Study On Programmed Cryopreservation Technology Of Three Important Marine Fish Embryos

Cryopreservation of fish gametes and embryos plays an important role in the preservation of seed resource, cryobiology, genetics breeding and aquaculture. This paper dealt with the factors affecting cryopreservation of marine fish embryos and programmed cryopreservation of three important marine fish embryos.Cryoprotectants which are low toxicity and high efficiency are foundation of successful cryopreservation of embryo. Survival rate of embryo in DS1 was the highest when embryos were exposed to different extenders for 18h. Toxicities of methanol, propylene glycol and n, n-dimethylformamide were lower than the others when sea perch and Japanese flounder embryos were exposed to 6 cryoprotectents, respectively. On the basis of this, toxicity experiment of the mixed cryoprotectant composed of methanol and propylene glycol were coinducted and the results indicated that mixed Cryoprotectants had toxicity neutralization effect and survival rate of embryo equilibrated in mixed cryoprotectant PM32 was the highest.The developmental stages of embryo and its quality were .also important factors affecting cryopreservation of embryos. The results indicated that heartbeat stage of sea perch embryo, tail bud stage of Japanese flounder and turbot embryo were suitable developmental stages for cryopreservation. Removal of membrane was one of efficient methods of improving penetrability of Cryoprotectants. When eggs and embryos of turbot were exposed to pronase E +EDTA, respectively, membrane of eggs could be removed, but membrane of embryos exposed to seawater could not be removed.Experiments on addition and dilution of cryoprotectant were carried out using four methods, respectively and the results indicated that two-step method, three-step method and four-step method had no significant differences(P > 0.05), thus, simpletwo-step method was selected for cryopreservation. Freezing points of cryoprotectants were determined using microcomputer freezer. During cooling, seeding at feasible temperature could certainly reduce supercooling degree and cryodamage of embryo.Optimal cooling rate of programmed cryopreservation of embryos was selected using microcomputer freezer and the results indicated that 2-3.5/min from room temperature to freezing point of cryoprotectant and 5-10/min from freezing point of cryoprotectant to the temperature quenching in liquid nitrogen (DSb) were suitable. In addition, relation between thawing temperature and thawing time was investigated in this paper and two-step thawing method was brought forward.Low temperature preservation and cryopreservation of tree marine fish embryos were examined using rapid cooling rate and slow cooling rate, respectively. Survival rate of 4.72% was obtained when turbot embryos were cryopreservated at -30 癈 for 30 min using cooling rate of 2 癈/min. When Japanese flounder embryos were cryopreservated in liquid nitrogen for 0.5~28h, 5 survival embryos were obtained and 4 of them hatched, one Japanese flounder fry without feeding survived for 6 days. Sea perch embryos were cryopreservated in liquid nitrogen for 0.5~24h, 4 survival embryos were also obtained and one of them hatched, furthermore the fry without feeding survived for 3 days. In summary, great progress was made in cryopreservation of marine fish embryos.