Basic media used for fertilization of oocytes were mKRB and mR1ECM. The mKRB was basically the same as that developed and used by Toyoda and Chang except that phenol red was omitted. The ingredients of mKRB and mR1ECM are shown in Table 1. Basically, mKRB and mR1ECM contain BSA and polyvinyl alcohol (PVA), respectively. In the present studies, mKRB with PVA (1.0 mg/ml) instead of BSA, and mR1ECM with BSA (4.0 mg/ml) instead of PVA, were also used and were named mKRB-PVA and mR1ECM-BSA, respectively. Culture medium for early development of rat embryos was mRlECM. All fertilization and culture media (each 400 ^l) were covered with paraffin oil (Nacalai Tesque Inc., Kyoto, Japan) and equilibrated in an atmosphere of 5% CO2 in air at 37°C overnight. The pH of all media was 7.4 after equilibration. antibiotic levaquin
Preparation of Sperm Suspension
According to a traditional procedure, a dense mass of spermatozoa from the epididymis of Wistar rats (10-12 mo old) was introduced into 400 ^l of mKRB, which had been covered with paraffin oil and equilibrated in an atmosphere of 5% CO2 in air at 37°C overnight. About 5 min after preparation, the sperm suspension (10-30 ^l) was transferred into a fertilization medium (400 ^l) to provide a final sperm concentration of 5-10 X 105 cells/ml. The diluted sperm suspension was preincubated in an atmosphere of 5% CO2 in air at 37°C for 5-6 h.