Study On Vitrification Method And Freezing Injury Mechanism Of Olive Flounder (Paralichthys Olivaceus) Embryos

In order to optimize the vitrification method for cryopreservation of olive flounder embryos, several aspects were studied with different stages olive flounder embryos in this paper, including toxicity of cryoprotectants, selecting of vitrification solutions, physical characteristics of cryoprotectants, chilling sensitivity of olive flounder embryos, freezing injury mechanism, freezing and thawing method.In the toxicity experiments, seven permeating and five non-permeating cryprotectants were tested. Result shows that the toxicity of permeating cryprotectants on tail-bud stage embryos is as follows: EG > EtOH > Gly > DMF > DMSO > MeOH > PG. In five non-permeating cryprotectants, the toxicity of PVP is the highest. When the proportion of PM (PM:MeOH=3:2) and DMSO is 3:1, the toxicity of combined cryoprotectants is the lowest to tail-bud stage embryos. The permeability, freezing point and lowest vitrifying concentration of PG, MeOH, DMSO and PMD were also studied. Compared with other cryoprotectants in the same concentration, MeOH has the lowest freezing point and the highest permeability, and PG's lowest vitrifying concentration is lower than other cryoprotectants.The survival rate of the embryos frozen at -20℃declined as longer freezing time, and increased as development of the embryos. The hatching rate of the embryos frozen at -1℃has the same trend of change with above, but the abnormality rate of the embryos frozen at -1℃increased when frozen for longer time.The comparison between droplets method and straw method was carried out. And the result shows that freezing and thawing time of droplets were shorter than those of straw. The vitrification rate and the intact embryo rate in droplets method were higher. And chilling injury of embryos was less serious in droplets method than that in straw method.The morphology of the embryos was observed through microscope. The yolk sac was slightly damaged after equilibrated with five-step method, and the embryonic body and yolk sac were most likely damaged in cryopreservation.The thawing method of the cryopreserved straw was studied. The vitrification rate of the straw thawed in steam (1cm above 50℃water) was significantly higher than that in water bath (37℃).The olive flounder embryos from tail-bud stage to heart beating stage were crryopreserved using selected vitrification solution (35% PMD + 5% Sucrose) and vitrified droplets method, and 4 survival embryos were obtained from 173 embryos after thawed in three experiments. All of them hatched, and the survival time of the 4 larvae varied from 11 days to 15 days.