摘要
通过两个试验对体外培养成熟的牛卵母细胞进行玻璃化冷冻。试验１用１０％丙二醇（ＰＧ）加上不同浓度（０％～２０％）的乙二醇（ＥＧ）作为细胞内玻璃化溶液（ＶＳ１），２５％ＰＧ＋２５％ＥＧ作为细胞外玻璃化溶液（ＶＳ２）对卵母细胞进行冷冻。结果卵母细胞的体外受精分裂率（０．９％～２３．４％）均显著低于对照组（７５．０％）。在处理组中，以１０％ＰＧ＋５％ＥＧ和１０％ＰＧ＋１０％ＥＧ作为ＶＳ１的效果较好，分裂率分别为２３．４％和２２．２％，均显著高于其他处理组。试验２以１０％ＰＧ＋５％ＥＧ作为ＶＳ１，２５％ＰＧ＋２５％ＥＧ作为ＶＳ２进行冷冻。解冻后在０．５ｍｏｌ／Ｌ或０．２５ｍｏｌ／Ｌ蔗糖溶液中１～２步或在含防冻剂的溶液中经２～３步稀释防冻剂。卵母细胞经体外受精后，分裂率在１８．２％～３０．６％之间。在０．２５ｍｏｌ／Ｌ蔗糖溶液中一步脱防冻剂的卵母细胞经体外受精、体外培养后，２５个早期胚胎中有３个发育成囊胚，其它组均未发现囊胚。这些结果表明玻璃化过程对卵母细胞造成了损伤。引起损伤的原因可能是玻璃化溶液的化学毒性作用及脱防冻剂过程中卵母细胞受到的渗透压损伤。
Two experiments were undertaken to cryopreserve bovine oocytes matured in vitro. In Exp. 1, the combination of 10% propylene glycol (PG) plus 0￣20% ethylene glycol (EG) in PBS was used as the intracellular vitrification solution (VS1) and that of 25 % PG+25 % EG as the extracellular vitrification solution (VS,). Oocytes were vitrified after exposure to 5% PG, 10% PG and VS1 each for 5 min, prior to VS2 for less than 20 seconds. The cleavage rate of the vitrified-thawed oocytes after in vitro fertilization (IVF)ranged from 0. 9% to 23. 4%, which were significantly lower than that of the controls (75. 0%, P<0. 01 ). Within the treatment groups, the combinations of 10% PG+5% EG and 10% PG+ 10% EG as VS1 produced the highest cleavage rates (23. 4% and 22. 2% resp.).In Exp 2, oocytes were vitrified using 10% PG+ 5 % EG as VS1 and 25 % PG+25% EG as VS2. After thawing of the oocytes,cryoprotectants were diluted either in sucrose solutions (0. 5 mol/L or 0. 25 mol/L) in 1￣2 steps or in diluted cryoprotectant solutions in 2￣3 steps. The cleavage rate of oocytes following IVF was in the range of 18. 2%￣30. 6%. Three blastocysts were obtained following culture of the 25 early embryos derived from oocytes after removal of cryoprotectants in 0. 25 mol/L sucrose for 10 min.
关键词
牛
玻璃化
冷冻
体外成熟
bovine oocytes
vitrification
cleavage rate
in vitro fertilization
