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The vertebrate mucosal barrier comprises physical and immune elements, as well as bioactive molecules, that protect organisms from pathogens. Vitamin D is a vital nutrient for animals and is involved in immune responses against invading pathogens. However, the effect of vitamin D on the mucosal barrier system of fish, particularly in the skin, remains unclear. Here, we elucidated the effect of vitamin D supplementation (15.2, 364.3, 782.5, 1167.9, 1573.8, and 1980.1 IU/kg) on the mucosal barrier system in the skin of grass carp (Ctenopharyngodon idella) challenged with Aeromonas hydrophila. Dietary vitamin D supplementation (1) alleviated A. hydrophila-induced skin lesions and inhibited oxidative damage by reducing levels of reactive oxygen species, malondialdehyde, and protein carbonyl; (2) improved the activities and transcription levels of antioxidant-related parameters and nuclear factor erythroid 2-related factor 2 signaling; (3) attenuated cell apoptosis by decreasing the mRNA and protein levels of apoptosis factors involved death receptor and mitochondrial pathway processes related to p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signaling; (4) improved tight junction protein expression by inhibiting myosin light-chain kinase signaling; and (5) enhanced immune barrier function by promoting antibacterial compound and immunoglobulin production, downregulating pro-inflammatory cytokine expression, and upregulating anti-inflammatory cytokines expression, which was correlated with nuclear factor kappa B and the target of rapamycin signaling pathways. Vitamin D intervention for mucosal barrier via multiple signaling correlated with vitamin D receptor a. Overall, these results indicate that vitamin D supplementation enhanced the skin mucosal barrier system against pathogen infection, improving the physical and immune barriers in fish. This finding highlights the viability of vitamin D in supporting sustainable aquaculture.

Deterioration of flesh quality has bad effects on consumer satisfaction. Therefore, effects of safe mannan-oligosaccharides (MOS) on flesh quality of grass carp (Ctenopharyngodon idella) muscle were studied. A total of 540 healthy fish (215.85 ± 0.30 g) were randomly divided into six groups and fed six separate diets with graded levels of MOS (0, 200, 400, 600, 800 and 1000 mg kgOptimal MOS could improve tenderness (27.4%), pH (5.5%) while decreasing cooking loss (16.6%) to enhance flesh quality. Meanwhile, optimal MOS improved flavor inosine 5'-monophosphate (IMP) of 11.8%, sweetness and umami-associated amino acid, healthy unsaturated fatty acid (UFA) of 14.9% and n-3 polyunsaturated fatty acids (n-3 PUFAs) especially C20:5n-3 (15.8%) and C22:6n-3 (38.3%). Furthermore, the mechanism that MOS affected pH, tenderness and cooking loss could be partly explained by the reduced lactate, cathepsin and oxidation, respectively. The enhanced flesh quality was also associated with enhanced antioxidant ability concerning improving antioxidant enzymes activities and the corresponding transcriptional levels, which were regulated through NF-E2-related factor 2 (Nrf2) and target of rapamycin (TOR) signaling. Based on pHDietary MOS is a promising alternative strategy to improve flesh quality of fish muscle. © 2022 Society of Chemical Industry.

Aflatoxin B1 (AFB1) is kind of a common mycotoxin in food and feedstuff. Aquafeeds are susceptible to contamination of AFB1. In teleost fish, the spleen and head kidney are key immune organ. Moreover, the fish skin is a critical mucosal barrier system. However, there was little study on the effects of dietary AFB1 on the immune response of these immune organs in fish. This study aimed to explore the impacts of oral AFB1 on the immune competence and its mechanisms in the skin, spleen, and head kidney of grass carp. Our work indicated that dietary AFB1 reduced antibacterial compounds and immunoglobulins contents, and decreased the transcription levels of antimicrobial peptides in grass carp immune organs. In addition, dietary AFB1 increased the transcription levels of pro-inflammatory cytokines and reduced the transcription levels of anti-inflammatory cytokines in the grass carp immune organs, which might be regulated by NF-κB and TOR signaling, respectively. Meanwhile, we evaluated the content of AFB1 in the grass carp diet should not exceed 29.48 μg/kg diet according to the levels of acid phosphatase and lysozyme. In summary, dietary AFB1 impaired immune response in grass carp skin, spleen, and head kidney.

An 8-week feeding trial was conducted to investigate the effects of dietary soy protein concentrate (SPC) levels on growth, blood biochemical indexes, non-specific immune enzyme activity, and nutrient apparent digestibility for juvenile Litopenaeus vannamei (initial weight 0.44 ± 0.002 g). They were formulated by replacing 0% (the control), 10%, 20%, 30%, 40%, 60%, and 80% of fish meal (FM) protein with SPC (designed FM, S11, S22, S33, S44, S55, and S66, respectively). With the replacement level of SPC in the diet was higher than 30%, weight gain (WG) and specific growth rate (SGR) for juvenile Litopenaeus vannamei significantly decreased (P

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 recent years, mannose oligosaccharide (MOS) as a functional additive is widely used in aquaculture, to enhance fish immunity. An evaluation of the effect of dietary MOS supplementation on the immune barrier function and related signaling molecules mechanism of grass carp (Ctenopharyngodon idella) was undertaken in the present study. Six diets with graded amounts of MOS supplementation (0, 200, 400, 600, 800, and 1000 mg/kg) were fed to 540 grass carp over 60 days. To examine the immune response and potential mechanisms of MOS supplementation on the intestine, a challenge test was conducted using injections ofAeromonas hydrophilafor 14 days. Results of the study on the optimal supplementation with MOS were found as follows (1) MOS enhances immunity partly related to increasing antibacterial substances content and antimicrobial peptides expression; (2) MOS attenuates inflammatory response partly related to regulating the dynamic balance of intestinal inflammatory cytokines; (3) MOS regulates immune barrier function may partly be related to modulating TLRs/MyD88/NFκB and TOR/S6K1/4EBP signalling pathways. Finally, the current study concluded that MOS supplementation could improve fish intestinal immune barrier function underAeromonas hydrophilainfected conditions.

In recent years, mannose oligosaccharide (MOS) as a functional additive is widely used in aquaculture, to enhance fish immunity. An evaluation of the effect of dietary MOS supplementation on the immune barrier function and related signaling molecules mechanism of grass carp (

This dissertation was primarily focused on the immune response, inflammatory response and molecular mechanisms in the skin, head kidney and spleen of grown-up grass carp (Ctenopharyngodon idella). Six iso-nitrogen diets differing in lysine concentrations (5.6, 8.5, 11.6, 14.4, 17.5 and 20.7 g/kg) were fed to 540 grass carp (164.85 ± 0.79 g) for 60 d. After that, grass carp were challenged by Aeromonas hydrophila for 6 d. This study revealed that lysine deficiency (1) suppressed the growth performance of the fish and decreased their ability to resist skin lesion morbidity, (2) impaired the immune organ's immune response by decreasing the gene expressions of mucin, liver-expressed antimicrobial peptide (LEAP)-2B, β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp, and (3) aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines (interferon γ2 [IFN-γ2], tumor necrosis factor α [TNF-α], interleukin [IL]-15, IL-17D, IL-12p40, IL-6 and IL-8) and down-regulating anti-inflammatory cytokines (IL-11, transforming growth factor β1 [TGF-β1], IL-10 and IL-4/13A), which were tightly correlated with signal transducer and activator of transcription (STAT)1 and STAT3 signaling pathway, respectively. The different phenomenon in the skin, spleen and head kidney of fish may be correlated with the difference in gene subtype. In addition, using quadratic regression analysis of percent weight gain (PWG), skin lesion morbidity, and the lysozyme activities in the spleen and head kidney, the dietary lysine requirements for grown-up grass carp were estimated to be 13.58, 13.51, 14.56 and 14.18 g/kg, respectively.

The immune function of immune organs is extremely crucial for maintaining organism health status, which ultimately affects fish growth. Our previous study has found that dietary supplementation of (2-carboxyethyl)dimethylsulfonium Bromide (Br-DMPT) in non-fish meal (NFM) diet could promote the growth of grass carp (Ctenopharyngodon idella), whereas the underlying reason or mechanism for this results is largely unclear. Herein, we further explored whether dietary supplementation of Br-DMPT promoted fish growth is connected with the enhanced immune function in the immune organs (the head kidney, spleen and skin). In this study, 540 fish (216.49 ± 0.29 g) were irregularly distributed to six groups with three replicates (30 fish replicate−1) and fed corresponding diets group containing a fish meal (FM) diet group and five different NFM diets supplemented with gradational Br-DMPT (0–520.0 mg/kg level) group for 60 days. After the 60-days feeding trial, 8 fish from each replicate were selected and then conducted a challenge test with A. hydrophila for 14 days. Our results indicated that in the NFM diets, appropriate Br-DMPT: (1) significantly decreased the morbidity of skin haemorrhage and lesion after A. hydrophila infection (P 0.05). Taken together, in non-fish meal diet, appropriate supplementation of Br-DMPT could improve the disease resistance capacity, non-specific immunity and ameliorate inflammation, and simultaneously could mitigate these adverse effects induced by the non-fish meal diet in fish immune organs. Moreover, this study may be helpful to decipher the underlying mechanisms of how Br-DMPT promote fish growth by immune organs and also provide scientific theoretical evidence for the future application of Br-DMPT as a new immunopotentiator in aquaculture industry.

Glycinin is a major protein and antinutritional factor of soybean. However, how dietary glycinin affect intestinal immune function of fish were largely unknown. In this study, we used juvenile grass carp as a model to investigate the impacts of glycinin on intestinal immune function of fish and involved mechanisms. We set three treatments including control, glycinin and glycinin + glutamine in this trial. For immune components, results revealed that compared with control group, glycinin group had lower acid phosphatase activities in the foregut, midgut and hindgut, lower C3 and C4 content, and lower mRNA abundances of IgM, IgZ, hepcidin, LEAP-2A, LEAP-2B and β-defensin-1 in the midgut and hindgut rather than foregut of grass carp. For pro-inflammatory cytokines and relevant signaling, glycinin elevated mRNA abundances of IL-1β, IL-8, IL-12p35, IL-12p40 and IL-17D in the midgut and IL-1β, IFN-γ2, IL-6, IL-8, IL-12p35, IL-12p40 and IL-17D in the hindgut, and increased protein abundances of PKC-ζ and nuclear NF-κB p65 in the midgut and hindgut in comparison to control. For anti-inflammatory cytokines and relevant signaling, glycinin reduced mRNA abundances of TGF-β1, TGF-β2, IL-4/13B (rather than IL-4/13A), IL-10 and IL-11 in the midgut and hindgut, and reduced p-mTOR (Ser 2448), p-S6K1 (Thr 389) and p-4EBP1 (Thr 37/46) protein abundances in the midgut and hindgut rather than foregut. Co-administration of glutamine with glycinin could partially enhance intestinal function and reduce intestinal inflammation compared with glycinin treatment. Concluded, glycinin decreased intestinal immune components and caused intestinal inflammation associated with PKC-ζ/NF-κB and mTORC1 signaling.

A 56 days feeding trial was conducted to investigate the effects of enzyme-treated soy protein (ETSP) supplementation in low protein diets on immune function of immune organs (head kidney, spleen and skin) in on-growing grass carp. A total of 540 on-growing grass carp (initial average weight: 325.72 ± 0.60 g) were fed six diets, which included a normal protein diet (28% crude protein) and five low protein diets (26% crude protein) supplemented with graded levels of ETSP (0.0, 0.8, 1.2, 1.6 and 2.0%). At the end of feeding period, a challenge test was performed by infection with Aeromonas hydrophila for two weeks. The results indicated that (1) reducing dietary protein content from 28 to 26% decreased antibacterial substances and aggravated inflammatory responses of above three immune organs; (2) under the condition of reducing protein level in diet, 0.8–1.2% ETSP supplementation reversed these above adverse effects on immune function of above three immune organs; (3) suitable ETSP supplementation-decreased inflammatory responses were partly associated with [IκB kinase β (IKKβ)/inhibitor of κBα (IκBα)/nuclear factor kappa B (NF-κB) p65 and p52 or NF-κB p65] signaling and [target of rapamycin (TOR)/(S6K1, 4E-BP)] signaling in above three immune organs. (4) On the basis of C3 content (head kidney), C4 content (spleen) and skin hemorrhage and lesion, the optimal ETSP supplementation levels in low protein diets were estimated to be 1.48%, 1.61% and 1.03%, respectively. In summary, ETSP supplementation in low protein diets improved immune function of head kidney, spleen and skin in on-growing grass carp.

This research investigates the influences of dietary mannan oligosaccharides (MOS) on the head-kidney and spleen immune function in on-growing grass carp (Ctenopharyngodon idella) and its related mechanism. Fish were fed during 60 days at different levels of MOS (0, 200, 400, 600, 800 and 1000 mg kg−1). Subsequently, 14 days after the feeding trial was injected intraperitoneally with Aeromonas hydrophila, the immune function was studied. The results are as follows: (1) appropriate MOS supplementation could increase the content of antibacterial compound and immunoglobulin (Ig), up-regulate antimicrobial peptides transcriptional levels in these two organs; (2) appropriate MOS supplementation attenuated inflammatory response in these two organs by regulating cytokines (pro- and anti-inflammatory cytokines) and related signalling pathways (NF-κB and TOR). The interesting points though, was, no differences were found in liver-expressed antimicrobial peptide (LEAP)-2A, interleukin (IL)-8, IL-4/13B, IκB kinase (IKK) α and nuclear factor kappa B (NF-κB) p52 gene expression in these two organs. Consequently, the present research suggests that MOS supplementation can enhance head-kidney and spleen immune function. Finally, we obtained these appropriate MOS dose (538.5 and 585.8 mg kg−1) by quadratic regression analysis of lysozyme activity (head-kidney) and phosphatase activity (spleen), respectively.

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp (Ctenopharyngodon idella) infected with Flavobacterium columnare. The fish were fed different concentrations of vitamin A diets for 10 weeks and then infected with F. columnare by immersion. We observed that optimal vitamin A significantly prevented gill rot morbidity in fish infected with F. columnare. Further investigations revealed that vitamin A boosted the gill immunity by increasing the contents of complements (C3 and C4), activities of acid phosphatase (ACP) and lysozyme, mRNAs of β-defensin-1, liver-expressed antimicrobial peptide 2A and 2B (LEAP-2A and LEAP-2B), hepcidin, and anti-inflammatory cytokines like transforming growth factor β1 (TGF-β1), TGF-β2, interleukin-10 (IL-10), and IL-11. It also enhanced the levels of various related signaling molecules including inhibitor protein κBα (IκBα), target of rapamycin (TOR), and ribosome protein S6 kinase 1 (S6K1) but downregulated the expression of pro-inflammatory cytokines including IL-1β, IL-8, tumor necrosis factor α (TNF-α), and interferon γ2 (IFN-γ2) and related signaling molecules including nuclear factor κB p65 (NF-κB p65) (rather than NF-κB p52), IκB kinase β (IKKβ), IKKγ (rather than IKKα), eIF4E-binding protein 1 (4E-BP1), and 4E-BP2 mRNA levels in fish gills. In addition, dietary vitamin A markedly lowered the concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC), increased both the activities and mRNAs of copper/zinc superoxide dismutase (Cu/ZnSOD), MnSOD, glutathione transferases (GSTs), glutathione peroxidase (GPx), and glutathione reductase (GR) associated with upregulation of NF-E2-related factor 2 (Nrf2) mRNAs and downregulation of Kelch-like-ECH-associated protein (Keap1a) and Keap1b mRNAs. Moreover, vitamin A decreased the mRNAs of different apoptotic mediators [caspases 8, 9, 3 (rather than 7)] associated with downregulation of signaling molecule p38 mitogen-activated protein kinase (p38MAPK) mRNAs in fish gills. Besides, vitamin A promoted tight junction (TJ) complex mRNAs [including claudin-b, -c, -3, -7, -12, occludin, and zonula occludens-1 (ZO-1)] that have been linked to the downregulation of myosin light chain kinase (MLCK) signaling. Taken together, the current study demonstrated for the first time that vitamin A markedly enhanced gill health associated with immune modulation and physical barrier protection. Based on protecting fish against gill rot morbidity, ACP activity, and against lipid peroxidation, optimum vitamin A concentrations in on-growing grass carp (262–997 g) were found to be 1,991, 2,188, and 2,934 IU/kg diet, respectively.

Mannan oligosaccharides (MOS) are a type of functional oligosaccharide which have received increased attention because of their beneficial effects on fish intestinal health. However, intestinal structural integrity is a necessary prerequisite for intestinal health. This study focused on exploring the protective effects of dietary MOS supplementation on the grass carp's (

This study aims to investigate the effects of threonine (Thr) on immunoregulation in vivo and in vitro of teleost grass carp (Ctenopharyngodon idella). Juveniles (9.53 ± 0.02 g) were reared for 8 weeks with respective Thr diet (3.99, 7.70, 10.72, 14.10, 17.96, and 21.66 g/kg) and then challenged with Aeromonas hydrophila for in vivo study. Macrophages isolated from head kidney were treated in vitro for 48 h with L-Thr (0, 0.5, 1.0, 2.0, 4.0, and 8.0 mM) after 6 h of lipopolysaccharide induction. The results showed that, compared with Thr deficiency (3.99 g/kg), the optimal dietary Thr (14.10g/kg) affected the immunocyte activation in the head kidney (HK) and spleen (SP) by downregulating the mRNA expressions of MHC-II and upregulating CD4 (not CD8), and it mediated the innate immune by enhancing the activities of lysozyme (LZ), acid phosphatase content of complement 3 (C3) and C4, increasing the mRNA abundances of hepcidin, liver expressed antimicrobial peptide-2A (LEAP-2A), LEAP-2B, β-defensin1, downregulating tumor necrosis factor α (TNF-α), IL-6, IL-1β, IL-12p35, IL-12p40, IL-17AF1, and IL-17D partly by attenuating RORγ1 transcriptional factor and nuclear factor kappa B p65 (NF-κBp65) signaling cascades [IKKβ/IκBα/NF-κBp65] and upregulating transforming growth factor β1 (TGF-β1), IL-4/13A, -4/13B, IL-10, and IL-22 partly by GATA-3. Besides these, the optimal dietary Thr regulated the adaptive immune by upregulating the mRNAs of immunoglobulin M (IgM) and IgZ (not IgD). Moreover, 2 mM Thr downregulated in vitro the mRNA abundances of colony stimulating factor-1, inducible nitric oxide synthase, mannose receptor 1, matrix metalloproteinase2 (MMP-2), and MMP-9 significantly (P < 0.05), indicating that Thr could attenuate the M1-type macrophages’ activation. Moreover, L-Thr downregulated the mRNA transcripts of TNF-α, IL-6, and IL-1β associated with impairing the SOCS1/STAT1 signaling and upregulated IL-10 and TGF-β1 partly by accentuating the SOCS3/STAT3 pathway. The above-mentioned observations suggested that Thr improved the immune status in the immune organs of fish by enhancing the immune defense and mediating the inflammation process. Finally, based on the immune indices of LZ activity in HK and C3 content in SP, the optimal Thr for immune enhancement in juvenile grass carp (9.53–53.43 g) was determined to be 15.70 g/kg diet (4.85 g/100 g protein) and 14.49 g/kg diet (4.47 g/100 g protein), respectively.

Flesh quality is evaluated according to nutritional value and sensory quality. Cinnamaldehyde (CIN) improves mammalian meat quality, but research relating this to aquaculture is scarce. In this study, five doses of CIN (0, 36, 72, 108, 144 mg/kg diet) were fed to grass carp (Ctenopharyngodon idella) for 60 days. The results show that CIN supplementation increased nutritional value by increasing crude protein content. CIN also improved the sensory quality by increasing the pH and collagen content, decreasing shear force, lactate, and cooking loss. These changes may be related to changes in muscle fiber growth by increasing myofiber diameter. The increased myofiber diameter induced by CIN is associated with TOR mRNA and protein levels, and down-regulated FOXO3a mRNA levels, which might be associated with PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling. Based on muscle crude protein content, optimal CIN supplementation dosage was 88.01 mg/kg.

We studied the effects of conjugated linoleic acid (CLA) on the amount of nutrients, flavour substances, and healthcare fatty acids, the physicochemical properties, and the potential molecular mechanisms in the muscles of sub-adult grass carp (

The present study aimed to explore the effects of phytic acid (PA) on the antimicrobial activity and inflammatory response in three immune organs (head kidney, spleen and skin) of on-growing grass carp (Ctenopharyngodon idella). To achieve this goal, we first conducted a 60-day growth trial by feeding fish with graded levels of PA (0, 0.8, 1.6, 2.4, 3.2 and 4.0%). Then, the fish were challenged with Aeromonas hydrophila for 6 days. Compared with the control group, the following results were obtained regarding supplementation with certain levels of PA in the diet. (1) There was an increase in skin haemorrhage and lesion morbidity in fish. (2) There was a decrease in activities or contents of immune factors, including lysozyme (LZ), complement 3 (C3), C4 and immunoglobulin M (IgM), and there was downregulation of gene expression levels of hepcidin, liver-expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, and β-defensin-1 in immune organs. (3) There was upregulation in the gene expression of the following pro-inflammatory cytokines: tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) (except in the spleen), interferon γ2 (IFN-γ2), IL-6 (except in the spleen), IL-8, IL-12p40, IL-15 and IL-17D. These changes were partly related to the nuclear factor kappa B (NF-κB) signalling pathway, but downregulation of mRNA levels of anti-inflammatory cytokines (transforming growth factor β1 (TGF-β1), TGF-β2, IL-413/A, IL-413/B, IL-10 (except in the skin) and IL-11) occurred in a manner partially related to the target of rapamycin (TOR) signalling pathway. Finally, based on the broken-line analysis of skin haemorrhage and lesion morbidity and IgM content in the head kidney, the maximum tolerance levels of PA for on-growing grass carp (120.56–452.00 g) were estimated to be 1.79 and 1.31% of the diet, 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 (SD0·36) 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 present study explored effects of L-glutamate (Glu) levels on growth, digestive and absorptive capability, and intestinal physical barrier functions of Jian carp (Cyprinus carpio). A total of 600 Jian carp (126.40 ± 0.21 g) were randomly distributed into 5 groups with 3 replicates each, fed diets containing graded levels of Glu (53.4 [control], 57.2, 60.6, 68.4, and 83.4 g/kg) for 63 d. Results showed compared with control diet, feed intake and percent weight gain (PWG) in fish fed 83.4 g of Glu/kg diet were increased and feed conversion ratio in fish fed 68.4 g of Glu/kg diet was decreased (P

The present study aimed to investigate the dynamic process of soybean β-conglycinin in digestion, absorption, and metabolism in the intestine of grass carp (Ctenopharyngodon idella). Fish fed with 80 g β-conglycinin/kg diet for 7 weeks, the intestinal digestive enzyme was extracted to hydrolyze β-conglycinin in vitro, the free amino acid and its metabolism product contents in intestinal segments were analyzed. The present study first found that β-conglycinin cannot be thoroughly digested by fish intestine digestive enzyme and produces new products (about 60- and 55-kDa polypeptides). The indigestible β-conglycinin further caused the free amino acid imbalance, especially caused free essential amino acid deficiency in the proximal intestine but excess in the distal intestine. Moreover, these results might be partly associated with the effect of β-conglycinin in amino acid transporters and tight junction-regulated paracellular pathway. Finally, dietary β-conglycinin increased the content of amino acid catabolism by-product ammonia while decreased the amino acid anabolism product carnosine content in the proximal intestine and distal intestine. Thus, the current study first and systemically explored the dynamic process of β-conglycinin in digestion, absorption, and metabolism, which further supported our previous study that dietary β-conglycinin suppressed fish growth and caused intestine injure.

This study investigated the effect of xylanase supplementation of a plant protein‐enriched diet on the growth performance and intestinal immune function of grass carp grow‐out (Ctenopharyngodon idella). Five hundred forty healthy grass carp grow‐out (232.74 ± 0.12 g) were fed diets containing graded levels of xylanase for 60 days. The fish were then challenged with Aeromonas hydrophila for 14 days. The results indicated that xylanase supplementation (a) improved fish growth performance and optimized the intestinal microflora; (b) increased lysozyme and acid phosphatase activities, complement and immunoglobulin M levels and antibacterial peptide hepcidin mRNA levels, thereby enhancing fish intestinal immune function; and (c) attenuated intestinal inflammation associated with the inhibition of the NF‐κB signalling pathway and activation of the TOR signalling pathway, resulting in the down‐regulation of pro‐inflammatory cytokines [except IL‐1β and IL‐12p40 in the distal intestine (DI) and IL‐6 in the proximal intestine (PI)] and up‐regulation of anti‐inflammatory cytokines [except IL‐4/13B and TGF‐β1 in the mid intestine (MI) and DI]. Finally, on the premise of a basal diet containing 45.8 g/kg araboxylans (AXs), the appropriate supplementation levels based on the ability to improve per cent weight gain (PWG) and prevent enteritis were estimated to be 1,527 and 1,608 U/kg, respectively.

To investigate the effect of dietary folic acid on fish flesh quality, muscle antioxidant status and the potential mechanism, young grass carp (Ctenopharyngodon idella) were fed diets containing 0.10–3.12 mg/kg diet of folic acid for 8 weeks. The results showed that optimum folic acid increased muscle contents of protein, lipid, pH, shear force, amino acids, unsaturated fatty acids, hydroxyproline and glutathione, whereas contents of moisture, cooking loss, lactate and saturated fatty acids and cathepsin L activity showed an opposite trend (p

Deoxynivalenol (DON) is a common mycotoxin existed in animal feed, and lead to significant economic loss due to its negative impacts on animal growth performance and animal health. The gill is a primary mucosal immune organ in teleosts, and the structural integrity of the gill is closely relevant with fish healthy growth. Hence, this study assessed the influences of DON on the gill structural integrity of juvenile grass carp, Ctenopharyngodan idella (initial average weight 12.17 ± 0.01 g), when offered with six different diets which contained various content of DON (27, 318, 636, 922, 1243 and 1515 μg/kg diet) for 60 days. Our research firstly systematically elaborated that DON caused histopathological lesions, oxidative injury, reduction of antioxidant ability, apoptosis as well as damages of tight junctions in fish gills. Comparing these data to the control, we found that DON at dose of more than 318 μg/kg diet led to oxidative injury, apoptosis and disruption of tight junctions in fish gill, which were likely to be relevant with Nrf2, JNK and MLCK signalling pathways, respectively. It was worth noting that DON was not found to affect the gene expressions of Keap1b (rather than Keap1a), claudin-b, claudin-3c and claudin-15b (not claudin-15a) in fish gills. Furthermore, based on MDA and T-AOC activities in the gill, the maximum permissible levels of DON were evaluated to be 375.60 as well as 412.91 μg/kg diet in grass carp, 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 catfishPelteobagrus vachelli♀ ×Leiocassis longirostris♂. A total of 1200 fish (14·19 (se0·13) 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 andγ-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 was carried out to investigate the effects of dietary pyridoxine (PN) on the antioxidant status, apoptosis, intercellular integrity and immune function of head kidney and spleen in young grass carp (Ctenopharyngodon idella). Results showed that compared with the optimal PN level, (a) PN deficiency decreased antioxidant enzyme activities and down‐regulated the mRNA levels of antioxidant enzymes, NF‐E2‐related factor 2 (Nrf2), myeloid cell leukaemia‐1 (Mcl‐1) and tight junction proteins, whereas it increased the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC), and up‐regulated the mRNA levels of cysteinyl aspartic acid‐protease (caspase), Fas ligand (FasL), p38 mitogen‐activated protein kinase (p38MAPK) and myosin light chain kinase (MLCK) in the head kidney and spleen of fish (p

The current study aimed to investigate the effects and mechanisms of dietary soybean β-conglycinin in immune function and oxidative damage among different intestinal segments of juvenile grass carp (Ctenopharyngodon idella). 240 fish (13.77 ± 0.10 g) were fed control or 8% β‐conglycinin diet for 7 weeks. Dietary β-conglycinin caused inconsistent suppression effects on the innate immune by decreasing complement component, lysozyme, antimicrobial peptide and acid phosphatase among different intestinal segments. Meanwhile, dietary β-conglycinin caused inflammation in the mid and distal intestine by raising pro-inflammatory cytokines and declining anti-inflammatory cytokines mRNA levels, while more serious in the distal intestine than in the mid intestine. Furthermore, dietary β-conglycinin regulating inflammatory cytokines might be associated with transcription factors nuclear factor-κB P65 (NF-κB P65) nucleus translocation and target of rapamycin (TOR) phosphorylation in the distal intestine but only related to TOR phosphorylation in the mid intestine. Interestingly, in the proximal intestine, dietary β-conglycinin decreased both pro-inflammatory and anti-inflammatory cytokines mRNA level, and did not affect NF-κB P65 nucleus translocation and TOR phosphorylation. For oxidative damage, dietary β-conglycinin exposure elevated both malondialdehyde (MDA) and protein carbonyl (PC) contents in the distal intestine, which might be attributed to the suppression of the Mn-SOD, catalase (CAT) and glutathione peroxidase (GPx) activities. In the mid intestine, dietary β-conglycinin only increased PC content in association with the low activities of CAT, GPx and glutathione peroxidase (GR). Unexpectedly, in the proximal intestine, dietary β-conglycinin did not significantly change MDA and PC contents while decreased antioxidant enzyme activities. Furtherly, dietary β-conglycinin affect the antioxidant enzyme activity might be regulated by the varying pattern of nuclear factor-erythroid 2-related factor 2 (Nrf2) nucleus translocation among these three intestinal segments. In summary, dietary β-conglycinin caused intestinal inflammation and oxidative damage in association with NF-κB, TOR and Nrf2 signaling molecules, which were varying among the three intestinal segments of grass carp.

Iron is an important mineral element for fish. In this study, we investigated the influences of dietary iron deficiency on intestinal immune function as well as underlying signaling of on-growing grass carp (Ctenopharyngodon idella). Fish were fed with six graded level of dietary iron for sixty days, and a fourteen days’ challenge test under infection of Aeromonas hydrophila thereafter. Results showed that compared with optimal iron level, iron deficiency increased enteritis morbidity, decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) concentrations and down-regulated mRNA levels of hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, Mucin2, β-defensin-1, anti-inflammatory cytokines transforming growth factor β1 (TGF-β1), TGF-β2, interleukin 4/13A (IL-4/13A), IL-4/13B, IL-10, IL-11 and IL-15, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated mRNA levels of pro-inflammatory cytokines IL-1β, interferon γ2 (IFN-γ2), IL-8, IL-12p35, IL-12p40 and IL-17D, nuclear factor kappa B (NF-κB) p65, IκB kinases α (IKKα), IKKβ and eIF4E-binding protein (4E-BP) in intestine of on-growing grass carp, indicating that iron deficiency impaired intestinal immune function of fish under infection of A. hydrophila. Besides, iron excess also increased enteritis morbidity and impaired immune function of fish under infection of A. hydrophila. In addition, the effect of ferrous fumarate on intestinal immune function of on-growing grass carp is more efficient than ferrous sulfate. Finally, based on ability against enteritis, LZ activities in mid intestine and distal intestine, we recommended adding 83.37, 86.71 and 85.39 mg iron/kg into diet, respectively.

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp (

The occurrence of immuno-compromised status in animals with zearalenone (ZEA) exposure may be a critical contributor to associated mucosal (gastrointestinal tract) diseases. However, it is difficult to assess the associated risks with limited reference data. This study comprehensively discussed the effects of ZEA on intestinal immune components, cytokines and molecular mechanism of juvenile grass carp infected with Aeromonas hydrophila. Specifically, the fish were fed six graded levels of dietary ZEA (0–2507 μg kg−1 diet) for 70 d. The results pointed out that the average residual amount of ZEA in the intestines increased with dose level after ZEA feeding. We further performed an infection assay using A. hydrophila. After 14 d, ZEA groups increased enteritis morbidity rate compared with controls. The acid phosphatase (ACP), lysozyme (LZ) activities and immunoglobulin M (IgM) content were significantly decreased in three intestinal segments. Furthermore, ZEA could reduce the transcription of β-defensin-1, Hepcidin, liver expressed antimicrobial peptide 2A/2B (LEAP-2A/2B) and Mucin-2. We next confirmed the loss of these immune components accompanied by the invasion of the intestinal barrier by bacteria, as indicated by activation of the nuclear factor κB (NF-κB) and the expression of downstream cytokines. Notably, the phosphorylated target of rapamycin (TOR) plays an important role in regulating these genes, thus indicating a possible target caused by ZEA. In summary, the extensive inhibition of immune components by ZEA promotes the spread of pathogens, which may increase the possibility of intestinal mucosa exposure and the risk of transforming disease.

Vitamin D

The objective of this study was to evaluate the efficacy of dietary Mannan oligosaccharides (MOS) supplementation on skin barrier function and the mechanism of on-growing grass carp (Ctenopharyngodon idella). Five hundred forty grass carp were fed for 60 days from the growing stage with six different levels of MOS diets (0, 200, 400, 600, 800, and 1,000 mg kg-1). At the end of the growth trial, the 14-day Aeromonas hydrophila challenge experiment has proceeded. The obtained data indicate that MOS could (1) decline skin lesion morbidity after being challenged by the pathogenic bacteria; (2) maintain physical barrier function via improving antioxidant ability, inhibiting excessive apoptosis, and strengthening the tight junction between the epithelial cell and the related signaling pathway (Nrf2/Keap1, p38MAPK, and MLCK); and (3) regulate immune barrier function by modulating the production of antimicrobial compound and expression of involved cytokines and the related signaling pathway (TOR and NFκB). Finally, we concluded that MOS supplementation reinforced the disease resistance and protected the fish skin barrier function from Aeromonas hydrophila infection.