| Cryopreservation of fish embryos plays a significant role in the preservation of seed resources, cryobiology, genetics breeding and aquaculture.Flounder (paralichthys olivaceus) is an important economic fish.In this article , taking flounder embryos as material,the key factors of vitrification and programmed freezing methods were studied on,including the appropriate embryo stage,cryoprotectants,length of straw,cryoprotectant volume in straw, and so on.Otherwise, the factors of microinjection method were studied on ,including cryoprotectants,toxicity of cryoprotectant microinjected, microinjection dosage of cryoprotectant,and so forth.Results showed that,among blastula stage,middle gastrula stage,closure stage of blastopore 15-20 somites stage, tail-bud stage , heart-beating stage ,pre-hatching stage ,embryo at tail-bud stage had the greatest resistance to cryoprotectant and the tail-bud stage was the appropriate stage for cryopreservation .At room temperature, the toxicity of the six kinds of cryoprotectants ,PG,MeOH,DMSO,EG,DMF,GLY , ranked that PG﹤MeOH﹤DMSO﹤DMF﹤EG﹤GLY. However ,when the temperature was 0℃,the toxicity of these cryoprotectants reduced and the rangement of toxicity was unchanged.Mixting PG and MeOH or PG and DMSO in a certain proportion, the toxicity of mixture was lower than the single one.And then , Mixting PM whose concentration was 4M with PVP,Dextran,Ficoll ,respectively, survival rate mixed with Ficoll was the highest,and the Dextran was the lowest.the toxicity of three cryoprotectants ranked that Ficoll﹤PVP﹤Dextran.But when PM was mixed with DMSO or DMF , survival rate with DMSO was higher ,and the toxicty was lower. Among three kinds of straws(length of 5cm,7.5cm,10cm), vitrificational effect of straw with 5 cm length was the best ,and the most appropriate loading cryoprotectant volume of staw was 50ul.The cryopreservation of fish embryos has not reached an applicational level.The main reason is the characteristics of fish embyros, such as big physical volume, the bad permeability of yolk syncytial layer, etc, which casued that traditional techniques for the incorporation of cryoprotectants could not make the cyroprotectants come into the inner part of embryos sufficiently and cryoprotectants could not protect embryos effectively. But the method microinjection used to incorporate cryoprotectants into the yolk sac of fishes embryos, could offset the shortage of traditional methods a certain extent and raise the protection degree of cryoprotectants to embryos. Several factors relating to cryopreservation of flounder Paralichthys olivaceus embryos by vitrification were studied: cryoprotectants, microinjection volumes of cryoprotectants, microinjection concentration of cryoprotectants,and the cooling sensitivity of embryos after being microinjected was analyzed. From the results, it indicated that, 1, the becoming microinjection volume was 750pl. the toxicity of five single-agent cryoprotectants sequenced as follows PVP>Sucrose,DMSO>MeOH>PG, among these cryoprotectants, the embyros injected with PG got the highest survival rate and hatching rate, with PVP got the lowest, however, there was a cryoprotectant mixture PM, its toxicity was much lower than PG and the embyros injected with it got higher survival rate and hatching rate than with PG. The toxicity of cryoprotectans increased as the concentration of its increased..The survival rate of embryos microinjected with 6M PM was 25.07±1.57% after being coolled in -20℃for 10 minutes by programmed cooling method, whenas ,the survival rate of controls dealt with five steps balance method was 20.88±2.84%, which indicated that the chilling sensitivity of embryos with microinjection decreased.Based on the above optimizational conditions,flounder embryos were cryopreserved in LN2 by vitrification and microinjection-vitrification.By vtrification ,seven times of resurgent embryos at tail-bud stage were obtained and one time at heart-beating stage.The number of all the resurgent embryos was 23,the average survival rate was 8.91%. Thereinto,14 survival embryos hatched ,the average hatching rate was 61.25%.Seven fries were normal and the longest survival time was 84h. In addition, the turbot embryos at heart beating stage were attempted being cryopreserved in LN2 by vitrificaiton with the optimum conditions above ,with the cryoprotectants of 35%PM and 0.8%的PVP in different proportion.One time of resurgent embryos was obtained ,and the number of survival embryos was 8 with the average survival rate of 21.05%. One of eight survival embryos hatched. Otherwise,microinjection was used to dealt with embryos for preservation in low temperature and cryopreservation in LN2.Though there was no survival embryo, the most embryos were transparent ,intact. With the dissection microscope ,it was found that the yelk shrinked and draped,the membrane of yelk was well basically ,no yelk flowed out ,no embryo body was injured. |
