2.7. Parthenogenetic activation of porcine oocytes

After IVM, Metaphase II (MII) oocytes from each group were chosen and rinsed with a calcium-free TLH/PVA medium. The process of Parthenogenetic activation (PA) was carried out using a standard method (30). MII oocytes were rinsed twice in a 280 mM mannitol solution containing 0.01 mM CaCl₂ and 0.05 mM MgCl₂. Between the chamber electrodes the MII oocytes were placed in 2 mL of a 260 mM mannitol solution containing 0.01 mM CaCl₂ and 0.05 mM MgCl₂. The chamber was connected to an electrical pulse generator (LF101; Nepa Gene, Chiba, Japan), and the MII oocytes were stimulated by two direct-current pulses of 60 s at 120 V/mm each. Following PA, activated oocytes from all groups were immediately transferred to an in vitro culture (IVC) medium (30), composed of porcine zygote medium containing 5 μg/mL cytochalasin B. The oocytes were incubated for 4 h at 39°C in a humid atmosphere composed of 5% CO₂ and 95% N₂. The oocytes were then rinsed twice in a fresh IVC medium and placed in 25 μL of fresh IVC medium, with 10 oocytes per droplet, and covered with mineral oil. After 48 h (day 2) and 96 h (day 4), the IVC culture medium was replaced with 10% fetal bovine serum. Embryos were cultivated for a total of 7 days at 39°C in a humid incubator consisting of 5% O₂, 5% CO₂, and 90% N₂. This experiment was performed thrice.