| Semen cryopreservation is not only a key link in modern artificial insemination technology in animal husbandry,but also a core technology for long-term preservation of excellent male animal genetic resources.However,the accumulation of excessive reactive oxygen species(ROS)in semen during cryopreservation induces oxidative stress in sperm,destroys sperm membrane structure,and even loses normal physiological functions and fertilization ability.Current research shows that adding antioxidants to semen diluent can inhibit the generation of ROS and improve the quality of semen cryopreservation.As an antioxidant and immune enhancer,astragalus polysaccharide(APS)has the characteristics of good immune activity,no drug resistance and high safety.It has begun to be used in veterinary clinics,animal husbandry production and feed industry.However,there is no research on the effect of APS on the semen cryopreservation effect of dairy goat.Therefore,in this experiment,different concentrations(0,0.1,0.2,0.3,0.4,0.5 g/L)of APS were added to the frozen diluent of dairy goat semen.After freezing and thawing,the sperm motility,antioxidant properties,sperm apoptosis levels and AMPK phosphorylation were measured,and the effects of APS on semen cryopreservation of dairy goat were analyzed,in order to provide a certain scientific basis for the research and development of new dairy goat semen cryopreservation diluent formula.The main results of this experiment are as follows:1.Adding different concentrations of APS to the frozen dilution of dairy goat semen can significantly improve sperm motility,acrosome integrity and plasma membrane integrity in dairy goats after frozen-thawed.The results showed that: 0.2 g/L,0.3 g/L,0.4 g/L APS treatments can significantly improve the sperm motility of dairy goats(P < 0.05),and the 0.2g/L APS treatment group had the highest sperm motility(52.89% ± 0.26%),significantly higher than the control group(P < 0.05).The 0.2 g/L APS treatment group had the highest acrosome integrity(50.31%)after frozen-thawed,which was significantly higher than other groups(P < 0.05);the 0.2 g/L APS treatment group had the highest plasma membrane integrity(55.34%),significantly higher than the control group(P < 0.05),but not significantly different from the 0.3 g/L APS treatment group(P > 0.05).The straight line velocity in the 0.2 g/L APS treatment group(45.84 μm/s)and the curvilinear velocity(59.46μm/s)were the highest,but it was not significantly different from the 0.3 g/L APS treatment group(P > 0.05).2.Adding different concentrations of APS to the frozen diluent of dairy goat semen can effectively increase the activities of T-AOC and antioxidant enzymes SOD and CAT after frozen-thawed of dairy goat sperm,and reduce the level of ROS and the content of MDA.The results showed that the sperm T-AOC content and CAT in the 0.2 g/L APS treatment group were significantly higher than those other treatment groups(P < 0.05);the 0.1 g/L,0.3g/L and 0.4 g/L APS treatment groups were significantly higher than the control group(P <0.05).The sperm SOD activity of the 0.2 g/L APS treatment group was significantly higher than that of the control group(P < 0.05);the sperm SOD activity of the 0.3 g/L APS treatment group and the 0.2 g/L APS treatment group were not significantly different(P >0.05),but higher than the control group(P < 0.05).The 0.2 g/L APS treatment group had the lowest MDA content and ROS level,which was significantly lower than that of the control group(P < 0.05).3.Adding different concentrations of APS to the frozen diluent of dairy goat semen can effectively increase the mitochondrial membrane potential and ATP content after sperm frozen-thawed,and the added concentration is 0.2 g/L,it can activate AMPK signaling pathway and reduce sperm apoptosis.The mitochondrial membrane potential of the 0.2 g/L and 0.3 g/L APS treatment groups were significantly increased(P < 0.05),but the difference between the two groups was not significant(P > 0.05).The sperm ATP content in the 0.2 g/L APS treatment group was significantly higher than that in the control group(P < 0.05),and the degree of apoptosis was significantly lower than that in the control group(P < 0.05),indicating that it can down-regulating the expression of the protein BAX,up-regulating the expression of apoptosis-inhibiting protein BCL-2,which significantly increased the anti-apoptotic level of sperm(P < 0.05).At the same time,compared with the control group,0.2 g/L APS can promote sperm AMPK phosphorylation(P < 0.05),improve the regulation of sperm AMPK signal pathway during frozen-thawed process,and effectively improve the effect of semen cryopreservation.Therefore,the results of this experiment show that APS as an exogenous antioxidant can improve the semen cryopreservation effect of dairy goat.When the concentration of APS is 0.2 g/L,it can significantly improve the quality and antioxidant capacity of sperm after frozen-thawed,increase sperm ATP levels and mitochondrial membrane potential,promote sperm AMPK phosphorylation,reduce sperm apoptosis,and improve semen cryopreservation effect. |
