Cryopreservation of pre-hatch embryos of zebrafish (Brachydanio rerio)

Abstract

The toxicity of five cryoprotectants – methanol, DMSO, glycerol, ethanediol and sucrose – on different development stages of zebrafish embryos was investigated. Embryos were exposed to a range of cryoprotectant concentrations for 30 min at room temperature. Post-heart beat stage embryos were more resistant to cryoprotectants than carly embryonic stages. The maximum non-toxic concentrations of cryoprotectants on heart beat stage embryos were 2 M methanol, 2 M DMSO, 1 M glycerol, 2 M ethanediol and 0.5 M sucrose. Gradual stepwise addition did not reduce toxicity. Heart beat stage embryos survived 2 M methanol at room temperature for up to 5 hours, whilst DMSO and ethanediol were toxic after 3 and 1 hour exposure respectively. The effect of the nature and concentration of cryoprotectant, equilibrium time, and cooling rate were investigated during cooling to –30 °C. Methanol was more effective than either DMSO or ethanediol for zebrafish embryo cryopreservation and 0.3 °C/min was found to be the optimum cooling rate. Two-step addition of cryoprotectants, with the higher concentration of cryoprotectants added at 0 °C, improved the results. The best embryo survivals obtained after cooling were 94% at – 10 °C, 72% at – 15 °C, 43% at –20 °C, and 8% at –25 °C, no embryos hatched following cooling to –30 °C. Ice formation within the egg was found to be the main factor affecting survival of the embryos.