Effects Of Dietary Methionine Hydroxy Analogue On Growth Performance,the Health Status Of Intestine,Organism And Gill,Flesh Quality And Potential Modulation Mechanism In Young Grass Carp (Ctenopharyngodon Idellus)

The object of this study is to investigate the effect of dietary methionine hydroxy analogue (MHA) on growth performance, the healthy status of intestine, organism and gill, flesh quality and potential modulation mechanism, as well as the optimal MHA supplementation level for young grass carp (Ctenopharyngodon idella). A total of 630 healthy grass carp (259.70±0.47 g) were randomly assigned to triplicate of seven dietary treatments (n=30), and the initial weight among each treatment is not significant (P> 0.05). Diets 1-6 were fed graded levels of MHA (0,2.4,4.4,6.4,8.5 and 10.5 g/kg diet) and one DL-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. At the end of the feeding trial, fish slected from each treatment at similar weight, were challenged with Aeromonas hydrophila for 14 days and Flavobacterium columnare for 3 days, respectively.The results of feeding trial indicated that optimal MHA supplementation significantly increased specific growth rate (SGR), percent weight gain (PWG), feed intake (F1) and feed efficiency (FE) of young grass carp, which promoted fish growth, simultaneously significantly increased liver and muscle glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activities, and decreased plasm amino content (PAC) (P< 0.05), which declined amino acids catabolism. The growth promotion of young grass carp by optimal MHA supplementation might be related to the improvement of organism immunity and intestinal and gill health status, which was contributed to enhancement of organism health. The present study indicated that optimal MHA supplementation increased head kidney index (HKI) and spleen index (SPI), lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents in the head kidney, spleen, intestine, gill and skin (P< 0.05). Meanwhile, optimal MHA supplementation down-regulated mRNA levels of pro-inflammatory cytokines tumor necrosis factor a (TNF-a), interferon y2 (IFN-y2), interleukin 1? (IL-1c), IL-8 (not in spleen and proximal intestine) and IL-12p35, and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2 (LEAP-2), hepcidin (not in spleen), and anti-inflammatory cytokines transforming growth factor ?1 (TGF-?1), IL-10, IL-12p40 and IL-4/13A in the head kidney, spleen, intestine, gill and skin (P< 0.05), which repressed inflammation and strengthened immune function of these immune organs. The repression of inflammation is related to NF-?B and TOR signaling pathways. Further investigation indicated that optimal MHA supplementation down-regulated signaling molecules p38 mitogen-activated protein kinase (p38MAPK), I?B kinase ? (IKK?), nuclear factor ?B p65 (NF-KBp65), c-Rel, eIF4E-binding proteinl (4E-BP1) and 4E-BP2, and up-regulated inhibitor of ?B? (I?B?), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) in the head kidney, spleen, intestine, gill and skin (P< 0.05). In addition, the present study indicated that optimal MHA supplementation decreased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents, and increased anti-superoxide anion (ASA) and anti-hydroxy radical (AHR) capacities, glutathione (GSH) contents and catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferases (GST) activities, and up-regulated mRNA levels of manganese superoxide dismutase (MnSOD), GPxl(a), GPxlb, GPx4(a), GSTP2 and GR, as well as signaling molecule NF-E2-related factor 2 (Nrf2), and down-regulated Kelch-like ECH-associating protein 1a (Keapla) mRNA levels in the head kidney, spleen, intestine, gill and skin of young grass carp, which elevated antioxidant capacity of these immune organs, simultaneously down-regulated mRNA levels of caspase-3, caspase-7, caspase-8 and caspase-9, as well as signaling molecules Fas ligand (FasL), apoptosis protease activating factor (Apaf-1), Bcl-2 associated X protein (Bax), c-Jun-N-terminal kinase (JNK), and up-regulated B cell leukemia-2 (Bcl-2) levels in the head kidney, spleen, intestine, gill and skin, which inhibited apoptosis in these immune organs and then maintained cellular structural integrity. Optimal MHA supplementation significantly up-regulated mRNA levels of occludin, claudin-b, claudin-c, claudin-3(c), claudin-7(a) (not in middle or distal intestine), claudin-12, claudin-15(a), ZO-1 (not in spleen) and ZO-2 (not in spleen), and down-regulated signaling molecule myosin light chain kinase (MLCK) mRNA levels in the head kidney, spleen, intestine, gill and skin, which maintained intercellular structural integrity. Meanwhile, compared to DLM, equal sulfur MHA showed lower mRNA levels of head kidney IL-6, IL-15, IL-17D, p38MAPK and 4E-BP1, spleen IL-15, IL-17D, c-Rel and 4E-BP1, as well as proximal intestinal TNF-a and IL-1?, simultaneously higher mRNA levels of head kidney IL-12p40 and L-11,spleen IL-12p40 and IL-4/13A, as well as proximal intestinal IL-10 (P<0.05), which suggested superior positive influence on the immune function of head kidney, spleen and intestine. Equal sulfur MHA showed lower mRNA levels of head kidney caspase-3,FasL, Apaf-1 and Bax, spleen caspase-3 and caspase-9, proximal intestinal caspase-3 and caspase-7, middle and distal intestinal caspase-3, as well as gill caspase-3 (P< 0.05), which suggested stronger repression on apoptosis in the head kidney, spleen, intestine and gill. Meanwhile, equal sulfur MHA showed higher head kidney GSH content, GR and SOD activities, and spleen GSH content, AHR, ASA, SOD, GR and GST activities, GPxla and Nrf2 mRNA levels, and lower MDA content in the head kidney, intestine and gill, as well as PC contents middle intestinal and gill (P< 0.05), which suggested higher antioxidant capacity and superior positive influence on cellular integrity of these immune organs. Equal sulfur MHA showed higher mRNA levels of head kidney claudin-c, ZO-1 and ZO-2, proximal intestinal occludin and gill ZO-1 and claudin-3(c) (P< 0.05), which suggested superior positive influence on maintaining intercellular integrity of head kidney, intestine and gill. Besides, MHA supplementation significantly increased flesh firmness, collagen content, water holding capacity, pH, protein, lipid and essential amino acids (methionine, aspartic acid, threonine, serine, glutamic acid, valine, leucine, isoleucine, phenylalanine and lysine) contents, polyunsaturated fatty acids (C16:1, C18:1c+t, C18:2c+t, C20:4 and C22:6) proportion, antioxidant enzymes (SOD, CAT, GPx and GR) activities and corresponding isoforms (MnSOD, CAT, GPxla, GPxlb, GPx4a, GPx4b and GR) mRNA levels, as well as signaling molecules CK2a, CK2?, TOR, S6K1 and Nrf2 mRNA levels(P< 0.05), and decreased lactate content, cathepsin B and L activities and signaling molecules Keap1a and Keap1b mRNA levels (P<0.05), suggesting that MHA could improve fish flesh quality. Based on PWG, erythrodermatitis, enteritis and gill-rot morbidity, and flesh cooking loss of young grass carp, the optimal MHA supplementation levels were 5.21,5.76,5.83,5.49 and 5.57 g/kg diet (basal methionine:4.01 g/kg diet and 14.3 g/kg protein), respectively.In conclusion, optimal MHA supplementation could improve growth and flesh quality of young grass carp. MHA promoting fish growth might be related to MHA improving fish healthy status via enhancing immune function and improving structural integrity of head kidney, spleen, skin, intestine and gill. MHA enhancing immune function of these immune organs might be via modulating p38/IKK?/I?Ba/NF-icB (p65 and c-Rel) and TOR (S6K1, 4E-BP1 and 4E-BP2) pathways to repress inflammation and up-regulating antimicrobial peptides and immune substances activities and contents. Simultaneously MHA improving structural integrity of these immune organs might be through modulating Keapla/Nrf2 signaling pathway to elevate antioxidant capacity, and JNK (FasL, Bcl-2 and Bax) signaling molecules to inhibit apoptosis, which maintained cellular integrity, simultaneously through MLCK signaling pathway to modulate tight junction proteins genes expression, which maintained intercellular integrity. The improvement of flesh quality might be related to increment of flesh firmness, water holding capacity and nutrients (essential amino acids and polyunsaturated fatty acids) deposition owing to MHA supplementation. MHA might be via modulating CK2/TOR/S6K1 signaling pathway to facilitate collagen synthesis, and via modulating Keap1/Nrf2 pathway to enhance antioxidant capacity and then increase polyunsaturated acids proportion. According to the effect on young grass carp growth, MHA was as 128% efficient as DLM, and according to the effect on fish organism health status, MHA was also superior to DLM, whereas according to the effect on flesh quality, MHA is as efficient as DLM.