Maintenance of Bovine Oocytes: MATERIALS AND METHODS(7)

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.

This entry was posted in Bovine Oocytes and tagged Antral Follicle Culture, Bovine Oocytes, Meiotic Arrest.