| With the rapid development of animal husbandry,the sheep raising model has gradually changed from grazing type to large-scale house feeding,and artificial insemination technology can greatly improve its breeding benefits.Semen preservation technology can make the artificial insemination of sheep no longer limited by region,insemination time,introduction of live animals,etc.,while the advantages and disadvantages of semen diluent directly determine the effect of semen preservation.Objective: Taking Sheep Semen as the test object,the different buffer substances,sugars and antioxidants in semen preservation solution were screened step by step.By analyzing the conventional indexes of semen quality and sperm antioxidant capacity,the better diluent and preservation methods to improve the low-temperature and cryopreservation of sheep semen were explored.Methods:(1)Low temperature preservation of sheep semen,first collect the semen of 5 healthy Duolang sheep rams by sham vaginal method,screen the diluents A1(EDTA + sodium citrate),A2(Hepes+ sodium citrate),A3(sodium citrate),A4(Tris + citric acid)of different buffer systems,and then screen the diluents B1(glucose),B2(fructose),B3(glucose + fructose),B4(fructose + lactose),B5(glucose +lactose)of different sugars;then add tea polyphenols(0,0.4,0.6,0.8,1,1.2 mg/m L)and chlorogenic acid(0,0.04,0.06,0.08,0.1,0.12 mg/m L)based on the better B5 formula;Finally,estrus synchronization was performed in Hu sheep,semen was preserved with diluent of control group and better formula for artificial insemination,and its conception rate was measured to evaluate the preservation effect of better diluent.(2)Cryopreservation of sheep semen,based on the formula of cryopreservation diluent B5,glycerol was used as an antifreeze,and tea polyphenols(0,0.4,0.6,0.8,1,1.2 mg/m L)and chlorogenic acid(0,0.04,0.06,0.08,0.1,0.12 mg/m L)were added to set cryopreservation diluents to compare the cryopreservation effect of antioxidants on sheep semen by detecting sperm survival rate,motility,plasma membrane integrity rate,acrosome integrity rate,CAT activity,SOD activity,and the expression of freezing-related genes BCL-2,TUBB,HSP70,HSP90.Then the effects of three different thawing procedures,38℃ for 30 s,45℃ for 20 s,and 75℃ for 3 s,on the quality of frozen semen were compared on a 0.08 mg/m L chlorogenic acid basis.Finally,artificial insemination was performed with frozen semen stored in the control group and better process to detect the conception rate of frozen semen.Results:(1)When sheep semen was stored at low temperature,the diluent of "Tris + citric acid" could significantly improve the survival rate,motility,motility and plasma membrane integrity rate of semen.The quality of semen preservation was significantly higher in the "glucose + lactose " group than in the other groups.On the basis of this formula,the highest sperm survival rate,motility,plasma membrane integrity rate,acrosome integrity rate,CAT activity and SOD activity after preservation were significantly higher with the addition of 1.0 mg/m L tea polyphenols or 0.08 mg/m L chlorogenic acid than in the control group,and the conception rates of semen after cryopreservation were 73.33% and 76.67% in the two groups,respectively.(2)when sheep semen was cryopreserved,the diluent containing 1.0 mg/m L tea polyphenols group or 0.08 mg/m L chlorogenic acid group,after cryopreservation of semen in liquid nitrogen,the sperm survival rate,motility,plasma membrane integrity rate,acrosome integrity rate,CAT activity and SOD activity,as well as BCL-2,HSP70,and HSP90 gene expression were significantly higher than those in the control group.On the thawing method of frozen semen,the thawing procedure at 38℃ for 30 s had a better effect on post-frozen sperm thawing;the conception rates of cryopreserved semen supplemented with antioxidants in the two groups were 52% and 56%,respectively.Conclusion:(1)Tris + citric acid,glucose + lactose based diluent supplemented with 1.0 mg/m L tea polyphenols or 0.08 mg/m L chlorogenic acid can improve the effect of sheep semen cryopreservation.(2)using glycerol as an antifreeze and also adding 1.0 mg/m L tea polyphenols or 0.08 mg/m L chlorogenic acid as a freezing diluent,which is conducive to the cryopreservation of sheep semen;the thawing procedure at38℃ for 30 s has a better effect on post-freezing sperm thawing. |
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