recipients

Abstract

Se comparó el desarrollo in vitro de ovocitos obtenidos de ovarios de vaca de matadero, madurados, fecundados y cultivados in vitro bajo dos sistemas. Los ovocitos fueron cultivados en un medio de maduración a 39 °C, 5 % de CO2 y humedad relativa de 95 % durante 22 horas. Posteriormente, fueron incubados con espermatozoides seleccionados a través de una gradiente discontinua de Percoll. La tasa de maduración nuclear y fecundación fueron de 93,7 % (74/79) y 76,9 % (50/65) respectivamente. Un total de 252 ovocitos fecundados fueron cultivados in vitro. El porcentaje de desarrollo in vitro a las 2 días post-inseminación (embriones de 4-8 células) fue de 62,7 % (64/102) para los cigotos cultivados con células oviductales y de 67 % (100/150) para los cultivados en medio condicionado (P0,05). El porcentaje de desarrollo de mórulas fue de 17,6 % (18/102) para los cigotos cultivados con células oviductales y de 13,3 % (20/150) para los cultivados con medio condicionado (P0,05). Se obtuvo una tasa de desarrollo del 15,7 % (16/102) de blastocistos para aquellos cigotos cultivados con células oviductales. No se obtuvo blastocistos a partir de cigotos cultivados en medio condicionado. Cuatro blastocistos fueron transferidos a dos hembras receptoras. A los 42 y 57 días se encontró la presencia de un feto en cada hembra

The in vitro development of matured and fertilized bovine oocytes was compared between two culture systems. Oocytes were collected by aspiration of follicles of 3-8 mm in diameter using an 18g needle. After morphological selection the oocytes were incubated at 39 0C, 5 % C02 y 95 % relative humidity, during 22 hours. Afterwards, oocytes were incubated with spermatozoa selected by Percoll gradient system. The rate of nuclear maturation and fertilization was 93,7 % (74/79) and 76,9 % (50/65), respectively. A total of 252 zygotes were cultured, 102 with oviductal cells and 150 in conditioned medium. The in vitro development on day 2 of culture (4- or 8-cell embryos) was 62,7 % (64/102) for the zygotes co-cultured with oviductal cells and 66,7 % (100/150) for the zygotes cultured in conditioned medium. The development to the morula stage was 17,6 % (18/102) for the zygotes co-cultured with oviductal cells and 13,3 % (20/150) for the zygotes cultured in conditioned medium. A statiscally significant difference was not found in the development of 4, 8-cell embryos or morula. The development of embryos up to the blastocyst stage was 15,7 % (16/102) for the zygotes co-cultured with oviductal cells. Two blastocysts were transferred to the uterine horn ipsilateral to the CL in two recipients by non-surgical embryo transfer. Pregnancy was confirmed by ultrasonography at 42 and 57 days detecting the presence of one conceptus in each animal. This work has shown that in vitro inseminated of bovine oocytes with espermatozoa prepared with modified BO and co-cultured with oviductal cells, can develop to the blastocysts stage, unlike those that were cultured with conditioned medium. Finally, it is important to mention that this is the first communication in Chile of pregnancy after in vitro fertilization of bovine oocytes