Effect And Mechanism Of Kojic Acid On In Vitro Fertilizaton Of Boar Semen Preservation At Room Temperature

The preservation of boar semen at room temperature is a main step of artificial insemination,and bacterial loading is one of the main obstacles faced by this part.During boar semen preservation at room temperature,the bacterial loading resulted in the decrease of sperm quality parameters and oxidative stress.The amount of reactive oxygen species(ROS)in boar semen is a double-edged sword.A small amount of ROS in boar semen can maintain sperm capacitation and other metabolic functions.However,excessive ROS could lead to sperm DNA breakage,sperm membrane integrity destruction,damage to mitochondria,etc.,affecting the normal physiological functions of sperm.Kojic acid as a natural bacteria extract with antioxidant and antibacterial ability.Previous study indicated that the addition of kojic acid can improve the quality and total antioxidant capacity of boar semen by inhibiting the growth of bacteria when preserved at room temperature.In order to explore the effect of kojic acid on the preservation of boar semen at room temperature on days 5,the effects of kojic acid on in vitro fertilization parameters,mitochondrial vacuolation and pyknosis,anti-apoptotic proteins,pro-apoptotic proteins,capacitated sperm motility parameters,protein tyrosine phosphorylation and sperm capacitation were explored.The main results of this study are as follows:1.The addition of 0.04 mg/mL kojic acid significantly increased the number of oocytes-bound sperm,monospermy rate,cleavage rate and blastocyst rate(P<0.05).What’s more,the optimal in vitro artificial fertilization of the sperm-oocyte concentration is 2.0×10~4,which can significantly inhibit multiple spermatozoa and promote early embryonic development(P<0.05).2.Adding 0.04 mg/mL kojic acid can reduce the mitochondrial vacuolar rate and pyknosis rate,inhibit the release of cytochrome C from sperm mitochondria to cytoplasm(P<0.05),by reducing the production of reactive oxygen species.Thus,the relative expression level of sperm apoptotic protein(Bcl-2)was increased(P<0.05),and the relative expression levels of anti-apoptotic proteins(Bax,Caspase-3 and Caspase-9)were decreased(P<0.05).3.The motor parameters of sperm after capacitation were significantly improved,the tyrosine phosphorylation level of sperm-capacitance-related proteins and increase the proportion of successful capacitated sperm were increased,and also,the sperm acrosome reaction was inhibited(P<0.05)after adding 0.04 mg/mL kojic acid to the boar semen dilution.In summary,the addition of 0.04 mg/mL kojic acid significantly increased sperm-oocyte binding and early embryonic development in vitro.The mechanisms were related to sperm protein tyrosine phosphorylation levels increased and pro-apoptotic protein expression reduced.