In Vitro Maturation of Bovine Cumulus Enclosed Primary Oocytes and Their Subsequent In Vitro Fertilization and Cleavage

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Abstract
Cumulus enclosed primary oocytes from 2-4 mm bovine follicles were matured in vitro in Minimum Essential Medium containing (0, 0.1, 1, 10, 50 or 100 .mu.g/ml) or human chorionic gonadotropin (0, 0.1, 1 or 10 IU/ml) for 48 h at 37.degree. 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 FSH. The optimum FSH concentration for cumulus expansion was 1 .mu.g/ml, and this was used to mature oocytes individually or in groups of 5 for in vitro fertilization. Ejaculated bovine semen, extended 1:10 with yolk-TES [trismethylaminoethanesulfonic acid]-Tris extender and stored 24-48 h at 4.degree. 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 2-cell stage 48 h after insemination. Oocytes, inseminated individually, cleaved with a frequency of 5%; 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%. Primary oocytes matured in vitro to secondary oocytes were successfully fertilized in vitro and cleaved to at least the 2-cell stage in the Minimum Essential Medium. Individual differences between bulls in ability to fertilize in vitro were noted.