4.6. Somatic Cell Nuclear Transfer (SCNT) and Chaetocin Treatment

SCNT were performed as previously described [60]. MII oocytes in PB1 medium (DPBS supplemented with 4 mg/mL BSA, 75 µg/mL penicillin G, and 50 µg/mL streptomycin sulfate) containing 7.5 µg/mL cytochalasin B were cut using a sharp pipette, and then the first polar body and cytoplasm-containing chromosomes at metaphase II were removed using the squeezing method under an inverted microscope (DMI 3000B; LEICA, Wetzlar, Germany) equipped with a micromanipulator (NT-88-V3; Nikon Narishige, Tokyo, Japan). Porcine kidney cells were suitable for the donor cell resource to produce SCNT embryos, as they showed higher proliferation and blastocyst formation rates after SCNT compared to porcine fetal or ear fibroblast cells [61]. Donor cells were selected with good refractivity and placed into the perivitelline space. A single cell–oocyte couplet was placed between two parallel electrodes (CUY 5100-100; Nepa gene, Ichikawa, Japan) and activated by one direct current pulse of 0.24 kV/cm for 50 µs using an Electro Cell Fusion generator in fusion medium consisting of 280 mM mannitol containing 0.1 mM CaCl₂·2H₂O, 0.2 mM MgSO₄·7H₂O, and 0.01% polyvinyl alcohol (PVA), and incubated at 38.5 °C in 5% CO₂ in air. After 2 h, oocyte–cell couplets that were completely fused as observed under an inverted microscope were selected and activated by one direct current pulse of 1.2 kV/cm for 50 µs in activation medium consisting of 280 mM mannitol containing 0.1 mM CaCl₂·2H₂O, 0.2 mM MgSO₄·7H₂O, 0.01% PVA, and 0.5 mM HEPES, and then cultured in post-activation medium, which consisted of IVC medium supplemented with 5 µg/mL cytochalasin B and 2mM 6-dimethylaminopurine, for 4 h at 38.5 °C in 5% CO₂ in air. After activation, the activated embryos were transferred to IVC medium at 38.5 °C in 5% CO₂ in air.

To confirm the optimal conditions for chaetocin treatment during porcine SCNT embryo development, activated embryos were cultured in activation medium with various concentrations of chaetocin (0, 0.1, 0.5, and 1 nM) for 24 h after activation. The concentration that caused the highest percentage of embryos to develop (0.5 nM) was used for various durations of treatment (0, 24, 48, and 72 h) after activation. The cleavage and blastocyst rates were determined at 48 and 144 h, respectively.