【作者】 包华琼；

【导师】 李跃民； 王新庄；

【作者基本信息】 西南农业大学 ， 临床兽医学， 2003， 硕士

【摘要】 本实验探讨了冷冻保护剂、冷冻方法、卵母细胞发育阶段、冻前预处理方法等因素对牛卵母细胞冷冻效果的影响，目的在于筛选适合超低温冷冻保存牛卵母细胞的冷冻程序。通过这一途径可望建立卵母细胞种子库，并为体外受精、卵核移植和体细胞克隆研究与应用提供实验材料。研究结果如下： (1)冷冻保护剂对牛卵母细胞毒性实验表明：四种冷冻保护剂：二甲基亚砜(DMSO)、丙二醇(PROH)、乙二醇(EG)、甘油(GL)在室温(25℃)和培养温度(39℃)条件下与牛GV期卵母细胞作用3min，脱保护剂后进行体外成熟培养。表明：四种冷冻保护剂在低浓度室温(25℃)条件下对牛GV期卵母细胞毒性弱，但随浓度增加毒性增强；39℃时毒性明显增加，其中EG毒性最弱。四种冷冻保护剂的毒性由强至弱是：GL＞PROH＞DMSO＞EG。分别添加1.5mol/L冷冻保护剂(DMSO、PROH、EG、GL)程序化冷冻牛GV期卵母细胞时，EG和PROH对牛GV期卵母细胞的保护比GL、DMSO效果要好一些。 (2)采用程序化冷冻法、细管玻璃化法(Straw)、开放式拉管法(OPS)、毛细玻管法(GMP)冷冻牛GV期卵母细胞，解冻经体外成熟、体外受精后观察2-细胞卵裂率表明：程序化冷冻和Straw玻璃化冷冻的卵裂率分别为3.33％和8.33％，二者差异不显著(P＞0.05)；OPS冷冻和GMP冷冻卵裂率分别为36.84％和36.67％，二者差异不显著(P＞0.05)，但OPS冷冻和GMP冷冻与程序化冷冻、Straw玻璃化冷冻的卵裂率相比差异极显著(P＜0.01)。结果表明：玻璃化冷冻效果优于程序化冷冻，GMP和OPS优于Straw玻璃化冷冻，GMP方法冷冻效果与OPS方法相近。 (3)采用GMP法冷冻GV期、体外培养6h、18h、22h各发育阶段的卵母细胞，解冻后再培养24h、18h、6h和2h，经体外受精分析卵裂率。冷冻后GV期卵母细胞的卵裂率为36.67％，培养6h和18h的卵裂率分别为40.98％和41.27％，三者之间无显著差异(P＞0.05)，但与培养22h卵母细胞(52.38％)和对照(68.42％)差异极显著(P＜0.01)。于如每一出冷冰际名的研究 搞一体外成熟（IVM）卵母细胞冷冻后卵裂率为 52．38O，与对照（68．42O）差异不显著 （P＞0刀5）。冷冻不同发育阶段的牛卵母细胞的结果表明：体外成熟培养22h的卵母细胞冷冻效果好于 GV期、培养 6h、18h的卵母细胞，越接近成熟的卵母细胞冷冻效果越好。 （4）在卵母细胞玻璃化之前，分别采用 1OEG、3％EG、6oEG、10OEG、l／2 VS溶液预处理卜！smin，比较玻璃化冷冻后卵母细胞的体外成熟率和卵裂率。结果表明采用3％的EG稀溶液预处理玻璃化前牛卵母细胞可分别获得较高的卵裂率36．96％。 研究表明，采用GMP玻璃化冷冻法来保存牛GV期卵母细胞是可行的。更多还原

【Abstract】 The objective of this study is to provide an effective method to cryopreserve bovine GV oocytes for research and practice of gamete bank, in vitro fertilization, nuclear transfer and cell cloning. The study compared the effects of cryopreservation reagent , freezing method, the developmental stages of oocytes and equilibration method in bovine. The results as follows:Studies on cryoprotectants toxicity for bovine oocytes. Bovine GV oocytes were treated with four 1.5-7.2 mol/L cryoprotectants (DMSO, PROH, EG, GL) at 25 and 39 , then diluted cryoprotectants and compared the ratio of the maturation in vitro. The results demonstrated that the four cryoprotectants’ toxicity for bovine oocytes were low and increased depend on the concentration at 25 ; And those toxicity were evident at 39 , but EG’s is lowest. The toxicity from the high to low was GL>PROH>DMSO>EG. Using the programmed freezing method to freeze the bovine oocytes with 1.5 mol/L DMSCK PROH, EG, GL, the effects of EG and PROH were better than GL, DMSO.Using programmed freezing method, Straw vitrification, open-pulled-straw (OPS), glass micropipette (GMP) to freeze the bovine GV oocytes, Freezed and warmed oocytes were matrured and fertilized in vitro . The cleavage ratio was 3.33% and 8.33% respectively in programmed freezing method and Straw vitrification, and the latter was higher by 5.1% than the former, but the results was not different (P>0.05 ); The cleavage ratio using OPS and GMP was similar, 36.84% and 36.67% respectively, and the results was not different (P>0.05) ,but was significant different with programmed freezing method and Straw vitrification( PO.01 ) .This demonstrated that vitrification was better than programmed freezing method,that GMP and OPS was better than Straw vitrification, that the effect of GMP and OPS was similar.We study the effects of the developmental stages of oocytes. GV oocytes and ooctyes cultured 6h, 18h, 22h in vitro were vitrification using GMP. the cleavage ratio of warmed oocytes cultured 24h, 18h, 6h and 2h and fertilized in vitro was compared .The cleavage ratio of warmed GV oocytes was 36.67%, 40.98% and 41.27% after culture 6h and 18h. The three was not different (P>0.05).However, this was significant different compared with oocytes cultured 22h (52.38%) and the control (68.42%) (P>0.05) . The cleavage ratio of IVM freezed and thawed oocytes was 52.38%, and it was not different with the control (68.42%) (P>0.05).This results indicated the cyroperservation effectiveness of oocytes cultured 22h was better than that of GV and those oocytes cultured 6h and 18h. The more maturation of bovine oocytes were, the more cyroperservation effectiveness was.We study the effects of equilibration for vitrification. Prior to vitrification of bovine oocytes in the vilification solution, the oocytes were exposed to equilibration solution (1%EG, 3%EG, 6%EG, 10%EG, 1/2 VS solution) for 1-1 5min, the results indicated that effectiveness of 3%EG is best than others, whose cleavages and maturation ratio in vitro were high.This suggested the diluted solution equilibrium can improve the cyroperservation effectiveness before vitrification.The conclusions proved that GMP vitrification procedure can be used to cyro-perserve bovine GV oocytes.更多还原