After washing, groups of 15-30 COC
were transferred to 1 ml TCM-199 (Gibco BRL, Grand Island,
NY) supplemented with 10 $zg/ml LH, 5 Rg/ml FSH,
1 ,ag/ml estradiol, 200 1iM pyruvate, 50 Rpg/ml gentamicin,
and 10% estrous cow serum, and cultured for 20 h at 39°C
in an atmosphere of 5% CO2 in air and 100% humidity [19].