| The quality of pig semen is an important factor affecting the reproductive performance of sows and directly determines the economic benefits of artificial insemination stations.However,the problem of poor quality of boar semen is very prominent in China’s swine artificial insemination stations and is one of the main reasons for the elimination of breeding boars.Studies have confirmed that the high content of docosahexaenoic acid(DHA)in boar sperm fatty acids has an important impact on sperm quality.The addition of appropriate amount of DHA to the diet has the effect of improving the semen quality of boars.The use of DHA to improve semen quality has been widely reported,but the effect of different concentrations of DHA and GML complexes on semen quality is not known.Therefore,in this experiment,the effects of different levels of DHA and GML complexes on sperm fatty acid composition,semen quality,oxidative stress status,inflammatory response,intestinal permeability and intestinal flora of boars were investigated by adding different levels of DHA and GML complexes to the diets of breeding boars.46 Duroc boars aged 19-24 months were randomly divided into 4 groups: the control group was fed the basal diet,and the three treatment groups were fed DHA and GML complexes at 0.18%(treatment group 1),0.24%(treatment group 2)and 0.30%(treatment group 3),respectively,with a 4:1ratio of DHA to GML in the complexes.0w,2w,4w,8w,10 w,12w,16 w,10w,12 w,16w,10 w,12w and 16 w were collected,10 w,12w and 16 w semen,and determined semen volume,sperm density,sperm motility and total sperm count;calculated 4～16w malformation rate,sperm membrane integrity and functional sperm count;analyzed the fatty acid composition of 16 thw sperm by gas chromatography;collected 16 w venous blood and prepared serum.Superoxide dismutase(SOD)and malondialdehyde(MDA)contents of 16 w serum,semen serum and sperm were measured,as well as interleukin-1β(IL-1β),interleukin-6(IL-6),D-latic acid(D-LAC),Fecal calprotectin(FC),Tumor necrosis factor-α(TNF-α),Diamine oxidase(DAO),.The 16 w swine feces were collected and the fecal-like flora was determined using 16 S r RNA sequencing technology.The main test results are as follows:(1)Effect on fatty acid composition: at the 16 th week of the testing period,the DHA content in Treat 2 and Treat 3 was significantly higher than that in the Con(P<0.01),but there was no difference between Treat 1 and Con;all treatment groups had higher levels of n-3 PUFA and PUFA compared to the Con(P<0.01),but the content of n-6PUFA,SFA and MUFA and the n 6:n-3 ratio were not significantly different compared with the Con.In addition,lauric acid(C12:0)was not detected in this study.(2)Effect on fresh semen conventional semen quality: There was no interaction between diet treatment and feeding time on boar semen volume and sperm density,but there was an interaction on total sperm count(P<0.05),motility(P<0.05),abnormal sperm rate(P<0.01)and functional sperm count(P<0.05).The effect of diet treatment on semen volume and density was not significant.Compared with the Con,there was no significant effect of diet treatment on total sperm count,sperm malformation rate and functional sperm count from 0w to 10w;at 12 thw,only the total sperm count and functional sperm count of Treat 3 was significantly higher than Con(P<0.01),and the rate of sperm malformation in Treat group was significantly lower than that in Con(P<0.01);At 16 weeks,the total sperm count and functional sperm count of the three treatment groups were significantly higher than those of the Con(P<0.05),and the abnormal sperm rate was also significantly lower than that of the Con(P<0.05).In terms of sperm motility,the dietary treatments only had a significant effect on 10 w sperm motility.The sperm motility of Treat 1 and sperm motility of Treat 2 were significantly higher than those of the control group(P<0.01),and Treat 3 had significantly higher sperm motility than Treatment 2(P<0.01).The above results indicated that the dietary addition of DHA and GML complexes had the effect of improving the total sperm count,sperm motility,malformation rate and functional sperm count.(3)Effect on sperm motility parameters of fresh semen: dietary treatment had no significant effect on the ratio of local oscillating sperm ratio for 16 weeks.At 16 weeks,the rotational motility sperm ratio was significantly lower in treatment group 1 than in control group(P<0.05).The above results suggest that the rotational motile sperm ratio can be reduced by feeding boar diets supplemented with DHA and GML complex.(4)Effect on sperm motility parameters of semen during storage: the sperm motility parameters of 16 weeks semen storage for 24 hours and 72 hours showed that the fast Progressive motile sperm ratio in Treat 3 was significantly higher than that in Treat 1 at24 hours(P<0.05).At 72 h,compared with the Con,the rotational motility sperm ratio of Treat 1 was significantly lower than that of the Con(P<0.01),but there was no difference between Treat 2 and Treat 3 and the Con.The above results suggest that the addition of DHA and GML complex to the diet can increase the fast Progressive motile sperm ratio during storage and reduce the rotational motile sperm ratio.(5)Effect on sperm membrane functional integrity of fresh semen: at 4w,12 w and 16 w,the sperm membrane integrity of the level of each treatment group was significantly higher than that of the weekly group(P<0.01);at 10 w,the sperm membrane integrity of Treat 2 and Treat 3 was significantly higher compared to the Con(P<0.05),however,the s Perm membrane integrity of Treat 1 was not significantly different from that of the Con.These results suggest that the addition of DHA and GML complexes has the effect of improving sperm membrane integrity in boars.(6)Effects on SOD activity and MDA levels of serum,semen serum and sperm:compared with the Con,the diet treatment did not have a significant effect on serum SOD activity;however,the SOD activity of Treat 2 and Treat 3 was significantly higher than that of the Con(P<0.05);the SOD activity of sperm in Treat 1 and Treat 2 was highly significant greater than that of the Con(P<0.01);the diet treatment had no significant effect on the stored sperm SOD There was also no significant effect of diet treatment on post-storage sperm SOD activity.In terms of MDA,serum MDA levels were significantly lower in Treat 1 and 3 compared to the Con(P<0.01),but did not affect seminal serum MDA levels.After 24 h of storage,sperm MDA levels were highly significantly smaller in each treatment group than in the Con,and highly significantly smaller in Treat 1 and 2than in Treat 3(P<0.01).The above results indicate that the addition of DHA and GML complexes to the diet can increase the SOD activity of semen clear and spermatozoa in fresh semen and reduce the lipid oxidative damage of spermatozoa in fresh semen and short-term(24 h)stored semen.(7)Effect on serum inflammatory cytokines: There was no significant difference in serum levels of TNF-α and IL-1β in each treatment group compared with the Con.However,IL-6 levels were significantly lower in Treat 1 and Treat 2 than in the Con(P<0.01),but there was no difference in IL-6 levels in Treat 3 compared with the Con.The above results indicated that the addition of DHA and GML complexes mainly reduced the serum IL-6 level in boars.(8)Effects on intestinal inflammation and permeability: After dietary treatment,the DLAC levels in the treated groups did not differ from those in the Con;while the DAO levels in all treated groups were extremely significantly lower than those in the Con(P<0.01).Compared to the Con,the FC was significantly lower in Treat 1 and Treat 2(P<0.05).(9)Effect on intestinal flora: principal co-ordinates analysis(PCo A)based on bray＿cutis distance showed a significant separation of intestinal flora structure in the Con compared to the Treat 2(P<0.01).Treat 2 Lentisphaeria order,Oligosphaerales order was enriched in the intestine,norank＿c＿Clostridia order was enriched in the intestine of control boars,at the genus level,Treat 2 Lactobacillus was significantly increased compared to Con,while the relative abundance of Streptococcus Treat 2 was highly significantly smaller than that of the Con;spearman correlation analysis showed that Lactobacillus spp.in the intestine showed a highly significant positive correlation(P<0.01)with sperm membrane integrity and a significant negative correlation(P<0.05)with cyclic motility of stored sperm for 72 h.(10)Effect on sperm DNA methylation: the level of DNA methylation was significantly lower in the Treat 2 compared to the Con after dietary treatment(P<0.05).In summary,the main conclusions of this experiment are:Positive effects on fresh and ambient preserved semen quality were observed in boars fed diets containing 0.24% DHA and 0.06% GML complexes for 10 w.This improvement could be attributed to the increase in sperm fatty acid content,which in turn improved the integrity of the sperm membrane.In addition,DHA and GML complexes may also play a role through the regulation of gut microbiology and barrier function,as well as the regulation of oxidative stress,inflammatory status and sperm DNA methylation in the organism. |
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