延边黄牛活体采卵体外受精胚胎程序化冷冻保存的研究

Study on programmed cryopreservation of in vitro fertilized embryos of oocyte recovery from live Yanbian yellow cows

为了探究延边黄牛活体采卵体外受精胚胎程序化冷冻时,冷冻保护剂种类、浓度、平衡时间及囊胚不同发育阶段对冷冻效果的影响,试验以延边黄牛活体采卵体外受精胚胎为研究对象,设置4组试验:试验一,以甘油作为冷冻保护剂,设置冷冻液中甘油的浓度分别为0.5,1.0,1.5,2.0,2.5,3.0 mol/L,统计各浓度下冷冻胚胎(囊胚)的存活率和孵化率;试验二,以乙二醇作为冷冻保护剂,设置冷冻液中乙二醇的浓度分别为0.5,1.0,1.5,2.0,2.5,3.0 mol/L,统计各浓度下冷冻胚胎(囊胚)的存活率和孵化率;试验三,以上述最佳冷冻液的最适浓度作为冷冻条件,设置胚胎在冷冻液中平衡时间分别5,10,15,20,25 min,统计各平衡时间下的冷冻胚胎(囊胚)的存活率和孵化率;试验四,以上述最佳冷冻保护剂的最适浓度和最佳平衡时间为条件,选择第7天囊胚和扩张囊胚进行冷冻试验,统计不同发育时期的胚胎的存活率和孵化率。结果表明:以甘油作为冷冻保护剂,浓度为2.0 mol/L时冷冻延边黄牛活体采卵体外受精胚胎的存活率和孵化率最高;以乙二醇作为冷冻保护剂,浓度为1.5 mol/L时冷冻延边黄牛活体采卵体外受精胚胎的存活率和孵化率最高;对延边黄牛活体采卵体外受精胚胎程序化冷冻时,甘油(2.0 mol/L)与乙二醇(1.5 mol/L)为冷冻保护剂冷冻的胚胎存活率与孵化率两者之间差异不显著(P>0.05),但甘油的效果好于乙二醇;以甘油(2.0 mol/L)作为冷冻保护剂,平衡时间为15 min时冷冻胚胎的存活率和孵化率最高,且扩张囊胚的存活率[(68.69±3.50)%]及孵化率[(37.37±7.00)%]要高于囊胚的存活率[(60.74±5.60)%]及孵化率[(31.85±8.98)%]。说明当甘油浓度为2.0 mol/L、乙二醇浓度为1.5 mol/L时,均能有效地保护胚胎不受低温环境的影响,但以甘油作为冷冻保护剂效果更好;且扩张囊胚在甘油冷冻保护剂中平衡15 min冷冻效果较好。

In order to explore the effects of cryoprotectant types,concentrations,equilibrium time and different development stages of blastocysts on the freezing effect in the process of programmed freezing of embryos from in vitro fertilization of oocyte recovery from live Yanbian yellow cows,in vitro fertilization embryos of Yanbian yellow cattle were used as the research object in this experiment.Four groups of experiments were set up.In the first experiment,glycerol was used as cryoprotectant,and the concentration of glycerol in the frozen solution was 0.5,1.0,1.5,2.0,2.5 and 3.0 mol/L,respectively.The survival rate and hatching rate of frozen embryo(blastocyst)were calculated at each concentration.In the second experiment,ethylene glycol was used as cryoprotectant,and the concentration of ethylene glycol in the frozen solution was 0.5,1.0,1.5,2.0,2.5 and 3.0 mol/L,respectively.The survival rate and hatching rate of frozen embryo(blastocyst)were calculated at each concentration.In the third experiment,the optimum concentration of the above-mentioned optimal freezing solution was used as the freezing condition,and the equilibrium time of embryos in the freezing solution was set to be 5,10,15,20,and 25 min,respectively.The survival rate and hatching rate of frozen embryos(blastocysts)under each equilibrium time were counted.In the fourth experiment,blastocyst and expanded blastocyst on day 7 were selected for freezing test under the condition of the optimum concentration and equilibrium time of the best cryoprotectant mentioned above,and the survival rate and hatching rate of embryos at different development stages were counted.The results showed that the survival rate and hatching rate of the frozen embryos were the highest when glycerol was used as cryopectant at 2.0 mol/L.The survival rate and hatching rate of the frozen embryos were the highest in the frozen embryo when 1.5 mol/L ethylene glycol was used as cryoprotectant.When glycerol(2.0 mol/L)and ethylene glycol(1.5 mol/L)were used as cryoprotectant,there was no significant difference in the survival rate and hatching rate of frozen embryos(P>0.05),but the effect of glycerol was better than that of ethylene glycol.When glycerol(2.0 mol/L)was used as cryopectant,the survival rate and hatching rate of frozen embryos were the highest in the balance time of 15 min.The survival rate([68.69±3.50]%)and the incubation rate([37.37±7.00]%)of expanded blastocysts were higher than those of expanded blastocysts([60.74±5.60]%)and hatching rate([31.85±8.98]%).The results indicated that both 2.0 mol/L glycerol and 1.5 mol/L ethylene glycol could effectively protect embryos from the effects of low temperature environment,but the effect of glycerol as a frozen protective agent was better.The best freezing effect was obtained when the expanded blastocyst was balanced in glycerol cryoprotectant for 15 min.