| With the popularization of artificial insemination technology in the swine industry,semen preservation technology is also concerned.The quality of sperm preservation directly affects the success rate of artificial insemination and the conception rate and litter size of the female.Semen diluent plays a role in protecting the quality of the sperm.Diluent components can maintain sperm motility and protect the integrity of sperm acrosome and plasma membrane.However.the components of the semen diluents on the market is relatively complicated,and the preservation time is not long,and the effect is not stable.In order to develop a diluent formulation with a stable high-efficiency preservation effect,six new porcine semen dilution formulations were designed in this study.These are a modified Modena-A formulation based on Modena.B formulations characterized by antioxidant activity,and C.formulations characterized by adding polymer adding agents.D formulation characterized by the use of a non-anaerobic glycolytic substrate for the sugar source.E formula which is mainly exercise-based,and the F formula to slow down the sperm deposition rate.Compared with the classic porcine semen thinner formula(Modena diluent)and the similar products in the market,the sperm viability,the integrity rate of plasma membrane,sperm deformity rate,the cleavage rate and the blastocyst rate of in vitro fertilization were compared.The sperm preservation effect of each formulation under room temperature and low temperature conditions was further optimized,and a formula of pig semen preservative with excellent effect was preliminarily successfully developed.Thinner products lay the foundation.The following are the main results of this study:1.Screening of the diluent formula for the preservation of semen at room temperature:The determination of the sperm motility,plasma membrane integrity,and sperm deformity rate,which were stored under normal temperature conditions for each formulation,showed that:The formula D has the best preservation effect,and the formula C The preservation effect was the second,and there was a significant difference in the preservation effect between the two after the 7th day(P<0.05).The sperm motility of the D-formula was still above 0.5 on the 9th day,and the plasma membrane integrity and sperm deformity rate were 41%and 74.4%,respectively.After the C formulation was stored to the 8th day,the sperm motility was above 0.5,and the integrity of the plasma membrane and sperm deformity rate were 33.6%and 69%,respectively.Although the preservation effect of formula A was lower than that of formula C and formula D,the viability was still above 0.5 on the 7th day of preservation,and the integrity rate of plasma membrane and sperm deformity rate were 25.3%and 54.5%,respectively.The preservation effect of F ormula E was lower than that of Formula A.The sperm notility was 0.52 on the 7th day of storage.The preservation effect of Formula B and Formula F was significantly lower than that of other formulas(P<0.05),and the sperm motility was 0.49 and 0.52 on the 4th day of preservation.2.The screening of the diluent formula under the condition of low temperature preservation of semen:D formula had the best preservation effect,and the preservation effect of C formula was the second,and there was no significant difference in the preservation effect of the two formulas between 1 and 6 days of preservation(P>0.05)..After the D formula was stored to the 7th day,the sperm motility was still above 0.5,and the plasma membrane integrity rate and sperm deformity rate were 46.4%and 78.2%,respectively.The C-formula was stored to the sixth day of sperm motility above 0.6,and the plasma membrane integrity and sperm deformity rate were 41.6%and 76.4%,respectively.Although the preservation effect of formula A was lower than that of formula C and formula D,the viability was still above 0.5 on the 6th day of preservation,and the integrity of the plasma membrane and sperm deformity rate were 34.4%and 69.6%,respectively.The sperm motility of E formulation was 0.52 at the 4th day of storage.The preservation effect of Formula B and Formula F was significantly lower than that of other formulas(P<0.05),and the sperm motility was 0.43 and 0.53 on the 4th day of storage.3.Formula optimization test:On the basis of selecting the best C formula and D formula through six formulations,the composition of the two formulations was optimized,and two formulations of T1 and T2 were designed and compared with the D formula and the S formula sold in the market,the results showed that the preservation effect of the T1 formula and the T2 formula was better than the D formula and the Cont formula.The preservation effect is good,T2 formula is better than T1 formula,and D formula has higher preservation effect than Cont formula.There was no significant difference in sperm motility,plasma membrane integrity and sperm deformity rate(P>0.05)in the preserved 1?3d,T1 formula and T2 formula.The sperm vitality of T2 formula at 4d and 5d was significantly higher than that of T1 formula(P<0.05).The sperm vitality,plasma membrane integrity rate and sperm deformity rate in the T2 group were significantly different from those of the Cont formula(P<0.05).4.Optimization of in vitro fertilization of semen preserved by the formula Verification:By comparing the splitting ratio of the fertilized eggs after in vitro fertilization with the T2 formulation and the Cont formula,Modena was stored to the 5th day of semen,it can be seen that the semen quality of the T2 formula is superior to the other two formulas.5.The influence of semen shaking frequency on the preservation of semen at room temperature:Shaking operation during different frequency intervals during the preservation of diluted semen,comparative determination of sperm motility,sperm plasma membrane integrity,sperm deformity rate and other indicators of different shaking times.It was shown that there was no significant difference in sperm motility,sperm plasma membrane integrity,and sperm deformity rate between the experimental groups that were incubated with the C formula and shaken once every 12h,1d,and 2d(P<0.05).the sperm motility,sperm membrane integrity rate and sperm deformity rate in the Modena base solution and the Cont formula every 12h,1d,2d and 3d in the experimental group were significantly decreased(P<0.05).This shows that the addition of the polymer X C formula can reduce the number of shaking,so that the management of dilute preservation of semen saves time and effort. |
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