Effects of electro-stimulation of somatic nuclear-transfer oocytes after electro-fusion on the following development in cattle

"The present study was undertaken to investigate the effects of fusion between an enucleated oocyte and a granulosa cell on the developmental competence in vitro and in vivo in cattle. In vitro-matured oocytes were enucleated after maturation culture for 18-19 h. A single granulosa cell was fused electrically with an enucleated oocyte by a single pulses of 30V/DC/150 microm for 25 microsec in Zimmerman fusion medium. Nuclear transferred oocytes were subjected to a single DC pulse of 20V/mm for 20 microsec, 15 to 30 minutes after fusion in order to examine the effect of activation after fusion on the development to the blastocyst stage. To determine the effect of the number of pulses for activation on the development, nuclear transferred oocytes were activated once at 15 to 30 min (single activation) or twice at 15 min-intervals (repeated activation) after fusion by a single DC pulse. After treatments, the reconstructed oocytes were immediately cultured in CR1aa medium containing 10 microg/ml cycloheximide for 5 h and then cultured in the same medium without cycloheximide. The nuclear transplanted embryos were cultured according to the method of Tominaga et al sup12). The cleavage rates on day 1 and developmental rates to blastocysts on day 7 were estimated. To assess the developmental ability of blastocysts to term, 2 embryos per cow were transferred to 3 Japanese black cows. (1) Both rates of cleavage and development to the blastocyst stage in the group of repeated activation were significantly higher than those in the group of nuclear transferred oocytes without activation. There was, however, no significant difference between groups of single and repeated activation. (2) All recipient cows became pregnant. One recipient aborted twin fetuses on day 103 of gestation. A single fetus was obtained from a hydrallantoic cow by Caesarian section on day 232, and twin stillborn calves were delivered by vaginal delivery on day 270 of gestation. Body weight of the single calf on day 232 was 20.0kg, and those of the stillborn twin calves were 25.7kg and 17.5kg. From the results of the present study, it was concluded that activation after fusion stimulates the developmental ability of nuclear transterred oocytes to the blastocyst stage."