In vitro maturation of bovine cumulus enclosed primary oocytes and their subsequent in vitro fertilization and cleavage.
Cumulus enclosed primary oocytes from 2 to 4-mm bovine follicles were matured in vitro in Minimum Essential Medium containing follicle-stimulating hormone (0, .1, 1, 10, 50, or 100 micrograms/ml) or human chorionic gonadotropin (0, .1, 1, or 10 IU/ml) for 48 h at 37 degrees C under paraffin oil. Cumulus mass expansion comparable to that seen in vivo occurred in 18% of the control oocytes, 39% of those cultured in human chorionic gonadotropin, and 56% of those cultured in follicle-stimulating hormone. The optimum follicle-stimulating hormone concentration for cumulus expansion was 1 microgram/ml, and this was then used to mature oocytes individually or in groups of 5 for in vitro fertilization. Ejaculated bovine semen, extended 1:10 with yolk-TES-Tris extender and stored 24 to 48 h at 4 degrees C, was warmed, washed once with Minimum Essential Medium, and 500,000 motile sperm/ml were used to inseminate the matured oocyte-cumulus cell complexes. Criteria for fertilization was cleavage to the two-cell stage 48 h after insemination. Oocytes, inseminated individually, cleaved with a frequency of 5%, whereas 15% of those inseminated in groups of 5 cleaved, perhaps as the result of cumulus factors enhancing capacitation. The cleavage rate for the parthenogenetic control with killed spermatozoa was 0%. Therefore, primary oocytes matured in vitro to secondary oocytes were successfully fertilized in vitro and cleaved to at least the two-cell stage in the Minimum Essential Medium. Individual differences between bulls in ability to fertilize in vitro were noted.
D000818:Animals; D002417:Cattle; D002455:Cell Division; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003470:Culture Media; D005260:Female; D005307:Fertilization in Vitro; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D066298:In Vitro Techniques; D008540:Meiosis; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development*; D006080:Ovarian Follicle / Q000166:cytology; D015053:Zygote / Q000166:cytology*
D006063:Chorionic Gonadotropin; D003470:Culture Media; D005640:Follicle Stimulating Hormone