Nonsorted semen was separated using a standard Percoll gradient (2 mL 45% Percoll over 2 mL 90% Percoll) in a 15 mL conical tube. Semenwas centrifuged for 20 minutes at 600 g, and supernatant removed to just above the pellet. Resuspended sperm were washed in 4 mL H-CDM-1
by centrifugation for 10 minutes at 600 g. Sperm concentration was determined with a hemacytometer, and sperm was suspended to 5 106/mL to result in a final concentration of 5 105 sperm per mL for fertilization. Fertilization of COC with nonsorted sperm was performed
in four-well dishes with 440 mL of F-CDM, 50 COC in 10 mL F-CDM, and 50 mL of sperm suspension.