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

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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.