Chorulon

Bovine ovaries were obtained from a commercial slaughterhouse and transported in warm sterile saline [0.9% (w/v) NaCl] for about 3 h. In the laboratory the ovaries were washed several times again with sterile saline to remove blood and debris. Cumulus-oocyte complexes (COCs) were collected by aspiration of follicles with diameter between 2 and 8 mm using syringe and needle.
For in vitro maturation (IVM), groups of 25–30 oocytes were placed in 90 μL drops of the maturation medium (TCM-199 bicarbonate supplemented with 10% of FBS, 0.5 μg/mL FSH [Folltropin-V, Bioniche, Belleville, Canada], 100 IU/mL hCG [Chorulon, Merck Animal Health, Boxmeer, The Netherlands]) under mineral oil. Maturation of COCs followed in an incubator at 38.5 °C, 5% CO₂ in air and high humidity for 24 h.
Frozen commercial semen straws from 2 bulls were thawed in a water bath at 37 °C for 30 s and their contents were mixed and centrifuged on a discontinuous Percoll gradient (45% and 90%) as a strategy to avoid the influence of the bull in the kinetics of embryo development. Sperm concentration was adjusted to 1 × 10⁶ sperm/mL. Matured COCs were transferred to 90 μL drops of fertilization media^{19 (link)} and covered with mineral oil. Oocytes and sperm were incubated at the same conditions of air and temperature for 18 h. This study did not require direct handling of animals.

When comCOCs or expCOCs were subjected to IVM, they underwent four washes in drops of HH medium without IBMX and NPPC followed by four washes in the IVM medium. The IVM medium consisted of Medium 199 supplemented with 22 µg/ml sodium pyruvate, 4 IU/ml hCG (Chorulon^®; Merck & Co., Inc., Rahway, NJ, USA), 50 µg/ml gentamicin, and 100 µl/ml fetal bovine serum (Patel et al., 2007 (link)). COCs were cultured in 10 µl drops of IVM medium in groups of 3–5 for 22 h at 38.5°C in a 5% CO₂ atmosphere with 100% humidity.
To assess the nuclear maturation of COCs after IVM, our statistical power analysis limited us to groups of 3–5 COCs per drop from each heifer unless otherwise specified. However, a preliminary study was conducted to aspirate COCs from ovaries collected from cattle at a local abattoir (West Michigan Beef Co LLC, Hudsonville, MI, USA) to determine if 3 or 5 COCs per drop was representative of the proportion of oocytes reaching metaphase II (MII) during IVM. Chi square analysis indicated that groups of 3 (n = 55) or 5 (n = 31) COCs per drop did indeed produce similar proportions of MII oocytes during IVM (mean ± SEM, 71 ± 2% versus 80 ± 6%, respectively, P ≥ 0.85). Data for heifers that had <3 COCs recovered by oocyte retrieval were not subjected to IVM and were omitted from statistical analysis.