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

本研究的目的是建立兔原核期受精卵的冷冻保存方法。在1.5M乙二醇+0.1M蔗糖的存在下，常规的两步冷冻受精卵的形态存活率和囊胚发育率分别为74%和52%。只有4%（18/414）的冻融受精卵随后进行DNA显微注射，发育成正常后代，但据我们所知，它们是第一批来自冻存原核受精卵的兔后代（Hochi et al.，摩尔Reprod.偏差，2001年）。接下来，一些超快速冷却程序，已被报道是有效的牛卵母细胞冷冻保存，适用于原核阶段的兔受精卵。当以Cryotop为冷冻装置，以2.7M乙二醇+2.3M DMSO +0.5M蔗糖为冷冻保护剂时，受精卵的形态存活率最高，为100%，囊胚发育率最高，为51%。玻璃化加温的受精卵导致每次转移产生36%的后代，而新鲜对照受精卵以53%的比率发育成后代（Hochi等人，Theriogenology，2003）。本文建立的原核期兔受精卵的Cryotop玻璃化冷冻方法（体内存活率为36%），对原核显微注射外源DNA系统地生产转基因兔具有重要的实用价值。

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.