The Cryopreservation Research Of Several Fish Germplasm Resources

Since the1950s, Blaxter firstly succeeded in the cryopreservation of the Pacificherring (Clupea harengus). So far, the cryopreservation technology of germplasmresources has achieved relative excellent accomplishment and is increasingly matures.To date, sperm from200freshwater and more than40marine species has beensucceeded in the cryopreservation, and established cryobank. But a unified methodapplicable to all fish species has not yet been acquired, therefore, fish spermcryopreservation technology remain to be developed. In the same period, researchcryopreservation of fish embryo has been begun. With the increasing maturity andimprovement of biotechnology and new techniques, the development of fish embryocryopreservation research also had rapidly promoted.Starry flounder (Platichthys stellatus, Pallas1788) belong to the subfamilyPlatichthys of the family Pleuronectidae in the order Pleuronectiformes, is distributedin Bering Strait, Chukchi Sea, Alaska, Canada’s northern coast, and North Americaand southern California. Meanwhile, there is distribution in Canada, the United States,Russia, China, North Korea, South Korea, and Japan. But, in recent years, it was lessreport that natural population of starry flounder is found in China, therefore, theindividual is small and the produce of sperm is relative less, which limits to expandproduction in the artificial breeding and crossbreeding.Groupers are a protogynous hermaphrodite, warm water, and sea water species.There are about100populations in the world mainly distributed in the tropical andsubtropical zone waters. In China, it is distributed in the East China Sea and SouthChina Sea. Giant grouper (Epinephelus lanceolatus) and seven-band grouper(Epinephelus septemfasciatus) belong to the subfamily Epinephelinae of the familySerranidea in the order Perciformes. Giant grouper large size, rapid growth, is a new species of aquaculture industry. But, the number of male fish is few, and theproduction of sperm is limited, which lead reality cannot meet the practical needs.Moreover, reproductive isolation also obstructs the development of grouperinterspecific hybridization.Seven-band grouper is an important captured and cultured fish that the onlydistributed in the yellow sea in China. In2013, our lab has successful incryopreservation seven-band grouper sperm. To date, due to palindromia and spreadnervous necrosis disease recurrence of seven-band grouper, the production is sufferedaffected seriously. Therefore, in view of above the problems, study of fish germplasmpreservation is feasible methods that solves and keep sustainable development of theaquaculture and the protection of fish genetic diversity.In present study, the sperm from starry flounder and giant grouper wascryopreserved. In the study of starry flounder sperm cryopreservation, we made selectfor sperm diluents (species, composition, and osmotic pressure), suitable temperatureof seawater, and dilution degree of frozen sperm. It indicated: Ts-2and SFs-4as adiluent,10%DMSO as cryoprotectant, post-thaw sperm motility was the highest with12℃seawater, and that the rate of fertilization of frozen sperm was the highest in the20times diluted. Moreover, using computer aided sperm analysis (CASA) analyzeand test for motion parameters from frozen sperm in the Ts-2and SFs-4. And itindicated that the motion parameters of frozen sperm with SFs-4significantly higherthan the ones in Ts-2.In the study of giant grouper sperm cryopreservation, we selected for spermdiluents species, the suitable concentration of DMSO and FBS, which indicated thatELS3and ELRS3as diluents,15%DMSO and10%FBS as cryoprotectant, the spermcryopreservation effect was best, post-thaw sperm motility was the highest.Then, in the study, seven-band grouper embryos were cryopreserved. From thecryoprotectant species and different cryoprotectants combination using to embryotoxicity effect, selected the lowest toxicity optimum vitrification solution forcryopreservation. The results indicated that the embryos hatchability was the highestwith35%PMG3+5%Trehalose (PMG3T) before cryopreservation, was31.76%, then 6floating embryos in the78frozen embryos after cryopreservation, and one embryoproduced hatched larva fish.In addition, we measured6enzymes, superoxide dismutase (SOD), creatinekinase (CK), Na+/K+-ATPase, lactate dehydrogenase(LDH), malondialdehyde (MDA),glutathione peroxidase (GSH-Px) activity of embryos that were dealt with PM、PMG3、 PMG3T and cryopreserved, the results indicated that except for theconcentration of MDA enzymes activity increasing, other five enzymes activitydecreased, which indicated cryopreservation and vitrification solution have effects onembryonic cells antioxidant system, lead that the content of energy metabolismenzymes in the cell and some antioxidant enzymes decrease, peroxidation damage,consequently, which reduces the rate of embryo survival. Therefore, through thedetermination of related enzyme activity of embryos before and after cryopreservation,the research results provide a theoretical basis for cryopreservation of seven-bandgrouper embryos.In addition, in order to better explore the mechanism of marine fish embryocryopreservation, we made preliminary research experiments for seawater Oryziasmelastigma embryos, but has yet to make a systematic study, remains to be continuedin future studies.