In vitro fertilization of bovine oocytes in a chemically defined, protein- free medium varying the bicarbonate concentration

Bovine cumulus-enclosed oocytes were matured in culture, freed from cumulus cells, and inseminated with frozen-thawed spermatozoa in a chemically defined protein-free medium containing 5 mM caffeine and 10 μg/ml heparin. No penetration of oocytes was observed in the medium without polyvinylalcohol (PVA); but when the medium was supplemented with 0.1-5 mg/ml PVA, penetration rates (9-16%) significantly increased. Sperm motility was also stimulated during incubation for 2 h in the presence of PVA. In the medium with 1 mg/ml PVA, a high penetration rate (24 of 62 = 39%) was observed at a sperm concentration of 10 x 10⁶ cells/ml. When the bicarbonate concentration was changed in the fertilization medium containing 1 mg/ml PVA and 10 x 10⁶ spermatozoa/ml, a high penetration rate (47 of 67 = 70%) and a high proportion (44 of 47 = 94%) of oocytes in which male and female pronuclei had developed were obtained at 46 mM NaHCO₃. However, the penetration rate (58- 95%), the incidence of pronuclear formation (64-96%), and the proportion of polyspermy (9-21%) varied according to the animal (five different bulls). Spermatozoa obtained from two bulls started to penetrate oocytes 5 h after insemination in the presence of 46 mM NaHCO₃. This is the first report indicating that induction of capacitation of bull spermatozoa and penetration of oocytes matured in culture are possible in a chemically defined, protein- free medium.