培养条件对牛卵母细胞体外受精后胚胎发育的影响

Effects of culture system on developmental competence of bovine oocytes following maturation,fertilization and culture in vitro

以屠宰场牛卵巢为材料,采用体外成熟、体外受精、早期胚胎的体外培养等方法,研究了牛卵泡液(BFF)浓度、颗粒细胞单层(GCM)和培养微滴大小对牛卵母细胞体外发育潜力的影响.结果表明:(1)与未添加BFF的对照组相比,成熟培养液中分别加入10%,20%和40%的混合BFF,均可明显促进卵母细胞受精后的发育(P<0.05),但20%组和40%组出现卵母细胞或胚胎粘连.因此,成熟培养液中以添加10%的混合BFF较为适宜.(2)添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无显著影响(P>0.05);但添加GCM的2级、3级卵母细胞受精后的卵裂率、6～8细胞发育率和囊胚率分别显著高于未添加组(P<0.05).(3)将取自每头牛的约20枚卵母细胞,分别置于不同大小的微滴(30,50,100和200 μL)中培养、受精,结果表明,30和50 μL组的囊胚发育率显著高于100和200 μL组(P<0.05).

Experiments were conducted to study the effects of bovine follicular fluid （BFF） concentration, granulose cell monolayer （GCM） and medium drop size on developmental competence of bovine oocytes derived from abattoir ovaries following in vitro maturation,fertilization and culture. In experiment 1,BFF of （10%,20%,and 40%） different concentrations from all follicles （〉2 mm） were supplemented to the maturation medium. The results show that different concentration＇s of BFF could support in vitro maturation of COCs and subsequent development capacity,however, 20% and 40% BFF could seriously cause oocytes and embryos to adhere together. Therefore,adding 10% of mixing BFF in maturation medium could be the best choice. Experiment 2 shows that the presence of granulosa cells during maturation and culture did not affect the cleavage rate,6-8 cells rate and blastocyst rates （P〉0. 05）of oocytes in grade 1. However,the cleavage rate,6-8 cells rate and blastocyst rates in grade 2 and grade 3 were higher （P〈0.05） when COCs were cultured in the presence of GCM than when cultured in the absence of GCM. In experiment 3,about 20 oocytes derived from every cow were cultured and fertilized in different sizes of medium drops （30,50,100 and 200 μL）. The blastocyst developmental rates of 30 and 50 μL groups were higher than 100 and 200μL treatments（P〈0.05）.