Studies On Electric Fusion Of Somatic Cell Cloning In Yanbian Yellow Cattle

Successfully somatic cell nuclear transfer(NT) has been achieved in cattle,as reported by the birth of offspring;however,the efficiency of cloning is generally lower.It is a long-term task to improve the efficiency of nuclear transfer.Now,there are many techniques need to be improved.The fusion of reconstructed oocyte is highly important.We can produce more available embryos if we improve the efficiency of fusion.It is obvious that the fusion efficiency is one of the keys on success of nuclear transfer;it can directly affect the NT efficiency.In this research,we use YanBian Yellow Cattle ear skin's fibroblast cells as donor cells and use MⅡenucleated oocytes as recipient cells.We do the experiment by means of acupressure enucleated oocytes putting donor cells into yolk space and use electric fused.We investigated different cell fusion mediums,different methods of fusion and suitable conditions of fusion.We discussed the best conditions of fusion for somatic cell cloning in Yanbian Yellow Cattle.The result is evaluated as fused rate,cleavage rate and the development rate of reconstructed embryos in vitro.They are shown that:1.The fused rates and cleavage rates of sucrose cell fusion medium with 025M,0.28M,0.30M three different concentrations were no significant differences.the development rates of 4-8cell,0.28M was the best,it is significantly higher than the group of 0.25M(51.01%vs 32.95%,p＜0.05)and no significant differences from 0.30M(40.12%,p＞0.05).2.The concentrations of D-sorbitol cell fusion medium with0.25M,0.28M,0.30M in fused rates and cleavage rates were no significant differences.The development rates of 16cell,the group of 0.28M was no significant differences from 0.30M(20.68%vs 17.45%,p＞0.05)and it is significantly higher than the the group of 0.25M(9.25%,p＜0.05).3.The concentration of mannitol with 0.28M,the fused rate,cleavage rate and the development rate of 4-8cell and 16cell are 89.54%,82.64%,61.29%and23.70%,respectively;but the three groups were no significant differences(p＞0.05).4.The effects of the concentrations of sucrose,D-sorbitol and mannitol cell fusion medium with 0.28M on nuclear transfer efficiency.The fused rate were no significant differences(88.44%vs 86.83%vs 86.85%p＞0.05).Cleavage rates(87.98%vs 86.06%) and the rates of development into blastocyst stage(22.16%vs19.91%)of sucrose and mannitol cell fusion medium were no significant differences(p＞0.05),but there are significantly higher than D-sorbitol(78.64%and 10.14%,p＜0.05).5.The concentration of sucrose cell fusion medium with 0.28M.the fused rate,cleavage rate and the rate of development into blastocyst stage of the group of fusion concentration gradient method were significantly higher than the group of general fusion method(92.98%vs 88.55%;91.07%vs 80.96%;40.06%vs 30.19%p＜0.05).6.The concentration of mannitol cell fusion medium with 0.28M.the fused rate,cleavage rate and the rate of development into blastocyst stage of the group of fusion concentration gradient method were significantly higher than the group of general fusion method(91.22%vs 84.89%;88.02%vs 80.42%;39.51%vs 29.19%,p＜0.05).7.Electric field strength in fusion with 1100V/cm,1200v/cm and 1300v/cm,the fused rate are 87.66%,89.07%and 90.14%,respectively,there were no significant differences between them(p＞0.05),but there were significantly higher than the group of 1000V/cm (81.83%,p＜0.05),Cleavage rate of 1100v/cm and 1200v/cm were significantlyhigher than the group of 1300V/cm(85.07%vs 86.47%vs 76.96%,p＜0.05),they were not significant different with 1000v/cm(80.85%,p＞0.05).In the rate of development into blastocyst stage,1200v/cm is the highest(29.59%),it was not significant different with 1100v/cm(28.89%,p＞0.05),but it was significant higher than1000v/cm and 1300v/cm(19.46%and 14.61%,p＜0.05).8.The fused rate and cleavage rate and the rate of development into blastocyst stage of double direct current pulses are 88.03%,86.35%and 31.63%,respectively.it is the best suitable electric pulse times in somatic cell cloning in Yanbian Yellow cattle useing fusion concentration gradient method.9.The fused rate of electric pulse time with 40μs had no significant difference with 20μs,80μs(86.10%vs 83.30%vs 84.60%,p＞0.05),had significant difference with 10μs (79.60%,p＜0.05).The cleavage rate(88.54%,83.61%vs 74.21%,65.63%,p＞0.05)and the rate of development into blastocyst stage(32.92%vs 30.38%vs 20.88%vs 19.62%,p＞0.05)of 20μs,40μs had significant differences with 10μs,80μs.Conclusion:The 0.28M sucrose fusion medium is the best fusion medium for YanBian Yellow Cattle somatic cell cloning.Use fusion concentration gradient method can effectively improve the efficiency of YanBian Yellow Cattle somatic cell cloning.Double direct current pulses of 1200V/cm for 40μs separated are the best for somatic cell cloning in YanBian Yellow Cattle using fusion concentration gradient method.