Effects Of Royal Jelly Supplementation In Diluent On Sheep Semen Quality During Liquid Storage At 4℃

Liquid preservation at 4°C is a main way for semen preservation,which plays an important role in artificial insemination.The oxidative damage during this process is one of factors affecting semen quality.Royal jelly is well-known natural antioxidant,which can reduce cellular ROS content.The effects and mechanisms of royal jelly on semen preservation remain undefined.The objective of this study was to determine the influence of supplementation of different concentration of royal jelly on diluent on semen preservation quality.This study can provide a basis for the research and application of royal jelly in liquid storage of sheep sperm at 4°C.Experiment 1: Different concentration of royal jelly(from 0%,0.25%,0.5%,0.75%,to1%)were added into Tris-fructose-citric acid diluent.The sperm motility,viability,motion parameters,plasma membrane integrity,mitochondrial activity and enzymatic activity of antioxidant were detected after storage at 4°C for 7 days.The results showed that in the middle storage period(3 d-4 d),the sperm movement capacity and plasma membrane integrity of the 0.75% group were significantly greater than control group(P < 0.05).The sperm mitochondrial activity of each treatment groups was greater than that of the control group(P < 0.05).In the late storage period(5 d-6 d),sperm viability,motility,plasma membrane integrity of 0.5% group and 0.75% group were significantly greater than those of the control group(P < 0.05).The sperm motion parameter and mitochondrial activity of each treatment group were significantly greater than those of the control group(P < 0.05).After the 7 day of storage,mitochondrial activity in 0.5% supplementation group was significantly greater than that of the 0.25% group(P < 0.05),but lower than that of 0.75%group(P < 0.05).The motion parameter in 0.25%,0.5%,1% groups were higher than those of the control group(P < 0.05),but lower than those in 0.75% group(P < 0.05).Meanwhile,on the 3rd and 5th day of storage,T-AOC of each treatment groups significantly was greater than that of the control group(P < 0.05),whereas MDA content was lower than that of the control group(P < 0.05).The SOD activity of 0.75% group was greater than that of the control group(P < 0.05).Furthermore,On the 7th day of storage,the T-AOC of each treatment group were greater than that of the control group(P < 0.05),while MDA content were reduced(P < 0.05).The T-AOC of 0.75% group was greater than that of the other treatment group(P < 0.05),and the MDA content was significantly lower than that of the other treatment group(P < 0.05).Experiment 2: Western blot was used to detect the expression of Glut 3 and Glut 8related to sperm energy metabolism from 0 to 3 days during storage at 4°C.The results showed that Glut 3 and Glut 8 abundances were gradually decreased with the extension of storage time.The expression level of Glut 8 at 0 day of storage was greater than that at day3 of storage(P < 0.05).The appropriate concentration was added to the liquid storage dilution with 0.75% royal jelly,the expression levels of Glut 3 and Glut 8 on the 3rd day of storage were significantly increased(P < 0.05).Conclusion: Adding 0.75% of royal jelly in sheep semen diluent can effectively remove lipid peroxides produced during storage,protect plasma membrane integrity,maintain mitochondrial activity,and reduce the loss of sperm energy metabolism related proteins Glut 3 and Glut 8,promote its energy metabolism and motility,extend storage time,and improve semen quality.