IVM and IVF of Bovine Oocytes
Oocyte collection, IVM, and IVF were carried out as previously described. Briefly, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (25 mm in diameter) on bovine ovaries obtained from a slaughterhouse. After washing of COC with Hepes-buffered Tyrode’s medium (TALP-Hepes ), only oocytes with 34 layers of intact and unexpanded cumulus cells were selected; these were then cultured for 22 h with maturation medium (Hepes-buffered TCM199 [Sigma Chemical Co., St. Louis, MO] + 10% heat-inactivated fetal bovine serum [Gibco Life Technologies, Grand Island, NY] supplemented with 0.2 mM sodium pyruvate, 0.02 U/ml porcine FSH [Sigma], 1 ^g/ml estradiol-17p [Sigma], and 50 ^g/ml gen-tamicin sulfate [Sigma]) at 39°C in a humidified atmosphere containing 5% CO2 in air. flovent inhaler
For IVF, motile spermatozoa were obtained by centrifugation (700 X g for 20 min) of frozen-thawed semen on a 45%/90% Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient. The sperm pellet was resuspended with modified Brackett and Oliphant’s isotonic medium without BSA but supplemented with 50 ^g/ml gentamicin sulfate (mBO) and then washed by centrifugation at 500 X g for 5 min. COC were coincubated with spermatozoa in 100-^l droplets of mBO supplemented with 2.5 mM theophylline, 5 ^g/ml heparin (Sigma), and 3 mg/ml fatty acid-free BSA (Sigma) for 20 h at 39°C in a humidified atmosphere containing 5% CO2 in air. The final concentration in fertilization drops was 5 X 106 spermatozoa/ml.