This 62-d research aimed to evaluate the effects of dietary lysine levels (DLL) and salinity on growth performance and nutrition metabolism of genetically improved farmed tilapia (GIFT) juveniles (Oreochromis niloticus). Six diets with lysine supplementation (1·34, 1·70, 2·03, 2·41, 2·72 and 3·04 % of DM) were formulated under different cultured salinities in a two-factorial design. The results indicated that supplemental lysine improved the specific growth rate (SGR) and weight gain (WG) and decreased the feed conversion ratio (FCR). Meanwhile, the fish had higher SGR and WG and lower FCR at 8 ‰ salinity. Except for moisture, the whole-body protein, lipid and ash content of GIFT were increased by 8 ‰ salinity, which showed that DLL (1·34 %) increased the whole-body fat content and DLL (2·41 %) increased whole-body protein content. Appropriate DLL up-regulated mRNA levels of protein metabolism-related genes such as target of rapamycin, 4EBP-1 and S6 kinase 1. However, 0 ‰ salinity reduced these protein metabolism-related genes mRNA levels, while proper DLL could improve glycolysis and gluconeogenesis mRNA levels but decrease lipogenesis-related genes mRNA levels in liver. 0 ‰ salinity improved GLUT2, glucokinase and G6 Pase mRNA levels; however, sterol regulatory element-binding protein 1 and fatty acid synthase mRNA levels were higher at 8 ‰ salinity. Moreover, 8 ‰ salinity also increased plasma total protein and cholesterol levels and decreased glucose levels. These results indicated that the recommended range of lysine requirement under different salinity was 2·03–2·20 % (0 ‰) and 2·20–2·41 % (8 ‰) and 8 ‰ salinity resulted in higher lysine requirements due to changes in the related nutrient metabolism, which might provide useful information for designing more effective feed formulations for GIFT cultured in different salinity environment.

To assess the role of dietary creatine on myofibre characteristics and protein synthesis in muscle, we fed grass carp (Ctenopharyngodon idellus, initial body weight: 88·47 ± 1·44 g) creatine-supplemented diets (1·84, 5·91, 8·48 and 15·44 g/kg diet) for 8 weeks. Creatine supplementation did not affect growth performance, but significantly increased creatine contents in muscle and liver. At 8·48 g/kg, creatine decreased the activities of alanine transaminase and aspartate aminotransferase in serum and improved hardness and chewiness of muscle due to shorter myofibre mean diameter, higher myofibre density and the frequencies of the diameters of classes I and III and collagen content, longer sarcomere length and upregulated mRNA levels of slow myosin heavy chains. Creatine supplementation upregulated the mRNA expressions of myogenic regulatory factors. The 8·48 g/kg creatine-supplemented diet significantly increased the contents of protein, total amino acids (AA), essential AA and free flavour AAs in muscle, the protein levels of insulin-like growth factor I, myogenic differentiation antigen and PPAR-γ coactlvator-1α in muscle and stimulated the phosphorylation of target of rapamycin (TOR) pathway in muscle. In summary, 8·48 mg/kg creatine improved fish health and skeletal muscle growth and increased hardness and protein synthesis in muscle of grass carp by affecting myofibre characteristics and the TOR signalling pathway. A second-order regression model revealed that the optimal dietary creatine supplementation of grass carp ranges between 8·48 and 12·04 g/kg.

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.

A 6-week growth trial was conducted to evaluate the influences of dietary valine (Val) levels on growth, protein utilisation, immunity, antioxidant status and gut micromorphology of juvenile hybrid groupers. Seven isoenergetic, isoproteic and isolipidic diets were formulated to contain graded Val levels (1·21, 1·32, 1·45, 1·58, 1·69, 1·82 and 1·94 %, DM basis). Each experimental diet was hand-fed to triplicate groups of twelve hybrid grouper juveniles. Results showed that weight gain percentage (WG%), protein productive value, protein efficiency ratio, and feed efficiency were increased as dietary Val level increased, reaching a peak value at 1·58 % dietary Val. The quadratic regression analysis of WG% against dietary Val levels indicated that the optimum dietary Val requirement for hybrid groupers was estimated to be 1·56 %. Gut micromorphology and expression of growth hormone in pituitary, insulin-like growth factor 1, target of rapamycin and S6 kinase 1 in liver were significantly affected by dietary Val levels. In serum, fish fed 1·58 % dietary Val had higher superoxide dismutase, catalase, lysozyme activities and IgM concentrations than fish fed other dietary Val levels. Fish fed 1·58 % dietary Val had higher expression of NF-E2-related factor 2 in head kidney than fish fed other dietary Val levels. Generally, the optimum dietary Val requirement for maximal growth of hybrid groupers was estimated to be 1·56 % of DM, corresponding to 3·16 % of dietary protein, and dietary Val levels affected growth, protein utilisation, immunity and antioxidant status in hybrid groupers.

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.

An 8-week feeding experiment was conducted to investigate the effects of dl-methionine (Met) supplementation in a low-fishmeal diet on growth, key gene expressions of amino acid transporters and target of rapamycin (TOR) pathway in juvenile cobia, Rachycentron canadum. Seven isonitrogenous and isolipidic diets were formulated, containing 0·72, 0·90, 1·00, 1·24, 1·41, 1·63 and 1·86 % Met. Weight gain and specific growth rates increased gradually with Met levels of up to 1·24 % and then decreased gradually. In dorsal muscle, mRNA levels of ASCT2 in the 1·00 % Met group were significantly up-regulated compared with 0·72, 1·63, and 1·86 %. The insulin-like growth factor-I (IGF-I) mRNA levels in the dorsal muscle of fish fed 1·00 and 1·24 % Met were higher than those in fish fed other Met levels. In addition, fish fed 1·24 % Met showed the highest mRNA levels of TOR and phosphorylation of TOR on Ser2448. The phosphorylation of ribosomal p70-S6 kinase (S6K) on Ser371 in the dorsal muscle of fish fed 1·86 % Met was higher than those in the 0·72 % group. In conclusion, straight broken-line analysis of weight gain rate against dietary Met level indicates that the optimal Met requirement for juvenile cobia is 1·24 % (of DM, or 2·71 % dietary protein). Met supplementation in a low-fishmeal diet increased cobia growth via a mechanism that can partly be attributed to Met’s ability to affect the TOR/S6K signalling pathway by enhancing ASCT2 and IGF-I transcription in cobia dorsal muscle.

An 8-week growth trial was conducted to evaluate the effects of dietary arginine (Arg) levels on growth, gut morphology, oxidation resistance and immunity of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂) juveniles. Seven isoenergetic (1465 kJ (350 kcal)/100-g DM), isoproteic (53·5 % of DM) and isolipidic (7 % of DM) experimental diets were formulated to contain graded Arg levels ranging from 1·9 to 4·7 % (dry weight) at approximately 0·5 % increments. Each diet was randomly assigned to triplicate groups of 16 juvenile fish (average initial body weight: 11·7 (sd 0·1) g) and was administered twice daily (08.00 and 16.00 hours). After the growth trial, all remaining fish were fed their prescribed diets for 2 d and then exposed to 4·5 mg Cu2+/l water for 36 h. Results showed that growth performance and feed utilisation of experimental fish were significantly affected by different dietary Arg levels. Weight gain % (WG%) of fish was increased as dietary Arg increased, reaching a peak value at 3·8 % dietary Arg level, and when dietary Arg level increased to 4·7 % WG% was reduced. Fish fed 1·9 and 2·2 % dietary Arg levels had higher daily feed intake compared with fish fed other dietary Arg levels. Feed conversion ratios in fish fed 1·9, 2·2, 2·7 and 4·7 % dietary Arg levels were higher than those in fish fed 3·1, 3·8 and 4·1 % dietary Arg levels. Protein efficiency ratio and protein productive value (PPV) increased with an increase in dietary Arg, up to a peak value at 3·8 % dietary Arg level, above which these parameters declined. On the basis of quadratic regression analysis of weight gain % (WG%) or PPV against dietary Arg levels, the optimal dietary Arg requirement for hybrid grouper was estimated to be 3·65 %. Fish fed 3·8 % dietary Arg had higher whole-body and muscle protein contents compared with fish fed other dietary Arg levels. Fish fed 3·8 and 4·1 % dietary Arg levels had higher levels of mRNA for insulin-like growth factor-I and target of rapamycin in the liver compared with fish fed other dietary Arg levels. Hepatic S6 kinase 1 mRNA expression in fish fed 3·8 % dietary Arg level was higher than that in fish fed any of the other dietary Arg levels. Gut morphology, hepatic antioxidant indices and immune indices in serum and head kidney were significantly influenced by dietary Arg levels. In conclusion, the optimal dietary Arg requirement for hybrid grouper was estimated to be 3·65 %, and suitable dietary Arg supplementations improved gut morphology and oxidation resistance of hybrid grouper.

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.

In this study, we chose a carnivorous fish, turbot (Scophthalmus maximus L.), to examine its nutrient-sensing and metabolic responses after ingestion of diets with fishmeal (FM), or 45 % of FM replaced by soyabean meal (34·6 % dry diet) balanced with or without essential amino acids (EAA) to match the amino acid profile of FM diet for 30 d. After a 1-month feeding trial, fish growth, feed efficiency and nutrient retention were markedly reduced by soyabean meal-incorporated (SMI) diets. Compared with the FM diet, SMI led to a reduction of postprandial influx of free amino acids, hypoactivated target of rapamycin signalling and a hyperactivated amino acid response pathway after refeeding, a status associated with reduced protein synthesis, impaired postprandial glycolysis and lipogenesis. These differential effects were not ameliorated by matching an EAA profile of soyabean meal to that of the FM diet through dietary amino acid supplementation. Therefore, this study demonstrated that the FM diet and SMI diets led to distinct nutrient-sensing responses, which in turn modulated metabolism and determined the utilisation efficiency of diets. Our results provide a new molecular explanation for the role of nutrient sensing in the inferior performance of aquafeeds in which FM is replaced by soyabean meal.

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 test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6·33 (se 0·03) g, were fed with diets containing graded concentrations of arginine, namely, 9·8 (control), 12·7, 16·1, 18·5, 21·9 and 24·5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0·05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na+/K+-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0·05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18·0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.