| The effective activation of dual sperm cloned embryos is one of the key steps for the development of cloned embryo, because the initial development of nuclear transfer depends on the effective activation and it will affect the embryo development rate and blastocyst rate directly. The efficiency of nuclear transplantation has been increased with improving of activation methods in recent years. Activation methods of sperm cloned embryos may be different from the conventional differentiated somatic cell cloned embryos, because the genetic material of sperm cloned embryos all comes from the male parent. The optimal activation method of sperm cloned embryos should be selected from the available activation method to improve embryo developmental rate and it is necessary to improve the activation method. Therefore, it is urgent to study the activation method to improving the method of dual sperm cloned embryos. So far, the method of dual sperm cloned embryos has already made great achievements, but its efficiency is still low, maybe due to the reconstructed embryo is not fully activated. And the same concentration and activation time of the same activator had different results. The solution of these problems will greatly improve the success rate of dual sperm cloned embryos. This study was undertaken to explore the methods, such as A23187、6-DMAP、CB for activation of dual sperm cloned embryos, and the effect on development after activation, in order to obtain an optimal method for activation of dual sperm cloned embryos. Four conclusions were drawed based on the analysis of experimental data.1) Different concentrations and activation time of calcium ionophore A23187 had different effect on the dual sperm cloned embryos:the dual sperm cloned embryos rested and rehabilitated for 2 hours were treated for lmin,5min, 10min in different concentrations (0.05μM、0.1μM、0.15μM) of calcium ionophore A23187 in SOF. Then these embryos were cultured in SOF without A23187. Cleavage and blastocyst rate were observed. We found that calcium ionophore time had a significant effect on the cleavage rate (P<0.05) and had no significant effect on the blastocyst rate (P>0.05). The early embryo development first increases and then decreases with the extension of time and when the role time was more than 5min, the embryo development rate started to decline. Different concentrations had a significant effect on the embryo development rate (P<0.05) and the higher cleavage rate was obtained with the higher concentration. Cleavage rate was not high,regardless of the length of time when concentration was low,however cleavage rate was significantly decreased,as the activation time extended at high concentration.No blastocyst was produced at low concentration, just received a small amount of blastocyst, when the concentration was 0.1μM and 0.5μM.2) Different concentrations of 6-DMAP had different effect on the dual sperm cloned embryos:the dual sperm cloned embryos were treated 5min in 0.1μM A23187,5min in 0.5μM Ionomycin and 5min in Mixture of A23187 and Ionomycin. Then these embryos were activated 4hours in different concentrations (1 mM/L、2 mM/L、4 mM/L、6 mM/L) of 6-DMAP respectively. We found that the stronger activation effect and the more development rate were obtained with the increasing concentrations when concentration of 6-DMAP was less than 4 mM. With the increasing concentration, the reconstructed embryo cleavage rate and blastocyst rate gradually decreased when 6-DMAP was greater than 4 mM. We compared the activation method of Ion+6-DMAP and A23187 +6-DMAP when 6-DMAP was 4 mM and found that A23187+6-DMAP had a better activation results and embryo cleavage and blastocyst rates were 18.2% and10.9%.3) Activation effect of the dual sperm cloned embryos was better when cytochalasin (cytochalasinB, CB) was supplemented for activation. The cleavage rate of reconstructed embryos treated with cytochalasin was extremely significant higher than that without cytochalasin (P<0.01) and blastocyst rate of reconstructed embryos treated in activation solution with cytochalasin was significant higher than that without cytochalasin(P<0.05). The cleavage rate of reconstructed embryos treated with A23187+6-DMAP+CB was extremely significant higher than Ionomycin+6-DMAP+CB (P<0.01), but blastocyst rate was lower.4) Activation effect of the dual sperm cloned embryos used the method of pre-activation was observed and the results indicated that cleavage rates of activation group and control group were 12.5% and 21.7%, and the difference was extremely significant (P<0.01). The blastocyst rate of the control group were significantly higher than activation group (P<0.05) and blastocyst rate were 5.0% and 8.7% respectively.The result indicated that we do not need the pre-activation on enucleated oocytes during dual sperm clone. |
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