Study On The Mechanism Of Cellulose Mediated Intestinal Microorganism And Its Metabolites Regulating Intestinal Health In Different Feeding Fish

Fish meal replacement is the necessary way for the healthy and sustainable development of aquaculture.With its advantages of stable supply and low price,plant-based protein source has became the main alternative source of fish meal,and its added proportion in fish feed has increased significantly.The cell wall of plant feed material contains abundant cellulose.At present,there is no unified definition for it,which generally refers to plant cell wall components in feed that cannot be digested by animal intestinal digestive enzymes.High dosage addition will reduce nutrient digestibility and energy intake,which is inhibited the growth and health of fish.However,in recent years,many studies have found that cellulose can not be digested by digestive enzymes secreted by fish,but which can produce SCFA by intestinal microorganism.However,the microecological nutrition effect of cellulose mediated microorganism is unknown.Based on previous studies,a low-bacteria environment in fish intestinal tract was constructed by using complex antibiotics（500mg/kg meropenem+500mg/kg vancomycin+40mg/kg gentamicin）in this study.Combined with 16S r DNA sequencing and metabolomics technology,the studied the effects of different gradient feed dietary fiber DF（0%,2.5%,5%,7.5%,10%Unicell PF200 plant DF（cellulose content≥99%））and antibiotics（0,1040mg/kg）on herbivores（Grass Carp Ctenopharyngodon Idella,GC）,omnivorous（Tilapia Oreochromis niloticus,T）and carnivorous（Rainbow trout Oncorhynchus mykiss,RT）fish intestinal microflora and their metabolites,to evaluate the microecological effects of cellulose mediated intestinal microflora in different feeding fish.To explore the mechanism of cellulose mediated intestinal microorganism and its metabolites differentially regulating intestinal health of different feeding fish.1.The mechanism of cellulose mediated intestinal microorganism and metabolites regulating intestinal health of herbivorous fishThe grass carp basal diet（30%crude protein,5%crude fat）were formulated,which contained fish meal,casein and gelatin were used as main protein sources,and soybean oil and fish oil were used as the main fat source.The 9-week feeding experiment was conducted,and results were as follows:In the normal intestinal microorganism groups,the final weight,body weight gain,daily gain coefficient and food intake of grass carp were all the highest at 5%DF.According to the quadratic regression model,the DF requirement of grass carp was 4.97%.The contents of succinic acid and taurine were increased in serum of grass carp,and were involved in the ERK1/2 signal transduction pathway,and the protein expressions of ERK2 and p-ERK1/2 in hindgut had no significant changes.The content of acetic acid dominates in SCFA.The villus length,muscular layer thickness,crypt depth of foregut and CAT activity of hindgut of grass carp were significantly increased,while MDA of hindgut content was significantly decreased.Among them,the contents of butyric acid and isovaleric acid,and the gene expression level of foregut EGF,the activities of Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP of foregut were increased at 5%DF.The gene expression level of foregut IGF-1R was the highest at 10%DF.The activities of trypsin and CK of foregut and GSH content of hindgut were increased at 5～7.5%DF.The relative length and weight of intestinal tract were increased at 5～10%DF.The gene expression level of SGLT-1 of foregut was increased at 7.5～10%DF.The intestinal epithelial cells of grass carp are tightly connected and the structure is complete,and the microvilli are arranged closely and neatly.The gene expression level of Occludin,Claudin-3,Claudin-12,ZO-1 of foregut,the content of C3,C4,TGF-β,IL-8 and IL-10 gene expression levels of hindgut were increased.While the contents of TNF-αof midgut,IL-6 and IL-12 of hindgut,and the gene expression levels of IL-8 and IFN-γof hindgut were decreased.Among them,the gene expression level of MUC2 of hindgut was highest and the content of foregut SIg T was increased,while IL-1βcontent of hindgut were decreased at 5%DF.The abundance of Fusobacteria,Bacteroidetes,Aeromonas,and Brevundimonas was the highest,while the abundance of Firmicutes was the lowest at 5%DF.The gene expression level of TNF-αof hindgut was decreased at 7.5%DF.The contents of foregut C4 and midgut IL-10 were increased,while midgut IL-8 content were decreased at10%DF.Foregut TRE and PIg R gene expression were increased at 5～7.5%DF.The contents of foregut TGF-βcontent,SIg T and PIg R of midgut,PIg R and IL-10 of hindgut and the gene expression level of hindgut TGF-βwere increased,while the content of IL-1β,IL-6 of midgut and hindgut IL-1βgene expression were decreased at 5～10%DF.The midgut IFN-γcontent decreased at 7.5～10%DF.In the low intestinal microorganism groups,the final weight,body weight gain and daily gain coefficients of grass carp were maximum at 5%DF,and the DF requirement of grass carp was 5.53%according to the quadratic regression model.The contents of glutathione and adenosine in serum of grass carp increased,and were involved in ERK1/2 signal transduction pathway,but the expression of ERK2and p-ERK1/2 proteins in hindgut had no significant changes.The villus length,muscular layer thickness,crypt depth,and the activities of foregut Na⁺-K⁺-ATP,Ca²⁺-Mg²⁺-ATP were increased,while hindgut MDA content was decreased.Among them,the contents of acetic acid and isovaleric acid were highest at2.5%DF.The content of propionic acid was highest,and the gene expression level of SGLT-1,trypsin activity of foregut,GSH content and GSH-Px activity of hindgut were increased at 5%DF.CAT activity of foregut was increased at 2.5～7.5%DF.The CK activity of foregut was increased at 5～7.5%DF.The relative intestinal weight was increased at 5～10%DF.The relative length of intestinal tract was increased at 7.5～10%DF.With the DF level increasing,the gene expression level of Occludin and ZO-1,the content of midgut IL-6,LZM and MUC2 of hindgut were increased,while the gene expression levels of IFN-γand TNF-αof hindgut were decreased in DF treatment groups.Among them,the gene expression level of IL-10 of hindgut was increased at 2.5%DF.The contents of IL-10 and TGF-βof foregut and midgut IL-10 were the highest at 5%DF,while the gene expression level of IL-1βof hindgut was lowest.The contents of C3 and C4 of hindgut were increased,while the contents of midgut TNF-αand hindhut ET-1and hindgut gene expression levels of IL-8 were decreased,and the abundance of Firmicutes and Aeromonas were the highest at 5%DF.The content of midgut PIg R was the highest at 7.5%DF.The content of foregut ET-1,IL-10 and TGF-βof hindgut were increased,while midgut IFN-γcontent was decreased at 5～7.5%DF.The content of midgut SIg T and hindgut TGF-βgene expression were increased at 5～10%DF.The content of IL-1βof hindgut was increased at 7.5～10%DF,while the contents of foregut IL-12 and midgut ET-1 were decreased.In conclusion,5～10%DF promotes the growth of grass carp attributed to that which can promotes substances increased that involved in body metabolism,and improve intestinal digestion and absorption function and barrier function.And cellulose does not mediate intestinal microorganism and its metabolites to regulate intestinal health of herbivorous fish.2.The mechanism of cellulose mediated intestinal microorganism and metabolites regulating intestinal health of omnivorous fishThe tilapia basal diet（32%crude protein,5%crude fat）were formulated,which contained fish meal,casein and gelatin were used as main protein sources,and soybean oil used as the main fat source.The 9-week feeding experiment was conducted,and results were as follows:In the normal intestinal microorganism groups,the final weight,body weight gain and daily gain coefficients of tilapia were increased,while the feed coefficient was decreased.The contents of arginine and lysine in serum of tilapia increased,and were involved in the signal transduction pathway of ERK1/2and P38 MAPK,and the protein expression of hindgut P38MARK decreased at 10%DF.The villus height and crypt depth,the activities of Na⁺-K⁺-ATP,trypsin and AKP of foregut were increased,while ROS activity and MDA content of hindgut were decreased.Among them,valeric acid content was highest,and the relative length of intestinal tract and the gene expression level EGF of foregut were increased at 10%DF.The gene expression level of GLUT-2 of foregut was increased at 2.5～5%DF.The acetic acid content,the gene expression levels of IGF-1,IGF-1R and SGLT-1 of foregut,the activities of Ca²⁺-Mg²⁺-ATP and CK of foregut,and the activities of SOD,CAT and GSH of hindgut were increased at 5～10%DF.The contents of isovaleric acid and caproic acid and GSH-Px content of hindgut were increased at 7.5～10%DF.The structure of tight intercellular junctions was complete,microvilli were arranged neatly.The TRE and the gene expression level of Clacudin-3 of foregut,and the gene expression level of MUC2 of hindgut was increased,while the contents of foregut IL-6,IFN-γand IL-6 of midgut were decreased.At the same time,the abundance of Proteobacteria,Verrucobacteria and Brevundimonas were increased,while Firmicutes decreased.Among them,the content of midgut PIg R was increased at 5%DF.The gene expression level of foregut ZO-1 and the contents of ET-1 and PIg R of hindgut were increased at 7.5%DF.The IL-10 content of foregut was increased at 10%DF.The gene expression level of Clacudin-12 and PIg R content of foregut were increased,while the contents of IL-1βand IL-6 of hindgut were decreased at 5～7.5%DF.The contents of C3 and C4 and the gene expression level of IL-10 of hindgut were increased,while the contents of foregut IL-12 and hindgut IL-12 and the gene expression level of IL-1β,IL-8 and IFN-γof hindgut were decreased at 5～10%DF.The gene expression level of Occludin of foregut,the contents of LZM,IL-10 and TGF-βof hindgut,and the gene expression level of TGF-β,SIg T,PIg R of hindgut were increased,while the content of foregut IL-8,midgut ET-1 and hindgut TNF-αwere decreased.In the low intestinal microorganism groups,final weight,body weight gain,daily gain coefficient and food intake of tilapia were increased at 5～10%DF.The contents of arginine and lysine in serum of tilapia increased,and were involved in the signal transduction pathway of ERK1/2 and P38 MAPK.The protein expression of P38MARK of hindgut decreased at 10%DF.The muscle layer thickness,crypt depth,Ca²⁺-Mg²⁺-ATP and AKP activities of foregut and SOD activity of hindgut were increased,while ROS activity and MDA content of hindgut were decreased.Among them,the villus height,and the gene expression level of IGF-1R and DMT1 of foregut,the activities of Na⁺-K⁺-ATP,trypsin and CK of foregut,and the activities of CAT,GSH,and GSH-Px content of hindgut were increased at 5～10%DF.With the DF level increasing,the foregut epithelial microvilli of tilapia were slightly damaged.The TRE of foregut,the contents of MUC2 and IL-10 and MUC2 gene expression of hindgut were increased,while the gene expression level of Clacudin-3 of foregut,and the gene expression level of TNF-αand IFN-γof hindgut were decreased.At the same time,the abundance of Firmicutes,Verrucomonas and Brevundimonas were increased,while Bacteroides decreased.Among them,the contents of midgut PIg R,SIg T and IFN-γof hindgut were increased at 2.5%DF.The contents of IL-1β,IL-8 and IL-12 of midgut were increased at5%DF.The gene expression level of hindgut SIg T was increased,while IL-1βwas decreased at 10%DF.The contents of ET-1,TGF-βand IL-6 of midgut and PIg R,IL-1βand IL-6 of hindgut were increased at2.5～5%DF.The gene expression level of ZO-1 of foregut and midgut IL-10 content were increased at2.5～7.5%DF.The contents of midgut IFN-γand hindgut ET-1 were increased at 5～7.5%DF.The content of LZM was increased,while hindgut IL-12 content decreased at 5～10%DF.The C4 content and TGF-βgene expression level of hindgut were increased,while the gene expression level of Clacudin-12 and IL-12 content of foregut were decreased at 7.5～10%DF.In conclusion,2.5～10%DF promotes the growth of tilapia attributed to that which can promotes substances increased that involved in inhibited inflammation and enhanced antioxidant,and improve intestinal digestion and absorption function and barrier function.And cellulose does not mediate intestinal microorganism and its metabolites to regulate intestinal health of omnivorous fish.3.The mechanism of cellulose mediated intestinal microorganism and metabolites regulating intestinal health of carnivorous fishThe rainbow trout basal diet（40%crude protein,15%crude fat）were formulated,which contained fish meal,casein and gelatin were used as main protein sources,and soybean oil and fish oil used as the main fat source.The 9-week feeding experiment was conducted,and results were as follows:In the normal intestinal microorganism groups,the intake and intake rate of rainbow trout were showed an increasing trend.The contents of niacin and adenosine in the serum of rainbow trout decreased,and were involved in the ERK1/2 signal transduction pathway,and the protein expression of ERK2 and p-ERK1/2 of hindgut had no significant changes.Acetic acid content was absolutely dominant in the content of SCFA.The villus height,muscular layer thickness and crypt depth of foregut were decreased,as well as the activities of Na⁺-K⁺-ATP,Ca²⁺-Mg²⁺-ATP,trypsin,AKP and CK and the gene expression level of SGLT-1 of foregut were decreased.Among them,caproic acid content was lowest at 5%DF.The intestinal relative weight,GLUT-2 gene expression level of foregut,and GSH content and the activities of CAT and GSH-Px of hindgut were decreased,while hindgut MAD content was increased at 5～10%DF.The gene expression level of EGF and DMT1 of foregut and hindgut SOD activity were decreased at7.5～10%DF.The tight junction structure between intestinal epithelial cells was incomplete,and the tight junction of intestinal epithelial cells was damaged.The contents of SIg T,IL-10,TGF-βof midgut,the gene expression level of MUC2,TGF-βand SIg T of hindgut were decreased,while the contents of IL-1βand IL-6 of midgut,and the gene expression level of IL-1βand TNF-αof hindgut were increased.The abundance of Firmicutes and Streptococcus were increased,while Verrucococcus decreased.Among them,the contents of foregut ET-1 and midgut PIg R were decreased,while the contents of IFN-γ,IL-1βand IL-12 of foregut,midgut TNF-αand hindgut IL-1βwere increased at 10%DF.The TRE,the gene expression level of Occludin,Claudin-3,Claudin-12,ZO-1 of foregut,the contents of LZM,MUC2,C4,ET-1,SIg T,PIg R of hindgut,and the gene expression level of hindgut PIg R were decreased,while the contents of foregut IL-8,IL-8 and IL-12 of midgut,and IL-6,IL-8 and IL-12 of hindgut,and hindgut IL-8 gene expression were increased at 5～10%DF.The contents of SIg T,PIg R and IL-10 of foregut,C3,IL-10 and TGF-βof hindgut and gene expression of hindgut IL-10 were decreased,while the contents of foregut IL-6 and midgut IFN-γ,and gene expression of hindgut IFN-γwere increased at 7.5～10%DF.In the low intestinal microorganism groups,with the DF level increasing,the final weight,body weight gain,daily gain coefficient,food intake and feeding rate of rainbow trout showed decline trend.The contents of succinic acid and taurine in the serum of rainbow trout were decreased,and were involved in the ERK1/2 signal transduction pathway,and the protein expression of ERK2 and p-ERK1/2 of hindgut had no significant change.The villus height,muscular layer thickness and crypt depth of foregut,relative intestinal weight,the gene expression level of EFG,IGF-1,DMT1 and SGLT-1 of foregut,the activities of Na⁺-K⁺-ATP,Ca²⁺-Mg²⁺-ATP,trypsin,AKP,CAT and GSH of foregut were decreased.Among them,the contents of acetic acid and valerate were decreased at 7.5%DF.The relative length of intestinal tract decreased at 10%DF.The gene expression level of foregut IGF-1R,the activities of foregut CK and SOD,GSH-Px of hindgut and hindgut MDA content were decreased at 5～10%DF.With the DF level increasing,the tight junction structure between intestinal epithelial cells of steelhead was incomplete and damaged.The gene expression level of Claudin-3,Claudin-12 and ZO-1 of foregut,the contents of LZM and C3 of hindgut,and the gene expression level of MUC2,TGF-β,IFN-γand PIg R of hindgut were decreased.The contents of IL-12 and TNF-αof foregut,hindgut IL-6,and the gene expression level of IL-1β,IL-8 and TNF-αof hindgut were increased.Meanwhile,the abundance of Firmicutes was increased,while Proteobacteria and Brevundimonas decreased.Among them,the midgut TGF-βcontent was increased,while hindgut ET-1 content was decreased at 10%DF.The contents of IFN-γof hindgut was increased,while the contents of C4,SIg T and PIg R of hindgut were decreased at 5～7.5%.The TRE,Occludin gene expression level and the contents of TGF-βof foregut,the content of midgut SIg T,and the content of MUC2 and IL-10 gene expression of hindgut were decreased,while the contents of IL-1βand IL-6 of foregut and IL-1β,IL-6,IL-8 and TNF-αof midgut were increased at 5～10%DF.The contents of IFN-γand IL-8 of foregut,midgut IFN-γ,and IL-1βand IL-8 of hindgut were increased at 7.5～10%DF.In conclusion,DF can inhibit the growth of rainbow trout attributed to that which can inhibit substances increased that involved in improving growth performance,and inhibit intestinal digestion and absorption function and barrier function.And cellulose does not mediate intestinal microorganism and its metabolites to regulate intestinal health of carnivorous fish.