Effects Of Three Antioxidants On Boar Semen Preservation

With the development of artificial insemination technology,there is a growing demand for pig semen.However,due to the particularity of large amount of unsaturated fatty acids in the cells of pig sperm,it is difficult to be widely used in production.Therefore,it is necessary to add antioxidants into pig semen to improve the quality of sperm.Tanshinone IIA,ganoderma lucidum polysaccharide and rutin are all natural and effective antioxidants,which have not been used in the study on the preservation of pig semen.This experiment use the long white boar semen as test object,adding different concentration of tanshinone IIA,ganoderma lucidum polysaccharides and rutin in the pig semen diluent,after 17? room temperature preservation and freezing-thawing using computer-assisted Sperm analyzer(Computer-aided Sperm Analys,CASA),a vigor of the microscope to detect Sperm,swelling in the low permeability test of Sperm plasma membrane integrity,isothiocyanate labeled peanut agglutinin(FITC-PNA)test of Sperm acrosome integrity,rhodamine 123(Rh123)and propyl iodide organism(PI)to detect Sperm mitochondrial activity,Antioxidant enzyme activities such as superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)were detected with enzyme activity kit,ROS content was detected with fluorescence probe dcfh-da,and MDA level was detected with lipid oxidation kit.Finally,the rutin test group with better sperm quality after freezing and thawing was selected to carry out in vitro fertilization experiment.The results are as follows:1.The addition of tanshinone IIA with a certain concentration in the normal temperature diluent of pig semen can significantly improve sperm motility,plasma membrane integrity and acrosome integrity after 6 days of preservation(P < 0.05).When the concentration of tanshinone IIA was 0.9mM,the sperm viability and acrosomal integrity were significantly improved,reaching 51.43±0.91% and 58.20±1.13%,respectively.2.In the pig semen diluent frozen,add 1.2 mM of tanshinone IIA,can significantly enhance the vitality of the sperm(60.36±1.97%),living rate(53.38±3.64%),mitochondrial activity and acrosome integrity,significantly reduce the MDA content and increase of antioxidant enzyme activity of SOD and GSH-Px enzyme activity(P < 0.05).Meanwhile,the addition of tanshinone IIA did not play a role in protecting the integrity of sperm plasma membrane and improving the activity of CAT enzyme.3.Adding ganoderma lucidum polysaccharides to the normal temperature diluent of pig semen can improve the quality of sperm.When 0.4mg/mL of ganoderma lucidum polysaccharide was added,after 6d of preservation,the sperm viability and acrosome integrity was also significantly improved(P < 0.05).However,in terms of plasma membrane integrity the addition of 0.2-0.4mg/mL ganoderma lucidum polysaccharide can significantly improve the plasma membrane integrity.4.The addition of 0.8mg/mL of ganoderma lucidum polysaccharide to the frozen diluter of pig semen can significantly improve the activity,plasma membrane integrity,acrosome integrity,antioxidant enzyme CAT and GSH-Px of thawed sperm(P < 0.05),but it has no significant effect on improving mitochondrial activity.The addition of 0.4-0.8mg/mL of ganoderma lucidum polysaccharide can significantly improve the SOD enzyme activity and reduce the MDA content of the product of lipid peroxidation reaction(P < 0.05).5.Adding certain concentration of rutin to the normal temperature diluent of pig semen can significantly improve sperm motility,mitochondrial activity,plasma membrane integrity and acrosomal integrity(P < 0.05).When adding 0.4mM of rutin,the preservation effect is the best.6.Addition of 0.6mm rutin in pig sperm freezing diluent can significantly improve the motility of sperm after thawing,improve the physiological characteristics of sperm(P < 0.05),and significantly reduce the lipid peroxidation of sperm(P < 0.05).In vitro fertilization,the addition of 0.6mM rutin significantly increased the total effective fertility rate(P < 0.05).In conclusion,adding antioxidants tanshinone IIA,ganoderma lucidum polysaccharides and rutin in pigs under normal temperature preservation diluent can significantly increase the living rate of sperm plasma membrane and acrosome integrity,significantly improve the physical characteristics of cryopreservation diluents and activity of antioxidant enzymes,and reduce the occurrence of lipid peroxidation level.