Effect Of Porcine Serum Exosomes On Preservation Of Porcine Semen At Room Temperature

Maintaining sperm quality during livestock semen storage helps to enhance the effect of artificial insemination.During semen preservation,the redox homeostasis in the system is unbalanced,and excessive ROS accumulation causes lipid peroxidation,destroys sperm membrane structure and function,and even induces DNA damage in sperm nucleus,leading to sperm death.In order to effectively avoid the damage of sperm in the process of preservation,some exogenous protective agents are often added in the process of boar semen preservation,such as egg yolk,polysaccharide and so on.If the substances from pigs can be added,and the same or better preservation effect can be obtained as exogenous protective agents,so as to avoid the potential negative impact of exogenous substances on sperm or offspring,this substance will be very valuable.Porcine serum is rich in sources.As an extracellular vesicles,its serum exosomes load many bioactive proteins and lipids,and can be used as protective agents of their own sources to participate in the preservation of porcine semen in vitro ? In this study,exosomes from castrated boars serum(cbsEXO)and exosomes from sow serum(ssEXO)were added to boar semen to compare their effects on semen quality,and then the possible mechanism was analyzed.The results are as follows:(1)Separation and characterization of cbsEXO and ssEXO.500 m L serum exosomes were extracted from 4 months castrated boars and 3.5～4 years sows by ultracentrifugation.Scanning electron microscopy(SEM)and Nano Sight analyzer showed that cbsEXO and ssEXO were spherical,and the particle size was 30～200 nm.Both cbsEXO and ssEXO were loaded with exosome marker proteins CD63,CD9 and Alix by Western blot.(2)The effects of cbsEXO and ssEXO on semen quality were compared.In the cbsEXO group,when the concentration of exosomes added was four times that of the original seminal plasma(cbs Exo-4),the linear motility of sperm was the best;in the ssEXO group,the effect was the best when the added exosome concentration was 16 times(ss Exo-16)of the original seminal plasma.More importantly,from day 5,the plasma membrane integrity,acrosome integrity,mitochondrial membrane potential and ATP content of sperm in the exosome-added groups(cbs Exo-4 and ss Exo-16)were significantly higher than those in the control group(P < 0.05).These results showed that the addition of serum exosomes to semen during normal temperature preservation could delay the decline of semen quality.(3)In the normal temperature preservation of porcine semen,cbsEXO-4 and ssEXO-16 can significantly improve the antioxidant activity of sperm.After adding cbsEXO-4 and ssEXO-16 to boar semen diluents at room temperature,the contents of reactive oxygen species(ROS)and malondialdehyde(MDA)in sperm of the experimental group were significantly lower than those of the control group from the fifth day(P < 0.05),while the activities of total antioxidant capacity(T-AOC)and total glutathione peroxidase(GPX)were significantly higher than those of the control group(P < 0.05).The results showed that the addition of serum exosomes to semen during normal temperature preservation could help reduce the oxidative stress of sperm and improve the antioxidant capacity of sperm.(4)The zona pellucida binding experiment was carried out after the incubation of exosomes with sperm,and it was found that the number of sperm binding to zona pellucida in the cbsEXO-4 group was greater than that in the ssEXO-16 group,and both were greater than those in the control group.Therefore,the addition of porcine serum exosomes can promote the combination of sperm and egg zona pellucida.In addition,in order to further explore the way of serum exosomes affecting sperm,Di I was used to label the co-incubation of exosomes and sperm in this study.Combined with the SEM results,it was found that exosomes could bind to the head and tail of sperm,indicating that exosomes could directly bind to the sperm surface,reconstruct the sperm membrane structure to play a role and affect sperm function.In summary,the results of this study showed that the addition of cbsEXO and ssEXO to the diluent of boar semen stored at room temperature could effectively improve the semen quality,and the improvement effect of cbsEXO-4 was greater than that of ssEXO-16.cbsEXO and ssEXO play a protective role by directly binding to sperm surface,reconstructing sperm membrane structure and improving sperm antioxidant capacity and fertilization ability.