Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival.

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.