Porcine Oocyte Isolation and Culture

Porcine ovaries were collected from Hongteng slaughterhouse (Chenggong Ruide Food Co., Ltd, Kunming, Yunnan Province, China) with the permission to use animal parts for this study. The ovaries were transported to the laboratory at 25°C to 30°C in 0.9% (w/v) NaCl solution supplemented with 75 mg/mL potassium penicillin G and 50 mg/mL streptomycin sulfate. Cumulus-oocyte complexes were obtained from follicles 3 mm to 6 mm in diameter using an 18-gauge needle connected to a 10 mL disposable syringe. Cumulus-oocyte complexes with at least three layers of compacted cumulus cells were selected, and approximately 50 oocytes were cultured in 200 µL drops of TCM-199 medium supplemented with 0.1 mg/mL pyruvic acid, 0.1 mg/mL l-cysteine hydrochloride monohydrate, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 mg/mL potassium penicillin G, 50 mg/mL streptomycin sulfate, and 10 IU/mL eCG and hCG (Teikoku Zouki Co., Tokyo, Japan) at 38.5°C in an atmosphere with 5% CO₂ (100% humidity) (APC-30D, ASTEC, Japan).