Title: 1 SHORT TERM CHILLED STORAGE OF ZEBRAFISH (Danio rerio) EMBRYOS IN 2 CRYOPROTECTANT AS AN ALTERNATIVE TO CRYOPRESERVATION

23 As zebrafish embryos never been cryopreserved, we developed a protocol to store zebrafish embryos 24 (50% epiboly5.3 hpf) for up to 18 h at 0° C. Initial experiments to optimise cryoprotectant solution 25 demonstrated improved embryo hatching rate following chilling at 0°C for 18 h with 1 M MeOH + 26 0.1 M sucrose (56 ± 5%) compared to other combination of methanol (0.2-, 0.5 M) and sucrose (0.05-, 27 0.1 M). This combination of cryoprotectants that protects against chilling injury was further tested to 28 assess its impact on sox gene and protein expression. Significant decreases in sox3 gene expression 29 were observed in hatched embryos that had been chilled for 18 h in 1 M MeOH + 0.1 sucrose 30 compared to non-chilled controls, however expression of both sox2 and sox3 proteins was unaffected. 31 Significant decreases in sox2 protein expression were however observed in embryos that had been 32 chilled without cryoprotectants and these embryos also had lower hatching rates than those chilled 33 with the optimal cryoprotectant solution. We therefore conclude that the cryoprotectant combination 34 of 1 M MeOH + 0.1 M sucrose facilitates chilled storage of early stage (50% epiboly) zebrafish 35 embryos for up to 18 h without compromising transcriptional response. 36