Cryoprotectants Protect Medaka (Oryzias Latipes) Embryos from Chilling Injury

This study was conducted to investigate the effect of six cryoprotectants (dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), ethylene glycol (EG), 1,2propylene glycol (PG) and N, N-dimethylformamide (DMF) on the survival of medaka (Oryzias lapites) embryos at low temperatures (0 and -5°C). Firstly, the embryos at 8 to 16-cell stages were exposed to different concentrations (1 to 4 mol/L) of DMSO, Gly, MeOH, EG, PG and DMF for 40min at 26°C. After removal of the cryoprotectants (CPAs), the embryo survivals were assessed by their development into live fries following 9 day of culture. The results showed that the higher concentration of the CPA, the lower survival of the embryos; and that the toxicity of the six CPAs to medaka embryos is in the order of PG < MeOH = DMSO < Gly < EG < DMF (P<0.05). Secondly, based on the results obtained above, embryos at 8 to 16-cell stages or other stages were exposed to 2 mol/L of PG, MeOH or DMSO for up to 180 min at 0°C and up to 80 min at -5°C respectively. The 8 to 16-cell embryos treated with MeOH at low temperatures showed highest survival. Thirdly, when embryos at different stages were treated with 2 mol/L of MeOH at -5°C for 60 min, 16-somite stage embryos showed highest survival, followed by 4-somite, neurula, 50% epiboly, blastula, 32-cell and 8 to 16-cell embryos. These results demonstrated that PG had the lowest toxicity to medaka embryos among the six permeable CPAs at 26°C, whereas MeOH showed highest cryoprotective efficiency under chilling conditions and chilling injury decreased gradually with the development of medaka embryos.