| Semen cryopreservation technology is one of the important links in the animal reproduction and breeding system,and the use of this technology can achieve long-term stable preservation of animal sperm.However,in the process of semen freezing,sperm cells are very susceptible to oxidative damage,which damages their plasma membrane structure and DNA integrity to varying degrees,resulting in reduced sperm motility and motility after thawing.It ultimately affects the application effect of artificial insemination using frozen semen.Reduced glutathione(GSH)is an important antioxidant,which can induce glutathione peroxidase(GSH-Px)to effectively scavenge reactive oxygen species(ROS)and maintain the stability of sperm nuclear structure by protecting disulfide bonds.At present,it has outstanding application effect in the cryopreservation of semen such as cattle and pigs,but the application research in the cryopreservation of dairy goat semen is still blank.Therefore,in this study,different concentrations(0,1.0,2.0,3.0 and 4.0 mmol/L)of GSH were added to the frozen diluent of dairy goat semen for semen freezing,and a computer-assisted sperm analysis system was used.Comparative analysis of sperm total motility and viability,acrosome integrity,plasma membrane integrity,antioxidant indexes and energy metabolism with multi-function microplate reader and other instruments,to evaluate the effect of GSH on dairy goats The effect of semen cryopreservation and its optimal concentration in cryopreservation diluent are expected to provide a scientific basis for the improvement of dairy goat semen cryopreservation.The main results of this study are as follows:1.GSH at different concentrations(0,1.0,2.0,3.0 and 4.0 mmol/L)was added to the frozen diluent of dairy goat semen for semen freezing.The results showed that when the concentration was 1.0,2.0 and 3.0 mmol/L,the total motility of thawed sperm could be significantly improved(P < 0.05).Among them,when the concentration was 2.0 mmol/L,the effect was the most significant,and the sperm total motility after thawing was 62.14%.At the same time,when the addition concentration was 1.0,2.0 and 3.0 mmol/L,the acrosome integrity rate,head plasma membrane integrity rate and tail plasma membrane integrity rate of sperm after thawing were also significantly higher than those in the control group(P < 0.05).Among them,the addition concentration of 2.0 mmol/L has the most significant effect.The acrosome integrity rate,head plasma membrane integrity rate and tail plasma membrane integrity rate of thawed sperm reached 70.87%,48.06% and 37.62%,respectively.2.Different concentrations(0,1.0,2.0,3.0 and 4.0 mmol/L)of GSH were added to the frozen diluent of dairy goat semen for semen freezing,which had different effects on the antioxidant properties of thawed sperm.Among them,when the concentration was 1.0 and2.0 mmol/L,the total antioxidant capacity(T-AOC),catalase(CAT)activity,superoxide dismutase(SOD)activity and Glutathione peroxidase(GSH-PX)activity of thawed dairy goat sperm could be significantly improved(P < 0.05),while the content of malondialdehyde(MDA)and the level of reactive oxygen species(ROS)were significantly decreased(P < 0.05).And compared with the control group,the effect was most significant when the concentration was 2.0 mmol/L(P < 0.05),the total antioxidant capacity(T-AOC),catalase(CAT)activity,superoxide dismutase(SOD)activity and glutathione peroxidase(GSH-PX)activity of sperm after thawing reached 11.20 U/m L,19.81 U/m L,218.23 U/m L and 133.63 U/m L respectively,while the content of malondialdehyde(MDA)and reactive oxygen species(ROS)levels were only 5.05 nmol/L and 395.58 U/m L3.Different concentrations(0,1.0,2.0,3.0 and 4.0 mmol/L)of GSH were added to the frozen diluent of dairy goat semen for semen freezing,which had different effects on the energy metabolism of sperm after thawing: Among them,the addition of concentrations of1.0,2.0 and 3.0 mmol/L could significantly increase the content of adenosine monophosphate(ATP)and the level of mitochondrial membrane potential(MMP)in thawed sperm(P < 0.05).And compared with the control group,when the concentration was 2.0 mmol/L,the content of adenosine phosphorylase(ATP)and the level of mitochondrial membrane potential(MMP)in sperm increased most significantly(P < 0.05),reaching 0.62 μmol/m L and 6.09ΔΨM respectively.However,there were no significant differences in the content of adenosine phosphorylase(ATP)and mitochondrial membrane potential(MMP)levels in sperm after thawing between the two groups with concentrations of 1.0 and 3.0 mmol/L(P > 0.05).When the added concentration was increased to 4.0mmol/L,the content of adenosine monophosphate(ATP)and the level of mitochondrial membrane potential(MMP)in sperm after thawing were not significantly different from those in the control group(P > 0.05).This study shows that adding an appropriate amount of reduced glutathione to the frozen dilution of dairy goat semen can significantly improve the sperm motility,plasma membrane integrity and acrosome integrity after thawing.At the same time,it can effectively improve the antioxidant properties of sperm during cryopreservation,and improve the energy metabolism of sperm,thereby improving the cryopreservation effect of dairy goat semen.And the effect of reduced glutathione was the most significant when the concentration of reduced glutathione was 2.0 mmol/L. |
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