Effects Of EGTA And W7 On Liquid Preservation Of Boar Semen

Artificial insemination,which is widely used technology in modern animal breeding to avoid the spread of disease and save costs,can improve livestock reproduction and modify the traditional production methods.The quality of boar semen,which affects the conception rate and litter size of sows directly,has great influence on artificial insemination.Therefore,it is particularly,important to properly preserve boar semen in vitro.There are many factors that can influence semen quality such as:preservation solution,storage temperature,and pH.Room temperature liquid preservation of semen is a commonly used method in pig reproduction.At this temperature,metabolism of spermatozoa still exists and ROS accumulates,thus the acrosome reaction and capacitation of sperm are tended to happen.Calcium,which binds to calmodulin and activates the Ca²⁺/CaM signaling pathway,provoke sperm to occur capacitation and result in hyperactivation.The Ca²⁺/CaM complex regulates the activity of a variety of enzymes,causing the phosphorylation of proteins,particularly tyrosine phosphorylation,which is a capacitation marker.Calcium regulates protein tyrosine phosphorylation via Ca²⁺/CaM signaling and cAMP/PKA signaling pathways.In order to inhibit the capacitation of spermatozoa during preservation at room temperature,two experiments were designed in this study.The first experiment was to clarify the effect of EGTA,a chelating agent,on liquid preserved sperm quality by adding EGTA,to the Modena to chelate calcium ions and prevent the extracellular calcium ions from entering the cell.In the second experiment,the calmodulin antagonist W7 was added to the extender.W7 can compete with the CaM binding protein directly,and inactivate the CaM-dependent binding protein,the Ca²⁺/CaM signal pathway was hindered,thus protein phosphorylation and capacitation of sperm were inhibited.Firstly,different concentration of EGTA（0（control）,1.5,3,6,12 mM）and 6.3 mM EDTA were added to the Modena preservation solution respectively.It was found that 3 mM EGTA was more suitable for sperm preservation than 6.3 mM EDTA.It was further confirmed that the addition of chelators to suppress capacitation can improve the quality of the sperm and prolong the survival time.Secondly,0（control）,25,50,75,and 100μM W7 were added to the preservation solution,and the extender with 50μM W7 was beneficial for the preservation of boar semen.Moreover the effect of EGTA and W7 on the ROS production of sperm preserved 5 days were examined.ROS levels and capacitated spermatozoa in EGTA and W7 treated groups were reduced significantly compared to the control group.And ROS levels of EGTA groups were significantly lower than that of the EDTA group.Finally,effects of EGTA and W7 on sperm fertilization were examined.From the results of fertilization and blastocyst rate,it was seen that calcium ion chelators and calmodulin antagonist W7,could effectively controlled sperm capacitation and improve sperm quality and fertilization ability.Moreover,the fertilization and blastocyst rate of EGTA treated groups were significantly higher than the EDTA group.Through the above experimental results,the following conclusions can be drawn:（1）The extender with 3 mM EGTA is contribute to the preservation of boar semen.（2）The extender with 3 mM EGTA is better than EDTA to preserve boar semen at room temperature.（3）The extender with 50μM W7 is beneficial for the preservation of boar semen.（4）The extender with EGTA and W7,respectively,can effectively reduce the production of ROS to the preservation of boar semen,which is beneficial to sperm preservation in vitro.（5）Fertilization of sperm preserved by adding 3 mM EGTA or 50μM W7 dilution,is beneficial to maintain the fertilization capacity.