| Methodology of handmade somatic cell cloning in cattle was studiedelementarily. Oocytes were enucleated by bisection in the studies. The resultshowed that there was no significant difference in survival rate of oocytesbetween handmade and micromanipulation bisection(85.6%vs 90.3%,P>0.05).When two cytoplasts and one granulosa cell were fused by PEG, the fusionrate in 50% of PEG was significantly different with that of 45%, 55% and60% (39.3% vs 0%,39.3% vs 16.7%,39.3% vs 5.3%, respectively, P<0.01)There was no significant difference between Parameter Two (AC 3.0V, 12μs;DC 100 V,30μs×1) and Parameter One (AC 3.0V,12μs; DC 90 V,30μs×1)in one-step electrofusion (63.4% vs 55.2%,P>0.05). In two-step electrofusion,however, there was significant difference in fusion rate of oocytes (55.6% vs73.2%,P<0.01) by increasing DC from 60v and 40v to70v and 50v withconstant AC. There was significant difference in blastocyst rate of clonedembryos (11.2% vs 0%,P<0.01) derived from fusion by Parameter Two and50% PEG; but there was no significant difference (11.2% vs 4.9%,P>0.05)between Parameter Two and Parameter Four. When zona-free oocytes were activated by different concentration of ionomycin, blastocyst developmentrates in activation by 2μmol/L of ionomycin was siginificant difference to5μmol/L or 0.5μmol/L (29.4% vs 8.3%, 29.4% vs 9.4%, respectively,P<0.01);however, there was no significant difference between 2μmol/L and3.5μmol/L treatments (29.4% vs 19.1%,P>0.05). There was no significantdifference in cleavage rate of zona-free PA embryos (61.3% vs 71.7%,P>0.05)between culturing in WOW and microdrops, but the blastocyst developmentrate (44.0% vs 8.3%, P<0.01) was significantly different. The result alsoindicated that, in handmade somatic cell cloning, survival rate of cattleoocytes enucleated by handmade bisection can be as high as bymicromanipulation. In conclusions, cytoplasts and donor cells can be fusedefficiently by one-step electrofusion or two-step electrofusion; theconcentration of ionomycin should be lower than usual (5μmol/L); WOW wasbetter than microdrops for culturing zona-free embryos in vitro. |
