STUDIES ON PRODUCTION AND EFFICIENT UTILIZATION OF LIVESTOCK EMBRYOS BY IN VITRO FERTILIZATION AND MICROMANIPULATION IV. NUCLEAR TRANSPLANTATION AND ELECTROFUSION FOR CLONING IN BOVINE FOLLICULAR OOCYTES

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.