| Oocyte vitrification technique is simple,economic,practical and reliable. Vitrification technology to reduce feeding,breeding costs,the expansion of animal populations.At the same time can also be for other biological technologies such as gender identification,in vitro fertilization,nuclear transfer and transgenic provide oocytes available.As a result of pig oocytes more abundant lipid content,low temperature is extremely sensitive to relatively slow research.The test pig oocytes targeted balance from time to frozen,refrigerated and frozen carrier liquid,such as foundation,the study and improvement of vitrification methods to improve pig oocytes vitrification effect.Experiment 1:In order to detect the equiliberation of the different time period of pig GV oocytes impact.GV of pig oocytes in TCM-199 media solution containing 10% ethylene glycol, espectively 3min balance and 4min;in 20% EG, respectively,and equiliberation 2min,3min in 30% EG vitrification fluid equiliberation after 30s into the semi-straw, frozen in liquid nitrogen, thawed when 0.5M,0.25M and 0M sucrose solution,respectively,to deal with the 5min,the control group oocytes frozen directly without in vitro maturation of cultured.It was found that the equiliberation be 5min and 7min,respectively,56.81±0.63%,47.61±4.02% survival rate,10.50±1.43% and 5.02±1.64% maturity rate, the differences were significant (P <0.05); both with the control group survival rate of 93.99±0.27% and maturity rate of 69.95±1.72% difference was extremely significant (P<0.01).Description of the equiliberation 5min better than 7min,vitrification of oocytes in harm's way.Experiment 2:In order to test different vitrification media on the basis of pig GV oocytes impact.Vitrification solution,respectively,the use of TCM-199 and Dulbecco's Phosphate Buffered Saline,DPBS solution,based on different solution were added in 10% EG,20% EG,30% of the EG as cryoprotectant solution.Oocytes were equiliberation 3min,2min,30s into a semi-straw,frozen in liquid nitrogen.When thawed 0.5M, 0.25M and 0M sucrose solution,respectively, to deal with the 5min,the control group without oocytes in vitro maturation of frozen culture directly.The results showed that,TCM199 and the survival rate of DPBS were 52.90±1.98% and 55.27±1.24%,maturity rate of 9.96±1.15%,10.52±0.75% (P>0.05),no significant differences. The survival rate of two and the control group 99.43±0.99% and maturity difference 68.18±2.59% was significantly different (P<0.01).Description DPBS and TCM-199 as the vitrification media phase of pig GV oocytes the effect of the same period for pig GV oocytes vitrification preservation,Vitrification of porcine GV oocytes in harm's way.Experiment 3:In order to test different dilution methods of pig GV stage oocytes impact.GV of pig oocytes in TCM199 medeia solution containing 10%, 20% and 30% respectively of the EG to deal with 3min,2min and 30s,the thaw,respectively,using three-step dilution (that is,in containing 0.5mol/L,0.25mol/L,0mol/L sucrose solution, respectively,to deal with 5min,three-step cryoprotectant removal),two-step dilution (that is,in containing 0.5mol/L,0.25mol/L sucrose solution,respectively,to deal with 5min,two-step cryoprotectant removal),one-step dilution (that is,containing 0.5mol/L sucrose solution to deal with the 5min,step cryoprotectant removal)cryoprotectant removal.It was found that three-step dilution,two-step dilution survival were 53.49±3.57%,51.68±1.67%,and one-step dilution 39.68%±4.23% significantly (P<0.05),the maturity rate of three-step method 10.03±0.67%,and one-step dilution,two-step dilution and 6.7±0.13%,2.15±1.84% significantly (P<0.05).Note the effect of three-step dilution is superior to two-step and one-step dilution.Experiment 4:In order to explore the use of a new type of carrier Cryosheet vitrification cryopreservation of porcine oocytes feasibility.To Cryosheet,the glass Glass micropipette,and Open pulled straw of pig GV oocytes for vitrification cryopreservation. GV of pig oocytes in TCM199 medium containing 10%,20% and 30% respectively of the EG to deal with 3min,2min and 30s,when thawed 0.5M, 0.25M and 0M sucrose solution,respectively to deal with the 5min.The results showed that survival rates were 56.92±2.19%,51.77±0.96%,52.48±1.33%,maturity rates were 11.13±1.13%,8.02±0.78%,6.4±1.27%,Cryosheet,GMP and OPS no significant difference (P>0.05).The results show that this new type of carrier by the author and the import vector Cryosheet compared to not only safe, reliable, low cost, simple production, but also effective cryopreservation of pig GV oocytes.To sum up,pig GV oocytes vitrification cryopreservation,the DPBS media solution to be able to get the effect of TCM199, in 10%, 20% EG solution, respectively, to deal with equiliberation of 3min, 2min, and then into the 30 % EG to deal with 30s,with three-step dilution to thaw,with better access to the development of capacity, carrier-made frozen oocyte cryopreservation Cryosheet to achieve good results.
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