| The objectives of this study were to analyze the effects of vitrification on the immature oocytesin the crucian carp （Carassius cuvieri）. The selection of three separating methods with survival andmature rate. The effects of pre-equilibrium time and temperature to crucian carp oocytes’ survivalrates were detected. The suitable vitrification solution and procedure were selected due to thesurvival rates, the activity of SOD and SDH in mitochondria and the level of the lipid peroxidationin the cell membrane by the content of MDA. And the factors affecting the viability of oocytes wereanalyzed.（1）The selection of three separating methods of crucian carp oocytes: Comparison of thesurvival and mature rate with three separating methods is found that enzymatic treatment is a simpleand rapid method for crucian carp oocytes in contrast to the mechanical method. Collagenase hasbeen reported to be capable of attacking native collagen without having an effect on related proteinsand not damaging epithelial tissue. So it’s more suitable for crucian carp oocytes’ separation and thebest treatment times and concentrations were0.4mg·mL-1and10min.（2）The selection of pre-equilibration time and temperature: Survival rates of cells weredetected after3steps pre-equilibration and elution, and found that survival rates were reducedgradually along with the pre-equilibrium time. so we choose10min as the best pre-equilibrium timein each step. In temperature selected experiment, we found survival rate of0℃was higher than20℃,so0℃was the suitable temperature.（3）The selection of the vitrification solution: VS4and VSd selected according to the trend ofsurvival rates, vitality of SDH and SOD, content of MDA. VS4, consisted of160g·L-1PG,100g·L-1DMSO,150g·L-1MeOH,120g·L-1acetamide,0.6M sucrose and30g·L-1PEG, as well as VSbwhich contain80g·L-1PG,110g·L-1MeOH,0.1M trehalose and40g·L-1PEG.（4）The effects of vitrification to oocytes: The survival rates, vitality of SDH and SOD,content of MDA showed that in the process of cryopreservation the descent of the viability ofoocytes and the increase of damage on oocyte membrane integrity. So vitrification can’t completelyavoid the chilling injury of oocytes at low temperature.In conclusion, the optimum procedure of crucian carp oocytes vitrification was suggested asfollows: pre-equilibration with3steps for10min in each step at0℃, treating with VS4or VSd, plunging directly into LN2, thawing in37℃water and stepwise dilution with sucrose. |
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