Effect Of Quercetin On Cryopreservation Of Goat Semen

Goat semen is susceptible to oxidative stress damage during preservation.Reducing the storage temperature to 4-5℃can maintain the viability of goat sperm,slow down sperm metabolism and inhibit the production of toxic metabolites.Quercetin is a flavonol compound widely existing in plants with a variety of biological activities.It has antioxidant,anti-inflammatory,antibacterial,anti-allergic and other biological functions.However,there are few reports on the application of quercetin in cryopreservation of goat semen.Therefore,in this study,goats were selected as the research object,and quercetin with concentrations of10,25,50,100μmol/L was added into the semen dilution of goats.The sperm motility parameters,acrosome integrity,plasma membrane integrity,and antioxidant capacity of sperm were evaluated to explore the protective effect of quercetin on goat semen.Meanwhile,the expressions of phosphorylated modified proteins PTP and P-PKAs in sperm were detected by western blotting,so as to explore the mechanism of the effect of quercetin on the phosphorylated modified proteins in cryopreservation of goat sperm,providing a theoretical basis for the study of the preservation of goat semen.The results of this study are as follows:1.Effects of quercetin on sperm motility parameters of goatsQuercetin supplementation in semen dilution significantly improved sperm motility,viability,plasma membrane integrity and acrosome integrity during cryopreservation in goat semen.On the third day of cryopreserved,sperm motility and viability of 25μmol/L quercetin treatment group were the highest,which were 72.07%±1.38%and 76.08%±1.77%,respectively,significantly higher than those of control group（P<0.05）.The plasma membrane integrity rate and acrosome integrity rate of sperm in 50μmol/L quercetin treatment group were the highest,which were 67.61%±1.12%and 68.19%±0.51%,respectively,and there were significant differences compared with control group（P<0.05）.There was no significant difference in sperm viability between 25μmol/L and 50μmol/L treatment groups during storage（P>0.05）.The results showed that the addition of 25μmol/L and 50μmol/L quercetin in diluent could effectively improve the cryopreservation viability parameters of goat sperm.2.Effects of quercetin on antioxidant indices of goat spermQuercetin supplementation in semen dilution significantly improved the ability of goat sperm to resist oxidative stress during cryopreservation,reduced the ROS and MDA content,increased the T-AOC,SOD and CAT contents,and increased the ATP level during cryopreservation.The levels of T-AOC,SOD,CAT and ATP in 50μmol/L quercetin treatment group were 5.57 U/m L,98.73 U/m L,3.97 U/m L and 0.80μmol/L respectively,which were significantly higher than those in control group（P<0.05）.The content of MDA in sperm was 6.36 nmol/m L,significantly lower than that in control group（P<0.05）.On the5th day of semen preservation,the relative content of ROS in 10μmol/L,25μmol/L and 50μmol/L quercetin treatment groups was significantly lower than that in control group（P<0.05）,and the relative content of ROS in 50μmol/L quercetin treatment group was the lowest.The results showed that adding 50μmol/L quercetin to semen diluent could significantly reduce intracellular ROS toxicity and effectively relieve oxidative stress damage of sperm induced by low temperature storage.3.Regulation of quercetin on phosphorylated protein in goat spermThe levels of P-PKAs and PTP in sperm were detected by Western blot.The results showed that adding 50μmol/L quercetin in diluent significantly increased the level of sperm phosphorylated modified protein and was significantly higher than that in other groups（P<0.05）.Addition of 300μmol/L H₂O₂ to the dilution significantly promoted sperm protein dephosphorylation（P<0.05）.Dephosphorylation of sperm protein was significantly inhibited in quercetin treatment group with 50μmol/L（P<0.05）.Quercetin in the dilution inhibits the dephosphorylation of proteins induced by excess ROS generated during cryopreservation via the c AMP-PKA signaling pathway.In conclusion,adding 50μmol/L quercetin to semen diluent of goat can significantly improve sperm motility parameters and antioxidant capacity during cryopreservation of semen.Quercetin can inhibit dephosphorylation of sperm protein induced by excess ROS in spermatocytes through the c AMP-PKA signaling pathway.