Effects Of Clostridium Butyricum On Growth,Immunity And Intestinal Microecology Of Common Carp(Cyprinus Carpio L.)

Common carp(Cyprinus carpio L.)is an omnivorous benthic fish.It has become an important economical farmed fish in China because of its adaptability,cold tolerance,hypoxia tolerance,sweetness,flatness,non-toxicity and wide range of culture.However,with the rapid development of high-density culture technology and the deterioration of the culture environment,aquatic diseases have experienced explosive growth,which has hindered the healthy development of the aquaculture industry.Clostridium butyricum,as a probiotic,has been proven to improve animal growth performance,enhance animal immunity,and depress intestinal inflammation in humans and livestock.However,the effects of Clostridium butyricum on the growth performance,immunity defense and intestinal flora structure of the common carp are not fully understood yet.In this experiment,360 healthy common carp(89.89±0.77 g)were selected and randomly divided into 3 groups.Each group included 4 replicates with 30 fish.The basal diet group was as the normal control(NC,0%),and the lyophilized powder of Clostridium butyricum with the mass fraction of 0.125%(LCB),0.5%(HCB)was added into the basal diet as the treatment groups.After 60 days of experiment time,enzyme activity assay,HE staining,AB-PAS staining,scanning electron microscopy,real-time PCR,gas chromatography and 16 S rRNA high-throughput sequencing were performed.The indexes of growth performance,serum biochemical parameters,intestinal tissue integrity,short-chain fatty acid content and intestinal flora abundance and diversity were investigated.The results were as followings:(1)Effect of Clostridium butyricum on the growth performance,serum biochemistry and enzyme activity of the common carp(Cyprinus carpio L.)Compared with the control group,the weight gain rate(WGR),specific growth rate(SGR),feed coefficient(FCR),fatness(CF),and spleen body index(SI)of the experimental group were not significant difference(P>0.05),and there were also no significant difference in serum alkaline phosphatase(ALP)and malondialdehyde(MDA)activity(P>0.05).The activities of total antioxidant enzymes(T-AOC),catalase(CAT)and lysozyme(LZM)in serum were significantly increased(P<0.05)with respect to the NC,but total protein(TP),albumin(Alb),globulin(GLB),alanine aminotransferase(ALT)and aspartateaminotransferase(AST)showed no difference(P>0.05).(2)Effect of Clostridium butyricum on the immune related enzyme activity and intestinal tissue morphology of the common carp(Cyprinus carpio L.)Compared with the NC group,the CAT and LZM activities were significantly increased in the HCB group(P<0.05).MDA decreased with the increasing concentration of Clostridium butyricum,but no significant difference was observed(P>0.05).Also,the intestinal villus height and muscle thickness increased in the Clostridium butyricum treatment group,but there was no significant difference(P>0.05).By measuring the pH value,it was found that the intestinal pH showed the tendency of decreasing,although no significant difference(P>0.05).The results of the scanning electron microscopy showed that the intestinal microvilli injury was more obvious in the LCB group than that in the NC group;the intestinal microvilli in the HCB group was smoother.(3)Effect of Clostridium butyricum on the expression of immune genes in head kidney,liver,spleen and midgut of common carp(Cyprinus carpio L.)In the head kidney tissue of common carp,compared with the NC group,the group HCB expression of IL-1? levels were significantly decreased(P<0.05),IL-10 and GPx mRNA expression levels were significantly increased(P<0.05).In liver tissue,the mRNA expression levels of GPx,GST and TGF-?were not significantly different among the groups(P>0.05),but the expression level of pro-inflammatory factor IL-1? was no significant decreased(P>0.05)in the HCB group,and the anti-inflammatory factor IL-10 was increased but no significant(P>0.05).In the spleen tissue,compared with the NC group,the expression levels of pro-inflammatory factors IL-1? and TGF-? in the experimental group were decreased,but the difference was not significant(P>0.05).In the midgut tissues,the expression levels of IL-10,TLR-2and MyD-88 in the HCB group were significantly higher than those in the NC group(P<0.05).Furthermore,the expression levels of ZO-1 and Occludin expression levels were significantly increased(P<0.05)in the experimental group.(4)Effect of Clostridium butyricum on the intestinal microbeta and short-chain fatty acid content of common carp(Cyprinus carpio L.)The addition of Clostridium butyricum to the feed increased the abundance and diversity of the intestinal microbiota.Compared with the NC group,at the phylum level,the abundance of Fusobacteriawere significantly decreased(P<0.05)and Bacteroidetes were significantly increased(P<0.05)in the experimental group;at the genus level,the abundance of norank_f__Porphyromonadaceae and Bacteroides in the experimental group was significantly increased(P<0.05),and Cetobacterium was significantly decreased(P<0.05).The addition of Clostridium butyricum promoted the production of propionic acid and n-butyric acid,but there was no significant change in the total short-chain fatty acid(SCFAs)content compared to the control(P>0.05).As to the correlation analysis,the expression level of the anti-inflammatory factor IL-10 in the intestine was positively correlated with the abundance of Bacteroidetes,Bacteroides and norank_f__Porphyromonadaceae.And there was a negative correlation between the expression levels of Fusobacteria,Cetobacterium and pro-inflammatory factors IL-1?.It is suggested that Clostridium butyricum may enhance the level of host immunity by regulating the structure of the intestinal microbeta.In summary,the appropriate concentration of Clostridium butyricum in the feed can improve the immunity of the the common carp by regulating the structure of the intestinal microbiota,increasing the content of short-chain fatty acids,and enhance intestinal mucosal barrier function.