Effect And Mechanism Of Clostridium Butyricum On Growth Performance And Gut Function Of Weaned Piglets

Production of piglets has always been a speed-limiting link in pig production,and weaning stress leads to a decrease of growth performance of piglets and an increase of post-weaning diarrhea(PWD)and mortality rate,especially under the background of antibiotics prohibition.Improvement of intestinal function,reduction of PWD of piglets,and increase their growth performance by nutritional modulation became an urgent issue to be solved.In this study,the effects of Clostridium butyrate and its main metabolite butyrate on the growth performance and intestinal function of weaned piglets were studied through the feeding experiment of weaned piglets and the experiment of piglets challenged with lipopolysaccharide(LPS).We aimed to reveal the mechanisms underlying the improvement the intestinal function of weanling piglets by the clostridium butyrate via 16S r RNA gene sequencing combined with targeted metabolomics technology.This thesis mainly carries out the following four parts.1.Effects of addition of different levels of Clostridium butyrate(C.butyricum)on growth performance,nutrient digestibility,intestinal morphology,and host immunity of weaned pigletsOver 28 days,120 piglets with an average body weight of 8.09±0.01 kg were allocated to four treatments,with six replicates(five piglets)per treatment.The dietary treatments were CON(basal diet);AGP(basal diet containing 0.075 g/kg Chlortetracycline,0.055 g/kg Kitasamycin,and 0.01 g/kg Virginiamycin);CBN[basal diet containing 2.5×10~8 colony-forming unit(CFU)/kg C.butyricum];and CBH(basal diet containing 2.5×10~9 CFU/kg C.butyricum).On day 21,one piglet from each replicate was selected and slaughtered.During days 1-14,the ratio of feed to weight gain(F/G)in the CBN,CBH,and AGP groups were significantly lower than that in the CON group(P<0.05),and during days 15-28 and 1-28,the F/G in the CBN group was significantly lower than that in the CON groups(P<0.05).The fecal scores in the CBN group during days 1-14,15-28,and 1-28 were significantly lower than that in the CON group(P<0.05).In comparison with the CON and AGP groups,the apparent digestibility of dry matter,organic matter,and total carbohydrate in the CBH group were significantly increased(P<0.05).Villi height(VH)and crypt depth(CD)significantly improved as well(P<0.05).Diet with C.butyricum contributed to decrease serum pro-inflammatory cytokines(IL-1?,IL-6,and TNF?)(P<0.05)and increase serum level of immunoglobulins(Ig A,Ig M,Ig G),anti-inflammatory cytokines(IL-2),and serum antioxidant ability(GSH-PX activity and hydroxyl radical inhibition ability)(P<0.05).Therefore,addition of C.butyricum can improve the growth performance,antioxidant status,and intestinal morphology of piglets,and decrease their inflammation risk.2.Effects of addition of different levels of C.butyricumon colonic microbiota,metabolites,and intestinal barrier functionThe experimental design and animal feeding management in this study were same as the first experiment.The results showed that the colonic concentrations of acetic,propionic,butyric,and total short chain fatty acids(SCFAs)in the CBH group and acetic acid concentration in the CBN group were significantly higher than those in the CON group(P<0.05).Microbial diversity analysis revealed significant differences in microbiota between colonic chyme and mucus,and Alpha diversity in the mucus was higher than that in the chyme(P<0.05).The addition of C.butyricum increased the relative abundance of Bifidobacterium(P<0.05)and Butyricicoccus(P>0.05)in colonic chyme and Fournierella(P<0.05),Lactobacillus,and Butyricicoccus(P>0.05)in mucosa,but decreased the relative abundance of Sarcina,Blautia,and Actinobacillus(P>0.05)in chyme and Actinobacillus and Campylobacter in mucosa(P>0.05).ZO-1 expression in the CBN group was slightly higher than that in the CON group(P>0.05).However,compared with the CBH group,the m RNA expression level of Occludin-1 and ZO-1 in the CBN group was significantly increased(P<0.05),but the number of lymphocytes in epithelium was significantly decreased(P<0.05).These results demonstrate a significant difference between different niche,and different levels of C.butyricum in diet decrease the relative abundance of harmful bacteria and increase colonic SCFAs.3.Effects of butyric acid on growth performance,intestinal morphology,jejunal microbiota and inflammation of weaned piglets challenged by LPSBecause diet supplemented Clostridium butyrate increased the apparent digestibility of feed nutrients and improved the morphology of small intestine,a third experiement was performed.This experiment on piglets induced with LPS was carried out to further study the effect of C.butyricum metabolites on growth performance and intestinal function.Eighteen healthy weanling male piglets(9.10±0.15 kg)at 30-day were selected and randomly divided into three groups according to their body weight,with six piglets(six replicates)per group.The three treatments were CON(control group fed basal diet),LPS(group fed basal diet and induced with injection of LPS),and BT-LPS(group fed basal diet supplemented with protected butyrate and induced with injection of LPS).On days 19 and 21 of the experiment,piglets in the LPS group and the BT-LPS group were intraperitoneal injected with LPS,and the injection dose was 100 g/kg body weight.On day 21,after 4 h of challenging LPS,all piglets were slaughtered.During the experimental period,butyrate alleviated the decreasing of ADG of piglets,the increasing of clinical scores,and the decreasing of VH,CD,and V/C in the jejunum and ileum induced by LPS(P<0.05).Compared with the LPS group,protected butyrate addition in diet significantly increased the jejunum level of IL-10(P<0.05)and numerically reduced the level of pro-inflammatory factors(IL-1?,IL-6,IL-8,and TNF-?).Compared with the CON group,LPS inducing decreased the relative abundance of unidentified?Clostridiales and Lactobacillus(P>0.05)and increased the relative abundance of Romboutsia and unidentified?Enterobacteriaceae(P>0.05).Compared with the LPS group,the relative abundance of unidentified?Clostridiales and Lactobacillus in the BT-LPS group was respectively increased by 15.44%and 6.43%,but the relative abundance of Romboutsia and unidentified?Enterobacteriaceae was decreased by 7.49%and10.66%,respectively.Taken together,these results indicated that butyrate can reduce the relative abundance of facultative anaerobe,enhance absolute anaerobic bacteria,decrease jejunal inflammation,and improved intestinal morphological structure.4.Butyrate alleviates piglet colitis induced by LPS challenge by regulating colonic microbiota and energy metabolism in gut-liver axisBased on the results that Clostridium butyrate addition increased the concentration of SCFAs in the colon and reduced the body inflammation of piglets,in this study,we aimed to reveal the mechanism of Clostridium butyrate alleviating colitis through its main metabolite butyrate by combining with 16S r RNA gene sequencing and targeted energy metabolomics technology.Butyrate administration significantly decreased LPS-induced a rise of colonic histological scores of piglets(P<0.05),and reduced the susceptibility to LPS-induced severe inflammatory response by increasing anti-inflammation cytokines(IL-10 and TGF-?)and decreasing pro-inflammation cytokines(IL-1?,IL-6,IL-8,and TNF-?)(P<0.05).Compared with the CON group,LPS induced a significant increase of hypoxia inducible factor1?(HIF-1?),cyclooxygenase-2(COX-2),and inducible nitric oxide synthase(i NOS)in colonic mucosa(P<0.05),but butyrate supplementation significantly decreased those three proteins content(P<0.05).Diet with butyrate significantly increased the relative abundance of Faecalibacterium and Lactobacillus by enhancing the TCA cycle.Lactobacillus and Faecalibacterium were negatively correlated with IL-6(P<0.05),while Faecalibacterium was positively correlated with TGF-?,but negatively correlated with IL-1?(P<0.05).The protected butyrate addition significantly increased the concentration of butyric acid in the colon and portal venous circulation(P<0.05),and enhanced the TCA cycle in the gut-liver axis by mobilizing amino acids and B group vitamins as coenzymes,which was mainly manifested as increased levels of oxaloacetic acid and oxaloglutaric acid in the colon and increased levels of citric acid,cis-aconitic acid,isocitric acid in the liver(P<0.05).Meanwhile,LPS enhanced fatty acid synthesis in this process(P<0.05),while protected butyrate addition reversed this trend,which was reflected by reducing acylcarnitines(P<0.05).Butyrate reshaped colonic microbiota and energy metabolism in gut-liver axis by increasing anti-inflammatory cytokine levels and suppressing HIF-1?,and its downstream reaction elements(COX-2 and i NOS)improved colonic immune homeostasis and morphology.In conclusion,dietary supplementation with C.butyricumon increased feed conversion rate,improved intestinal morphology,enhanced antioxidant capacity,and reduced inflammation of weaned piglets.The application effect of C.butyricum was better at a dosage of 2.5×10~8 CFU/kg compared with the high dosage group.There is a significant difference in microbial diversity between colonic chyme and mucus,and dietary supplementation with C.butyricum increased the relative abundance of Bifidobacterium in colonic chyme.Butyrate improved the LPS-induced higher clinical score and small intestinal morphological damage of piglets,and inhibited the excessive proliferation of facultative anaerobes and some bacteria in the low relative abundance.Combined with 16S r RNA gene sequencing and targeted energy metabolome,it was revealed that butyrate enhanced the TCA cycle of the gut-liver axis and reshaped the colonic microbiota,which contributed to improve colonic immune homeostasis and reduce host inflammation.