Parthenogenetic Activation of Oocytes

After 45 h of IVM culture, COCs were transferred to 400 µL of 0.1% hyaluronidase for pipetting about 30 times, and then oocytes and granulosa cells were separated. The oocytes were selected and put into the activation solution (300 mM mannitol containing 0.5 mM HEPES, 0.05 mM CaCl2 2H2O, 0.1 mM MgSO4 7H2O, and 0.01% polyvinyl alcohol), followed by parthenogenetic activation using two direct-current (DC) pulses of 120 V for 60 µs. Next, the parthenogenetically activated oocytes were transferred to IVC medium containing 7.5 mg/mL cytochalasin B at 38.5 °C in an atmosphere of 5% CO₂ for three hours, thereby inhibiting the discharge of the second polar body. Oocytes were washed four to five times with IVC after three hours of cytochalasin B treatment. About 40 to 50 oocytes were placed in each well of the four-well plate with 50 µL IVC medium with or without (0 µM, control group) CHE (TargetMol, Boston, MA, USA) that was finally dissolved with IVC at concentrations of 0, 0.1, 1, and 3 µM and then cultured at 38.5 °C in an atmosphere of 5% CO₂ for seven days. On day 7, there were about 10–20 blastocysts in each group, and the experiment in each group was repeated at least three times.