Culture of Embryos
After 20 h of coincubation with sperm, presumptive zygotes were stripped of cumulus cells by vortexing for 4 min in 1 ml of TALP-Hepes, then washed three times with culture medium. A chemically defined medium (mSOF) containing 3 mg/ml polyvinyl alcohol (Sigma) was used for the basal culture medium as previously described. Presumptive zygotes were cultured in 30-^l droplets covered with paraffin oil at 39°C in a humidified atmosphere containing 5% CO2:5% 02:90% N2. Each droplet contained approximately 25 presumptive zygotes. At 120 h postinsemination, embryos were transferred to fresh medium containing 2.0 mM glucose and then cultured at 39°C in a humidified atmosphere containing 5% CO2:5% 02:90%N2. buy birth control online
Determination of Cell Number
Cell number was determined by an air-drying method as described previously. Briefly, embryos were put into a hypotonic solution (0.9% sodium citrate supplemented with 0.3% fetal calf serum) for 15 min. Then they were treated with fixative I (methanol:acetic acid:distilled water, 10:3:7) and fixative II (methanol:acetic acid, 3:1). After staining with 2% Giemsa solution, the total cell numbers, including metaphase plates but excluding pyknotic nuclei, were counted under a brightfield microscope.