Hagedorn, M., Kleinhans, F.W., Wildt, D.E., and Rall, W.F. (1997) Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, Brachydanio rerio. Cryobiology. 34(3):251-263.
The zebrafish (Brachydanio rerio) was used as a model for basic studies of the chilling sensitivity, permeability and toxicity of cryoprotectants. In both intact and dechorionated embryos, early-stage embryos (1.25, 1.5, 1.75, and 2 h) were more susceptible (P < 0.05) to chilling injury at 0 degrees C than late-stage embryos (50, 75, and 100% epiboly and three- somite stage). Moreover, enzymatic removal of the chorion did not alter (P > 0.05) this pattern of sensitivity to chilling. Eight-hour zebrafish embryos tolerated short-term exposures to temperatures ranging from 4 to 23 degrees C for 3.5 h with no detrimental developmental effects. The permeability of dechorionated embryos to cryoprotectants was examined by measuring the kinetics of volumetric change at various developmental stages (16 cells to six somites or ca. 1.25 to 14 h postfertilization) at 28.5 degrees C. The dechorionated zebrafish embryo is composed of two complex cellular compartments (i.e., a large yolk and the developing blastoderm). From 40 to 100% epiboly, the volumes of yolk and blastoderm remained constant, ca. 82 and 18%, respectively. However, these volumes changed rapidly after epiboly. For example, at the six-somite stage, the yolk composed 61% of the total volume, whereas the blastoderm composed 39%. When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurement of volume changes indicated no permeation of the cryoprotectants. However, some permeation was observed for six-somite embryos immersed in a 2.0 M methanol solution, but not for 3-somite embryos. For up to 30 min at room temperature, these cryoprotectant solutions were toxic to zebrafish embryos; however, 1.5 M glycerol and ethylene glycol solutions were. We conclude that the complex nature of the zebrafish embryo reduces the effectiveness and predictive value of light microscopical measurements for cryoprotectant permeability studies.