2.3. In Vitro Maturation of Porcine Oocytes

Porcine ovaries were obtained from a local slaughterhouse. Cumulus-oocyte complexes (COCs) were aspirated from ovarian follicles sized 3–6 mm, divided into groups of 100–150 in 4-well culture dishes (Nunc, Roskilde, Denmark), and matured in TCM-199 medium (Mediatech, Inc., Manassas, VA, USA) supplemented with 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 μg/mL FSH, 0.5 μg/mL LH, 10 ng/mL EGF, 10% porcine follicular fluid, 75 μg/mL penicillin G and 50 μg/mL streptomycin. The COCs were incubated at 38.5 °C, with 5% CO₂ in the air. After 22 h of culture, the COCs were transferred into TCM199 medium without FSH and LH and then cultured for an additional 22 h at 38.5 °C and 5% CO₂ in the air.