| Hu sheep is a famous prolific sheep breed in the world,which has the advantages of polyestrous cycle,two litters a year,and rapid growth and development.With the large-scale popularization and application of artificial insemination(AI)technology of Hu sheep,there is an increasing demand for Hu sheep semen from breeding enterprises and farmers.However,at present,the problems such as short preservation time of Hu sheep semen at room temperature,low sperm progressive motility and low conception rate have seriously affected the further expansion of Hu sheep breeding scale in our country.Therefore,the highly efficient and stable diluent formula and preservation temperature of Hu sheep semen become the key,and the addition of antioxidants has become the effective research direction of Hu sheep semen preservation.In this experiment,Taurine(Tau)is selected as an antioxidant.To explore its effect on the preservation of Hu sheep semen at room temperature,firstly,the formula with the best preservation effect was selected from five diluents preserved at room temperature,and then the optimum temperature for preservation of Hu sheep semen at room temperature was selected.Finally,Tau of 10,20,40,80 and 100 mM was added to this formula.The effects of Tau on of motility parameters such as sperm progressive motility,functional integrity such as plasma membrane integrity,oxidative stress parameters such as Malondialdehyde(MDA)content,mitochondrial membrane potential and fertility rate on the preservation of Hu sheep semen at room temperature were analyzed.The main results of this study are as follows1.Among the five diluents preserved at room temperature,the diluent A has the best preservation effect,which is mainly composed of 15.35 g Tris,8.20 g citric acid,10.00 g fructose,0.16 g penicillin and 0.35 g streptomycin sulfate dissolved in sterilized ultra-pure water of 500 mL.The sperm total motility,progressive motility,acrosome integrity rate,Straight line velocity(VSL),Curvilinear velocity(VCL),Average path velocity(VAP),Amplitude of lateral head displacement(ALH)and Average motion degree(MAD)of diluent A were the highest and decreased slowly.The effective survival time of sperm preserved by diluent A was 3.08 days,which was significantly higher than that of the other four diluents(P≤0.05).Compared with other diluents,the acrosome integrity rate of diluent A within 2～6 days was significantly higher than that of other diluents(P≤0.05).Compared with other diluents,the plasma membrane integrity rate of diluent A was the highest within 1～4 days of preservation.Therefore,the formula A had a good effect on the preservation of Hu sheep semen at room temperature,and the diluent A was used as the basic diluent for follow-up research.2.Using diluent A,the diluted semen was stored at 15℃,20℃ and 25℃,respectively.The progressive motility of sperm stored at 25℃ was the highest within 2 days.The progressive motility of sperm stored at 20℃ was the highest within 3～5 days.The progressive motility of sperm preserved at 15℃ was significantly higher than that of other groups within 6～9 days(P≤0.05).The effective survival time and total survival time of sperm stored at 15℃ were 5.67 days and 18.73 days respectively,which were significantly higher than those of other groups(P≤0.05).The intact rate of plasma membrane of sperm stored at 15℃ for 1 day was significantly higher than that of other groups(P≤0.05).The plasma membrane integrity rate and acrosome integrity rate of sperm stored at 15℃ and 20℃ were significantly higher than those stored at 25℃ within 3 days(P≤0.05).The intact rate of plasma membrane of sperm stored at 15℃ for 7 days was significantly higher than that of other groups(P≤0.05).After 5 days of preservation,the Reactive oxygen species(ROS)level of sperm stored at 15℃ was significantly lower than that in 25℃group(P≤0.05),Superoxide dismutase(SOD)activity was significantly higher than that in other groups(P≤0.05).Therefore,15℃ was the optimum temperature for semen preservation of Hu sheep at room temperature,and 15℃was used as the semen preservation temperature for follow-up study3.Based on the diluent A,10,20,40,80 and 100 mM Tau were added to the diluent,respectively.The results showed that with the increase of Tau concentration,sperm total motility,progressive motility,plasma membrane integrity rate,acrosome integrity rate,Total antioxidant capacity(T-AOC),SOD activity,Catalase(CAT)activity and mitochondrial membrane potential increased at first and then decreased,while the content of MDA in semen showed the opposite trend,and the content of ROS in sperm decreased gradually.When the concentration was 20mM,it could significantly improve the sperm plasma membrane integrity rate,acrosome integrity rate,SOD activity,CAT activity,T-AOC,mitochondrial membrane potential and total motility,progressive motility,VSL and other motility parameters(P≤0.05).It could also reduce the ROS content of sperm and the MDA content of semen on the 5th day after preservation(P≤0.05).The fertility rate was 75.00%using 20 mM Tau semen stored at room temperature for AI.Therefore,20 mM Tau is the optimum concentration for Hu sheep semen preservation at room temperature. |
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