Thawed oocytes were placed into separate 10 µL manipulation droplets of G-MOPS with 5% HSA and covered with tissue culture oil. After the first polar body of the oocytes reached 12 o’clock, partial zonapellucida dissection (PZD) was performed before enucleation (Ding et al., 2015). Then, they were placed into separate 10 µL manipulation droplets of G-MOPS medium (containing 7.5 µg/mL cytochalasin B, 5% HSA) in a glass-bottom dish at 37°C for 10 min. The spindle was aspirated into the pipette with a minimal amount of cytoplasm and surrounding plasma membrane using Spindle View (Cri Inc.). Enucleated oocytes were rinsed with G-MOPS medium containing 5% HSA and incubated in G-IVF medium with 10% HSA at 37°C in 6% CO2, 5% O2, and 89% N2 for 60 min before fusion. PB1 was aspirated out of ZP by the pipette before enucleated oocyte fused with donor cells. Donor cells were resuspended in a drop containing HVJ-E extract (Cosmo Bio, USA) and were inserted into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were kept in the manipulation medium until cell fusion was confirmed, and then the reconstructed oocytes were transferred into G-IVF medium (10% HSA) and incubated for 1 h before activation.