Differential scanning calorimetry studies of intraembryonic freezing and cryoprotectant penetration in zebrafish (Danio rerio) embryos

Nucleation temperatures of intraembryonic water and cryoprotectant penetration in zebrafish embryos were studied using differential scanning calorimetry. The effects of embryo developmental stage, dechorionation, partial removal of yolk, cooling rate, and cryoprotectant treatment on the temperatures of intraembryonic freezing were investigated. Embryo stages were found to have a significant effect on the nucleation temperatures of intact embryos. Freeze onset temperatures of -11.9 +/- 1.5, -15.6 +/- 0.3, and -20.5 +/- 0.1 degrees C were obtained for intact embryos at 6-somite, prim-6, and high-pec stages, respectively. After dechorionation, the freeze onset temperatures of intraembryonic water shifted to significantly lower temperatures, being -23.5 +/- 0.8, -18.7 +/- 0.7, -24.9 +/- 0.8 degrees C for 6-somite, prim-6, and high-pec stages, respectively. Yolk-reduced high-pec stage embryos showed significantly lower nucleation temperatures with an average onset at -27.9 +/- 0.4 degrees C. The effect of cryoprotectant treatment on the nucleation temperatures of intraembryonic water varies among different embryo stages and different cryoprotectants. Thirty-minute treatment with 2 M methanol significantly decreased the nucleation temperatures of dechorionated 6-somite embryos whilst no temperature decrease was observed for prim-6 or yolk-reduced high-pec embryos. Thirty-minute exposure to 1 M propylene glycol did not significantly affect the nucleation temperatures of dechorionated 6-somite, prim-6, or yolk-reduced high-pec embryos. In order to increase the permeability of embryos to cryoprotectants, the yolk sacs of dechorionated embryos at 6-somite or prim-6 embryos were punctured with a sharp micro-needle before exposure to cryoprotectants. The punctured prim-6 embryos showed significantly lower temperatures of intraembryonic freezing after 30 min of exposure to 2 M methanol following the multi-punctures. The nucleation temperatures of punctured 6-somite or prim-6 embryos were also decreased significantly after exposure to 1 M propylene glycol for 30 min. These results suggested that in intact embryos, intraembryonic freezing appeared to be seeded by the external ice in the perivitelline fluid and that in dechorionated embryos (in the absence of external water) intraembryonic freezing was more likely a consequence of heterogeneous nucleation. Methanol was demonstrated to show a limited degree of penetration into prim-6 stage embryos, but it did not penetrate later-stage embryos such as prim-6 and yolk-reduced high-pec. No propylene glycol permeation was observed for embryos at all stages. However, multi-punctures of yolk resulted in the permeation of both cryoprotectants into prim-6 embryos and propylene glycol permeation into 6-somite embryos. These findings may have important implications in overcoming the problem associated with the low membrane permeability of zebrafish embryos to cryoprotectants.