Porcine oocyte collection and in vitro maturation

Pig ovaries were obtained from a local abattoir (NH Livestock Cooperation Association, Nonsan City, Chungnam Province, Korea) where we had acquired permission, kept at 35°C in PBS supplemented with 50 μg/ml streptomycin sulfate and 100 IU/ml penicillin, and transported to the laboratory within 3 h. Cumulus-oocytes complexes (COCs) were aspirated from antral follicles (3 to 6 mm in diameter) using a 10-ml disposable syringe fixed with an 18-gauge needle. Oocytes with a uniform ooplasm and more than three layers of cumulus cells were selected for in vitro maturation (IVM, Figure A in S1 File). For IVM, ~ 50 COCs in 500 μl maturation medium were cultured in each well of a four-well multi dish, at 38.5°C in saturated-humidity air containing 5% CO₂. The maturation medium used for porcine oocyte maturation was TCM 199 supplemented with 3.5 mM D-glucose, 0.57 mM L-cysteine, 0.91 mM sodium pyruvate, 75 μg/ml penicillin, 50 μg/ml streptomycin, 10 ng/ml epidermal growth factor (EGF), 10 IU/ml pregnant mare serum gonadotropin (PMSG), 10 IU/ml human chorionic gonadotropin (hCG), and 10% porcine follicular fluid. After 22 h IVM, the COCs were washed and transferred to the same maturation medium without hormones for further culture.