Effects Of Dietary Iron Level On The Growth Performance, Intestinal Health, Body Health, Gill Health And Flesh Quality,as Well As The Possible Mechanisms In Young Grass Carp (Ctenopharyngodon Idella)

This study investigated the effects of iron level on the growth performance,disease resistance,intestine health,body health,gill health and flesh quality,as well as the possible mechanisms in young grass carp(Ctenopharyngodon idella),and determined the comparison between ferrous fumarate and ferrous sulfate sources as well as the optimal dietary iron requirements for young grass carp.A total of 630 healthy grass carp(242.32±0.58 g)were fed six diets containing graded levels of iron(12.15,35.38,63.47,86.43,111.09,136.37 and 73.50 mg/kg diet)for 60 days to investigate the effects of iron on growth performance and flesh quality of young grass carp.After growth trial,fish were challenged with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days to investigate the effects of iron level on the immune function,apoptosis related parameters,antioxidant ability and tight junction protein as well as its potential mechanisms in intestine,head kidney,spleen,skin and gill of young grass carp.The results showed that compared with iron deficiency(12.15 mg iron/kg diet),optimal iron level increased percent weight gain(PWG),feed intake(FI),feed efficiency(FE)and specific growth rate(SGR)(P<0.05),suggesting that iron could improve the growth performance of young grass carp.Optimal iron level decreased enteritis,skin hemorrhage and lesion,and gill rot morbidity(P<0.05),suggesting that iron could increase the disease resistance of young grass carp.The ability against enteritis,skin hemorrhage and lesion,and gill rot morbidity are related to the intestinal health,body health and gill health.In this study,optimal iron level increased lysozyme(LZ)and acid phosphatase activities(ACP),complement 3,complement 4 and immunoglobulin M(IgM)contents(P<0.05),up-regulated the mRNA levels of hepcidin,liver-expressed antimicrobial peptide 2(LEAP-2),β-defensin-1,anti-inflammatory cytokines,inhibitor of KBα(IκBα),target of rapamycin(TOR)and ribosomal protein S6 kinases 1(S6K1)(P<0.05),whereas down-regulated pro-inflammatory cytokines,nuclear factor kappa B(NF-κB)p65,IκB kinase β(IKKβ),IκB kinase y(IKKy)and eIF4E-binding proteins 2(4E-BP2)mRNA levels in intestine,head kidney,spleen,skin and gill of young grass carp(P<0.05),suggesting that iron could alleviate the inflammation and enhance immune function of grass carp;Meanwhile,dietary iron 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),whereas down-regulated the mRNA levels of cysteinyl aspartic acid-protease(caspase 2,8,9),Fas ligand(FasL),apoptotic protease activating factor-1(Apaf-1)and Bcl-2 associated X protein(Bax)(P<0.05),suggesting that iron could inhibit the apoptosis,maintiaining structural integrity in intestine,head kidney,spleen,skin and gill of grass carp;Meanwhile,optimal iron level decreased malondialdehyde(MDA),protein carbonyl(PC)and reactive oxygen species(ROS)contents(P<0.05),increased the activities and mRNA levels of antioxidant enzymes glutathione peroxidase(GPx),glutathione-S-transferase(GST)and glutathione reductase(GR)(P<0.05),and up-regulated the mRNA levels of NF-E2-related factor 2(Nrf2)(P<0.05),down-regulated the mRNA levels of Kelch-like-ECH-associated protein 1b(Keap1b)in intestine,head kidney,spleen,skin and gill of young grass carp(P<0.05),suggesting that iron could enhance the antioxidant ability of grass carp;down-regulated the mRNA levels of myosin light chain kinase(MLCK)and claudin-12,and up-regulate the mRNA levels of tight junction protein occludin,zonula occludens(ZO),claudin-b,-c,-f,-7a and-7b(P<0.05)(P<0.05),suggesting that ironcould maintain the intercellular structural integrity in intestine,head kidney,spleen,skin and gill of grass carp.At the same time,optimal iron level increased pH value and hydroxyproline content,and decreased cooking lose,shear force,cathepsin B,cathepsin L and lactate contents in muscle of young grass carp;meanwhile,dietary iron increased crude protein,crude lipid,free amino acid and PUFA contents,suggesting that iron could enhance the flesh quality of grass carp;optimal iron level decreased MDA,PC and ROS(P<0.05),increased the activities and mRNA levels of CuZnSOD,MnSOD,GPx,GST and GR(P<0.05),and up-regulated the mRNA levels of CK2a,CK2β,TOR,S6K1 and Nrf2(P<0.05),down-regulated the mRNA levels of Keap1a and Keap1b in the muscle of young grass carp(P<0.05),suggesting that ironould enhance the antioxidant ability in muscle of grass carp.As stated above,optimal iron level could increase the growth performance and ability of against enteritis,skin hemorrhage and lesion and gill rot morbidity.The increased ability against enteritis,skin hemorrhage and lesion and gill rot morbidity of young grass carp are related to the enhanced intestinal health,body health ang gill health with the supplemention of dietary iron.Optimal iron level enhanced the intestinal immune,body immune and gill immune function of young grass carp were related to the increased antibacterial compounds production and attenuated inflammation under the regulation of down-regulated pro-inflammatory cytokines which is partially related to NF-κB signalling and up-regulated anti-inflammatory cytokines which is partially related to TOR signaling in intestine,head kidney,spleen,skin and gill of young grass carp.Dietary iron enhance the structural integrity in intestine,head kidney,spleen,skin and gill of young grass carp which is partially related to,(1)inhibit apoptosis which is partially related to up-regulated anti-apoptosis factors and down-regulated pro-apoptosis factors gene expression,(2)enhance the antioxidant ability which is partially related to the up-regulation of gene expression and activities of antioxidant enzyme under the regulation of Keapl/Nrf2 signalling,(3)enhance the intercellular structural integrity which his partially related to the gene expression of tight junction protein under the regulation of MLCK signaling.Meanwhile,iron improve flesh quality which was partialy related to the increased physical quality,nutrition quality and antioxidant ability in grass carp.In this study,based on the growth performance,ability against enteritis,skin hemorrhage and lesion and gill rot morbidity,the optimal iron level for young grass carp(242-756g)were calculated to be 75.65 mg/kg,87.03 mg/kg,83.37 mg/kg and 76.52 mg/kg diet.