山羊卵母细胞冷冻保存技术研究

Study on cryopreservation technology of goat oocytes

检测不同冷冻保护剂(PROH、DMSO、甘油)及不同冷冻方法(程序冷冻法、OPS玻璃化冷冻法)对山羊卵母细胞发育情况。结果表明,PROH(GV:85.3%;IVM:85.4%)和DMSO(GV:82.2%;IVM:85.2%)对各期卵母细胞的正常形态保护能力无显著性差异(P>0.05),但均显著高于甘油(GV:70.4%;IVM:75.5%)(P<0.05)。以PROH作为冷冻保护剂,其GV期卵母细胞成熟率(27.2%)均显著高于DMSO(18.9%)和甘油(10.1%)(P<0.05);IVM卵母细胞受精率(25.6%)也显著高于DMSO(18.7%)和甘油(14.1%)(P<0.05)。常规程序冷冻和OPS玻璃化冷冻法,GV期卵母细胞的成熟率分别为21.3%和29.9%,受精率分别为6.3%和9.1%;培养9 h卵母细胞的成熟率分别为20.5%和32.0%,受精率分别为5.1%和10.7%;IVM卵母细胞的受精率分别为21.4%和30.3%,2-细胞率分别为4.8%和10.5%;不论是常规程序冷冻还是OPS玻璃化冷冻,GV期和培养9 h卵母细胞的成熟率和受精率差异不显著(P>0.05),但受精率均低于IVM卵母细胞(P<0.05)。结果显示,PROH作为山羊卵母细胞的冷冻保护剂,其保护作用明显优于DMSO及甘油;OPS玻璃化冷冻效果要明显优于常规程序法。

The aim of this study was to investigate effect of different cryoprotectants and frozen methods on the developmental capacity of thawed goat oocytes.The development situations of Goat oocytes with different cryoprotectants（PROH,DMSO and glycerol） and frozen methods（conventional freezing procedure and OPS vitrification freezing procedure） were determined,respectively.The results showed that there was no significant difference（P0.05） between PROH（GV： 85.3%;IVM： 85.4%） and DMSO（GV： 82.2%;IVM： 85.2%） on the normal morphology protective ability of different phases of oocytes,while both of them were significantly higher（P0.05） than glycerol（GV：70.4%;IVM： 75.5%）.The maturation rate of GV oocytes was significantly higher in PROH group（27.2%） than that in DMSO（18.9%） and glycerol（10.1%） groups（P0.05）.The fertilization rate of IVM oocytes was significantly higher in PROH group（25.6%） than that in DMSO（18.7%） and glycerol（14.1%） groups（P0.05）.By means of conventional freezing procedures（slow cooling,rapid thawing,SL） and OPS vitrification freezing method,in GV oocytes,the maturation rate was 20.5%,29.9% respectively,the fertilization rate was 6.3%,9.1% respectively;in oocytes of 9 h,the maturation rate was 20.5%,32.0%,respectively.The fertilization rate was 5.1%,10.7%,respectively;In IVM oocytes,the fertilization rate was 21.4%,30.3%,respectively.The 2-cell rate was 4.8%,10.5%,respectively.The maturation rate and fertilization rate in GV and 9h oocytes had no significant difference in SL and OPS groups（P0.05）,while the fertilization rate of GV and 9 h oocytes was higher than that of IVM oocytes（P0.05）.The present study demonstrated that the protective effect of PROH,as a cryoprotectant for goat oocytes,was significantly better than DMSO and glycerol.OPS vitrification freezing procedure had better effect than conventional freezing procedure.