| As an excellent local sheep breed in China,Hu Sheep has excellent breed characteristics such as high reproduction rate,strong adaptability and suitable for large-scale housing,and can be developed and utilized as an excellent germplasm resource at a time when China’s large-scale meat sheep industry is developing rapidly.The development of the meat sheep industry must focus on improving reproductive efficiency,and the industrial promotion of artificial insemination technology is an important solution to this problem.Semen preservation technology,as an important part of artificial insemination technology,is key to ensuring that high quality semen is used for production.Semen low temperature preservation has the advantages of simple operation,low-cost investment,no time and geographical restrictions on the exchange of germplasm resources,and a better fertility rate in production practice,and is widely used by breeding subjects.However,changes in temperature during low temperature preservation can easily lead to cold shock and accumulation of reactive oxygen species in sperm,resulting in damage to the structural and functional integrity of sperm membranes and seriously affecting semen quality.Therefore,the study of antifreeze and antioxidant substances in semen low temperature preservation dilutions is of great importance.In this experiment,four types of antifreeze substances(soybean lecithin,glycerol,ethylene glycol and dimethyl sulfoxide.DMSO)were added to the base diluent with fructose-sodium citrate-penicillin-streptomycin,and the sperm viability,motility,velocity parameters(straight line velocity VSL,curvilinear velocity VCL,average path velocity VAP)and spatial displacement degree parameters(amplitude of lateral head displacement ALH,beat-cross frequency BCF,Average motion degree MAD)were detect by automatic sperm analysis systems(CASA),and the optimal concentration of each antifreeze substance was screened.Secondly,at the optimal concentration of each antifreeze substance,sperm kinetic parameters were measured by CASA,sperm plasma membrane integrity by hypotonic swelling assay(HOST)and sperm acrosome integrity by Giemsa staining to determine the best antifreeze substance for the low temperature preservation of Hu sheep semen.Next,different concentrations of the antioxidant α-lipoic acid(0,0.025,0.05,0.1,0.5 and 1 mM)were added to the dilutions screened for optimal addition of the best anti-freezing substance,and the spermatozoa were examined by measuring sperm kinetic parameters,plasma membrane integrity rate,acrosome integrity rate,and the reactive oxygen species(ROS)content,total antioxidant capacity(T-AOC),lipid oxidation malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,adenosine triphosphate(ATP)content and mitochondrial membrane potential(MMP),of sperm were measured using a multimode microplate detection system after 1d,3d,and 5d of storage to explore the effect of antioxidant lipoic acid on the low temperature storage of Hu sheep semen.The main results obtained in this study are as follows:1.In this study,the optimal concentrations of the four antifreeze substances were screened,and according to the results of semen preservation from 0 to 6 days,0.1%soybean lecithin was optimal concentration among the different concentrations of soybean lecithin(0%,0.1%,0.2%,0.3%,0.4%);among the different glycerol concentrations(0%,1%,2%,3%,4%,5%,6%),2%glycerol was the optimal concentrations for preservation;among the different ethylene glycol(0%,1%,3%,5%,7%,9%),5%ethylene glycol was the optimal concentrations for preservation;among the different DMSO concentrations(0%,0.1%,0.3%,0.5%,0.7%,0.9%),0.7%DMSO was the optimal concentrations for preservation.2.The results of comparing the optimal concentrations of four antifreeze substances(0.1%soybean lecithin,2%glycerol,5%ethylene glycol,0.7%DMSO)to determine the best antifreeze substance for low temperature preservation of Hu sheep semen:The results of sperm viability,motility and plasma membrane integrity from 1 to 6 days showed that the 0.1%soybean lecithin group had significantly higher sperm viability,motility,BCF,MAD and plasma membrane integrity than the other three groups(P<0.05).Sperm VSL was significantly higher in the 0.1%soybean lecithin group than in the other three groups at the 3rd,5th and 6th day of storage(P<0.05).At the 2nd,3rd,5th and 6th day of storage,sperm VCL and VAP were significantly higher in the 0.1%soybean lecithin group than in the other three groups(P<0.05).From 2nd to 6th day of storage,sperm ALH was significantly higher in the 0.1%soybean lecithin group than in the other three groups(P<0.05).The results showed that 0.1%soybean lecithin was the most effective anti-freezing substance in the cryopreservation dilution solution for Hu sheep semen.3.Based on the determination of the optimal anti-freezing substance and its optimal addition concentration,different concentrations of ALA were added,and after 8 days of low temperature preservation,the kinetic parameters,plasma membrane integrity rate and acrosome integrity rate of spermatozoa were compared and analyst to compare the effects of different concentrations of ALA on the motility performance and structural integrity of spermatozoa.The results showed that with the increase of ALA addition,the sperm viability,motility,velocity parameters,ALH,MAD,plasma membrane integrity and acrosome integrity showed a trend of first increasing and then decreasing.Relative to other concentrations,ALA at 0.1 mM significantly improved the kinetic parameters and structural integrity indices of cryopreserved sperm and provided better protection to sperm.4.Different concentrations of ALA were added to the basal dilution solution supplemented with 0.1%soybean lecithin,and the ROS content,T-AOC,MDA content,SOD activity,ATP content and MMP of spermatozoa were examined at 1 d,3 d and 5 d of semen storage to investigate the effects of ALA on the antioxidant properties and energy metabolism of spermatozoa.The results showed that the ROS and MDA contents of spermatozoa in the ALA group at 1 d,3 d and 5 d of storage were significantly lower than those in the control group(P<0.05),and the ROS and MDA contents of spermatozoa in the 0.1 mM group at 3 d and 5 d of storage were significantly lower than those in all other ALA groups(P<0.05);at 3 d and 5 d of storage,the T-AOC,SOD activity,ATP content and The T-AOC,SOD activity,ATP content and MMP of spermatozoa in each group showed a trend of increasing and then decreasing at 3 and 5 d of preservation.A comprehensive analysis of the results showed that ALA at a concentration of 0.1 mM significantly inhibited the production of ROS and MDA,increased the production of ATP and effectively improved the antioxidant properties and energy metabolism level of semen during cryopreservation.The concentration of ALA at 0.1 mM significantly inhibited the production of ROS and MDA,and effectively enhanced the antioxidant properties of semen during low temperature preservation.In conclusion,the addition of 0.1%soybean lecithin and 0.1 mM ALA to the diluent during the low temperature preservation of Hu Sheep semen can provide good protection to the sperm.0.1%soybean lecithin as an antifreeze substance can alleviate the effects of cold shock on sperm,maintain the structural integrity of sperm membranes and provide better protection to sperm.ALA at 0.1 mM can effectively enhance the antioxidant capacity of sperm,inhibit lipid peroxidation(LPO)reaction,improve the enzymatic activity of antioxidant enzymes,reduce the oxidative damage of sperm under low temperature preservation conditions,enhance the energy metabolism level of sperm,and positively contribute to the improvement of semen quality after cryopreservation. |
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