The Best Formulation Of Sheep Sperm Diluent And Its Dilution Ratio And Storage Temperature

Although sheep cryopreservation technology has been developed,the production cost is high,the sperm viability after freezing is poor,and the conception rate is low.Therefore,the preservation of sheep is mainly based on liquid preservation.Liquid storage is divided into normal temperature storage(15 ~ 25 °C)and cryogenic liquid storage(0 ~ 5 °C).At present,the dilute stock solution of sheep sperm is limited,the preservation time is short,and the dilution is mainly based on low-fold dilution.In order to make full use of excellent ram germ resources,the main purpose of this experiment is to obtain a good sheep semen dilution storage solution formula and the best preservation temperature and dilution ratio,to provide theoretical and technical basis for sheep semen preservation and transportation.In this experiment,the foreign fine varieties of Dubo Yang as the experimental object,based on the Tirs-sodium citrate-fructose-yellow formula(No.I liquid),in order to further optimize the basic formula,first explore the impact of different sugars on the diluent,use Tirs-citric acid-glucose-yolk dilution(II liquid)was diluted 1:2,1:5,1:10 in sheep semen,diluted and then stored at 4°C,10°C,and 18°C.The results showed that there was no significant difference in the preservation time of sheep semen at the same dilution temperature after the dilution of sheep semen with 1:2,1:5,and 1:10 respectively at 4°C,10°C and 18°C.At 4°C,10°C,and 18°C,there was no significant difference in sperm survival time when the two polysaccharide dilutions were 1:5 and 1:10,and the sperm survival time was significantly lower when the dilution ratio was 1:2.The dilution ratio is 1:5 and 1:10.At different dilution ratios,the survival time of sheep sperm at 4°C was significantly higher than that at 18°C;the effect of yolk substitutes on dilutions was further explored,and 0.35% soy lecithin was used to replace 10% egg yolk,Tirs-Citric acid-glucose-soy lecithin dilution(III solution)was diluted 1:5 and 1:10 in sheep's semen and stored at 4°C and 10°C,respectively.The results showed that lecithin dilution and yolk dilution After dilution of 1:5 and 1:10 respectively at 4°C and 10°C,there was no significant difference in sheep semen preservation time at the same dilution temperature and the same dilution.When the dilution ratio was 1:5 at 4°C and 10°C,the sperm survival time was significantly lower than that when the dilution ratio was 1:10.The sperm survival time at 4°C was significantly higher than that at 10°C at different dilution ratios.Time,so choose 1:10 as the optimal dilution,4 °C as the best storage temperature;then explore the effect of antioxidant vitamin B12 on the dilution,add 0.003g/L to Tris-citric acid-glucose-soy lecithin Vitamin B12,the results showed that the Tris-citric acid-glucose-yellow-vitamin B12 dilution(Formula IV)was diluted 1:10 in sheep semen and stored at 4°C.After adding vitamin B12,the sperm preservation time was longer than when vitamin B12 was not added.Significantly improved,;finally explored the effect of glycerol on the dilution,in the Tirs-citric acid-glucose-yolk-vitamin B12 based on the addition of 10% glycerol,studies have shown that adding glycerin at room temperature is harmful to sperm Therefore,due to the addition of glycerol,the sperm must first be diluted to 20 million/mL and placed in a refrigerator at 4°C to reduce the temperature to4°C.Then an equal volume of diluent containing glycerol is added.The results indicate that Tirs-citric acid-Portuguese Diluted ovine semen was diluted to 20 million cells/m L with a glucose-yellow-vitamin B12-glycerol dilution(formula V)and stored at 4°C.Diluent with glycerol added resulted in a significantly longer sperm storage time than unsupplemented glycerol dilutions.In the formula,after the sperm was stored on the fifth day,the spermatozoa acrosome integrity rate and membrane integrity rate were all higher than 50%,and the in vitro fertilization cleavage rate also reached about 50%.Completely meet the requirements of in vitro fertilization to determine formula V as the optimal formulation.Conclusion: Tirs-citric acid-glucose-yellow-vitamin B12-glycerol dilution(formula V)is the best.It can be used for dilution and cryopreservation of sheep sperm.