Different Types Of Non-starch Polysaccharides Mediate The Mechanism Of Intestinal Flora And Their Metabolites On The Regulation Of Intestinal Health In Largemouth Bass

Largemouth bass(Micropterus salmoides)has become one of the main economic farmed fish species in China because of its fast growth and delicious meat quality,which is widely favored by farmers and consumers.As a carnivorous fish,largemouth bass has a high demand for feed protein,and a high level of fishmeal is usually added to its commercial feed.However,increasingly higher fishmeal prices have limited the benefits of largemouth bass farming and the further development of the farming industry,forcing the addition of more cheap plant-based feed ingredients to its feed,bringing in high doses of non-starch polysaccharides(NSPs).NSPs are a kind of complex polysaccharides existing in plant cell walls,which can be divided into insoluble NSPs(INSP)and soluble NSPs(SNSP).Fish cannot use NSPs due to the lack of endogenous NSPs degrading enzymes,leading to the retention of NSPs in the intestine,which can not only directly affect the digestion and absorption of feed nutrients,but also regulate the intestinal health and physiological metabolism of fish mediated by the intestinal flora.Intestinal health is a prerequisite to ensure the rapid growth of fish.Therefore,it is meaningful to refine the mechanism of NSPs regulating the intestinal health of fish.This study explored the mechanism of different types of NSPs mediating intestinal microflora and their metabolites regulating intestinal health of largemouth bass from the perspective of nutritional physiology and molecular mechanism.The research results can not only provide strategies for improving the intestinal health of largemouth bass,but also provide data support for scientific preparation the commercial feed of largemouth bass.The main results are as follows:1.Effects of different dietary NSPs levels on the growth,intestinal microflora,metabolites and intestinal health of largemouth bassSeven isoproteic and isolipidic diets(crude protein 47.0%,crude lipid 12.5%)were prepared to contain 0%,3%,6%,9%,12%,15%and 18%purified NSPs(each100 g of NSPs contained 32.26 g of cellulose,19.35 g of arabinoxylan,4.52 g ofβ-glucan,5.16 g of mannans,and 38.71 g of pectin),respectively,and were named as control group,NSPs3,NSPs6,NSPs9,NSPs12,NSPs15 and NSPs18 groups.After feeding experimental fish for 8 weeks,(1)the final body weight(FBW),weight gain rate(WGR),specific growth rate(SGR),protein efficiency ratio(PER),protein deposition rate(PDR)and lipid deposition rate(LDR)in the NSPs12-NSPs18 group were significantly lower than those of the control group(P<0.05);(2)In the NSPs12-NSP18 groups,the length and width of intestinal villus,the muscular layer thickness and the goblet cells numbers increased significantly,while the activities of protease,alkaline phosphatase(AKP)and apparent digestibility of nutrients decreased significantly(P<0.05);(3)plasma lipopolysaccharide(LPS)and endotoxin(ET-1)contents and intestinal IL-8,IL-1β,TNF-α,Keap1,Caspase3,Bax and BAD expression levels in the NSPs15 and NSPs18 groups increased significantly,while the intestinal peroxidase(POD),catalase(CAT),superoxide dismutase(SOD)activities and intestinal Occludin,Caludin-1,Caludin-4,ZO-1,IL-10,TGF-β1,Nrf2,Bcl-2,Bcl-xl and BAG expression levels decreased significantly(P<0.05);(4)in the NSPs18 group,the Shannon,Simpson,Chao,ACE index and the abundance of Paraclostridium bifermentans and Mycoplasma moatsii was significantly decreased while the abundance of Plesiomonas shigelloides,[Clostridium]dakarense and Romboussia ilealis were significantly decreased(P<0.05);(5)the content of total short chain fatty acid(TSCFA),propionate,butyrate and valerate in the NSPs6 group was significantly increased,while those parameters in the NSPs18 group were significantly decreased(P<0.05);(6)the content of epinephrine,acetylcholine and 4-aminobutyric in the NSPs18group was significantly lower than that in the control group,while the content of dopamine and norepinephrine were significantly higher than those in the control group(P<0.05).These results showed that high-dose dietary NSPs reduced intestinal antioxidant function,intestinal floraα-diversity,and the content of beneficial metabolites of intestinal microbiota;but increased intestinal mucosal permeability and the abundance of pathogenic bacteria,and induced intestinal inflammation and apoptosis of intestinal epithelial cells,resulting in intestinal health damage.In contrast,low-dose dietary NSPs can improve intestinal health and have the potential as"prebiotics"for aquatic feed.Based on the regression analysis of growth performance,the dietary NSPs contents of largemouth bass should not exceed 5.51%.2.Effects of different solubility of NSPs on the growth performance of largemouth bassThis study aimed at exploring the differential in the mechanisms of different solubility NSPs(INSP and SNSP)mediating intestinal microflora regulating intestinal health of largemouth bass.Four isoproteic and isolipidic diets(crude protein 42.5%,crude lipid 13.7%)were prepared to contain 8%microcrystalline cellulose(control group),8%low viscous carboxymethyl cellulose(Lvs-CMC group),8%middle viscous carboxymethyl cellulose(Mvs-CMC group)and 8%high viscous carboxymethyl cellulose(Hvs-CMC group),respectively.After feeding experimental fish for 8 weeks,(1)the FBW,WGR,SGR,PER,PDR and LDR in carboxymethyl cellulose(CMC)groups were significantly lower than those in the control group(P<0.05);(2)the length of intestinal microvillus in the Lvs-CMC and Hvs-CMC groups decreased significantly,while the intestinal muscularis thickness and the goblet cells number in the Mvs-CMC and Hvs-CMC groups increased significantly(P<0.05);(3)the plasma diamine oxidase(DAO)activity and the plasma ET-1 content in the CMC groups,and the plasma LPS content in the Mvs-CMC and Hvs-CMC groups increased significantly(P<0.05);(4)intestinal Caludin-1,IL-10,TGF-β1 expression levels,GSH content and CAT activity in the CMC groups decreased significantly,while Keap1 expression level increased significantly(P<0.05);(5)intestinal Nrf2,IL-1βexpression levels and Ig M content in the Mvs-CMC and Hvs-CMC groups decreased significantly(P<0.05);(6)the OUT numbers,Sobs,Shannon and Simpson indices in the CMC groups decreased significantly(P<0.05);(7)the content of acetate in the Lvs-CMC and Hvs-CMC groups increased significantly,while the content of butyrate,L-Kynurenine,5-Hydroxyindole-3-acetic acid,glutamine,tryptophan,histidine,arginine and phenylalanine decreased significantly(P<0.05).These results showed that SNSP was the main antinutritional type of NSPs,and its antinutritional action intensity was proportional to its viscosity.SNSP can increase feed viscosity,reduce intestinal antioxidation and physical barrier function of intestinal mucosa,induce intestinal inflammation and colonization of pathogenic microorganisms,and damage intestinal health.3.Effects of different viscous guar gum on the growth,intestinal microflora,metabolites and intestinal health of largemouth bassThis study was aimed to explore the differential in the mechanisms of different viscous guar gum(galactomannan)mediating intestinal microflora regulating intestinal health of largemouth bass,and provide data support for developing guar gum as an aquatic feed binder.Four isoproteic and isolipidic diets(crude protein 42.5%,crude lipid 13.7%)were prepared to contain 8%microcrystalline cellulose(control group),8%low viscous guar gum(Lvs-GG group),8%middle viscous guar gum(Mvs-GG group)and 8%high viscous guar gum(Hvs-GG group),respectively.After feeding experimental fish for 8 weeks,(1)the FBW,WGR,SGR,PER,PDR and LDR in guar gum groups were significantly lower than those in the control group,and decreased significantly with the increased viscosity(P<0.05);(2)the intestinal villus length and the muscular layer thickness in the guar gum groups increased significantly,while the goblet cell numbers,intestinal protease,lipase and AKP activities in the Mvs-GG group and Hvs-GG group decreased significantly(P<0.05);(3)plasma LPS and ET-1contents in the Hvs-GG group increased significantly,while intestinal Caludin-1 and ZO-1 expression level decreased significantly(P<0.05);(4)intestinal CAT activity and Bax expression level decreased significantly;intestinal IL-1β,IL-8,TNF-α,Keap1and Nrf2 expression levels in the Hvs-GG group increased significantly,while intestinal IL-10,TGF-β,Bcl-xl,Caspase3 and BAD expression levels decreased significantly(P<0.05);(5)the Shannon and Simpson indices in the guar gum groups decreased significantly,while the number of OUT and Sobs index in Hvs-GG group decreased significantly(P<0.05);(6)the abundance of C.somerae in the guar gum increased significantly,while the abundance of Clostridium colicanis,Clostridium perfringens,R.lituseburensis and P.bifermentans decreased significantly(P<0.05);the abundance of P.shigelloides,C.dakarense and Mycoplasma moatsii in the Hvs-GG group increased significantly(P<0.05);(7)the content of TSCFA,acetate and propionate in the guar gum groups increased significantly,while the contents of butyrate in the Mvs-GG and Hvs-GG groups decreased significantly(P<0.05);(8)the content of 5-Hydroxyindole-3-acetic acid,glutamine and arginine in the Lvs-GG and Hvs-GG groups decreased significantly,while the histamine content increased significantly(P<0.05).These results showed that guar gum reduced intestinal antioxidant function,damaged intestinal mucosal mechanical barrier,induced intestinal inflammation and apoptosis of intestinal epithelial cells,and damaged intestinal health.The strength of anti-nutritional action of guar gum is directly proportional to its viscosity.When developing guar gum as a binder for aquatic feed,the negative effect of guar gum viscosity should be carefully considered.4.Effects of pectin with different esterification degrees on the growth,intestinal microflora,metabolites and intestinal health of largemouth bassThis study aimed to explore the differential mechanism of different esterification degree pectin mediated intestinal microflora regulating intestinal health of largemouth bass,and provide data support for the development of pectin as a functional feed material.Four isoproteic and isolipidic diets(crude protein 42.5%,crude lipid 13.7%)were prepared to contain 8%microcrystalline cellulose(control group),8%low methyl-esterified pectin(LMP group),8%middle methyl-esterified pectin(4%LMP+4%HMP,MMP group)and 8%high methyl-esterified pectin(HMP group),respectively.After feeding experimental fish for 8 weeks,(1)the PDR,PER,SGR,WGR and FBW in the HMP group increased significantly(P<0.05);(2)intestinal Na~+/K~+-ATP and AKP activities in the HMP group increased significantly(P<0.05);(3)the intestinal muscularis thickness in the LMP group increased significantly,while the intestinal villus length decreased significantly;the crypt depth in the pectin groups increased significantly,while the goblet cell numbers decreased significantly(P<0.05);(4)plasma LPS and D-LA contents in the HMP group decreased significantly,while intestinal ZO-1,Caludin-1,Caludin-4 and Occludin expression levels increased significantly(P<0.05);(5)the intestinal GPx and SOD activities,and the intestinal Keap1 and Nrf2 expression levels in the HMP group increased significantly(P<0.05);(6)intestinal Ig M and Ig R contents,and intestinal IL-10,Bcl-xl,BAG,Bcl-2 and BAD expression levels in the HMP group increased significantly,while intestinal TNF-α,IL-1β,IL-8,and Bax expression levels decreased significantly(P<0.05);(7)the OUT numbers,Sobs,Chao and ACE indices in the HMP group increased significantly(P<0.05);(8),the abundance of C.colicanis,C.perfringens,and C.dakarense in the LMP group decreased significantly,while the abundance of C.somerae increased significantly(P<0.05);additionally,the abundance of C.dakarense in the HMP group increased significantly,while the abundance of P.shigelloides decreased significantly(P<0.05);(9)the content of acetate,propionate,and TSCFA in the MMP group increased significantly,while the butyrate content decreased significantly in the LMP and MMP groups(P<0.05);(10)the content of epinephrine and 5-Hydroxyindole-3-acetic acid increased significantly in the LMP group,while the content of arginine,4-aminobutyric,L-tyrosine and phenylalanine decreased significantly(P<0.05);additionally,the content of norepinephrine,L-kynurenine,and choline decreased significantly in the LMP and HMP groups(P<0.05).These results show that HMP can enhance intestinal antioxidant,immune and mechanical barrier functions,alleviate intestinal inflammatory reaction,and enhance intestinal floraα-diversity,improve the structure of intestinal flora and intestinal health.In contrast,LMP can induce the colonization of pathogenic bacteria,reduce the abundance of beneficial bacteria and their metabolites in the intestine,and damage the intestinal health.In conclusion,the physiological effect of pectin is closely related to its esterification degrees,and HMP has more application potential than LMP.In conclusion,(1)the effect of dietary NSPs on the growth and intestinal health of largemouth bass is dose-dependent,and low-dose NSPs benefits for improving intestinal health and growth,while high-dose dietary NSPs exhibited the opposite results.Dietary NSPs levels should be controlled lower than 5.51%in the feed of largemouth bass.(2)SNSP is the main NSPs type that damages the intestinal health and growth of largemouth bass.SNSP induces intestinal inflammation and colonization of pathogenic bacteria by disturbing the composition of intestinal flora and its metabolites.(3)The strength of anti-nutritional effect of guar gum is proportional to its viscosity,and high viscosity guar gum causes dysbiosis of flora and its metabolites,impairs intestinal mucosal barrier function,and induces intestinal inflammation,and viscosity is the main anti-nutritional characteristic of SNSP.(4)Pectin is a beneficial SNSP that improves intestinal mucosal barrier and immune function and promotes intestinal health.The degree of esterification is a key factor affecting the physiological benefits of pectin,and HMP has a better effect on intestinal health than LMP.