Effects Of Clostridium Butyricum On Growth Performance,Immune Function And Intestinal Health In Weaned Piglets

The present study was conducted to investigate the effects of Clostridium butyricum?CB?on growth performance,immune function and function,volatile fatty acids?VFAs?and intestinal microflora in weaned piglets.Thereby,the mechanism of Clostridium butyricum as as an antibiotic substitute product was further explored.This study was divided into three experiments.In experiment 1,lipopolysaccharide?LPS?used to stimulate mouse macrophages?RAW264.7?to explore the regulatory effect of Clostridium butyricum and its metabolites on immune stress stimulated by LPS.In experiment 2,28-day-old weaned piglets were selected to explore the effects of Clostridium butyricumon growth performance,immune function,volatile fatty acids and intestinal microflora in weaned piglets.In experiment 3,28-day-old weaned piglets were selected to explore the effects of Clostridium butyricumon immunomodulatory,related mechanisms and intestinal microflora in LPS-challenged weaned piglets.Experiment 1:Suitable dosages of Clostridium butyricum,butyric acid and Clostridium butyricum fermentation broth were added to macrophages?RAW264.7?stimulated by LPS to explore the cell morphology,inflammatory factors and gene expression of GPR41/43 and MAPK signaling pathway in different treatment groups.The results showed that LPS impaired cell morphology and slowed down cell proliferation.The contents of LDH,IL-2 and TNF-?in cell supernatant in LPS group were significantly higher than those in negative control group?p<0.05?.The expressions of GPR41/43,ERK in LPS group were significantly lower than those in negative control group?p<0.05?,while the expressions of P38,JNK in LPS group were significantly higher?p<0.05?.Clostridium butyricum?1×10⁵cfu/mL?,butyric acid?25?g/mL?and Clostridium butyricum fermentation broth?0.4%?were helpful to restore cell morphology and proliferative activity damaged by LPS.The contents of LDH,IL-2 and TNF-?in supernatant of cells treated with Clostridium butyricum and its metabolites were significantly lower than those in LPS group?p<0.05?.After treatment and protect with Clostridium butyricum and its metabolites,the expressions of GPR41/43,ERK in cells were significantly higher than those in LPS group?p<0.05?,while the expressions of P38,JNK in LPS group were significantly lower?p<0.05?.The results indicated that Clostridium butyricum and its metabolites alleviated LPS-induced inflammatory response and reduced immune stress by changing the mRNA expressions of GPR41/43 and MAPK signaling pathway.Experiment 2:A total of 180 weaned piglets?Duroc×Landrace×Yorkshire?were randomLy assigned to three treatment groups,each group contained 6 pens per treatment,and 10 animals per pen.The treatment group 1 was the control group?Con?,fed with basic diet.The treatment group 2 was the CB group,fed with basic diet with 6×10⁹ cfu/kg Clostridium butyricum.The treatment group 3 was the probiotic control group?EF?,fed with basic diet with 1×10¹⁰ cfu/kg Enterococcus faecalis.The experiment lasted for 28 days.Piglets were weighed individually at the beginning and end of the experiment.Feed intake and the rate of diarrhea were counted.Average daily feed intake?ADFI?,average daily gain?ADG?,and feed to gain?F:G?for each pen were calculated.On day 14 and 28,six piglets per treatment were randomLy selected for blood sample collection to further analysis.At 28 d,one pig per pen were slaughtered and sampled.Colon contents were collected for the detection of VFAs and high throughput sequencing.Piglets fed Clostridium butyricum had higher ADG compared to the Con group?P<0.05?.A reduced F:G and diarrhea rate were observed in CB piglets?P<0.05?.CB piglets exhibited higher IgA,IgG and IgM levels?P<0.05?in day 28.CB piglets also had higher concentrations of acetic acid,propionic acid,butyric acid,isobutyric acid and isovaleric acid in their colon?p<0.05?.Result of high-throughput sequencing revealed that Clostridium butyricum affected bacterial quantity and diversity in the colon,and increased the abundance Megasphaera?elsdenii.CB piglets had a higher relative abundance of Clostridium butyricum?p<0.05?.The results showed that Clostridium butyricum enhanced the immune function of piglets,changed the composition of intestinal microorganisms and increased the content of VFAs in colon,thus improving the growth performance of weaned piglets.Experiment 3:A total of 180 weaned piglets?Duroc×Landrace×Yorkshire?were randomLy assigned to three treatment groups,each group contained 6 pens per treatment,and 10 animals per pen.The treatment group 1 was the control group?Con?,fed with basic diet.The treatment group 2 was the CB group,fed with basic diet with 6×10⁹ cfu/kg Clostridium butyricum.The treatment group 3 was the probiotic control group?EF?,fed with basic diet with 1×10¹⁰ cfu/kg Enterococcus faecalis.The experiment lasted for 28 days.At the end of the experiment,twelve piglets were selected from each group.Six of them were injected with 25?g/kg body weight LPS,and the other six were injected with saline of equal volume.At 1.5 and 3 hours after LPS or saline treatment,blood samples were collected for the determination of serum biochemical and immune indices.Then,12 piglets in each group were slaughtered.Jejunum and jejunum mucosa were taken to determine the intestinal morphology and the expression of immunecell signaling pathway protein.The results showed that Clostridium butyricum alleviated the immune stress induced by LPS stimulation in piglets,significantly reduced the levels of ALB,BUN,ALT,AST,IL-2 and TNF-?in serum induced by LPS stimulation,which also increased the levels of ACP,SOD,T-AOC,IgA,IgG,IgM and IFN-?in piglets?p<0.05?.Clostridium butyricum significantly increased the height of jejunal villi?p<0.05?,and alleviated the damage of LPS to jejunal villi.LPS stimulation significantly increased the expression of TLR4,MyD88 and NF-?B in jejunal mucosa?p<0.05?,while Clostridium butyricum inhibited the expression of TLR4,MyD88 and NF-?B protein?p<0.05?,and reduced theexpression of NF-?B p65 induced by LPS stimulation?p<0.05?.And Clostridium butyricum affected bacterial quantity and diversity in the colon contents,maintained the intestinal flora balance.The results indicated that Clostridium butyricum reduced liver injury and secretion of inflammatory factors induced by LPS,improved antioxidant capacity and protected intestinal function,which alleviated excessive immune stress by regulating intestinal TLR4 signaling pathway,and conducived the balance intestinal microflora.In conclusion,dietary supplementation with Clostridium butyricum improved the growth performance in weaned piglets,enhanced the immune function,contributed to the balance of intestinal microorganisms,increased the content of VFAs in intestinal tract,which reduced the liver damage and the secretion of inflammatory factors caused by LPS stimulation,and alleviated excessive immune stress by regulating the intestinal TLR4 signaling pathway.