Ncsu 23

These couplets were washed with 0.3 M mannitol solution containing 0.5 mM Hepes, 0.1 mM CaCl 2 , and 0.1 mM MgSO 4 for 4 times and transferred to a chamber containing two electrodes that were overlaid with fusion and activation solution. Couplets were fused/activated with a single DC pulse of 1.5 kV/cm for 30 μsec using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA). After activation treatment, the reconstructed embryos were washed three times with NCSU-23 (Petters et al. 1993) supplemented with 4 mg/mL fatty acid free BSA (Sigma-Aldrich Corp.), and cultured with NCSU-23 supplemented with 4 mg/mL fatty acid-free BSA (Sigma-Aldrich Corp.), 5 mg/mL cytochalasin B and 10 mg/mL cycloheximide (CHX) for 4 h at 38.5, 5% CO 2 in air, and then the oocytes were cultured without chemical CB and CHX embryo medium which had been previously covered with paraffin oil in a polystyrene culture dish and equilibrated at 38.5 in an atmosphere of 5% CO 2 in air.