Porcine Oocyte Maturation for Nuclear Transfer

In vitro-matured porcine oocytes were used as nuclear transfer recipients and prepared according to methods described previously (Liu et al. 2014 (link); Zhu et al. 2016 (link)). Briefly, cumulus-oocyte complexes (COCs) were aspirated from the follicles with sizes of 3–8 mm, and washed twice in PVA-TL-HEPES medium. The COCs were transferred into 200 µL drops of preheated maturation medium (bicarbonate-buffered TCM-199 supplemented with 0.1 % [w/v] polyvinyl alcohol [PVA], 3.05 mM d-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/mL epidermal growth factor [EGF], 0.5 µg/mL follicle-stimulating hormone [FSH], 0.5 µg/mL luteinizing hormone [LH], 0.0750 g/L penicillin G, 0.0500 g/L streptomycin and 10 % [v/v] porcine follicular fluid [PFF]; pH = 7.3), covered with mineral oil, and then incubated for 20–22 h at 38.5 °C in a Forma Series II water jacketed incubator (Thermo Scientific, Marietta, OH, USA) with humidified atmosphere of 5 % (v/v) CO₂ in air. Then, the COCs were cultured for an additional 20 h in the same medium without the gonadotropins. Following maturation, expanded cumulus cells were removed from the oocytes by vigorous pipetting in the presence of 0.1 % (w/v) hyaluronidase. Oocytes with an evenly granulated ooplasm and an extruded first polar body were selected and placed into the micromanipulation medium drop (containing donor cells and 7.5 µg/mL cytochalasin B) on a 60-mm cell culture dish (NUNC) covered with mineral oil for using as nuclear transfer recipients.