The maturation medium was TCM199 with Glutamax (Gibco BRL, Invitrogen) supplemented with 10% FBS (Gibco BRL), 0.25 mM sodium pyruvate, penicillin–streptomycin, 2 μg/ml FSH (Ovagen, Bodinco B.V.), 10 μg/ml LH and 1μg/ml β-estradiol. Immature oocytes were matured for 24 h.