Suzuki T, Boediono A, Takagi M, Saha S, Sumantri C
United Graduate School of Veterinary Sciences, Yamaguchi University, Japan.
Cryobiology. 1996 Oct;33(5):515-24. doi: 10.1006/cryo.1996.0055.
The objective of this study was to evaluate in vitro fertilization and cleavage rates of frozen-thawed bovine oocytes at the germinal vesicle (GV) stage. In mouse oocytes, spindle microtubule reorganization after GV breakdown is particularly sensitive to cold and readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. The effects of various permeating cryoprotective agents [1.8 M ethylene glycol (EG), 1.3 M ethylene glycol monomethyl ether (EME), and 1.6 M 1,2-propanediol (PROH)] and different concentrations of trehalose (T) and polyvinylpyrrolidone (PVP) on post-thaw developmental capacity were examined. When bovine GV oocytes were frozen slowly in mixtures of 1.8 M EG plus 5% PVP and 0.05 M T, almost 80% developed to metaphase II; 22.2% degenerated after in vitro maturation, and none of those that had been cryopreserved underwent parthenogenetic activation. The total fertilization rate was higher (P < 0.05) for oocytes frozen in a mixture of 1.8 M EG plus 0.05 M T or 0.1 M T than in a mixture of 1.8 M EG with or without 0.2 M T; however, there was no difference in the number of normally fertilized or polyspermic oocytes that had been frozen in various cryoprotective solutions. No significant difference was observed in subsequent development using EG, EME, and PROH for GV oocytes. The addition of 0.05 or 0.1 M trehalose to the freezing solution yielded significantly better cleavage and blastocyst rates than the solutions containing 0.2 M or no trehalose. For unfrozen controls, GV oocytes yielded significantly higher (P < 0.01) cleavage and blastocyst rates compared with frozen-thawed GV oocytes. It was found that 5% PVP had a beneficial effect compared with 10 or 20% concentrations for the development of blastocysts. Transfer of six blastocysts derived from frozen-thawed GV oocytes into three recipient heifers resulted in three pregnancies and the birth of one set of twins and one singleton calf.
本研究的目的是评估生发泡（GV）期冷冻解冻牛卵母细胞的体外受精和卵裂率。在小鼠卵母细胞中，GV期破裂后纺锤体微管重组对寒冷特别敏感，暴露于低温下很容易受损，这种损伤仅在第一次有丝分裂时才变得明显。研究了各种渗透性冷冻保护剂[1.8 M乙二醇（EG）、1.3 M乙二醇单甲醚（EME）和1.6 M 1,2 - 丙二醇（PROH）]以及不同浓度的海藻糖（T）和聚乙烯吡咯烷酮（PVP）对解冻后发育能力的影响。当牛GV期卵母细胞在1.8 M EG加5% PVP和0.05 M T的混合物中缓慢冷冻时，近80%发育至中期II；体外成熟后22.2%退化，冷冻保存的卵母细胞无一发生孤雌激活。在1.8 M EG加0.05 M T或0.1 M T的混合物中冷冻的卵母细胞总受精率高于在含或不含0.2 M T的1.8 M EG混合物中冷冻的卵母细胞（P < 0.05）；然而，在各种冷冻保护溶液中冷冻的正常受精或多精受精卵母细胞数量没有差异。对于GV期卵母细胞，使用EG、EME和PROH进行后续发育未观察到显著差异。在冷冻溶液中添加0.05或0.1 M海藻糖产生的卵裂率和囊胚率明显优于含0.2 M或不含海藻糖的溶液。对于未冷冻的对照，GV期卵母细胞产生的卵裂率和囊胚率显著高于冷冻解冻的GV期卵母细胞（P < 0.01）。发现5% PVP与10%或20%浓度相比对囊胚发育有有益作用。将6个源自冷冻解冻GV期卵母细胞的囊胚移植到3头受体母牛中，导致3次怀孕，产下1对双胞胎和1头单胎小牛。
