Studies On The Micromanipulation Solution Of Somatic Cell Cloning In YanBian Yellow Cattle

The excessive ROS(reactive oxygen species) produced in vivo and vitro of oocyte during micromanipulation is harmful for oocytes.Suitable antioxidants added into micromanipulation solution perhaps can eliminate the excessive ROS.It is also perhaps that caffeine added into micromanipulation solution can increase the ration of cleavage and later development of reconstructed embryos.Therefor,the experiment have researched effect of micromanipulation solution added with different material on the development of reconstructed embryos,discusssed how to improve the efficiency of Yambian Yellow Cattle somatic cell cloning,thereby lays the rationale for the transgenic clonging and medical clonging.The experimental result is as follows:In the first experiment,0μM,50μM,100μM,150μM VitamineE(V_E) were added in the micromanipulation solution respectively(include enucleation and injection micromaniputation solution),and mensurated the effect on the development of reconstructed embryos,thereby determined the best concentration of V_E added in the micromanipulation solution.In the cleavage ration and the rate of development to the blastocyst stage,100μM group was the highest(89.68%and 28.57%,p＜0.05),it was significant higher than 0μM group.100μM is the suitable concentration.In the second experiment,0mM,0.3mM,0.6mM,0.9mM,1.2mML-cysteine were added in the micromanipulation solution respectively.In the cleavage ration,0.9mM group was significant higher than 0μM and 1.20mM group(90.56%vs 81.18%and 80.95%,p＜0.05),there was no significantly different among other groups;in the rate of development to blastcyst stage, there was no significant different between 0.6mM and 0.9mM group,they both significant higher than other groups(p＜0.05).The 0.9mM is the suitable concentration.In the third experiment,0mM,7mM,14mM,21mM taurine were added in the micromanipulation solution respectively.In the cleavage ration and the rate of development to the hlastocyst stage,14μM group was the highest (92.93%and 37.68%,p＜0.05),it was significant higher than other groups. 14μM is the suitable concentration. The fourth experiment,divided into four groups.Contrast group:V_E group added with 100μM V_E;L-cysteine group added with 0.9mM L-cysteine;taurine group added with 14mM taurine.In the cleavage ration,there was no significantly different among V_E group,L-cysteine group and taurine group(87.83%,85.85%,90.50%vs 78.84%,P＜0.5),they all significant higher than contrast group;in the rate of development to blastcyst stage,taurine group was significant higher than other groups(38.44%,P＜0.5),there was no significant different between V_E group and L-cysteine group,they both significant higher than contrast group.The 14mM taurine is the suitable antioxidant added in the micromanipulation solution.In the fifth experiment,0mM,2.50mM,5.0mM,7.50mM caffeine were added in the injection micromanipulation solution respectively.In the cleavage ration and the rate of development to the blastocyst stage,5.0mM group was the highest(91.10%and 38.99%,p<0.05),it was significant higher than 0μM group.5.0mM is the suitable concentration.The sixth experiment,divided into four groups.Contrast group;the group added with taurine singly,the group added with caffeine singly,and both added group.Mensurated the effect on the development of reconstructed embryos,thereby determined the best adding method of the micromanipulation solution.In the cleavage ration and the rate of development to the blastocyst stage,there was no significant different among taurine group,caffeine group and both added group,(88.84%,89.19%,90.92% and36.78%,36.22%,38.90%,p＞0.05),they all significant higer than contrast group(p＜0.05).Because the caffeine is included the state regulation energetic drugs scope,it can't be used in practice widely.