EFFECT OF THE NUMBER OF GRANULOSA-CELLS ADDED TO CULTURE-MEDIUM FOR INVITRO MATURATION, FERTILIZATION AND DEVELOPMENT OF BOVINE OOCYTES

To induce both in vitro nuclear and cytoplasmic maturation of bovine follicular oocytes, effects were investigated of the presence or absence of cumulus cells, additions of different sera and different numbers of granulosa cells into the maturation medium on fertilizability and subsequent development to the blastocyst stage. A 2x7x3 factorial design experiment was conducted using 2 levels of cumulus cells (with or without cumulus), 7 levels of granulosa cells concentrations (0.0, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 x 10(6)/ml) and 3 levels of serum (no serum, fetal calf serum; FCS or estrous cow serum; ECS) for 24 hours. In vitro fertilization rates of cumulus-denuded oocytes were lower than those of cumulus-enclosed oocytes (P < 0.05). The fertilization rate of oocytes cultured in the medium supplemented with FCS was higher than that of oocytes cultured in the medium supplemented with ECS or in no serum. Oocytes cultured in medium supllemented with granulosa cells and FCS had higher rates of fertilization (P < 0.05) and subsequent development to the blastocyst stage (P < 0.05) than oocytes cultured without the addition of granulosa cells. The present results indicate that the presence of cumulus cells surrounding immature oocytes and the addition of granulosa cells (1.0 to 7.5 x 10(6)/ml) and FCS to the culture medium may be required to induce both nuclear and cytoplasmic maturation of bovine oocytes.