402. Cryopreservation of oocytes and follicular cells of the cane toad Bufo Marinus

Abstract

Amphibians are currently the most threatened of all vertebrate groups with more than 30% of all known species in decline, facing extinction or recently extinct. Cryobanking of amphibian germ cells and reproductive tissues could be used to manage threatened species and provide insurance against extinction. However, cryopreservation of fully developed amphibian oocytes and whole embryos has not been achieved due to technical problems freezing such large cellular structures. As an alternative approach, we investigated the feasibility of developing protocols for the slow-cool freezing, storage and retrieval of developmentally competent amphibian ovarian follicles containing Stage I and II oocytes which are much smaller in size than later developmental stages. Ovarian follicles from euthanased Cane Toads were incubated in cryodiluents containing either glycerol or DMSO to assess cryoprotectant toxicity and response to slow cooling freezing protocols. The fluorescent live cell stain SYBR 14 and its counter stain propidium iodide was used to score the proportion of viable follicle cells before and after cryopreservation. Cryoprotectant type, concentration and exposure time all had significant effects (P < 0.05) on the viability of follicle cells, with significant interactions between these variables. Overall, glycerol was less toxic to follicle cells than DMSO. At higher concentrations, glycerol exerted high osmotic stress on oocytes, and there was evidence that DMSO triggered apoptosis in oocytes. The most effective cryopreservation protocol for stage I and II oocyte follicles resulted in a post-thaw recovery of a mean 70% of viable follicular cells. This protocol involved cryopreservation in 15% v/v glycerol, inclusion of seeding and temperature holding periods during cryopreservation, coupled with rapid thawing in a 30°C water bath. The successful cryopreservation of intact follicles in this study indicates the potential to recover functional ovarian tissues post cryopreservation for continuation of amphibian oogenesis in vitro or in vivo.