Birth of offspring after transfer of Mongolian gerbil (Meriones unguiculatus) embryos cryopreserved by vitrification

Abstract

The Mongolian gerbil (Meriones unguiculatus) has been used as a laboratory species in many fields of research, including neurology, oncology, and parasitology. Although the cryopreservation of embryos has become a useful means to protect valuable genetic resources, its application to the Mongolian gerbil has not yet been reported. In this study, we investigated the in vitro and in vivo developmental competence of Mongolian gerbil embryos cryopreserved by vitrification. In vivo-fertilized embryos were vitrified on the day of collection using the ethylene glycol (EG)-based solutions EFS20 and EFS40, which contained 20% and 40% EG, respectively, in PB1 containing 30% (w/v) Ficoll 70 and 0.5 M sucrose. First, we compared one-step and two-step vitrification protocols. In the one-step method, the embryos were directly transferred into the vitrification solution (EFS40), whereas in the two-step method, the embryos were exposed serially to EFS20 and EFS40 and then vitrified. After liquefying (thawing), late two-cell embryos (collected on day 3) vitrified by the two-step method showed significantly better rates of in vitro development to the morula stage compared to those vitrified by the one-step method (65% vs. 5%, P < 0.0001). We then examined whether the same two-step method could be applied to early two-cell embryos (collected on day 2), four-cell embryos (day 4), morulae (day 5), and blastocysts (day 6). After liquefying, 87%-100% of the embryos were morphologically normal in all groups, and 23% and 96% developed to the compacted morula stage from early two- and four-cell embryos, respectively. After transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) developed to full-term offspring from vitrified and liquefied early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. This demonstrates that Mongolian gerbil embryos can be safely cryopreserved using EG-based vitrification solutions.