Effects Of Dietary DL-methionyl-DL-methionine On The Growth Performance,Intestinal Health,Gill Health And Body Health,as Well As The Mechanisms In Juvenile Grass Carp (Ctenopharyngodon Idella)

This study investigated the effects of DL-methionyl-DL-methionine(Met-Met)on the growth performance,intestinal health,gill health,body health and the potential mechanisms,as well as the optimal Met-Met supplementation for juvenile grass carp(Ctenopharyngodon idella).A total of 2520 health grass carp(10.10 ± 0.03 g)were fed one dite containing 2.50 g DL-Met/kg and six diets containing graded levels of Met-Met(0,0.79,1.44,1.84,2.22 and 2.85 g/kg diet)for 10 weeks.After growth trial,fish were challenged with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days to investigate the effects of Met-Met on intestine,head kidney(HK),spleen(SP),skin and gill structural integrity and immune function as well as the potential mechanisms,respectively.The results indicated that compared with methionine deficiency,optimal Met-Met supplementation increased percent weight gain(PWG),specific growth rate(SGR),feed intake(FI),feed efficiency(FE),muscle and hepatopancreas glutamate oxaloacetate transaminase(GOT)and glutamate pyruvate transaminase(GPT)activities,and decreased plasma amino content(PAC)(P<0.05),improving the growth performance of juvenile grass carp.Met-Met supplementation decreased enteritis,skin lesion and gill rot morbidity(P<0.05),increased intestinal,gill and body health.It might be partially related to improving structural integrity and immune function in the intestine,HK,SP,skin and gill of fish.The results of this study indicated that compared with methionine deficiency,optimal Met-Met supplementation decreased malondialdehyde(MDA),protein carbonyl(PC)and reactive oxygen species(ROS)contents(P<0.05),increased the capacities of anti-superoxide anion(ASA)and anti-hydroxy radical(AHR),content of glutathione(GSH)(rather than SP),the activities and mRNA levels of antioxidant enzymes manganese superoxide dismutase(MnSOD),catalase(CAT)(rather than gill),glutathione peroxidase(GPx),glutathione-S-transferase(GST)and glutathione reductase(GR)(P<0.05),which was associated with the up-regulation of NF-E2-related factor 2(Nrf2)mRNA levels(P<0.05)and down-regulation of Kelch-like-ECH-associated protein 1a(Keap1a)and Keap1b mRNA levels in the intestine,HK,SP,skin and gill of juvenile grass carp(P<0.05),enhancing fish against oxidative damage capacity and reducing the oxidative damage;Optimal dietary Met-Met supplementation down-regulated the mRNA levels of cysteinyl aspartic acid-protease(caspase-2,-3,-7,-8,-9),fatty acid synthetase ligand(FasL),apoptotic protease activating factor-1(Apaf-1),Bcl-2 associated X protein(Bax),c-Jun-N-terminal kinase(JNK)(rather than SP)and p38 mitogen-activated protein kinase(p38MAPK)(rather than gill)(P<0.05),up-regulated the mRNA levels of B-cell lymphoma protein-2(Bcl-2),inhibitor of apoptosis proteins(IAP)and myeloid cell leukemia-1(Mcl-1)(P<0.05),inhibiting the apoptosis to ensure the structural integrity in the intestine,HK,SP,skin and gill of fish;Optimal dietary Met-Met supplementation up-regulated the mRNA levels of tight junction protein occludin,zonula occludens 1(ZO-1)(rather than SP),ZO-2(rather than skin),claudin-b,-c(rather than PI or MI),-f,-3c,-7a,-7b,-11(rather than gill),-12,-15a and-15b,and down-regulated the mRNA levels of myosin light chain kinase(MLCK)(P<0.05),suggesting that Met-Met could maintain the intercellular structural integrity in the intestine,HK,SP,skin and gill of fish.In addition,compared with methionine deficiency,optimal Met-Met supplementation increased lysozyme(LZ)and acid phosphatase(ACP)activities,complement 3(C3),C4 and immunoglobulin M(IgM)contents(P<0.05),up-regulated the mRNA levels of hepcidin,liver-expressed antimicrobial peptide 2A(LEAP-2A),LEAP-2B,p-defensin-1 in the intestine,HK,SP,skin and gill of juvenile grass carp.Meanwhile,Met-Met down-regulated pro-inflammatory cytokines tumor necrosis factor ?(TNF-?),interferon y2(IFN-?2),interleukin 1?(IL-1?)(rather than gill),IL-8(rather than PI,MI or SP),IL-12p35,IL-12p40,IL-15,IL-17D(rather than SP)and nuclear factor kappa B p65(NF-?B p65),I?B kinase ?(IKK?),IKK?,eIF4E-binding proteins 1(4E-BP1)(rather than skin)and 4E-BP2 mRNA levels(P<0.05),up-regulated anti-inflammatory cytokines IL-4/13A,IL-4/13B,IL-6,IL-10(rather than gill),IL-11,transforming growth factor ?1(TGF-?1)and TGF-?2,inhibitor of ?B?(I?B?),target of rapamycin(TOR)and ribosomal protein S6 kinases 1(S6K1)mRNA levels(P<0.05),alleviating the inflammation and enhancing fish intestine,HK,SP,skin and gill immune function.In this study,based on growth performance,against desease resistance,intestine health,gill health and body health related parameters,Met-Met was better than DL-Met.As stated above,Met-Met supplementation could increase the growth performance of juvenile grass carp.The improvement of structural integrity by Met-Met might be partially related to the up-regulation of gene expression and activities of antioxidant enzyme by Nrf2 signaling,leading to the increased antioxidant capacity and decreased oxidative damage;the down-regulation of pro-apoptosis factors and up-regulation of anti-apoptosis factors gene expression to inhibit apoptosis;the down-regulation of MLCK signaling to up-regulate the gene expression of tight junction protein,leading to the improvement of intercellular stractural integrity in the intestine,HK,SP,skin and gill of juvenile grass carp.The improvement of immune function by Met-Met might be partially related to the increased of antimicrobial compounds production,antimicrobial peptide gene expression in the intestine,HK,SP,skin and gill of juvenile grass carp.Met-Met supplementation attenuated inflammation by NF-?B signaling to down-regulate pro-inflammatory cytokines and TOR signaling to up-regulate anti-inflammatory cytokines gene expression in the intestine,HK,SP,skin and gill of juvenile grass carp.Measwhile,as the basal diet containing 8.03 g/kg sulfur-containing amino acids(methionine and cysteine),based on the growth performance,protecting fish against the enteritis,skin lesion and gill rot morbidity,the optimal Met-Met supplementation for juvenile grass carp(10.10-126.39 g)were estimated to be 1.61,1.64,1.65 and 1.65 g/kg diet,respectively.