Electroporation enhances permeation of cryoprotectant (dimethyl sulfoxide) into Japanese whiting (Sillago japonica) embryos

Survival after cryopreservation has never been achieved with fish embryos, presumably because of insufficient cryoprotectant permeation before cooling. The objective was to determine the relative efficiency of electroporation for incorporation of cryoprotectant into Japanese whiting embryos and survival of electroporated embryos in DMSO after freeze-thawing. Embryos (somites stage) subjected to electroporation at 100, 200, or 300 V in artificial sea water had similar hatching rates (94%-96%) as untreated control embryos (0 V; 97%) and those treated with voltages between 400 and 900 had survival rates of 88% to 0%. Embryos (somites stage) electroporated at 300 V in 10%, 20%, or 30% DMSO/artificial sea water solutions had hatching rates of 94%, 88%, and 85%, respectively, and DMSO contents of 10, 30, and 78 mM. Embryos treated with higher voltages had higher DMSO uptake (up to 84 mM), but reduced survival (62%-6%). Pre-exposure of embryos to 10% DMSO for 20 minutes before electroporation improved DMSO uptake (116 mM). Embryos treated with DMSO and electroporated under the best conditions determined in this study did not resume development after attempted vitrification. We concluded that electroporation enhanced DMSO uptake by fish embryos, but concentrations obtained with this procedure alone were apparently insufficient to prevent internal ice formation during cooling and thawing.