Bovine Oocyte Maturation Protocol

The cattle, from which we obtained their ovaries, had been slaughtered for the public edible meat. Those ovaries were discarded without any utilization. Hence, an ethics statement in our paper was not required to use ovary for experiments as previously described [22 (link)–24 ]. Bovine ovaries were obtained from an abattoir, placed in saline containing a 0.1% solution of antibiotic/antimycotic (Gibco Laboratories, Grand Island, NY, USA: AB), and transported to the laboratory within 1–3 h. Oocyte collection and in vitro maturation of oocytes were carried out as previously described [25 ]. More than 30 ovaries from Japanese black and holstein cows were collected to permit appropriate an experimental replication. Oocytes with intact cumulus cells and evenly granulated cytoplasm were selected, washed and cultured in modified TCM-199 (m-TCM199) for 22–25 h at 39.0°C under 5% CO₂ in air with high humidity. m-TCM199 consisted of hepes-buffered medium 199 (Gibco) supplemented with 0.1% (w/v) polyvinyl alcohol (PVA;Sigma Chemical Co, St. Louis,MO.), 0.5 mM sodium pyruvate (Nacalai Tesque Inc., Kyoto, Japan), 1% AB, 0.02 AU/ml FSH (Antrin, Denka), and 1 μg/ml estradiol-17β (Sigma). The rates of in vitro maturation after 22–24 hrs were more than 95% in all experiments.