Effects Of Dietary Sodium Butyrate On Intestinal Health And The Potential Mechanisms In Young Grass Carp

The present study evaluated the effect of dietary sodium butyrate(SB)supplementation on the growth performance,intestine,head kidney,spleen,skin and gill health and the potential mechanisms in young grass carp(Ctenopharyngodon idella).A total of 540 grass carp(256.57 ± 0.71 g)were fed one powdery sodium butyrate(PSB)diet(1000.0 mg/kg diet)and five graded levels of microencapsulated sodium butyrate(MSB)diets:0.0(control),500.0,1000.0,1500.0 and 2000.0 mg/kg diet for 60 days to investigate the effect of SB supplementation on the fish growth performance,intestinal development,intestinal digestion capacity,intestinal bacteria,intestinal VFAs contents and flesh quality.After the feeding trial,two challenge test were conducted by injection fish with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days to explore the effect of SB on fish intestine[proximal intestine(PI),middle intestine(MI)and distal intestine(DI)],head kidney,spleen,skin and gill health,as well as the potential mechanisms.The results showed that optimal SB supplementation increased the grass carp growth performance[percent weight gain(PWG),specific growth rate(SGR),feed intake(FI)and feed efficiency(FE)],improved the intestinal development[intestinal weight(IW),intestinal length(IL)and intestinal somatic index(ISI)]and intestinal digestion capacity(trypsin,chymotrypsin,lipase and amylase activities,as well as folds height),increased lactobacillus amount and butyrate concentration,decreased Aeromonas and Escherichia coli amounts,as well as acetate and propionate concentrations.Further study showed that SB enhanced the ability of fish defensing against enteritis,skin lesion and gill rot after challenge test.The improvement of the ability of grass carp defensing against enteritis,skin lesion and gill rotare closely related to the maintenance of the intestine,head kidney,spleen,skin and gill structure integrity:(1)optimal SB supplementation decreased the reactive oxygen species(ROS),malondialdehyde(MDA)and protein carbonyl(PC)contents,increased copper/zinc superoxide dismutase(CuZnSOD)(rather than head kidney,spleen and skin),manganese superoxide dismutase(MnSOD),catalase(CAT),glutathione peroxidase 1a(GPx1a),GPx1b,GPx4a,GPx4b,glutathione S-transferases R(GSTR),GSTP1,GSTP2,GSTO1,GST02 and glutathione reductase(GR)mRNA levels and enzyme activities in the intestine,head kidney,spleen,skin and gill,up-regulated NF-E2-related factor 2(Nrf2)mRNA levels and down-regulated kelch-like-ECH-associated protein la(Keapla)(rather than PI,MI and DI)and Keap1b mRNA levels in the intestine,head kidney,spleen,skin and gill,enhanced the ability of antioxidative damage,maintained the intestine,head kidney,spleen,skin and gill cell structure integrity.Meanwhile,optimal SB supplementation prevented the DNA fragments in the intestine,head kidney,spleen,skin and gill,down-regulated c-Jun N-terminal protein kinase(JNK)(rather than skin),factor-related apoptosis ligand(FasL),pro-apoptotic protein apoptotic protease activating factor 1(Apaf-1)as well as B-cell lymphoma protein 2 associated X protein(Bax)(rather than skin),caspase-2,caspase-3(rather than PI),caspase-7,caspase-8(rather than PI)and caspase-9 mRNA levels,and up-regulated the anti-apoptotic protein B-cell lymphoma 2(Bcl-2)(rather than PI),inhibitor of apoptosis proteins(IAP)and myeloid cell leukemia 1(Mcl-1)mRNA level in the intestine,head kidney,spleen,skin and gill,down-regulated eIF4E-binding protein 1(4E-BP1)and 4E-BP2 mRNA level,and up-regulated the target of rapamycin(TOR)and ribosomal protein S6 kinase 1(S6K1)mRNA level in the intestine,suppressed intestine,head kidney,spleen,skin and gill cell apoptosis;(2)optimal SB supplementation up-regulated the mRNA levels of occludin(rather than spleen),zonula occludens-1(ZO-1),ZO-2,claudin-b,claudin-c,claudin-f,claudin-3c(rather than PI,head kidney,spleen,skin and gill),claudin-7a,claudin-7b and claudin-11,and down-regulated the mRNA levels of claudin-12(rather than head kidney,spleen,skin and gill),claudin-15a,claudin-15b and the molecule myosin light-chain kinase(MLCK)in the intestine,head kidney,spleen,skin and gill,thus maintained the intestine,head kidney,spleen,skin and gill structural integrity between cells.Besides,SB supplementation enhanced the ability of grass carp defensing against enteritis,skin lesion and gill rot which also related to enhancing grass carp intestine,head kidney,spleen,skin and gill immune function:(1)optimal SB supplementation increased immune related factors lysozyme(LZ)and acid phosphatase(ACP)activities,complement 3(C3),C4 and IgM contents,and up-regulated ?-defensin-1(rather than DI),hepcidin,liver expressed antimicrobial peptide 2A(LEAP-2A)(rather than intestine),LEAP-2B and Mucin2(rather than head kidney,spleen and skin)mRNA levels in the intestine,head kidney,spleen,skin and gill to improve immune function;(2)optimal SB supplementation down-regulated proinflammatory cytokine tumor necrosis factor a(TNF-a)(rather than spleen and gill),interferon y2(IFN-y2),interleukin-1?(IL-1?)(rather than PI,head kidney,spleen and skin),IL-6(rather than spleen and gill),IL-8(rather than head kidney,spleen and skin),IL-15(rather than PI),IL-17D(rather than PI),IL-12p35 and IL-12p40(rather than PI and MI)mRNA levels,and up-regulated anti-inflammatory cytokines IL-10(rather than skin and gill),IL-11(rather than PI),transforming growth factor ?1/?2(TGF-?1/?2)(rather than PI),IL-4/13A and IL-4/13B mRNA levels,and down-regulated pro-inflammatory signaling molecules I?B kinase a(IKKa)(rather than intestine),IKK?(rather than PI),IKKy,nuclear factor-KB p52(NF-?B p52)(rather than intestine),NF-?B p65 and c-Rel(rather than PI and MI)mRNA levels,while up-regulated inhibitor of ?B?(I?B?)mRNA levels in the intestine,head kidney,spleen,skin and gill,down-regulated p38 mitogen-activated protein kinases(p38MAPK)and MAPKK6 mRNA levels in the intestine,up-regulated TOR and S6K1 mRNA levels,and down-regulated 4E-BP1 and 4E-BP2 mRNA levels in the head kidney,spleen,skin and gill to attenuate the inflammatory response and strengthen grass carp intestine,head kidney,spleen,skin and gill immune function.Meanwhile,optimal SB supplementation improved the fish muscle quality through increase of the nutritional value(protein,amino acid,fatty acid contents),water holding capacity,pH and so on,and optimal SB supplementation enhanced muscle quality partly related to enhancing muscle antioxidant ability,optimal SB supplementation up-regulated Nrf2,down-regulated Keap1a and Keap1b mRNA levels,then increasedCuZnSOD,MnSOD,CAT,GPxla,GPxlb,GPx4a,GPx4b,GSTR,GSTP1,GSTP2,GSTO1,GST02 and GR mRNA levels and enzyme activities,decreased ROS content,reduced lipid peroxidation and protein oxidative damage.Furthermore,in this study,the effect of MSB on grass carp growth performance,intestine,head kidney,spleen,skin and gill health were superior to PSB.Taken together,current study indicated that optimal SB increased fish growth performance and intestinal development,and enhanced intestinal digestion capacity,optimized intestinal bacteria,and improved the ability of fish defensing against enteritis,skin lesion and gill rot which were closely related to the enhanced intestine,head kidney,spleen,skin and gill structural integrity and immune function:(1)SB might be via up-regulating Nrf2 signaling to enhance the CuZnSOD et al.antioxidant enzyme gene expressions and activities,thus enchanced the ability of antioxidative damage,and through up-regulating the anti-apoptotic protein Bcl-2,Mcl-1 and IAP,as well as down-regulating the pro-apoptotic protein Apaf-1 and Bax gene expressions to inhibit the cell apoptosis,maintained the intestine,head kidney,spleen,skin and gill cell structure integrity;(2)SB might be via down-regulating MLCK signaling to regulate the tight junction protein gene expression,thus maintained the structural integrity between cells;(3)SB might be via improving the antibacterial components activity and contents,up-regulating the antimicrobial peptide gene expression to enhance antibacterial ability,SB might be via down-regulating NF-?B p65 et al.signaling molecules to down-regulate the main pro-inflammatory cytokines and up-regulate the main anti-inflammatory cytokines gene expression,attenuated the inflammatory response and improved the grass carp intestine,head kidney,spleen,skin and gill immune function.Meanwhile,SB improved fish muscle nutritional value and water holding capacity,et al.to improve flesh quality,which might be relating to enhance of muscle antioxidant damage ability.Finally,based on growth perfomance,intestinal health,gill health,head kidney,spleen and skin health and flesh quality indexes,the optimal SB supplementation(MSB as SB source)of grass carp(256-781 g)were estimated to be 2266.7-283.0,196.4-548.7,307.4-427.7,278.9-387.5,298.9-430.8 mg/kg diet,respectively.