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

The toxicity of the cryoprotectant dimethyl sulfoxide (Me2SO) to isolated b lastomeres was examined in three fish species representative of distinct en vironments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odonte sthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects o f embryonic stage, Me2SO concentration, and cooling rate on the cryopreserv ation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomer es from the three species tolerated Me2SO concentrations up to 9% relativel y well for over 5 h but lost viability rapidly at 18%. Cells from later emb ryonic stages (512 or 1024 cells) were more tolerant of Me2SO than those fr om earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres aft er freezing and thawing, but the extent of the effect and the optimum condi tions 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% Me2SO. Sur vival rates for blastomeres cryopreserved under the most effective combinat ion 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. (C) 199 9 Academic Press.