Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

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Abstract
Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH .mu.g/ml + 5 .mu.g LH/ml + 1 .mu.g oestradiol/ml) and granulosa cells (5 .times. 106/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastoysts of bovine follicular oocytes. The maturation rates after 24 h culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.