Study On The Main Factors Affected The Efficiency Of The Goat Embryo Nucleus Transplant Technique

The major factors affected the efficiency of goat embryo nuclear transfer technique were discussed systematically in this study in order to establish an acceptable manipulation and production system of embryo nuclear transfer technique in our lab. The main contents of this study were: effect of different collecting methods on the quality and development potential of goat oocytes;effect of different maturation period on the quality and enucleation rate of goat oocytes;effect of different enucleation and injection methods on the development rate of goat embryo nuclear transfer embryos and the effect of different electric fusion and activation methods on the development rate of goat embryo nuclear transfer embryos.To collect oocytes from slaughtered goat ovaries, two methods, surface cutting and aspiration, were used for recovering of the usable cumulus-oocyte complexes (COCs) from surface visible follicles. The average total number of COCs recovered per ovary was 4.96 by surface cutting and 2.86 by aspiration. There is significant difference(P<0.05) between the two methods for collection of the COCs in goat, however, the maturation rate of collected oocytes was not different (P>0.05). We observed that goat oocytes cultured for 18 h in vitro would be matured, and the highest enucleation rate would be achieved after culturing 18-20 h in vitro. Modified McGrath-Solter method and modified Willadsen method were used to enucleate the goat oocytes matured 18-24 h in vitro, the enucleation rate were 82.00% and 84.68% respectively as determined under fluorescent microscope for the presence of chromatin after oocytes had been dyed in Hoechst33342. There was no difference between the two enucleation methods (P>0.05), and they had no effects on the development of reconstructed embryos up to morula stage (P>0.05). Two methods intracytoplamic injection and perivitelline injection were used in the experiment, the injection rate was 93.33% and 90.4% respectively, and there was no significant difference between them(P>0.05), after injection, 26.98% and 22.41% of the reconstructed embryos developed to the morula stage, and the difference was significant (P<0.05). Different electric strengths, different width of pulse and different repeats wereused to treat the goat reconstructed embryos, the results indicated that the highest electrofusion (81.67%) and the best development rate of morula stage (29.03%) were obtained by the pulse-strength 1.25KV/cm, pulse-width 30fis, 2 repeats.The reconstructed embryos were activated by electroactivation then cultured in Ionomycin and then in 6- Dinethylaminopurine. The activation rate (73.79%) and the development rate of morula stage (35.53%) were better than separated electroactivation. There was significant difference between them(P<0.05).