| The application of cryopreservation technology to the preservation of fish embryos could play a major role in seed production, genetic management of broodstock, and conservation of aquatic resources. Although investigators have examined fish cryobiology for over 50 years, successful cryopreservation of fish embryos has not been achieved. Red sea bream (Pagrosomus major) has a high commercial value in Chinese aquaculture production. Some attempts at red sea bream's embryo cryopreservation have been carried out, but successful cryopreservation is still far from being achieved. In order to achive successful cryopreservation, several parameters ( toxicity of cryoprotectants; membrane permeability; intraembryonic ice formation and so on) should be taken into account in the design of a cryopreservation protocol. In this paper, the cryoprotectant and osmotic tolerance , intraembryonic ice formation of embryos in red sea bream (Pagrosomus major) were analyzed expeimentally. The effect of sperm cryopreservation on sperm DNA stability in red sea bream was also investigated.Heart-beating stage embryos were incubated in five permeable cryoprotectants -- dimethyl sulfoxide (DMSO), glycerol (GLY), methanol (MeOH), 1,2-propanediol (PG), ethylene glycol (EG), in concentrations ranging from 5% to 30% for 10min, 30min and 1h. After treatment, embryos were washed and incubated in seawater until hatched. The toxicity of permeable cryoprotectants increased with concentration and exposure time. PG was best tolerated by the embryos. In group of non-permeable cryoprotectant--5%, 10% and 15% polyvinyl pyrrolidone (PVP) and PEG-8000, 5%, 10%, 15%, 20% sucrose for 10min and 30min, exposure to sucrose did not affect the hatching rate. The toxicity of PEG-8000 increased with concentration but did not have distinct difference between two exposure times. The toxicity of PVP increased with concentration and exposure time. The osmotic values of all solutions used in this study were measured. And the correlation between solution osmotic values and corresponding hatching rate of embryos were analyzed in each time group. The correlation between osmotic values of permeable cryoprotectants and corresponding hatching rate of embryos was significant (P<0.05) and the values of R2 of 10min, 30min and 1h group were 0.713, 0.685 and 0.725 respectively. The correlation between osmotic values of non-permeable cryoprotectants and corresponding hatching rate of embryos was not significant.Ice crystal damage was investigated by response of red sea bream embryos to rapid and slow cooling rates. The changes of heart-beating stage embryos were observed by a cryomicroscopy after immersing in five diffirent cryoprotectants (10%DMSO; 5%glycerol; 10%methanol; 20% 1, 2-propanediol; 10% ethylene glycol) for 30min. Three temperatures, oil globule turn to white, embryo flash, embryo turn to black, which represented oil globule ice formation (Toif); extra-embryo ice formation (Teif) and intraembryonic ice formation (Tiif), respectively were recorded. Cryomicroscopic observation revealed that Teif appeared after Tiif almost synchronously. Three values were reducing as the cooling rate increased. Different cryoprotectants show different effects on three temperatures. More significant decreasing of Tiif was shown by 20%1,2-propanediol. Two programmable freezing with slow rate and two vitrification protocols which used in the cryopreservation of embryos were simulated. Significant difference of the changing trend of temperatures (Toif, Teif, Tiif ) was not investigated between the groups of cooling rate with 3℃/min and the other two slow programmable cooling rate groups. Teif appeared after Tiif and ice formatted gradually in two simulated vitrification methods. Tiif were significantly decreased in two vitrification protocols with max cooling rate (-130℃/min). The morphologic change of post-thawed embryos displayed a relationship with the Tiif decreasing. A smaller decreasing contributed a good morphological of embryos. The use of methanol, 1,2-propanediol and vitrification in the cryopreservation of the red sea bream's embryos was advised.The DNA damage was investigated in cryopreserved sperm of red sea bream. We used SCGE to study DNA stability of red sea bream spermatozoa by measuring alkali-induced DNA strand breaking, to find out under cryopreservation whether any hereditable materials were damaged or not. The SCGE examines outside harm to cell nuclear DNA, and widely used to various cell nucleus DNA,including mammalian sperms, now fish. Pretreat the sperm (collection, cleaning, freezing, and thaw). Electrophorese and unwind it in the alkaline electrophoresis solution, dye it with EB for 10min, observe it under fluorescence microscope, count the proportion of nuclei with tails in about 50 nuclei at random, then calculate the comet rate. The comet rate is defined as the percentage of the number of nuclei with DNA damaged that would appear in the comet tail, to the total nuclei numbers. Each sample is measured 2-4 times repeatedly. We evaluate the damage and divide them into 5 grades: no damage (G0), slightly damaged (G1), mildly damaged (G2), heavily damaged (G3), and totally damaged (G4). We found that most sperm with DNA damage are in the grades G1 and G2, and few were totally damaged nuclei. The measured comet rates in different cryoprotectant (DMSO) concentrations of 5%; 10%; 18%; 20%; 25% and 30% were: 33.47%±8.95%; 35.91%±19.44%; 48.95%±8.90%; 43.33%±11.19%; 55.80%±38.94%; and 81.99%±19.05% respectively. The comet rate of the fresh sperm was 31.43 %±2.68%. Compared with the DNA dam age states of red sea bream frozen sperm and the fresh sperm, the result indicates that the frozen sperm DNA in 30% of DMSO had remarkable diference with the fresh sperm (P<0.05).
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