玻璃化冷冻水牛MII期卵母细胞的初步研究

Preliminary Study on Vitrification of Metaphase II Buffalo Oocytes

为建立玻璃化冷冻保存水牛MⅡ期卵母细胞的有效程序,试验比较了3种冷冻保护剂组合(Ⅰ:20%乙二醇+20%二甲基亚砜;Ⅱ:25%乙二醇+25%二甲基亚砜;III:25%乙二醇+25%甘油)和3种玻璃化冷冻方法(毛细玻璃管法、铜环法及半麦管法)对水牛MⅡ期卵母细胞的毒性和冷冻效果的影响。结果表明,Ⅲ组的卵母细胞形态正常率显著低于Ⅰ和Ⅱ组(P<0.05),且III组的存活率也显著低于Ⅰ组(P<0.05),而Ⅰ和Ⅱ组的卵母细胞形态正常率和存活率没有显著差异(P>0.05)。进一步孤雌激活发现,Ⅰ组的卵裂率显著高于Ⅲ组(29.17±4.81%,5.56±11.11%,P<0.05),Ⅰ组的囊胚发育率显著高于Ⅱ和Ⅲ组(8.33±2.23%,2.43±2.87%,1.86±3.71%,P<0.05),然而Ⅱ和Ⅲ组间的卵裂率和囊胚发育率没有差别(P>0.05)。采用铜环法的回收率显著高于半麦管法(P<0.05),但和毛细玻璃管法之间差异不显著(P>0.05),冻后卵母细胞孤雌激活后的卵裂率和囊胚发育率差异不显著(P>0.05)。3种冷冻保护剂中Ⅰ组的细胞毒性作用最小,Ⅲ组的细胞毒性作用最大;铜环法和毛细玻璃管法能有效冷冻保存水牛的MⅡ期卵母细胞。

To establish an effective vitrification of Metaphase Ⅱ （MⅡ） buffalo oocytes,the experiments was performed to compare the toxicity of three combinations of cryoprotectants （I：20% ethylene glycol （EG）＋20% dimethyl sulfoxide（DMSO）; Ⅱ：25% EG＋25% DMSO;Ⅲ：EG 25%＋25% glycerol （GL） employed MⅡ buffalo oocytes. A further study was taken to compare the cryopreservation effects of glass micropipette vitrification （GMP）, cryoloop vitrification （CLV） and hemistraw method. The results showed that the morphological normal rate of group Ⅰ and group Ⅱ were significantly lower than that of group Ⅲ（P〈0.05）, and the survival rate of group Ⅲ was lower than that of group Ⅰ（P〈0.05）, but the morphological normal rate and survival rate between group Ⅰ and group Ⅱ had no significantly difference （P〉0.05）. By means of parthenogenetieall activation, the cleavage rate of group I was higher than that of group Ⅲ（29.17±4.81% vs 5.56±11.11%,P〈0.05）, and blastocyst development rate of group I was also significantly higher than that of group Ⅱ and group Ⅲ（8.33±2.23% vs 2.43±2.87,1.86±3.71%, P〈0.05）, but the both rates were not significantly different between group I and group Ⅱ （P〉0.05）. The reclaimed rate of CLV method was significantly higher than that of hemistraw method （P〈0.05）, but there were no significance difference between the GMP and hemistraw （P〉0.05）. The cleavage and blastocyst rate among GMP, CLV and hemistraw were not significantly different（P〉0.05） .In conclusion, the toxicity of I group I was the smallest and the group Ⅲthe biggest. MⅡ buffalo oocytes could be vitrified effectively by CLV and GMP methods.