INFLUENCE OF CUMULUS CELLS ON IN-VITRO FERTILIZATION OF BOVINE OOCYTES DERIVED FROM IN-VITRO MATURATION

The effect of the cumulus cells on in vitro fertilization (IVF) in cattle was evaluated. Frozen-thawed bovine spermatozoa were treated with 5 mM caffeine and 10 mug heparin ml-1 for sperm capacitation. Bovine oocytes with or without cumulus cells after maturation in culture were inseminated by different concentrations of spermatozoa. When the concentration of (0.8-1.5) x 10(6) spermatozoa ml-1 was employed, the penetration rate was significantly (P < 0.001) higher in cumulus-intact (87%, 130/149) than in cumulus-free (57%, 101/178) oocytes. However, such differences were not observed with sperm concentrations of (2.0-3.0) x 10(6) and (3.5-5.0) x 10(6) spermatozoa ml-1 (92% (69/75) vs. 86% (68/79) and 93% (67/72) vs. 98% (73/80) respectively). The polyspermy was significantly (P < 0.05) higher in cumulus-intact than in cumulus-free oocytes in all sperm concentrations (25% (17/68) vs. 10% (4/41), 29% (20/69) vs. 16% (11/68) and 31% (21/67) vs. 19% (14/73) respectively). When oocytes with or without cumulus cells were inseminated by a concentration of (2.0-3.0) x 10(6) spermatozoa ml-1, the penetration rate was almost completed at 12 h after insemination in cumulus-intact oocytes (83%, 80/96), whereas the sperm penetration was delayed in cumulus-free oocytes (64%, 42/66). There was a tendency to decrease the polyspermy when spermatozoa were removed from the oocyte surface at 8 and 12 h compared with at 16, 20 and 24 h after insemination in both cumulus-intact and cumulus-free oocytes. These results indicate that the cumulus cells participated in the mechanisms of bovine sperm capacitation and acrosome reaction and that the polyspermy was directly affected by the number of capacitated and acrosome reacting sperm on the oocyte surface. These results suggest that the preferential sperm concentration was (0.8-1.5) x 10(6) spermatozoa ml-1 in the present IVF system, and that removal of sperm-cumulus complexes from the oocyte surface at 12 h after insemination may prevent the increase of polyspermy, especially by use of higher sperm concentrations.