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

This study investigated the effects of protein on the growth performance, disease resistance, intestinal health, body health, gill health and flesh quality, as well as its mechanisms in young grass carp ?Ctenopharyngodon idella?, and evaluated the optimal dietary protein levels for young grass carp based on different indicators. A total of 540 health grass carp ?264.11±0.76 g? were fed six diets containing graded levels of protein (143.1,176.7,217.2,257.5,292.2 and 322.8 g digestible protein kg^-1 diet) for 8 weeks After growth trial, fish were challenged with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days to investigate the effects of protein on the immune function, against oxidative damage capacity, apoptosis related parameters, tight junction protein of intestine, head kidney, spleen and skin and gill, as well as its potential mechanisms, respectively.The results indicated that compared with low (143.1 g digestible protein kg^-1 diet) or high (322.8 g digestible protein kg^-1 diet) levels of protein, optimal protein level increased percent weight gain ?PWG?, feed intake ?FI?, feed efficiency ?FE? and specific growth rate ?SGR? ?P< 0.05?, suggesting that protein could improve the growth performance of young grass carp. Optimal protein level enhanced the survival rate after challenge, and decreased enteritis, skin hemorrhage and lesion and gill rot morbidity?P<0.05?, suggesting that protein could increase the capacity of against enteritis, skin hemorrhage and lesion and gill rot morbidity of young grass carp. The capacity of against enteritis, skin hemorrhage and lesion and gill rot morbidity are related to the intestinal health, body health and gill health. The results of this study indicated that optimal protein level increased lysozyme and acid phosphatase activities, complement 3 and complement 4 contents ?P< 0.05?, up-regulated the mRNA levels of liver-expressed antimicrobial peptide 2 ?LEAP-2?, Hepcidin, ?-defensin, anti-inflammatory cytokines transforming growth factor ?1 ?TGF-?1? and TGF-?2, inhibitor of KBa ?I?B??, target of rapamycin ?TOR? and ribosomal protein S6 kinases 1 ?S6K1? ?P< 0.05?, whereas down-regulated pro-inflammatory cytokines tumor necrosis factor ? ?TNF-??, interleukin 1? (IL^-10), IL^-12p35 and interferon y2 ?IFN-y2?, nuclear factor kappa B ?NF-?B? P65, I?B kinase ? ?IKK??, I?B kinase y ?IKKy? and eIF4E-binding proteins 2 ?4E-BP2? mRNA levels in the intestine, head kidney, spleen, skin and gill of young grass carp ?P<0.05?, suggesting that protein could alleviate the inflammation and enhance fish immune function of mucosal immune and body immune function. Meanwhile, optimal protein level decreased reactive oxygen species, malondialdehyde and protein carbonyl 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 lb ?Keaplb? in the intestine, head kidney, spleen, skin and gill of young grass carp ?P<0.05?, suggesting that protein could enhance the capacity of against oxidative damage and reduce the oxidative damage; 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 3,8,9?, fatty acid synthetase ligand ?FasL?, apoptotic protease activating factor^-1 (Apaf^-1) and Bcl-2 associated X protein ?Bax? ?P< 0.05?, suggesting that protein could inhibit the apoptosis to ensure the structural integrity of intestine, head kidney, spleen, skin and gill; partly through down-regulating the mRNA levels of myosin light chain kinase ?MLCK? to up-regulate the mRNA levels of tight junction protein occludin, zonula occludens 1 (ZO^-1), claudin b,7a, 7b,12 and 15a ?P< 0.05? ?P< 0.05?, suggesting that protein could maintain the intercellular structural integrity of intestine, head kidney, spleen, skin and gill. At the same time, optimal protein level increased muscle protein, lipid and hydroxyproline contents, ?AA and fillet fatty acids composition, shear force and pH; decreased muscle cooking loss, lactate content, cathepsins B and L activities of young grass carp, suggesting that protein could enhance the flesh quality. Meanwhile, optimal protein level decreased reactive oxygen species, malondialdehyde and protein carbonyl contents ?P< 0.05?, increased the activities and mRNA levels of CuZnSOD, MnSOD, ASA, AHR, GPx, GST and GR ?P< 0.05?, and up-regulated the mRNA levels of Nrf2, CK2a, CK2?, TOR and S6K1 ?P< 0.05?, down-regulated the mRNA levels of Keap1a and Keap1b in the muscle of young grass carp ?P< 0.05?, suggesting that protein could enhance the capacity of against oxidative damage of muscle and reduce the oxidative damage to ensure the structural integrity of muscle.As stated above, optimal protein level could increase the growth performance and the capacity of against enteritis, skin hemorrhage and lesion and gill rot morbidity. The increased capacity of against enteritis, skin hemorrhage and lesion and gill rot morbidity are related to protein enhanced intestinal health, body health ang gill health by improving immune function and structural integrity of young grass carp. Optimal protein level enhanced the intestinal mucocal immune, body immune and gill immune function were related to the increased antibacterial compounds production and attenuated inflammation by TOR signalling to up-regulate anti-inflammatory cytokines and NF-?B signalling to down-regulate pro-inflammatory cytokines in the intestine, head kidney, spleen, skin and gill of young grass carp. The improvement of structural integrity by protein was related to the up-regulation of gene expression and activities of antioxidant enzyme by Nrf2 signalling, leading to the increased antioxidant capacity and decreased oxidative damage; by up-regulating anti-apoptosis factors and down-regulating pro-apoptosis factors gene expression to inhibit apoptosis; by MLCK signalling to affect the gene expression of tight junction protein, leading to the improvement of intercellular stractural integrity in the intestine, head kidney, spleen, skin and gill of young grass carp. Meanwhile, the improvement of flesh quality by protein was partly related to the increase of antioxidant capacity in fish. In this study, based on the growth performance and disease resistance, the optimal protein levels for young grass carp ?264 g-787,g? were estimated to be 286.82 g kg^-1 (250.66 g digestible protein kg^-1),283.06 g kg^-1 (245.88 g digestible protein kg^-1), 292.10 g kg^-1 (255.47 g digestible protein kg^-1),294.53 g kg^-1 (258.88 g digestible protein kg^-1),241.45 g kg^-1(217.68 g digestible protein kg^-1) and 286.65 g kg^-1 (253.73 g digestible protein kg^-1) diet.