Effects Of Tea Polyphenol And Chitosan On The Quality Of Boar Semen During Normal Temperature Preservation

Reactive oxygen species(ROS) produced by boar semen during storage at room temperature. In normal circumstances, there is an equilibrium between the generation of ROS and antioxidant strategies of spermatozoa, leaving only a critical amount of ROS reguired for normal sperm functions, such as capacitation, acrosome reaction and fusion with the oocyte membrane. Excessive ROS will attack sperm membrane, lead to lipid peroxidation,destruction the function of spermatozoa. Besides, bacterium can also damage to spermatozoa during preservation, affect the function of spermatozoa, and reduce the pregnancy rate. In order to improve the effect of boar semen during preservation at room temperature,superaddition with antioxidant and antimicrobial such as tea polyphenol(TPP) and chitosan(CS) were added in the extender of boar semen during preservation at room temperature, and sperm motility, plasma membrane integrity, acrosome integrity,malondialdehyde(MDA) and total antioxidative capacity(T-AOC) activity were measured to determine the optimum concentration and the antibacterial mechanism. The results were as following:1. Different concentrations(0.01,0.02,0.04,0.08 and 0.16 g/L) of TPP were added into the basic extender, the result showed that the semen sample diluted with basic extender containing 0.04 g/L TPP could improve the effect of semen preservation effectively. After 6days preservation, the sperm motility, plasma membrane integrity and acrosome integrity were 60.02%, 46.40%, 75.25% respectively(P<0.05), MDA was 2.39 nmol/L lower than other groups(P < 0.05), while no statistical difference was observed for T-AOC(2.31 U/m L)activity compare with others( P>0.05).In conclusion, TPP could inhibite the growth of bacterium in semen.The growth rate of bacterium were significantly lower than the control group during 4 ~ 6 day( P<0.05), this indicated TPP(0.04 g/L) is helpful for semen preservation at room temperature preservation.2. Different concentrations(0.05,0.1,0.2,0.4 and 0.8 g/L) of CS were added into the basic extender. With the increasing of the concentration of CS in boar semen extender during preservation at room temperature, the effect of semen preservation was firstly increased and then decreased. After 6 days preservation, sperm motility which supplemented with 0.1 and0.2 g/L CS were significantly higher than other groups, and the the sperm motility with 0.1g/L CS was 61.56%, higer than the group with 0.2 g/L CS, but the difference was not significant(P>0.05). Besides, basic extender supplemented with 0.1 g/L CS could significantly improve the semen plasma membrane integrity, acrosome integrity and T-AOC concentrations, reduced the MDA content and the count of bacterial colonies in semen(P<0.05). In conclusion, basic extender supplemented with CS could improve the quality of boar semen during storage at room temperature, and 0.1 g/L CS was the optimum concentration.3. After 6 days preservation, TPP(0.04 g/L) and CS(0.05 g/L) was the best combination in boar semen extender, the sperm motility, plasma membrane integrity were 67.4% and54.07% respectively, higher than the groups added TPP or CS individually. Compared with the control group, the compatibility of TPP and CS could reduce the count of bacterial colonies, enhanced the activity of SOD, CAT and GSH-PX in boar semen(P<0.05), but compared with the added TPP or CS individually, GSH-PX activity improvement was not significant difference between them(P>0.05). Combined use the optimum concentration(0.01g/L and 0.04 g/L) that added TPP and CS individually, the results indicated that the count of bacterial colonies in semen were reduced(P<0.05),but sperm motility and plasma membrane integrity were lower than other groups(P<0.05).4. E. coli was treated with TPP-CS and the results showed that E. coli cell membrane shrinkage or even rupture, and contents spilled. At the same time, it can be observed that some particulates enter into bacterial intracellular, some bacteria also appear plaque.Determination of the total bacterial proteins by SDS-PAGE, after 2 hours treated, 20 k Da and50~70 k Da protein bands decreased significantly. After 6 hours, protein band keep stability but decreased at different extents compared with the control group. There is scarcely protein band below 35 k Da, These results indicated that the E. coli protein expression was severely inhibited. In conclusion, the combination of TPP(0.04 g/L) and CS(0.05 g/L) in extender could damage E. coli by destructing the cell membrane, further destructing the cytoplasm and nucleus then hinder the expression of protein.