Effects Of Epicatechin On Boar Semen Cryopreservation

The development of semen preservation technology in vitro has provided technical support for the successful application of artificial insemination technology in livestock.Cryopreservation,as an important technical means of livestock breed improvement,can expand the utilization rate of superior breeding boars.However,due to the physical,chemical and oxidative damage caused by cold shock in the freezing process,sperm motility decreased after frozen-thawed,affecting their fertilization ability.Current studies have shown that the main cause of sperm quality decline after frozen-thawed is oxidative stress induced by excessive accumulation of reactive oxygen species(ROS).During in vitro preservation,polyunsaturated fatty acids in the plasma membrane of boar sperm are vulnerable to oxidative damage,which leads to damage of cell membrane and DNA integrity and induces sperm apoptosis.At present,one of the measures to prevent the excessive production of ROS in sperm cryopreservation is to increase the antioxidant capacity of sperm by adding exogenous antioxidants into the diluent.Epicatechin,as a natural antioxidant,has a polyhydroxyl structure and can bind free radicals to achieve antioxidant effect.Although more and more reports have been published about the freezing protective effects of antioxidants,the application of epicatechin in cryopreservation of boar semen is still lacking.In this experiment,different concentrations of epicatechin were added into the frozen diluent of boar semen.Sperm motility,motor performance,antioxidant property and cell apoptosis after freezing and thawing were detected.To evaluate the cryopreservation effect of epicatechin and determine the optimal concentration of epicatechin in boar semen cryopreservation diluent.1.The addition of epicatechin in freezing extender can effectively improve the motility,athletic performance,plasma membrane and acrosomal integrity of boar sperm after frozen-thawed.Compared with the other treatment groups,the extender supplemented with75 μmol/L epicatechin had the best preservation effect.After frozen-thawed,sperm motility(48.17% ± 0.67%),curve velocity(37.71 ± 1.04 μm/s),beat cross frequency(3.55 ± 0.12Hz),plasma membrane integrity(49.70% ± 1.78%)and acrosomal integrity(50.45% ±1.56%)were significantly increased(P < 0.05).When epicatechin concentration was 200μmol/L,compared with the control group,there were no significant differences in each quality index(P > 0.05).In addition,there was no significant differences in all quality indexes among 25 μmol/L,50 μmol/L and 100 μmol/L epicatechin groups(P > 0.05).The results indicated that the addition of 75 μmol/L epicatechin in extender could effectively improve the quality of boar sperm after frozen-thawed and had protective effect on the cryopreservation of boar sperm.2.The addition of epicatechin in frozen extender could effectively increase the total antioxidant level of boar sperm after frozen-thawed.When the concentration of epicatechin was 75 μmol/L,T-AOC of sperm was 0.40 μmol/m L,SOD,CAT and GPx activities were4.48 U/m L,25.67 U/m L and 19.93 μg/m L,respectively,ROS level(28.2%)and MDA content(1.72 nmol/m L)were significantly lower than those in the control group(P < 0.05).When epicatechin concentration was 200 μmol/L,there was no significant differences between the two groups(P > 0.05).Therefore,adding 75 μmol/L epicatechin in frozen extender could improve the antioxidant capacity of boar sperm after frozen-thawed.3.The addition of epicatechin in frozen extender could increase the mitochondrial activity and ATP content of boar sperm and reduce the level of sperm apoptosis.Compared with the other treatment groups,mitochondrial membrane potential and ATP content in 75μmol/L epicatechin group was significantly increased(P < 0.05),reaching 18.94% and 16.79μmol/L.The level of apoptosis was significantly decreased(P <0.05),reaching 19.4%.Compared with the control group,epicatechin concentration of 25 μmol/L,50 μmol/L and200 μmol/L groups had no significant difference in each index(P > 0.05).Compared with the control group,the supplementation of 75 μmol/L epicatechin significantly increased the relative expression level of Cyt C and BCL-2 in sperm mitochondria(P < 0.05).Among them,the expression of Cyt C in mitochondria of epicatechin group was not significantly different from that of fresh semen(P > 0.05),while the expression of BCL-2 was significantly lower than that of fresh semen(P < 0.05).Compared with the control group,the supplementation of 75 μmol/L epicatechin significantly decreased the relative expression level of Cyt C in the cytoplasm,Caspase-9 and Caspase-3(P < 0.05).The expression level of Caspase-3 was not significantly different from that of fresh semen(P > 0.05).The expression level of sperm BAX protein in 75 μmol/L epicatechin group was lower than that in control group,but the difference was not significant(P > 0.05).These results indicated that epicatechin could inhibit apoptosis regulated by mitochondrial Cyt C and Caspase pathway,thus effectively improving the cryopreservation effect of boar sperm.In conclusion,this experimental study found that 75 μmol/L epicatechin was added into frozen of boar semen can significantly improve the quality of boar sperm after frozen-thawed,reduce the oxidative damage induced by ROS,effectively restrain the occurrence of apoptosis,thus improve the effect of the boar semen cryopreservation,epicatechin in boar semen cryopreservation provide theoretical basis for the application.