| Semen preservation plays an important role in artificial insemination technology,which can improve the utilization rate of excellent varieties and promote the genetic improvement of livestock.At present,sheep semen cryopreservation still has problems such as short preservation time and low conception rate.In order to reduce the oxidative damage to sperm in the cryopreservation of sheep semen,improve the preservation quality and prolong the preservation time,Tempol(superoxide dismutase derivative)and Apigenin(natural flavonoid antioxidant)were added to the sheep cryopreservation dilution.The effects of these two antioxidants on the cryopreservation of sheep semen were investigated,which shed light on the cryopreservation and application of sheep semen.In present study,Tris-fructose-citric acid served as base dilution,it was cooled gradually,and supplemented with 0,2.5,5,10,20,100 ?M Tempol and 0,5,10,15,20,100 ?M Apigenin respectively.Then Tempol and Apigenin dilution were combined,including 0+15,2.5+15,5+15,5+0,5+10,2.5+10 ?M Tempol+Apigenin combination,stored at 5° C for 7 d,The sperm motility index and plasma membrane integrity were detected every 24 hand mitochondrial activity,superoxide dismutase(SOD)activity and malondialdehyde(MDA)content in the supernatant were measured every 48 h.The results showed: 1.Adding a certain concentration of Tempol to the base diluent can improve the quality of semen stored at low temperature.Among them,the sperm motility and viability of the 5 ?M treatment group for3-7 days were significantly higher than those of the control group(P < 0.05).As for 3-5 days the mitochondrial activity of the 5 ?M treatment group was significantly higher than that of the control group.P < 0.05);F-type sperm in the 5 ?M treatment group was significantly higher than that in the control group observed for 4-6 days(P < 0.05).During this period,other treatment groups were also higher than the control group,but the difference between the groups was not significant.(P > 0.05).In addition,the SOD activity of each treatment group was significantly higher than that of the control group during the cryopreservation period(P <0.05).The MDA content of each treatment group was lower than that of the control group during the storage period,and the difference between the 5 ?M group and the control group was significantely high(P < 0.05).These results indicated the optimal concentration of Tempol was 5 ?M,in which the sperm motility was still 46.77%,the plasma membrane integrity rate was 66.40%,and the mitochondria activity was 45.46% after 7 days reservation at low temperature.2.Adding a certain concentration of Apigenin to the base diluent can improve the quality of semen stored at low temperature.Among them,the sperm motility,viability and mitochondrial activity of the 15 ?M treatment group were significantly higher than those of the control group(P < 0.05).After storage for 2-6 days,the F-type sperm of the 15 ?M treatment group was significantly higher(P < 0.05);during this period,the other treatment groups were also higher than the control group,but the difference between the groups was not significant(P > 0.05).In addition,the SOD activity of each treatment group was significantly higher than that of the control group during the cryopreservation period(P < 0.05).The MDA content of each treatment group was lower than that of the control group during the storage period,and the difference between the 15 ?M group and the control group was significant(P< 0.05).As a result,the optimal concentration of Apigenin was 15 ?M.After 7 days of storage,the sperm motility was still 52.24%,the plasma membrane integrity rate was 47.80%,and the mitochondrial activity was 57.15%.3.The combined addition of Tempol and Apigenin in the base dilution produced a ynergistic effect.The combined effect of the 2.5+15 ?M,5+10 ?M and 2.5+10 ?M Tempol+Apigenin during cryopreservation is significantly better than the single addition.Sperm stored at 2.5+15 ?M,5+10 ?M and 2.5+10 ?M,2.5+15 ?M Tempol+Apigenin had higher sperm motility,and 2.5+15 ?M Tempol+Apigenin was significantly higher than thecontrol group(P < 0.05);The F-type spermatozoa in the 2.5+15 ?M group was significantly higher than the two control groups(P < 0.05).The SOD activity in the 2.5+15 ?M Tempol+Apigenin was significantly higher than that in the 0+15 ?M group(P < 0.05).The content was significantly lower than the 0+15 ?M Tempol+Apigenin control group(P < 0.05).The results showed that the best combination was 2.5+15 ?M Tempol+Apigenin.After 7 days,the sperm motility still reached 63.89%,the plasma membrane integrity rate reached 55.72%,and the mitochondrial activity reached 54.08%.The 5+15 ?M treatment group had the lowest or even less than two additions to the single antioxidant group. |
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