Study On Cryopreservation Of Boar Semen

Research on the cryopreservation of boar spermatozoa was developed with the application of swine artificial insemination,and was thought as one important milestone in the history of swine industry.A "bank of boar spermatozoa" without the limitation of space-time by semen freezing could provide boar semen from an excellent boar for a lot of sows.In addition,the cryopreservation of boar spermatozoa also could provide a safeguard of biosafety for swine breeding.It was well-known that there is still a big gap between the cryopreservation of boar spermatozoa and the cryopreservation of bovine spermatozoa.So the cryopreservation of boar spermatozoa was still not applied widely in swine industry for some time to come.In order to improve the quality of frozen-thawed boar semen,two ways including freezing methods and freezing extenders were studied in this present study.The spermatozoa parameters(including spermatozoa motion),acrosome integrity,capacitated spermatozoa,membrane integrity,and DNA integrity were evaluated by computer-aided sperm analysis(CASA) system,Fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining,Chlortetracycline(CTC) staining,hypo-osmotic swelling test (HOST) and single cell gel electrophoresis(SCGE),respectively.In this present study,five experiments were carried out.Experiment 1,the effect of N,N-Dimethylformamide(DMF) on freezing boar spermatozoa.Previous studies showed that DMF could provide better protection for freezing spermatozoa than those of glycerol.However,few reports on freezing boar spermatozoa used DMF was found.We attempted to use a mixing cryoprotectant with DMF and glycerol in varing proportions.The results showed that the effect of freezing extender supplemented with 3%DMF was superior to those of freezing extender supplemented with 3%glycerol,and the mixed cryoprotectant(1%glycerol and 2%DMF) provided better protection for freezing boar spermatozoa than those of other treatment groups(P＜0.05).Experiment 2,the effect of centrifugation of egg yolk and dialysis of boar semen during cooling process on freezing boar spermatozoa.Egg yolk was used to protect boar spermatozoa against cold shock due to Low density lipoproteins(LDL) and phospholipid of it's during cool process.Large particulate of egg yolk was thought as a disadvantage factor for cryopreservation and evaluation of spermatozoa motion by CASA system;the role of boar semen plasma should not be neglected due to its property comparing with other domestic animals.So we centrifuged the egg yolk and dialyzed boar semen during cool process to remove the large particulate and the small molecules respectively which was harmful to spermatozoa comparing with the control treatments.Results: centrifugation of egg yolk and dialysis of boar semen during cool process could improve the effect on freezing boar spermatozoa comparing the control treatments respectively (P＜0.05).However,further studies are needed to reduce its cost and complexity.Experiment 3,the protective effect of rhodiola polysaccharide on freezing boar spermatozoa.As aqueous extract from Chinese Herb,rhodiola polysaccharide had strong scavenging activity against superoxide anion radical(O^2-) and inhibited lipid peroxidation. However,there are apparently no published data regarding the use of to prevent formation of ROS during cryopreservation of boar spermatozoa.Based on TCG extender, the effect of different concentrations or methods of rhodiola polysaccharide on freezing boar spermatozoa was evaluated by previous test methods and enzyme assay.Results:1. rhodiola polysaccharide could provide better antioxidant protection for boar spermatozoa during cooling and thaw-thawed process;2.the effect of rhodiola polysaccharide added inⅠextender was better than added inⅡextender and the proper concentration of it was 6 mg/L.Experiment 4,the effect of soy lecithin on freezing boar spermatozoa.Soy lecithin extracting from Soybean was similar with the functional ingredients of egg yolk.It was reported that freezing extender containing soy lecithin could provide better protection for bovine spermatozoa.However,few relative reports on boar were found.We investigated the effect of soy lecithin on cryopreserved boar semen compared with the treatments with 20%egg yolk.Results:1.Soy lecithin could provide better protection than that of 20% egg yolk;2.The proper concentration of soy lecithin was 6%,and the motility,the percentage of acrosome integrity,membrane integrity and DNA integrity were higher than those of 20%egg yolk treatment(P＜0.05).Experiment 5,the effect of glutamine on on freezing boar spermatozoa.Amino acids are the basic components of proteins and can provide energy for cellular metabolism in vivo.Studies have shown that,as one of Amino acids,glutamine could provide better protection for freezing spermatozoa of domestic animals.However,few reports on boar spermatozoa were found.Based on 9%LDL extender,the effect of different concentrations of glutamine on freezing boar spermatozoa was tested in this present study. Results:glutamine could provide better protection for boar spermatozoa during cryopreservation,and the proper concentration was 40 mM.