| The livestock industry is the basic industry of national economy and the pillar industry of rural economy.Swine industry is an important part of livestock industry,which has great significance in the development of national economy.Artificial insemination(AI)plays an important role in the development of modern swine industry,and it is an important technology to promote the quality of swine product,breed improvement and the industrialization of swine production.Semen preservation is acrucial part in AI.When AI is performed within a relatively short period of time after semen collection,boar semen is usually preserved at room temperature to ensure sperm motility and fertilization potential in vitro.However,with the prolongation of preservation time,the quality of semen decreases rapidly,which limits the application of the preservation of boar semen at room temperature.In addition,although the status changes of boar sperm during preservation at room temperature have been conducted in some studies,the molecular mechanism of mitochondrial dependent apoptosis induced by reactive oxygen species(ROS)underlying preservation at room temperature of boar sperm is still unclear.Therefore,this study analyzed the correlation between boar semen quality and ROS level during preservation at room temperature and explored discussed the effect of ROS-induced mitochondrial damage on boar sperm apoptosis,as well as the effect of curcumin and N-acetyl-L-cysteine(NAC)on sperm apoptosis.The main results of this study are as follows:1.In this study,boar semen was preserved at room temperature with the home made dilution for 9 d,and the sperm motility,plasma membrane and acrosome integrity as well as other indexes were measured in different stages during preservation.In the process of preservation at room temperature,with the extension of storage time,the quality of boar semen decreased in time dependent.There was negative correlation between ROS level and semen quality.Sperm motility,acrosomal integrity,plasma membrane integrity and antioxidant capacity were significantly decreased.The sperm motility rate had no significant change at 1 d.A significant reduction(P < 0.05)of sperm motility at 3 d was detected compared with that at 0 d and 3 d under preservation at room temperature,and sperm motility was reduced to 65.45% ± 1.13% at 7 d,which was close to 60%.The acrosome integrity and plasma membrane integrity of sperm were lower at 7 d than these at 0 d(P <0.05).And these were 64.45% ± 2.23%和 65.78% ± 2.95% at 7 d respectively.The effective storage time of semen was 7 d.ROS level at 3 d was significantly higher than that at 0 d and1 d(P < 0.05).ROS level at 7 d was significantly higher than that at 5 d(P < 0.05),and the level of ROS increased faster.The activities of T-AOC and antioxidant enzymes(CAT,SOD and GPX)in sperm at 7 d were significantly lower than these at 0 d(P < 0.05),while the content of MDA increased(P < 0.05).ATP content at 7 d was also lower than that at 0 d(P <0.05).2.The apoptosis level of sperm in different stages of boar semen preservation at room temperature was measured,which indicated that ROS-induced apoptosis occurred in the process of sperm preservation at room temperature.ROS level was significantly correlated with mitochondrial membrane potential(r =-0.979),ATP content(r =-0.804),sperm apoptosis level(r = 0.949)and mitochondrial T-AOC(r =-0.789).It caused lipid peroxidation of sperm and MDA increase significantly at 3 d and 7 d(P < 0.05).The antioxidant enzymes in mitochondria of sperm were insufficient to remove excessive ROS,resulting in that the activities of T-AOC and antioxidant enzymes(CAT,SOD and GPX)in mitochondria at 7 d were significantly lower than those at 0 d(P < 0.05).Mitochondrial transmembrane potential at 3 d and 7 d were lower than that at 0 d significantly(P < 0.05).Mitochondrial membrane structure was damaged and function decreased,leading to sperm apoptosis.This caused sperm apoptosis increased significantly at 3 d and 7 d(P < 0.05).3.In order to study the effect of curcumin and NAC on the apoptosis of boar sperm when preserved at room temperature,different concentration curcumin(0,5,10,15 and 20μmol/L)and NAC(0,12.5,25,50 and 100 μmol/L)were added to the semen extender.And the semens were preserved at room temperature for 3 d respectively.The results showed that adding 15 μmol/L curcumin and 50 μmol/L NAC to the semen extender could increase the sperm motility,and the sperm motility rate was 84.37% ± 0.42% and 86.24% ± 1.12% at 3 d.It indicated that the optimal concentration of curcumin was 15 μmol/L.ROS level in the group with 15 μmol/L curcumin or 50 μmol/L NAC was lower than that in the control group(P < 0.05)and mitochondrial membrane potential was higher than that in the control group(P < 0.05).Through apoptotic pathway analysis,we found that the release of cytochrome C(Cyt C)in mitochondria into cytoplasm was repressed(P < 0.05).Then the expression levels of apoptosis-related proteins were detected by Western Blot.The results showed that the expression of Cleaved caspase-3,Cleaved caspase-9 and pro-apoptotic protein Bax in curcumin and NAC group were lower than those in the control group(P < 0.05).The relative expression of anti-apoptotic protein BCL-2 in curcumin and NAC group was higher than that in the control group(P < 0.05).Thus,the apoptosis of sperm was inhibited(P < 0.05).In conclusion,sperm quality gradually decreased during preservation of boar semen at room temperature.Overproduction of ROS reduced mitochondrial antioxidant capacity,impaired mitochondrial membrane structure and function,decrease ATP content,and then led to sperm apoptosis.The addition of 15 μmol/L curcumin and 50 μmol/L NAC to semen extender could increase the mitochondrial transmembrane potential.Then the expression of pro-apoptotic protein BAX was inhibited.The expression of anti-apoptotic protein BCL-2was increased.And the release of Cyt C in mitochondria into cytoplasm and the activation of Caspases pathway was inhibited by removing ROS.and the apoptosis of sperm was inhibited. |
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