In vitro Maturation of Porcine Oocyte

Porcine in vitro maturation (IVM) was performed based on procedures as described previously (Ding et al., 2017; Cao et al., 2019, 2020). Peripuberty porcine ovaries of crossbreeds (Landrace × Yorkshire × Duroc) were collected from a local slaughterhouse and transported to the laboratory at 28–35°C in physiological saline solution. Ovaries were quickly washed in saline, and the follicles with 3 to 6 mm in diameter were aspirated using a sterile 10-ml syringe. Cumulus-oocyte complexes (COCs) within the follicular fluid were settled down at 38.5°C for 15 min. COCs with more than three layers of cumulus cells and homogeneous ooplasm were selected for subsequent experiments using a stereomicroscope. After washing three times in IVM medium, appropriately 50 COCs were transferred to 400 μl IVM medium (TCM-199 supplemented with 5% FBS, 10% porcine follicular fluid, 10 IU/ml eCG, 5 IU/ml hCG, 100 ng/ml L-cysteine, 10 ng/ml EGF, 0.23 ng/ml melatonin, 2.03 × 10^–5 ng/ml LIF, 2 × 10^–5 ng/ml IGF, 1.4 × 10^–5 ng/ml FGF2, 100 U/ml penicillin, and 100 mg/ml streptomycin) covered with mineral oil in four-well plates and cultured for 42–44 h at 38.5°C, 5% CO₂ in air with saturated humidity. After maturation, cumulus cells surrounding oocytes were removed by gentle pipetting in 1 mg/ml hyaluronidase in DPBS without Ca²⁺ and Mg²⁺. Only the matured oocytes that possess an extruded first polar body and uniform ooplasm were selected for subsequent experiments.