| Purpose: The survival time of sperm in vitro limits the efficiency of utilizing superior male animals.Therefore,improving the storage time and quality in vitro becomes a crucrial step in enhancing the effectivenedd of artificial insemination.Punicalagin and epicatechin are two different antioxidants.Exploring the impact of these antioxidants on the preservation of sheep can provide a basis for improving the reproductive capacity of excellent male sheep and preserving genetic resources.Methods:(1)Preservation of Duolang sheep at room temperature with different antioxidants: Firstly,three healthy Duolang male sheep were collected by the artificiall vagina.Sheep diluent mainly composed of "Tris+glucose+fructose" was used to prepare sheep diluent added with punicalagin(10,12.5,15,17.5,20μmol/L)and epicatechin(250,300,375,450,600 μmol/L),respectively.The preservation effect of Duolang sheep at room temperature was comprehensively evaluated by measuring sperm viability,motility,acrosome integrity,plasma membrane integrity,as well as the activity of superoxide dismutase(SOD)and the content of catalase(CAT),which are antioxidants.(2)Preservation of Duolang sheep at low temperature with different antioxidants: Egg yolk(10%)was added to the diluent at room temperature,and then sheep diluent was added with punicalagin(10,12.5,15,17.5,20 μmol/L)and epicatechin(250,300,375,450,600 μmol/L)were prepared,respectively.The preservation effect of Duolang sheep at low temperatures was comprehensively evaluated by measuring sperm viability,motility,acrosome integrity,plasma membrane integrity,as well as the activity of superoxide dismutase(SOD)and the content of catalase(CAT),which are antioxidants.(3)Cryopreservation of Duolang sheep with different antioxidants added: 5% glycerol was added to the low-temperature preservation diluent as a cryoprotectant,and sheep diluent was added with punicalagin(10,12.5,15,17.5,20 μmol/L)and epicatechin(250,300,375,450,600 μmol/L)was prepared,respectively.The preservation effect of cryopreservation was evaluated by measuring sperm viability,motility,acrosome integrity,plasma membrane integrity,as well as the activity of superoxide dismutase(SOD)and the content of catalase(CAT),and the cryopreservation effect was further judged by artificial insemination of Hu sheep ewes in the same estrus period,and the pregnancy rate was detected.Results: The results showed that(1)When Duolang sheep were preserved at room temperature,sheep diluent added with 15 μmol/L punicalagin or 375 μmol/L epicatechin had the highest sperm viability,motility,acrosome integrity,plasma membrane integrity,CAT activity,and SOD activity,which were all significantly higher than the control group.The motility of sperm in both groups on the third day after preservation was42.82% and 51.55%,respectively.(2)When Duolang sheep were preserved at low temperatures,sheep diluent added with 15 μmol/L punicalagin or 375 μmol/L epicatechin had the highest sperm viability,motility,acrosome integrity,plasma membrane integrity,CAT activity,and SOD activity,which were all significantly higher than the control group.The motility of sperm in both groups on theResults: The results showed that(1)the sperm viability,viability,acrosome integrity,plasma membrane integrity,CAT activity,and SOD activity were the highest when the semen dilution of "Tris+ glucose +fructose" was supplemented with 15 μmol/L gratin or 375 μmol/L epicatechin,respectively.The sperm motility of the two groups was 42.82% and 51.55%,respectively,on day 3 after preservation.(2)The sperm viability,motility,acrosome integrity,plasma membrane integrity,CAT activity,and SOD activity of the sperm were also the highest and significantly higher than those of the control group after the dilution of 15μmol/L again or 375 μmol/L epicatechin groups were added,respectively.Sperm motility was 57.93% and58.14% on the 7th day after preservation.(3)When the semen of Duolang sheep was cryopreserved,the dilution of 15 μmol/L angrein or 375 μmol/L epicatechin groups were added,respectively.After cryopreserved semen of liquid nitrogen cryopreserved,sperm viability,acrosome integrity,plasma membrane integrity,CAT activity,and SOD activity were the highest,and were significantly higher than those of the control group.The cryopreservation rate of semen with antioxidants was 55% and 60% in the two groups.Conclusions:(1)Adding 15 μmol/L again or 375 μmol/L epicatechin to sheep semen base diluent mainly "Tris+ glucose + fructose" can improve the preservation effect of sheep at room temperature.(2)The survival time of ovine sperm was prolonged by adding 15 μmol/L anti garnet or 375 μmol/L epicatechin in low-temperature diluent with the yolk as cryoprotectant.(3)Glycerol was used as an antifreeze agent,and15 μmol/L angaratine or 375 μmol/L epicatechin was also added as freezing diluent,which was beneficial to the cryopreservation of Duolang sheep semen... |
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