| At present,the Italian Mediterranean buffalo is recognized as one of the best varieties in the type of river milk buffalo in the world because of its excellent high milk production and milk quality.In 2014,Guang Xi introduced the variety of buffaloes,due to the cryopreservation formula,freezing process,individual differences and other factors,its semen cryopreservation is not very well,which may greatly affect their reproductive rate.Therefore,looking for effective antioxidants to improve the effect of semen cryopreservation,which has important significance on improving the utilization efficiency of breeding buffalo.In the study,the proportion was renewed and some new ingredients were added on the basis of traditional diluent.By comparing the effects of different ingredients,finally a new formula was proposed,and then MLT,GSH,and VE were added to the modified formula to discuss the effect of different antioxidants on semen cryopreservation.The result showed that.1.The research about the improvement semen freezing diluent formula of Mediterranean buffalo.On the basis of frozen diluent which are commonly used in buffalo semen,first the effective components of energy material,antibacterial substances and antifreezing substances were compared respectively.The study found that,the effect of yolk was better than fresh milk in cryopreservation,the effect of the combination of 3%glycerol and 2%ethylene glycol was better than 5%glycerol,5%ethylene glycol and 5%DMSO;Penicillin and Gentamicin's effect was obviously better than others.Secondly,BSA was introduced as a new protective agent,compared with the blank control group,0.9 mg/mL of BSA significantly improved the buffalo sperm vitality,and reduced the rate of deformity(P<0.05),Finally,the improved semen diluent formula was:0.10 g/mL Glucose,0.0006 g/mL Sodium citrate,0.0011 g/mL Penicillin,0.0006 g/mL Gentamycin,0.09 mg/mL BSA,3%Glycerin,2%ethylene glycol,20%yolk,75%distilled water.2.The effects of melatonin,glutathione,and vitamin E on semen cryopreservation of Mediterranean buffalo.When melatonin(MLT)was added to the frozen diluent,compared with the control group which didn't add MLT,0.1 mg/mL MLT significantly improved the zygote divides rate and the activity of GSH-Px(P<0.05),but there were no significant differences in the sperm vitality,deformity rate and movement performance(P>0.05),0.2 mg/mL and 0.4 mg/mL MLT both increased sperm vitality,acrosome intact rate,activity of GSH-Px,zygote division rate and blastocyst rate significantly(P<0.05),0.2 mg/mL MLT also reduced the rate of sperm deformity significantly.0.8 mg/mL MLT reduced the sperm vitality simultaneously(P<0.05),and there's no significant difference on the sperm motion performance,zygote division rate and blastocyst rate(P>0.05).When glutathione(GSH)was added to the frozen diluent,compared to the group which didn't add GSH,though 0.1 mM GSH increased the sperm forward motility and plasma membrane integrity rate significantly,but it reduced the deformity rate(P<0.05),and it had no obvious effect on sperm vitality and seaborne acrosome intact rate(P>0.05).0.2 mM GSH improved sperm vitality,sperm movement performance,plasma membrane integrity rate,activity of GSH-Px and blastocyst rate(P<0.05),it reduced deformity rate and MDA level significantly(P<0.05).0.4 mM GSH increased sperm deformity rate,the activity of GSH-Px,fertilization rate and blastocyst hatching rate obviously(P<0.05),and it had no significant influence on acrosome intact rate and the activity of SOD(P>0.05).0.8 mM GSH increased sperm deformity rate significantly(P<0.05),and it had no significant effects on sperm movement performance,antioxidant enzyme activity and blastocyst rate(P>0.05).When vitamin E(VE)was added to the frozen diluent,compared to the group which didn't add GSH,0.1 mg/mL,0.2 mg/mL VE had no obvious effect in sperm vitality(P>0.05).0.2 mg/mL and 0.4 mg/mL VE not only increased the sperm activity of GSH-Px,plasma membrane integrity and acrosome intact rate significantly(P<0.05),but also reduced MDA level(P<0.05).0.4 mg/mL and 0.8 mg/mL VE both significantly increased the sperm vitality(P<0.05),and 0.4 mg/mL VE reduced the rate of sperm deformity significantly while 0.8 mg/mL VE increased deformity rate(P<0.05),by the way,0.8 mg/mL VE increased the zygote divides rate obviously(P<0.05),but it had no significant effects on the blastocyst rate and blastocyst hatching rate(P>0.05).3.The effect of compatibility of three of kinds of antioxidants on semen cryopreservation of Mediterranean buffalo.To understand the synergy effect of two or more antioxidants,the effective concentrations of each type of antioxidant was screened according to their single effect,which namely MLT(0.2 mg/mL,0.4 mg/mL),GSH(0.1 mM and 0.2 mM)and VE(0.2 mg/mL,0.4 mg/mL),and then 12 compatibility combinations were formed by the style of every two concentrations combined together.The study found that,to a certain extent,0.2 mg/mL VE + 0.1 mM GSH,0.4 mg/mL VE + 0.2 mg/mL MLT,0.2 mg/mL MLT + 0.1 mM GSH improved sperm vitality and decreased the rate of sperm deformity,among which,0.2 mg/mL MLT + 0.1 mM GSH and 0.4 mg/mL VE + 0.2 mg/mL MLT were more outstanding.Compatibility of three combinations significantly increased the activity of sperm linearity,swing,prior to shipment,acrosome intact rate,plasma membrane integrity rate(P<0.05),and especially,0.2 mg/mL MLT +0.1 mM GSH effect was better than the other one.Compatibility of three combinations improved the zygote divides rate significantly(P<0.05),0.4 mg/mL VE + 0.4 mg/mL MLT and 0.2 mg/mL MLT + 0.1 mM GSH had significant differences on the blastocyst rate(P<0.05).0.4 mg/mL VE + 0.2 mg/mL MLT and 0.2 mg/mL VE + 0.1 mM GSH increased the blastocyst hatching rate significantly(P<0.05).In conclusion,0.2 mg/mL MLT + 0.1 mM GSH and 0.4 mg/mL VE + 0.2 mg/mL MLT was better than that 0.2 mg/mL VE+ 0.1 mM GSH.The above results showed that 0.2 mg/mL MLT,0.2 mM GSH and 0.04 mg/mL VE have better effect in cryopreservation when added them individually.Meanwhile,the combination types of 0.4 mg/mL VE+0.2 mg/mL MLT or 0.2 mg/mL MLT+0.1 mM GSH had significant differences on semen cryopreservation of Mediterranean buffalo. |
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