| Semen preservation is one of the key steps of artificial insemination.This technology can achieve the cross regional and cross time breeding of semen,and is significant for livestock breeding and variety improvement.But at present,semen preservation has some problems,such as poor preservation effect and short storage time,and these problems restricts the development of semen preservation.Therefore,improving the preservation effect of goat semen is of great significance to promote the breeding of sheep and increase the productivity of goat,and the lipid peroxidation in sperm fa’sheng is an important reason for the poor preservation of semen.To screen out a more efficient formulation of diluent and find antioxidants that play a positive role in semen preservation。First,compared the preservation effects of five different formula goat cryopreservation diluents on goat semen.Then used the best diluents as for base diluents,separately add apple polyphenols with 10,20,40,60,80 mg/L;separately add grape seed proanthocyanidins with 10,30,50,70 mg/L。Detection of sperm viability,acrosome integrity,plasma membrane integrity,mitochondrial activity,total antioxidant capacity(T-AOC)and malondialdehyde(MDA),to explore the effect of two antioxidants on cryopreservation of semen.Finally,used apple polyphenols + grape seed proanthocyanidins were added to different concentrations: 40+0 mg/L,0+30 mg/L,20+10 mg/L,20+30 mg/L,40+10 mg/L,40+30 mg/L,to explore the synergistic effect of two kinds of antioxidants.The main results of this study are as follows:1.Five different formulations of goat Semen Cryopreservation diluent were selected,and the best quality of “diluent 2”.After five days preservation,the survival rate of sperm in “diluent 2” could reach 45.39%,the complete rate of acrosome up to 61.86%,the intact rate of sperm plasma membrane up to 47.30%,the mitochondrial activity is 43.3%,which were significantly higher than those of other experimental groups(P<0.05).The second is “diluent 5”,the sperm motility,acrosome integrity and mitochondrial activity in “diluent 5” were significantly greater than those in the other test groups except for “diluent 2”(P<0.05).The lowest index of semen preservation in the five diluent was “diluent 1”,and all indexes were lower than those in other test groups,and the difference was significant(P<0.05).The best preservation “diluent 2” was selected as the basic diluent for further study.The relationship between sperm viability and acrosome integrity was the highest,was significantly positive correlation(r=0.932,P<0.001),and the sperm viability was positively correlated with the integrity of plasma membrane(r=0.876,P<0.01).2.The “diluent 2” was used as the base diluent for the experiment,and 10,20,40,60,80 mg/L apple polyphenols were added respectively.The results showed that all indexes except semen preservation increased first and then decreased,except for MDA,MDA opposite.In the five groups,the best effect of semen preservation was in group 40 mg/L,and all indexes detected after five days preservation,were significantly higher than those in other groups(P<0.05),the survival rate of sperm could reach 49.31%,the complete rate of acrosome up to 66.23%,the intact rate of sperm plasma membrane up to 49.91%,the mitochondrial activity is 49.56%.The preservation effect of 80 mg/L test group was the worst,and all indexes in semen were lower than those in other groups,and the three indexes of acrosome integrity,mitochondrial activity and T-AOC were significantly lower than those in the blank control group(P<0.05).3.The “diluent 2” was used as the base diluent to test,and 10,30,50,70 mg/L grape seed proanthocyanidins were added respectively.The results showed that all indexes except semen preservation increased first and then decreased,except for MDA,MDA opposite.In each treatment group,the best preservation effect of semen was group 30 mg/L,and all indexes except MDA were the highest during storage.After five days preservation,sperm viability,acrosome integrity,mitochondrial activity and T-AOC were significantly different(P<0.05),the survival rate of sperm could reach 51.46%,the complete rate of acrosome up to 67.55%,the intact rate of sperm plasma membrane up to 50.97%,the mitochondrial activity is 50.78%.70 mg/L test group has the lowest preservation quality compared with other test group.4.The “diluent 2” was used as the base diluent to test.Apple apple polyphenols+grape seed proanthocyanidins were added to different concentrations.The results showed that all indexes of spermatozoa except MDA in 20+10 mg/L,20+30 mg/L and 40+10 mg/L groups were increased to varying degrees compared with the control group added alone,the content of MDA was lower than that of the control group.The best preservation effect was 20+30 mg/L test group,and the sperm viability,plasma membrane integrity,acrosome integrity and T-AOC were higher than those of other experimental groups after five days preservation,and the difference was significant(P<0.05),the survival rate of sperm could reach 56.29.%,the complete rate of acrosome up to 70.03%,the intact rate of sperm plasma membrane up to 55.20%,the mitochondrial activity is 43.3%.In summary,of the five diluent formulations,"formula 2" has the best preservation effect,the sperm viability is still 45.42% after five days preservation,the acrosome integrity rate is up to 61.26%,the integrity rate of the plasma membrane is 47.24%,the mitochondrial activity is 43.3%,and the sperm viability is significantly positively correlated with the integrity of the acrosome,and the sperm viability is positively correlated with the integrity of the plasma membrane;Both apple polyphenols and grape seed proanthocyanidins could effectively improve the cryopreservation quality of goat spermatozoon.The optimum concentration of apple polyphenols was 40 mg/L,after five days preservation,the sperm viability reached 49.31%,the complete rate of acrosome reached 66.23%,the integrity rate of plasma membrane was 49.91%,the mitochondrial activity is 49.56%;The optimum concentration of proanthocyanidins was 30 mg/L,after five days preservation,the sperm viability was 51.46%,the complete rate of acrosome was 67.55%,the integrity rate of plasma membrane was 50.97%,the mitochondrial activity is 50.78%,and the two kinds of antioxidants had synergistic effect.The optimum combination concentration of apple polyphenols + grape seed proanthocyanidins was 20+30 mg/L,the sperm viability reached 56.29% after five days preservation,the integrity rate of acrosome was 70.03%,and the integrity rate of plasma membrane was 55.20%,the mitochondrial activity is 53.53%. |
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