The present study evaluated effects of dietary supplementation with tryptophan (Trp) on muscle growth, protein synthesis and antioxidant capacity in hybrid catfish Pelteobagrus vachelli♀ × Leiocassis longirostris♂. Fish were fed six different diets containing 2·6 (control), 3·1, 3·7, 4·2, 4·7 and 5·6 g Trp/kg diet for 56 d, respectively. Results showed that dietary Trp significantly (1) improved muscle protein content, fibre density and frequency of fibre diameter; (2) up-regulated the mRNA levels of PCNA, myf5, MyoD1, MyoG, MRF4, IGF-I, IGF-II, IGF-IR, PIK3Ca, TOR, 4EBP1 and S6K1; (3) increased phosphorylation levels of AKT, TOR and S6K1; (4) decreased contents of MDA and PC, and increased activities of CAT, GST, GR, ASA and AHR; (5) up-regulated mRNA levels of CuZnSOD, CAT, GST, GPx, GCLC and Nrf2, and decreased Keap1 mRNA level; (6) increased nuclear Nrf2 protein level and the intranuclear antioxidant response element-binding ability, and reduced Keap1 protein level. These results indicated that dietary Trp improved muscle growth, protein synthesis as well as antioxidant capacity, which might be partly related to myogenic regulatory factors, IGF/PIK3Ca/AKT/TOR and Keap1/Nrf2 signalling pathways. Finally, based on the quadratic regression analysis of muscle protein and MDA contents, the optimal Trp requirements of hybrid catfish (21·82–39·64 g) were estimated to be 3·94 and 3·93 g Trp/kg diet (9·57 and 9·54 g/kg of dietary protein), respectively.

In the current research, a 60-d experiment was conducted with the purpose of exploring the impacts of methionine (Met) on growth performance, muscle nutritive deposition, muscle fibre growth and type I collagen synthesis as well as the related signalling pathway. Six diets (iso-nitrogenous) differing in Met concentrations (2·54, 4·85, 7·43, 10·12, 12·40 and 15·11 g/kg diets) were fed to 540 grass carp (178·47 (SD 0·36) g). Results showed (P < 0·05) that compared with Met deficiency, optimal level of dietary Met (1) increased feed intake, feed efficiency, specific growth rate and percentage weight gain (PWG); (2) increased fish muscle protein, lipid and free amino acid contents and improved fish muscle fatty acid profile as well as increased protein content in part associated with the target of rapamycin complex 1 (TORC1)/S6K1 signalling pathway; (3) increased the frequency distribution of muscle fibre with >50 µm of diameter; (4) increased type I collagen synthesis partly related to the transforming growth factor-β1/Smads and CK2/TORC1 signalling pathways. In conclusion, dietary Met improved muscle growth, which might be due to the regulation of muscle nutritive deposition, muscle fibre growth and type I collagen synthesis-related signal molecules. Finally, according to PWG and muscle collagen content, the Met requirements for on-growing grass carp (178–626 g) were estimated to be 9·56 g/kg diet (33·26 g/kg protein of diet) and 9·28 g/kg diet (32·29 g/kg of dietary protein), respectively.

The experiment was conducted to investigate the effects of dietary threonine (Thr) on growth performance and muscle growth, protein synthesis and antioxidant-related signalling pathways of hybrid catfish Pelteobagrus vachelli♀ × Leiocassis longirostris♂. A total of 1200 fish (14·19 (se 0·13) g) were randomly distributed into six groups with four replicates each, fed six diets with graded level of Thr (9·5, 11·5, 13·5, 15·4, 17·4 and 19·3 g/kg diets) for 56 d. Results showed (P < 0·05) that dietary Thr (1) increased percentage weight gain, specific growth rate, feed efficiency and protein efficiency ratio; (2) up-regulated growth hormone (GH), insulin-like growth factor 1 (IGF-1), proliferating cell nuclear antigen, myogenic regulation factors (MyoD, Myf5, MyoG and Mrf4) and myosin heavy chain (MyHC) mRNA levels; (3) increased muscle protein content via regulating the protein kinase B/target of rapamycin signalling pathway and (4) decreased malondialdehyde and protein carbonyl contents, increased catalase, glutathione-S-transferase, glutathione reductase and GSH activities, up-regulated mRNA levels of antioxidant enzymes related to NFE2-related factor 2 and γ-glutamylcysteine ligase catalytic subunit. These results suggest that Thr has a potential role to improve muscle growth and protein synthesis, which might be due to the regulation of GH-IGF system, muscle growth-related gene, antioxidative capacity and protein synthesis-related signalling pathways. Based on the quadratic regression analysis of specific growth rate, the Thr requirement of hybrid catfish (14·19–25·77 g) was estimated to be 13·77 g/kg of the diet (33·40 g/kg of dietary protein).

The present study investigated the effects of condensed tannins (CT) on intestinal immune function in on-growing grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp were fed six diets containing different levels of CT (0, 10·00, 20·00, 30·00, 40·00 and 50·00 g/kg diet) for 70 d and then challenged with Aeromonas hydrophila for 14 d. The results showed that, compared with the control group, dietary CT (1) induced intestinal histopathological lesions and aggravated enteritis; (2) decreased lysozyme and acid phosphatase activities, complement 3 (C3), C4 and IgM contents and down-regulated the Hepcidin, liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, Mucin2 and β-defensin-1 mRNA levels in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) (P < 0·05); (3) down-regulated the mRNA levels of anti-inflammatory cytokines transforming growth factor (TGF)-β1, TGF-β2 (not in MI and DI), IL-4/13A (not IL-4/13B), IL-10 and IL-11 partly correlated with target of rapamycin (TOR) signalling; and (4) up-regulated the mRNA levels of pro-inflammatory cytokines interferon-γ2, IL-1β, IL-6, IL-8 (not in PI), IL-12p35, IL-12p40, IL-15 and IL-17D partly related to NF-κB signalling in the intestine of on-growing grass carp. Overall, the results indicated that CT could impair the intestinal immune function, and its potential regulation mechanisms were partly associated with the TOR and NF-κB signalling pathways. Finally, based on the percentage weight gain and enteritis morbidity, the maximum allowable levels of CT for on-growing grass carp (232·22–890·11 g) were estimated to be 18·6 and 17·4 g/kg diet, respectively.

This study aimed to investigate the impacts of dietary threonine on intestinal immunity and inflammation in juvenile grass carp. Six iso-nitrogenous semi-purified diets containing graded levels of threonine (3·99–21·66 g threonine/kg) were formulated and fed to fishes for 8 weeks, and then challenged with Aeromonas hydrophila for 14 d. Results showed that, compared with optimum threonine supplementation, threonine deficiency (1) decreased the ability of fish against enteritis, intestinal lysozyme activities (except in the distal intestine), acid phosphatase activities, complement 3 (C3) and C4 contents and IgM contents (except in the proximal intestine (PI)), and it down-regulated the transcript abundances of liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, hepcidin, IgZ, IgM and β-defensin1 (except in the PI) (P<0·05); (2) could up-regulate intestinal pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and IL-17D mRNA levels partly related to NF-κB signalling; (3) could down-regulate intestinal anti-inflammatory cytokine transforming growth factor (TGF)-β1, TGF-β2, IL-4/13A (not IL-4/13B) and IL-10 mRNA levels partly by target of rapamycin signalling. Finally, on the basis of the specific growth rate, against the enteritis morbidity and IgM contents, the optimum threonine requirements were estimated to be 14·53 g threonine/kg diet (4·48 g threonine/100 g protein), 15.05 g threonine/kg diet (4·64 g threonine/100 g protein) and 15·17 g threonine/kg diet (4·68 g threonine/100 g protein), respectively.

The present study investigated the effects of dietary vitamin A on immune function in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of young grass carp (Ctenopharyngodon idella). Fish were fed graded levels of dietary vitamin A for 10 weeks, and then a challenge test using an injection of Aeromonas hydrophila was conducted for 14 d. The results showed that, compared with the optimum vitamin A level, vitamin A deficiency significantly decreased fish growth performance, increased enteritis morbidity, decreased intestinal innate humoral immune response and aggravated intestinal inflammation. However, liver-expressed antimicrobial peptide 2A/B mRNA in the DI and IL-6, IL-17D, IL-10, transforming growth factor (TGF)-β1 and TGF-β2 mRNA in the PI were not affected by vitamin A levels. Meanwhile, vitamin A deficiency disturbed inflammatory cytokines in the PI, MI and DI, which might be partly linked to p38 mitogen-activated protein kinase (p38MAPK) signalling and NF-κB canonical signalling pathway (IκB kinase β (IKKβ), IKKγ, inhibitor of κBα, NF-κB p65 and c-Rel) rather than NF-κB non-canonical signalling pathway (NF-κB p52 and IKKα). However, the signalling molecules NF-κB p65 and p38MAPK did not participate in regulating cytokines in the PI. These results suggested that vitamin A deficiency decreased fish growth and impaired intestinal immune function, and that different immune responses in the PI, MI and DI were mediated partly by NF-κB canonical signalling and p38MAPK signalling pathways. On the basis of percentage of weight gain, to protect fish against enteritis morbidity and acid phosphatase activity, the optimum dietary vitamin A levels were estimated to be 0·664, 0·707 and 0·722 mg /kg, respectively.

The objective of this study was to determine the protective effect of glutamate (Glu) in Cu-induced oxidative injury in fish intestine in vivo and enterocytes in vitro. The results indicated that exposure to 6 mg/l Cu for 72 h induced the production of reactive oxygen species, thereby increasing protein oxidation and lipid peroxidation in enterocytes of grass carp in vitro. Cells exposed to Cu alone resulted in a significant increase in lactate dehydrogenase release, which is accompanied by depletions of antioxidants, including total superoxide dismutase (T-SOD), glutathione S-transferase (GST), glutathione reductase (GR), anti-superoxide anion (ASA), anti-hydroxy radical (AHR) activities and GSH content. Pre-treatment with Glu remarkably prevented the toxic effects of Cu on the T-SOD, GST, GR, AHR, and ASA activities and GSH content in enterocytes. However, Cu induced an adaptive increase in the activities of catalase and glutathione peroxidase (GPx). Glu supplementation further increased GPx activity in enterocytes. Interestingly, the experiment in vivo showed that Glu pre-supplementation significantly elevated SOD, GPx, GST, GR, ASA and AHR activities, as well as GSH content. Further results showed that pre-treatment with Glu could alleviate Cu-induced oxidative injury by elevating antioxidant enzyme activities through regulating the expression of NF-E2-related nuclear factor 2 (Nrf2) mRNA. Together, these results indicated that Glu could attenuate Cu-induced cellular oxidative damage in fish intestine, likely mediated through Nrf2 signalling pathways regulating mRNA expressions of antioxidant enzyme genes and synthesis of GSH.

This study investigated the effects of glycinin on the growth, intestinal oxidative status, tight junction components, cytokines and apoptosis signalling factors of fish. The results showed that an 80 g/kg diet of glycinin exposure for 42 d caused poor growth performance and depressed intestinal growth and function of juvenile Jian carp (Cyprinus carpio var. Jian). Meanwhile, dietary glycinin exposure induced increases in lipid peroxidation and protein oxidation; it caused reductions in superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities; and it increased MnSOD, CuZnSOD, GPx1b and GPx4a mRNA levels, suggesting an adaptive mechanism against stress in the intestines of fish. However, dietary glycinin exposure decreased both the activity and mRNA levels of nine isoforms of glutathione-S-transferase (GST) (α, μ, π, ρ, θ, κ, mGST1, mGST2 and mGST3), indicating toxicity to this enzyme activity and corresponding isoform gene expressions. In addition, glycinin exposure caused partial disruption of intestinal cell–cell tight junction components, disturbances of cytokines and induced apoptosis signalling in the distal intestines>mid intestines>proximal intestines of fish. Glycinin exposure also disturbed the mRNA levels of intestinal-related signalling factors Nrf2, Keap1a, Keap1b, eleven isoforms of protein kinase C and target of rapamycin/4E-BP. Interestingly, glutamine was observed to partially block those negative influences. In conclusion, this study indicates that dietary glycinin exposure causes intestinal oxidative damage and disruption of intestinal physical barriers and functions and reduces fish growth, but glutamine can reverse those negative effects in fish. This study provides some information on the mechanism of glycinin-induced negative effects.

The present study was conducted to investigate the anti-inflammatory effect of vitamin D both in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in enterocytes in vitro. In primary enterocytes, exposure to 10 mg lipopolysaccharide (LPS)/l increased lactate dehydrogenase activity in the culture medium (P<0·05) and resulted in a significant loss of cell viability (P<0·05). LPS exposure increased (P<0·05) the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8), which was decreased by pre-treatment with 1,25-dihydroxyvitamin D (1,25D3) in a dose-dependent manner (P<0·05). Further results showed that pre-treatment with 1,25D3 down-regulated Toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (Myd88) and NF-κB p65 mRNA expression (P<0·05), suggesting potential mechanisms against LPS-induced inflammatory response. In vivo, intraperitoneal injection of LPS significantly increased TNF-α, IL-1β, IL-6 and IL-8 mRNA expression in the intestine of carp (P<0·05). Pre-treatment of fish with vitamin D3 protected the fish intestine from the LPS-induced increase of TNF-α, IL-1β, IL-6 and IL-8 mainly by downregulating TLR4, Myd88 and NF-κB p65 mRNA expression (P<0·05). These observations suggest that vitamin D could inhibit LPS-induced inflammatory response in juvenile Jian carp in vivo and in enterocytes in vitro. The anti-inflammatory effect of vitamin D is mediated at least in part by TLR4-Myd88 signalling pathways in the intestine and enterocytes of juvenile Jian carp.

The Neoproterozoic mafic rocks in western Hunan, South China, form a NNE-striking mafic rock belt for which outcrops are found predominantly in Guzhang, Qianyang and Tongdao. Samples from Qianyang and Tongdao yielded ion microprobe U–Pb zircon ages of 747±18 Ma and 772±11 Ma, respectively. The mafic rocks are geochemically divided into two subtypes. Ultramafic rocks from Tongdao are depleted in Nb and Ti, with decoupled Nd–Hf isotopes, and geochemical features similar to the c. 761 Ma mafic–ultramafic rocks from Longsheng, northern Guangxi. Their εNd(t) value is −2.91, implying an enriched mantle source. Alkaline mafic rocks from Qianyang and Guzhang have high values of TiO2, total alkali, some high field strength elements and (La/Yb)N, and low Zr/Nb, La/Nb, Sm/Nd and 143Nd/144Nd ratios as well as coupled Nd–Hf isotopes. They are geochemically similar to ocean island basalts and show fractional crystallization of Fe–Ti oxides, olivine and pyroxene in the mafic magma. The c. 760 Ma mafic rocks in western Hunan may be the products of post-orogenic magmatism. After the Jinningian (Sibao) orogenic process, the upwelling of the deep asthenospheric mantle caused by the break-off and detachment of the subducted oceanic slab led to extension in the area. The extension might have taken place earlier in the Tongdao and Longsheng areas, which led to the partial melting of the lithospheric mantle that had been metasomatized during early oceanic subduction to generate a relatively large amount of sub-alkaline rocks. However, the less alkaline mafic rocks in Qianyang and Guzhang might have been generated in the relatively later stage of the extension, and may have resulted from a small degree of partial melting of the asthenospheric mantle.