抄録
The first successful cryopreservation of teleost embryos was reported in the Japanese flounder (Chen and Tian, 2005). To develop optimum protocols for cryopreservation of flounder embryos, we examined the toxicity of cryoprotectants to embryos, their water-and cryoprotectant-permeability, and survival of cryopreserved embryos. To examine the toxicity of the cryoprotectants (DMSO, ethylene glycol (EG), methanol (MeOH) and propylene glycol (PG)), embryos at the blastula, gastrula, tail bud and pre-hatch stages were exposed to cryoprotectant solutions at 15°C and their survival was assessed by their ability to hatch. To determine the permeability, embryos were exposed to solutions with a cryoprotectant or sucrose at 25°C, and relative volume changes were examined. MeOH and PG were less toxic than DMSO and EG to embryos at all the stages. Of the four stages, embryos at the tail bud stage showed the highest hatching rate after exposure to MeOH and PG. When embryos were suspended in cryoprotectant or sucrose solutions, they showed minimal volume change, suggesting low water- and cryoprotectant-permeability. When embryos were cryopreserved with vitrification solutions (FVS1 and FVS3) containing MeOH and PG (13% + 20% and 18% + 27%, respectively), embryos at all stages examined became opaque during cooling, indicating intracellular ice formation during cooling. The low-membrane permeability of Japanese flounder embryos might make them difficult to be cryopreserved. Although Chen and Tian reported successful cryopreservation of flounder embryos using FVS1 and FVS3, our study failed to repeat their results.
