In Vitro Fertilization of Bovine Oocytes in a Chemically Defined, Protein-Free Medium Varying the Bicarbonate Concentration1

Abstract

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 micrograms/ml heparin. No penetration of oocytes was observed in the medium without polyvinyl-alcohol (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 10(6) cells/ml. When the bicarbonate concentration was changed in the fertilization medium containing 1 mg/ml PVA and 10 x 10(6) 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.