This study was designed to determine the effect of electric field strength, duration and fusion buffer in fusion parameters on the rate of membrane fusion between the somatic cell and cytoplast for Korean cattle (HanWoo) somatic cell nuclear transfer (SCNT) procedure. Following electrofusion, effect of 5 or 10 μM Ca2+-ionophore of activation treatment on subsequent development was also evaluated. Cell fusion rates were significantly increased from 23.1% at 20 V/mm to 59.7% at 26 V/mm and 52.9% at 27 V/mm (p<0.05). Due to higher cytoplasmic membrane rupture or cellular lysis, overall efficiency was decreased when the strength was increased to 30 V/mm (18.5%) and 40 V/mm (6.3%) and the fusion rate was also decreased when the strength was at 25 V/mm or below. The optimal duration of electric stimulation was significantly higher in 25 μs than 20 and 30 μs (18.5% versus 9.3% and 6.3%, respectively, p<0.05). Two nonelectrolyte fusion buffers, Zimmermann’s (0.28 M sucrose) and 0.28 M mannitol solution for cell fusion, were used for donor cell and ooplast fusion and the fusion rate was significantly higher in Zimmermann’s cell fusion buffer than in 0.28 M mannitol (91.1% versus 48.4%, respectively, p<0.05). The cleavage and blastocyst formation rates of SCNT bovine embryos activated by 5 μM Ca2+-ionophore was significantly higher than the rates of the embryos activated with 10 μM of Ca2+-ionophore (70.0% versus 42.9% and 22.5% versus 14.3%, respectively; p<0.05). This result is the reverse to that of parthenotes which shows significantly higher cleavage and blastocyst rates in 10 μM Ca2+-ionophore than 5 μM counterpart (65.6% versus 40.3% and 19.5% versus 9.7%, respectively; p<0.05). In conclusion, SCNT couplet fusion by single pulse of 26 V/mm for 25 μs in Zimmermann’s fusion buffer followed by artificial activation with 5 μM Ca2+-ionophore are suggested as optimal fusion and activation methods in Korean cattle SCNT protocol.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Chemicals    Oocyte Recovery and In Vitro Maturation (IVM)    Parthenogenetic Activation and IVC    Preparation of Donor Cells    Enucleation and Nuclear Transfer    Electrofusion and Activation    Experimental Designs    Statistical Analysis   RESULTS    Effect of the Concentration of Ca2+-ionophore duringthe Activation on In Vitro Development of Bovine Parthenotes    Effects of Electric Pulse Strength and Duration on theFusion of SCNT Couplets    Comparison of Two Nonelectrolyte Fusion Buffers forCell Fusion    Effect of the Concentration of Ca2+-ionophore duringthe Activation on In Vitro Development of SCNT BovineEmbryos   DISCUSSION   ACKNOWLEDGMENT   REFERENCES

• Sewoong Kim(Cellular Reprogramming and Embryo Biotechnology Lab, Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul 110-749, Korea)
• Daehwan Kim(Cellular Reprogramming and Embryo Biotechnology Lab, Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul 110-749, Korea)
• Yeon-Gil Jung(ET Biotech, Daegu 712-714, Korea Sangho Roh)
• Sangho Roh(Cellular Reprogramming and Embryo Biotechnology Lab, Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul 110-749, Korea) Corresponding author