不同形态卵母细胞和培养方式对牛体外胚胎生产的影响

Effects of different oocyte morphology and culture methods on bovine embryo production in vitro

研究旨在优化牛体外胚胎生产体系,进一步提高囊胚的生产效率。试验随机选取假设受精的卵丘-卵母细胞复合体(COCs)10枚及全部自然裸卵和透明状COCs,分别于平衡至少2 h的100μL CR1aa培养微滴中进行体外培养,探索不同形态卵母细胞对牛体外胚胎生产的影响。另选择30枚具有3层以上正常形态的COCs进行体外受精,随机分为3组,各组分别用胚胎培养液CR1aa、CR1aa+10%FBS以及采用子宫角表皮细胞共培养方式进行孵化,探索不同培养方式对牛体外胚胎生产的影响。结果显示,CR1aa、CR1aa+10%FBS和CR1aa+子宫角表皮细胞单层共培养的胚胎卵裂率和囊胚率差异均不显著(P>0.05),但在数值上子宫角表皮细胞单层培养在卵裂率方面具有一定优势。不同形态卵母细胞的体外受精效果差异较大,仅具有良好形态COCs的卵子受精后有可能发育到囊胚阶段;自然裸卵虽然可以体外受精,但卵裂率显著低于COCs(P<0.05);透明状COCs无法受精,且体外受精48 h时观察基本均已退化完毕。研究表明,试验所用FBS和子宫角表皮细胞单层对体系的优化效果不明显。自然裸卵和透明状COCs难以有效产出可移植胚胎,在繁殖应用中可不做选择。

The purpose of the study was to optimize the production system of bovine embryos in vitro and further improve the production efficiency of blastocysts.The experiment randomly selected ten COCs and all natural naked eggs,transparent COCs that were supposed to be fertilized,and cultured them in vitro in 100μL CR1aa culture droplets that were balanced for at least 2 h respectively,to explore the effects of different morphology of oocytes on bovine in vitro embryo production.Thirty COCs with more than three layers of normal morphology were selected for in vitro fertilization,and were randomly divided into three groups.Each group was incubated with embryo culture medium CR1aa,CR1aa+10%FBS and co cultured with uterine horn epidermal cells to explore the effects of different culture methods on bovine embryos in vitro.The results showed that there was no significant difference in the cleavage rate and blastocyst rate of embryos co cultured with CR1aa,CR1aa+10%FBS and CR1aa+uterine horn epidermal cells monolayer(P>0.05),but in terms of numerical value,the cleavage rate of uterine horn epidermal cells monolayer culture had certain advantages.The effect of in vitro fertilization of oocytes with different morphologies is quite different,and only the oocytes with good morphologies of COCs may develop to the blastocyst stage after fertilization.Although natural naked eggs can be fertilized in vitro,the cleavage rate was significantly lower than that of COCs(P<0.05).Transparent COCs could not be fertilized,and had basically degenerated after 48 h of in vitro fertilization.The study indicates that the FBS used in the experiment and the monolayer of uterine horn epidermal cells had no obvious effect on the optimization of the system.It is difficult to effectively produce transplantable embryos from natural naked eggs and transparent COCs,so there is no choice in reproductive applications.