Cryopreservation of pronuclear-stage rabbit zygotes, applicable for production of transgenic rabbits

本研究旨在建立兔原核期受精卵的冷冻保存方法。用1.5M乙二醇+0.1M蔗糖对受精卵进行常规二步冷冻，形态成活率和囊胚发育率分别为74%和52%。只有4%(18/414)的冷冻解冻受精卵发育成正常后代，但据我们所知，他们是第一批来自冷冻保存的原核受精卵的兔子后代(Hochi等，Mol)。是Reprod。Dev.，2001)。其次，对牛卵母细胞进行了超快速冷冻，并对其进行了冷冻处理。以2.7M乙二醇+2.3M二甲基亚砜+0.5M蔗糖为CPA进行冷冻保存，获得了100%的形态存活率和51%的囊胚发育率。玻璃化冷冻的受精卵每次移植产生36%的后代，而新鲜对照受精卵发育成后代的比率为53%(Hochi等人，Therigenology，2003)。本文建立的兔原核期受精卵(活体存活率为36%)的冷冻玻璃化冷冻方法，对转基因兔的系统生产具有重要的实际意义。

The objective of this study was to develop cryopreservation method of pronuclear-stage rabbit zygotes. Conventional 2-step freezing of zygotes in the presence of 1.5M ethylene glycol + 0.1M sucrose resulted in morphological survival and blastocyst developmental rates of 74 and 52%, respectively. Only 4% (18/414) of the frozen-thawed zygotes followed by DNA microinjection developed into normal offspring, but to our best knowledge they were the first rabbit offspring derived from cryopreserved pronuclear zygotes (Hochi et al., Mol. Reprod. Dev., 2001). Next, some ultra-rapid cooling procedures, that have been reported to be effective in cryopreservation of bovine oocytes, were applied for the pro nuclear-stage rabbit zygotes. When the zygotes were cryopreserved using both Cryotop as a device and 2.7M ethylene glycol + 2.3M DMSO + 0.5M sucrose as CPAs, the maximum morphological survival rate of 100% and blastocyst developmental rate of 51% were obtained. The vitrified-warmed zygotes resulted in production of offspring at 36% per transfer, while fresh control zygotes developed to offspring at the rate of 53% (Hochi et al., Theriogenology, 2003). The Cryotop vitrification method established here for pronuclear-stage rabbit zygotes (in vivo survival 36%) is obviously of practical importance in systematic production of transgenic rabbits by pronuclear microiniection of foreign DNA.