In Vitro Embryo Culture
At 46-48 h after coincubation of the spermatozoa and oocytes, cleaved embryos with at least 4 cells were selected, washed twice in a Hepes-buffered synthetic oviductal fluid (Hepes-SOF) medium (pH 7.4), and transferred to 20-^l droplets of synthetic oviductal fluid (SOF) medium supplemented with 4 mg/ml Pentex crystalline BSA (Bayer, Elkhart, IN). Embryo culture was carried out in 35-mm cell culture dishes at 39°C, in a humidified incubator with a gaseous atmosphere of 5% CO2:5% O2:90% N2.
Total Cell Counting
Blastocyst-stage embryos were incubated for 15 min in dissection medium containing 5 ^g/ml bisbenzimide (Hoechst 33258; Sigma). The embryos were then placed onto clean glass slides in 5-^l drops of DABCO (Sigma) under coverslips. Counting took place using an inverted, differential interference contrast microscope fitted with epi-fluorescence (Nikon, Garden City, NY). ventolin inhalers
Design of Experiments and Statistical Analysis
Experiment 1. The aim of this preliminary experiment was to establish a system for culture of bovine antral follicles in vitro. A total of 90 antral follicles were submerged in culture medium, without the use of netwell insert membrane. After 24-h culture, the oocytes were recovered; a proportion of them were fixed and stained for examination of nuclear morphology, and the others were transferred to the maturation medium.