Effect Of Silymarin On Boar Sperm During Liquid Preservation And Cryopreservation

With the improvement of artificial insemination technology and the development of livestock breeding,semen preservation technology becomes more and more important.Boar semen can be preserved in proper extenders for more than 12 days at room temperature.However,there are still lots of problems in this technology such as the complicated ingredient of extender,atypism of preservation and poor fertilization rate and litter size.A large number of studies showed that ROS produced and accumulated during the semen preservation plays pivotal role in reducing the quality of sperm and decreasing fertilization During the preservation of semen,the accumulation of ROS can cause sperm mitochondrial activity change,while attacking the unsaturated fatty acids of the plasma membrane,leading to lipid peroxidation,generating large amounts of lipid peroxides,and provoking mutations in the guanine site in the DNA sequence,thus resulting in DNA sequence damage,DNA double-strand breaks,telomere sequence damage,even destroying the chromosome structure,exacerbating sperm apoptosis.Therefore,it is important part of the preservation of boar semen to improve the antioxidative ability and quench ROS of sperm by adding antioxidants to the extender of semen.In order to study the effect of antioxidant silymarin on the preservation of semen,different consintrations of silymarin(12.5μM,25μM,50μM,100μM,and 200μM)were added in Modena extender,and various indicators including sperm motility and membrane integrity,acrosome integrity and mitochondrial activity were detected at room temperature.The results showed that 25μM silymarin could significantly improve sperm quality and prolong semen preservation time.Subsequently,in order to explore the mechanism of silymarin,the oxidative stress was induced by 200 M tert butyl peroxide,and the oxidative stress status of sperm,such as the sperm quality,MDA content,H2O2 lever,GSH content and the level of apoptosis were detected after treated by 25 silymarin It was found that silymarin can reverse the oxidative damage caused by tert-butyl hydroperoxide and improve sperm quality.To investigate the role of silymarin in semen freezing,we added silymarin at 12.5μM,25μM,50μM,100μM,200μM and 400μM,respectively,into the cryopreservation and thawing solution.The results showed that addition of 100μM silymarin significantly improved sperm motility,plasma membrane integrity,and decreased MDA content and H2O2 content,both in the frozen liquid and in the thawing liquid.Finally,we examined the effect of silymarin-treated spermatozoa on fertilization and embryonic development..The results showed that adding silymarin can increase the cleavage rate and blastocyst rate at room temperature.In cryopreservation,the addition of silymarin to the thawing solution can only increase the cleavage rate but have no effect on the embryonic development.In summary,the results appear as shown below:(1)Adding 25μM silymarin can improve the quality of spermatozoa,reduce the ROS level of spermatozoa,and increase the antioxidant capacity of spermatozoa when the semen is diluted at room temperature.(2)Addition of 25μM silymarin can reduce sperm oxidative damage caused by t-butyl hydroperoxide.(3)In cryopreservation,adding 100μM silymarin can improve sperm quality,reduce oxidative damage,and increase its antioxidant capacity.(4)Liquid stored spermatozoa supplemented with 25μM silymarin at room temperature can increase the cleavage rate and blastocyst rate of oocytes after in vitro fertilization.Cryopreserved spermatozoa supplemented with 100μM silymarin can only increase the cleavage rate of oocytes after in vitro fertilization,but has no effect on blastocyst rate.