A preliminary study of osmotic dehydration in zebrafish embryos: Implications for vitrification and ultra-fast laser warming

To date, traditional cryopreservation techniques have not been amenable to zebrafish (Danio rerio) embryos, due in part to their large yolky eggs, which have a low surface to volume ratio and limited permeability to water and cryoprotectants. However, recent vitrification and ultra-rapid warming studies in mice have demonstrated successful preservation by dehydrating 85% or more of their total water content. We hypothesized that this approach may help overcome the barriers to embryo cryopreservation among D. rerio. The purpose of this study was to determine the osmotic tolerance limit of D. rerio embryos under conditions relevant to cryopreservation. We found that embryos undergoing gastrulation (30%-70% epiboly) were particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20-22 somite, prim 5) survived 3 h in a 2 M sucrose solution but exhibit developmental delay, edema and trunk necrosis 2-4 days post-treatment.