玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体的影响

以冷冻环为载体,探讨玻璃化冷冻对猪体外成熟卵母细胞染色体与纺锤体影响。单用40%乙二醇(ethyleneglycol,EG)或20%EG与20%二甲基亚砜(dimethylsulphoxide,DMSO)联合作冷冻保护剂,用直投液氮或使用玻璃化冷冻仪法制冷冷冻猪体外成熟卵母细胞;解冻2h后固定并免疫荧光法染色纺锤体及染色体;挑选各试验组形态正常卵母细胞进行体外受精实验。结果表明,与单用EG以及EG和DMSO联合直投液氮方案比较,EG和DMSO联合应用并采用玻璃化冷冻仪制冷方案卵母细胞染色体正常率为30.1%,纺锤体正常率为37.2%,可明显降低卵母细胞染色体及纺锤体结构损伤(P<0.05),并明显提高卵母细胞的激活效果(P<0.05)。采用联合冷冻保护剂及玻璃化冷冻仪高速冷冻可较好维持猪卵母细胞染色体与纺锤体形态,但玻璃化冷冻明显影响猪卵母细胞体外受精后的发育能力。

Influence of Vitrification on the Chromosomes and Spindles of Porcine Oocytes Matured in Vitro

To investigate the influence of vitrification freezing on the chromosomes and spindles of porcine oocyte matured in vitro by using nylon cryoloop. Porcine oocytes were verified with ethylene glycol (EG) singly or EG combined with dimethyl sulphoxide (DMSO) as cryoprotectants, and cooled by taking oocytes directly into liquid nitrogen or by vitrification machine. After frozen porcine oocytes thawed, the microtubulin of the spindles and chromosomes were fixation and stained by immunofluorescent method. Normal morphological oocytes in every groups were used to fertilize in vitro. Comparatively, in protocol of EG combined with DMSO and at ultra-rapid cooling rate, the normal configuration of spindle and chromosomes rate of thawed porcine oocytes was remarkably higher than that of the two protocol of single EG used and EG combined with DMSO, As the same in the rate of oocytes activated. The protocol of EG combined with DMSO as cryoprotectants and with extremely high cooling rate can produce better effect on conservation of chromosomes and spindle configuration in vitrification of porcine oocytes, while the development ability of thawed oocytes are severely affected.