Effects Of Resveratrol On Apoptosis And Apoptotic Pathways Of Frozen Boar Spermatozoa

1.In order to investigate the effects of cryopreservation on boar spermatozoa apoptosis and apoptotic pathway, the flow cytometry was used to detect the apoptosis state and ROS level of boar frozen spermatozoa. ATP content was measured by luminometer, and the m RNA expression level of genes involved in different apoptotic pathways were measured by real-time quantitative RT-PCR method. The results showed that the motility, nornal acrosome rate and plasma membrane integrity rate of sperm after freezing were decreased greatly(P<0.05). The TUNEL-positive spermatozoa rate(80.4%) from freezing group were much higher than that of fresh group(9.7%)(P<0.05). The percentage of ROS-positive spermatozoa in freezing group(58.2%) was also obviously higher than that of fresh group(P<0.05). The cryopreservation caused a significant decrease of ATP concentration in freezing group(0.247 μmol?L-1) comparing with fresh group(0.926 μmol?L-1)(P<0.05). q RT-PCR results showed that the relative expression level from promoting apoptosis genes(TNF-α, Fas, Caspase, P53 and Bax) were up-regulated, and in which the expression of Fas, P53, TNF-α, caspase-8 and caspase-9 increased greatly after cryopreservation(P<0.05). The relative expression level from inhibiting apoptosis genes(BCL-2, BIRC-5, Mn SOD, Cu Zn SOD and SURVIVN) were down-regulated, and in which the expression of BIRC-5, Mn SOD and SURVIVN decreased greatly(P<0.05). The results indicated that cryopreservation could lead to boar spermatozoa apoptosis. Not only extrinsic death receptor but also intrinsic mitochondrial signaling pathway might play key roles in mediating spermatozoa apoptosis after cryopreservation.2. Adding resveratrol to the cryopreservation agents, this experiment was set the concentrations separately by 12.5 μM, 25 μM, 50 μM and 100 μM, and at the same time 0 μM group was set as a blank.Comparing post-thaw sperm motility, acrosome integrity rate and membrane integrity rate of each group,the results showed that 50 μM group was significantly higher than other groups(P <0.05) on sperm motility, membrane integrity rate and acrosome integrity rate.The quality of 50 μM group sperm increased with the resveratrol concentration rising, but the sperm quality was reduced significantly when the concentrations of resveratrol was overtop(≥100 μ M)(P<0.05). The following experiment was used 50 μ M concentration as optimal concentration of resveratrol.3.The sperm apoptotic rate(29.0%) of experimental group after Caspase fluorescent staining within resveratrol and the post-thawing sperm was significantly lower than the control group(86.3%)without resveratrol(P <0.05);ROS positive ratio(58.6%) of experimental group was also significantly lower than the control group(88.4%)(P <0.05).The relative expression level of caspase-8 was down-regulated and relative expression of Fas, P53, TNF-α, Bax, caspase-3, caspase-9 were significantly decreased(P <0.05); the relative expression level of Cu Zn SOD was increased, and the relative expression of Bcl-2, Mn SOD, SURVIVN were significantly increased(P <0.05). The results showed that resveratrol can effectively inhibit apoptosis of sperm, reduce the damage to post-thaw sperm with cryopreservation and improve sperm motility and quality with influencing on extrinsic death receptor but also intrinsic mitochondrial signaling pathway.