| Compared with the mature oocyte cryopreservation, cattle GV oocyte cryopreservation is very difficult. But in the long-distance transportation of oocyte, cryopreservation is needed. So, the development of GV oocyte cryopreservation have significant role on making full use of immature oocytes, expecially long-distance cryopreservation of oocyte. Trehalose, a non-reducing disaccharide, can form a glassy protecting layer for the alive cell under extreme conditions. Trehalose can be applied in embryo and oocyte cryopreservation as a non-permeable cryoprotectants instead of the sucrose in CPA in this study; some previous studies had showed that the NaCl, as the main ingredient in the conventional cryoprotectants buffer DPBS might cause some certain damage in the embryo cryopreservation. Therefore, choline chloride could be a proper substitute of NaCl in alleviating the potential damage of the sodium ions. In this study, the optimal substitution proportion for the trehalose and choline chloride to the sucrose and NaCl respectively were found through comparing with the development potential, survive rate of the oocytes, cell damage and expressions of apoptosis and cycle genes among different cryopreservation groups. The experimental results were listed as follows:1. To explore frozen effect of different trehalose concentrations on GV-stage oocytes vitrification(1) Added different concentrations of trehalose in the CPA to detect the survival rates. The FDA fluorescence intensity of the frozen group0.5M Trehalose was less than the0.5M sucrose group (39.8%vs52.5%, p<0.05), but the Fluorescence value of the0.5M sucrose group was less than the0.5M Trehalose group (63.6%vs70.1%, p<0.05), There was no significant difference between the other frozen groups.(2) The zona pellucida dissolution time of all frozen groups compared with the fresh group were significantly increased (p<0.05); The zona pellucida dissolution time of0.5M sucrose group (93±2.5min) was significantly less than the other frozen group, Excepting for0.5M Trehalose (95.5±2.6min),0.3M Trehalose group (93.3±3.8min)(p<0.05).(3) In the thawed oocytes viability experiments, compared with the C group, oocyte IVF embryo cleavage and blastocyst rates were significantly reduced in the frozen groups (p<0.05).1M Trehalose,0.8M Trehalose,0.1M Trehalose group cryopreserved oocytes could not develop to the blastocyst stage,0.5M sucrose group and0.5M Trehalose,0.3M Trehalose group IVF embryo cleavage and blastocyst rates difference were not significant (p>0.05), and0.5M sucrose (13.9±2.3%)and0.5M Trehalose (12.2±0.9%),0.3M Trehalose group (10.4±2.8%) embryo cleavage rates were significantly higher than the other groups (except the0.8M Trehalose group (7.7±4.3%))(p<0.05); the blastocyst rates of0.5M sucrose group (2.5±2.2%) and0.5M Trehalose (1.5±1.3%),0.3M Trehalose group (1.3±2.2%) were higher than the other groups, but the difference was not significant.(4) The spindle morphologies of maturated oocytes cultured in vitro was discovered. It was found that the concentration of0.5mol/L was the trehalose’s optimal concentration and the protective effection was varied with the concentration.Conlusion:The trehalose had the potiential in protecting the viability of the freezing oocytes and could substitute the sucrose in CPA buffer. Based on the results of the surviving rates, embryo development the0.5mol/L trehalose was the optimal concentration.2To explore the mechanism of bovine GV-stage oocytes injury by frozen(1) The oocyte apoptosis was detected by immunofluorescence staining method of Apoptosis detection kit, The results showed that:the oocyte nonapoptosis ratios of the0.5M sucrose group was (4.1±0.9%) and the0.5M trehalose group was (5.5±2.6%), the oocyte nonapoptosis ratios (p<0.05) were significantly lower than the fresh control group as (13.0±0.9%).(2) In the RT-PCR experiments, the mRNA relative expression levels of apoptosis related genes BAX inhibitor and BAX, as well as the cell cycle-related genes CDC2and CyclinBl was detected according to the RNA extracted from the oocytes. Then it was found that the mRNA expression level of the areapoptotic gene BAX and cycle related gene CDC2were increased, the other were overall down.Conlusion:Frozen can exacerbate the apoptosis of GV-stage oocytes,which is the main cause of freezing injury.2. Modified phosphate buffered saline (mPBS) in the bovine GV stage oocytes vitrification freezing effect evaluation(1) The choline chloride Alternate sodium chloride, the survival rate of0.5M sucrose group (24.2%), mPBS+0.5M sucrose group (42.2%) and mPBS+0.5M trehalose group (47.0%) was significantly (p<0.05) lower than the fresh group (90.4%). The difference between mPBS+0.5M sucrose group and mPBS+0.5M trehalose group was not significant statistically.(2) The zona pellucida dissolution time of the frozen groups were significantly longer than the fresh group (p<0.05); The0.5M sucrose group (103±4.16min) was significantly longer than the mPBS+0.5M trehalose (86.67±5.69min) and mPBS+0.5M sucrose group (89.00±9.54min)(p<0.05).(3) Oocytes matured were in vitro and in vitro fertilization,.It was discovered that the cleavage rates of fertilized embryosof the frozen groups were significantly lower than the fresh group (45.4±0.9%, p<0.05); the mPBS+0.5M sucrose group (28.4±0.5%) and mPBS+0.5M trehalose group (26.8±2.1%) were significantly higher than the0.5M sucrose group (14.2±2.6%)(p<0.05). The blastocysts rates of the0.5M sucrose group (2.3±2.0%), mPBS+0.5M sucrose group (3.2%±0.1%) were lower than the mPBS+0.5M trehalose group (3.9%±0.9%), but there was no significant difference (p>0.05) among the three groups statistically.(4) Compared with the0.5M sucrose group, mPBS+0.5M sucrose and mPBS+0.5M trehalose group could ease the downward trend of BAX inhibitorl mRNA expression levels. But it was found that although apoptosis and regulation cycle gene expression has been suppressed, compared with the fresh, the frozen groups leaded the normal expression of apoptosis and cell cycle genes.Conlusion:The sodium ions might hurt the GV stage oocytes during the process of cryopreservation. The choline chloride could act as a protector for the oocytes by fully substituting the NaCl which was used in DPBS. Moreover, together with the choline chloride, the0.5mol/L trehalose could reach its optimal effect in protecting the freezing oocytes. |
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