• Investigations into the cryopreservation of zebrafish (Brachydanio Rerio) embryos

      Zhang, Tiantian (University of BedfordshireUniversity of Bedfordshire, 1994-06)
      Cryopreservation of fish embryos was studied using zebrafish embryo as a model system. Cryoprotectant toxicity, chilling sensitivity and embryo membrane permeability were investigated with a view to developing optimum protocols for cryopreservation of the embryos using controlled slow cooling, non-freezing storage and vitrification. Methanol was found to be the most effective cryoprotectant for controlled slow cooling and non-freezing storage of zebrafish embryos with 11 %heart beat stage embryos surviving after controlled slow cooling to -25°C. Zebrafish embryos were found to be very chilling sensitive with early developmental stages being the most sensitive to chilling injury. Embryo developmental stages after closure of the blastopore (>12-h), especially post heart beat stages were much more resistant to cryoprotectant toxicity and chilling injury. Heart beat stage (27-h) embryos proved to be the best embryo developmental stage for controlled slow cooling and non-freezing storage. Dechorionated embryos are more sensitive to cryoprotectant toxicity and chilling injury. The sensitivity to chilling injury of zebrafish embryos limited the use of controlled slow cooling and non-freezing storage for long term cryopreservation. The attempts at cryopreservation of zebrafish embryos using vitrification produced no embryo survival, although up to 32 % embryos remained morphologically intact immediately after vitrification. Poor cryoprotectant permeation, dehydration and consequently ice formation within the egg are probably the main factors on effecting embryo survival. The results of zebrafish embryo permeability studies demonstrated that the chorion of the embryos was permeable to water and cryoprotectants, whilst the vitelline (plasma) membrane was an effective permeability barrier. The inability to achieve sufficient penetration of the vitelline membrane by cryoprotectants poses severe problems for long term cryopreservation, which need to be overcome, possibly by permeabilisation of the vitelline membrane, before successful cryopreservation can be achieve.