Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio)

The goal of this research was to examine the developmental effects on zebra fish embryos (Brachydanio rerio) when cryoprotectants were directly microin jected into the yolk. Our objectives were to: (i) determine the final conce ntration of propylene glycol (PG) and dimethyl sulfoxide (Me2SO) that the e mbryos could tolerate without causing teratogenic effects; (ii) determine i f the toxicity of Me2SO could be reduced by the simultaneous presence of va rious proportions of amides; and (iii) examine whether this intracellular c ryoprotectant incorporation could reduce the cryodamage to the yolk syncyti al layer (YSL) after vitrification trials. The rationale for conducting the se microinjection experiments was to overcome the permeability barrier of t he YSL. Intracellular PG produced better survival than Me2SO (P < 0.05). Em bryos tolerated both 10- and 30-nl microinjections of PC, yielding final co ncentrations of 2.3 and 5.0 M within the yolk, resulting in 70 +/- 3 and 35 +/- 4% survival at day 5, respectively. In similar experiments with Me2SO, survival was lower than PG at 60 +/- 4 and 14 +/- 4% at 2.4 and 52 M. Unli ke other cellular systems, the presence of amides, specifically acetamide o r formamide, did not reduce the toxicity of Me2SO in zebrafish embryos (P 5 0.05). During vitrification trials, we estimated a 25% dehydration of the yolk, yielding an effective PC concentration of 5.9 M. However, the incorpo ration of this vitrifiable concentration of PG was not sufficient to improv e the postthaw morphology of the YSL (P > 0.05). Clearly, other factors nee d to be examined in establishing a successful vitrification protocol for ze brafish embryos. (C) 2000 Academic Press.