| Owing to evolvement of natural environment and effect of human being production activities, the wildstock of some fishes is declining and some of them are on the edge of extinction. It has become an important task to protect environment and wild species. Cryopreservation of fish gametes and embryos is one important method of preserving germplasm, which has tremendous potential in application.Vitrification was firstly introduced into cryopreservation of marine fish embryos in the present study. We used the sea perch (Lateolabrax japonicus), flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximns) embryos as materials and investigated marine fish embryos vitrification technique systemically.In this paper, it was investigated that the toxicity of penetrative and nonpenetrative cryoprotectants, such as propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (DMSO), glycerol, N,N-Dimethyl formamide (DMF), ethylene glycol (EG), polyvinyl pyrrolidonum (PVP), ficoll, dextran and polyethylene glycol (PEG). This dissertation demonstrated that the propylene glycol and methanol had lower toxic effects on fish embryos than other cryoprotectants, and PVP was advantageous to the vitrification forming and could also prevent devitrification.The basic solution BS2 suitable for embryo vitrification was selected by choosing the salt component, determining the osmotic pressure and pH, and cultivating the embryos in the basic solution.On the basis of the toxicity research of cryoprotectants, 80 types of vitrification solutions were confected. VSD2 suitable for the cryopreservation of sea perch embryos and PM1, PM2, PM3 and PM4 suitable for flounder embryos and PMP1 suitable for turbot embryos were chosen by adjusting the ratio of cryoprotectants in BS2 and equilibrating embryos in vitrification solutions.The changes of freezing point and super-cooling point of a series of PM vitrificationsolutions was investigated in the process of slow freezing at a ratio of 5C/min. This experiment showed that the vitrification solution over 41% had not freezing point, while the vitrification solution under 40% had freezing point and could ice.The temperature changes of vitrification solutions during freezing and thawing were studied. It is found that freezing time was 15.46+1.38sec from 13C to -196.2 C, the freezing rate was 800'C/min, thawing time was 6.67+0.49sec-7.24+0.36sec at 35-45 C, and thawing rate was 1500-2000 C/min.The effects of sucrose, glucose, galactose, fucose on elution were compared. The elution time, concentration of elution solutions and elution method were also investigated. The results showed that one-step washing with 0.125mol/L sucrose obtained higher survival rate, and the elution time was 15min.The endurance abilities of the embryos of sea perch, flounder and turbot at different developing stages to vitrification solutions were studied. The result showed that sea perch at tail-bud stage, heart-beating stage and pre-hatching stage had more endurance abilities to VSD2, flounder at 4-5 somites, 16 - 20 somites and tail-bud stage had more endurance abilities to PM3, and turbot at 4-5 somites, 16-20 somites and tail-bud stage had more endurance abilities to PMP1. The feasible equilibration time of three marine fish embryos in vitrification solutions was determined, which guided the dealing process before freezing.Trough a series of researches, we developed a good technique for cryopreserving embryos of sea perch, flounder and turbot by vitrification, and obtained survival embryos and hatching larvae after thawing with the technique that we developed.In the course of vitrification of sea perch embryos at tail-bud, heart-beating and pre-hatching stage using VSD2, we got 4 survival embryos, one of which hatched. The survival rate was 2.17%-5.88% and survival time was 42-73h.During the vitrification experiment of flounder embryos using PM1 -PM4 and PMP, we obtained 21 survival embryos, 14 of which hatched. The survival rate was 1.64%-32.35% and survival time was 14- 108h.PMPl was used to cryop...
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