Fertilization in vitro of cattle follicular oocytes with ejaculated spermatozoa capacitated in a chemically defined...

Cattle follicular oocytes were collected from the ovarian follicles and cultured for 28 h in m-KRB solution in a CO2 incubator (5% CO2 in air at 37 degrees C, 95% humidity). After further culture for 15-18 h with spermatozoa, 78.6% of the oocytes had matured to the second metaphase. The highest fertilization rates (36-58%) were obtained when spermatozoa were collected 1 day before insemination, kept for 14-18 h at 20 degrees C in a test tube (10 X 10(8)/ml), preincubated in m-KRB solution (1-1.2 X 10(8)/ml) in a CO2 incubator for 0-12 h, and then inseminated at a concentration of 1.5-2.0 X 10(6)/ml. The optimum period of preincubation in the CO2 incubator was 8 h: 6/15 denuded oocytes (40%) were fertilized with spermatozoa preincubated for 8 h. No polyspermic fertilization was observed in any of the 139 penetrated oocytes. When spermatozoa were preincubated in the uteri of oestrous cows and gilts for 4-4.5 h instead of the m-KRB solution, the fertilization rates were 80 and 86%, respectively. However, in this system, 13 and 24% of the penetrated oocytes were polyspermic. These results indicate that ejaculated bull spermatozoa can be capacitated in a chemically defined isotonic medium, and about half of the oocytes matured in culture are normally fertilized in vitro.