CHILL SENSITIVITY AND CRYOPROTECTANT PERMEABILITY OF DECHORIONATED ZEBRAFISH EMBRYOS, BRACHYDANIO-RERIO

The zebrafish (Brachydanio rerio) was used as a model for basic studie s of the chilling sensitivity, permeability and toxicity of cryoprotec tants. 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% epibo ly and three-somite stage). Moreover, enzymatic removal of the chorion did not alter (P > 0.05) this pattern of sensitivity to chilling. Eig ht-hour zebrafish embryos tolerated short-term exposures to temperatur es ranging from 4 to 23 degrees C for 3.5 h with no detrimental develo pmental effects. The permeability of dechorionated embryos to cryoprot ectants was examined by measuring the kinetics of volumetric change at various develop mental 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 larg e yolk and the developing blastoderm). From 40 to 100% epiboly, the vo lumes of the yolk and blastoderm remained constant, ca 82 and 18%, res pectively. However, these volumes changed rapidly after epiboly. For e xample, at the six-somite stage, the yolk composed 61% of the total vo lume, whereas the blastoderm composed 39%. When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxi de and propylene glycol), osmometric measurement of volume changes ind icated no permeation of the cryoprotectants. However, some permeation was observed for six-somite embryos immersed in a 2.0 M methanol solut ion, but not for 3-somite embryos. For up to 30 min at room temperatur e, these cryoprotectant solutions were not toxic to zebrafish embryos; however, 1.5 M glycerol and ethylene glycol solutions were. We conclu de that the complex nature of the zebrafish embryo reduces the effecti veness and predictive value of light microscopical measurements for cr yoprotectant permeability studies. (C) 1997 Academic Press.