牛体外受精卵的冷冻保存研究

The Study of Freezing Storage of Bovine IVF Embryos

将胚胎分别以PBS+20%FCS+10%乙二醇和PBS+20%FCS+10%甘油为冷冻保护液,或者在PBS+20%FCS+10%乙二醇中添加或不加蔗糖为保护液,用常规冷冻方法进行冷冻处理.胚胎保存1～18天后将其解冻,并在发育用培养液TCM199+10%OCS(发情母牛血清)+丙酮酸钠中培养48小时.结果表明解冻胚的形态正常率和培养后的发育率在乙二醇处理组和甘油处理组中分别为81.2%(13/19)、56.2%(9/16)和84.6%(11/13)、46.2%(6/13),两组之间无量著差异.在添加蔗糖和不加蔗糖的处理组中,胚胎解冻后的形态正常率和培养后的发育率分别为75.0%(15/20)、55.0%(11/20)和83.3%(15/18)、61.1%(11/18),两组之间也无明曼差异.试验结果证明,用乙二醇为防冻剂对牛体外受精胚胎进行冷冻保存,其效果相当于或略优于甘油,在乙二醇处理组中,添加蔗糖与否并不影响其冷冻效果.

In present study,the morulae and blastocysts developed from bovine IVM and IVF embryos were frozen by using ethylene glycol (EG) and glycerol (Gly) as cryoprotectants,and the effect of sucrose in EG treatment on freezing of embryos was examined also.Method:The embryos were treated with 10% EG,and 10%Gly in PBS+20%FCS,and with or without sucrose in PBS+20%FCS+10%EG.Then the embryos were frozen by means of conventional method.After being 1～18 days stored in liquid nitrogen,the embryos were thawed and cultured in TCM-199+10%FCS+Na-Pyruvate for further development.The morphology of embryos thawed and development rate of the embryos after cultured for 48 hrs.were observed.Results:The rate of morphologically normal embryos thawed and the embryos developed were 81.2%(13/16),56.2%(9/16) in EG treatment group and 84.6%(11/13),46.2% (6/13) in Gly group,respectively.There were no significant differences between the two groups;In EG treatment group with and without sucrose,the percentage of morphologically normal embryos thawed and the development rate of embryos cultured were 75.0%(15/20),55.0%(11/20) and 83.3%(15/18), 61.1%(11/18) respectively.There were no significant differences between them either.The results indicated that the effect of EG on freezing of bovine IVF embryos was same as or slightly better than that of Gly.Adding sucrose in EG group didn't improve the freezing result.