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

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 early 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 degrees C. Methanol was more effective than either DMSO or ethanediol for zebrafish embryo cryopreservation and 0.3 degrees C/min was found to be the optimum cooling rate. Two-step addition of cryoprotectants, with the higher concentration of cryoprotectants added at WC, improved the results. The best embryo survivals obtained after cooling were 94% at - 10 degrees C, 72% at - 15 degrees C, 43% at - 20 degrees C, and 8% at -25 degrees C, no embryos hatched following cooling to - 30 degrees C. Ice formation within the egg was found to be the main factor affecting survival of the embryos.