Aflatoxin B1 inhibited the development of primary myoblasts of grass carp (Ctenopharyngodon idella) by degrading extracellular matrix.

According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is a common mycotoxin with high toxicity. Previous research has found that AFB1 inhibited zebrafish muscle development. However, the potential mechanism of AFB1 on fish muscle development is unknown, so it is necessary to conduct further investigation. In the present research, the primary myoblast of grass carp was used as a model, we treated myoblasts with AFB1 for 24 h. Our results found that 5 µM AFB1 significantly inhibited cell proliferation and migration (P < 0.05), and 10 µM AFB1 promoted lactate dehydrogenase (LDH) release (P < 0.05). Reactive oxygen species (ROS), protein carbonyl (PC) and malondialdehyde (MDA) levels were increased in 15, 5 and 10 µM AFB1 (P < 0.05), respectively. Catalase (CAT), glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activities were decreased in 10, 10 and 15 µM AFB1 (P < 0.05), respectively. Furthermore, 15 µM AFB1 induced oxidative damage by Nrf2 pathway, also induced apoptosis in primary myoblast of grass carp. Meanwhile, 15 µM AFB1 decreased MyoD gene and protein expression (P < 0.05). Importantly, 15 µM AFB1 decreased the protein expression of collagen Ⅰ and fibronectin (P < 0.05), and increased the protein levels of urokinase plasminogen activator (uPA), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and p38 mitogen-activated protein kinase (p38MAPK) (P < 0.05). As a result, our findings suggested that AFB1 damaged the cell morphology, induced oxidative damage and apoptosis, degraded ECM components, in turn inhibiting myoblast development by activating the p38MAPK/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase (MMPs)/extracellular matrix (ECM) signaling pathway.

Cellular antioxidant mechanism of mannan-oligosaccharides involving in enhancing flesh quality in grass carp (Ctenopharyngodon idella).

BACKGROUND: 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 kg-1 ) for 60 days. This study aimed at investigating the benefits of dietary MOS on flesh quality (fatty acids, amino acids and physicochemical properties) and the protection mechanism regarding antioxidant status. RESULTS: Optimal 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 pH24h , cooking loss, shear force and DHA (docosahexaenoic acid, C22:6n-3), optimal MOS levels for grass carp were estimated to be 442.75, 539.53, 594.73 and 539.53 mg kg-1 , respectively. CONCLUSION: Dietary MOS is a promising alternative strategy to improve flesh quality of fish muscle. © 2022 Society of Chemical Industry.

Acute nitrite exposure-induced oxidative damage, endoplasmic reticulum stress, autophagy and apoptosis caused gill tissue damage of grass carp (Ctenopharyngodon idella): Relieved by dietary protein.

Nitrite poses a serious threat to intensive aquaculture. Protein, as a major nutrient in animals, is vital for protecting animal tissues from damage. In this study, we investigated the protective effect of dietary protein on gill tissue structure and the underlying mechanisms in sub-adult grass carp (Ctenopharyngodon idella) exposed to nitrite stress. Six iso-energetic semi-purified diets containing different protein levels (16-31 %) were formulated, and fed to fish for 60 d. The fish were then exposed to a nitrite solution for 4 d. Histopathological observation and determination of related indices (serum glucose, serum cortisol, nitric oxide, peroxynitrite, reactive oxygen species, malondialdehyde, and protein carbonyl) showed that 22-25 % dietary protein significantly alleviated the nitrite-induced stress response, gill tissue damage and oxidative damage. Further research found that a suitable dietary protein suppressed the nitrite-induced endoplasmic reticulum stress (ERS) 78 kDa glucose-regulated protein (GRP78) related signaling pathway which possibly activated autophagy and apoptosis. Interestingly, we discovered that proper dietary protein reduced autophagy, probably through unc-51-like kinase 1 (Ulk1), BCL-2-interacting myosin-like coiled-coil protein (Beclin1), autophagy-related gene 5 (Atg5), Atg12, microtubule-associated protein1 light chain 3 (LC3), BCL-2 interacting protein 3 (BNIP3) and autophagy receptor P62 (p62). We also found that an appropriate dietary protein inhibited nitrite-induced apoptosis via mitochondrial and death receptor pathways. In summary, our findings are the first to demonstrate that 22-25 % of dietary protein levels can play a protective role against nitrite-induced gill injury.

Effects of dietary methionine on growth performance, muscle nutritive deposition, muscle fibre growth and type I collagen synthesis of on-growing grass carp (Ctenopharyngodon idella).

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-ß1/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.

Zearalenone induces immuno-compromised status via TOR/NF/κB pathway and aggravates the spread of Aeromonas hydrophila to grass carp gut (Ctenopharyngodon idella).

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 ß-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.

Condensed tannins decreased the growth performance and impaired intestinal immune function in on-growing grass carp (Ctenopharyngodon idella).

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 ß-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)-ß1, TGF-ß2 (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-1ß, 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.

The regulatory effects of pyridoxine deficiency on the grass carp (Ctenopharyngodon idella) gill barriers immunity, apoptosis, antioxidant, and tight junction challenged with Flavobacterium columnar.

The effects of dietary pyridoxine (PN) on the gill immunity, apoptosis, antioxidant and tight junction of grass cap (Ctenopharyngodon idella) were investigated in this study. Fish were fed semi-purified diets containing graded levels of PN for 10 weeks, and then challenged with Flavobacterium columnare by bath immersion exposure for 3 days. The results indicated that compared with the optimal PN level, PN deficiency resulted in a decline in the antimicrobial compound production of gill. In addition, PN deficiency up-regulated the pro-inflammatory cytokines and down-regulated the anti-inflammatory cytokines gene expression, which might be associated with the enhanced nuclear factor κB p65 and the inhibited target of rapamycin signalling pathways, respectively, suggesting that PN deficiency could impair gill immune barrier function. Furthermore, PN deficiency (1) induced cell apoptosis, which may be partly associated with the (apoptotic protease activating factor-1, Bcl-2 associated X protein)/caspase-9 and c-Rel/tumor necrosis factor α (rather than FasL)/caspase-8 mediated apoptosis pathway. (2) Inhibited Kelch-like ECH-associating protein 1a/NF-E2-related factor 2 mRNA expression, decreased the mRNA expression and activities of antioxidant enzymes, increased the levels of reactive oxygen species, protein carbonyl and malondialdehyde. (3) Increased the mRNA expression level of myosin light chain kinase, which may be result in the down-regulation of tight junction complexes such as zonula occludens 1, occludin and claudins (expect claudin-12 and claudin-15). These results suggest that PN deficiency could impair gill physical barrier function. In summary, dietary PN deficiency could cause the impairment of gill barrier function associated with immunity, apoptosis, antioxidant and tight junction, which may result in the increased the susceptibility of fish to pathogenic bacteria. Moreover, based on the gill rot morbidity, LZ activity and MDA content, the dietary PN requirements for grass cap were estimated to be 4.85, 4.78 and 4.77 mg kg-1 diet, respectively.

Dietary phytic acid weakened the antimicrobial activity and aggravated the inflammatory status of head kidney, spleen and skin in on-growing grass carp (Ctenopharyngodon idella).

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 ß-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 1ß (IL-1ß) (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 ß1 (TGF-ß1), TGF-ß2, 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.

Inconsistently impairment of immune function and structural integrity of head kidney and spleen by vitamin A deficiency in grass carp (Ctenopharyngodon idella).

To investigate effects of vitamin A (VA) on fish immune function and structural integrity in the head kidney and spleen of fish, total of 540 on-growing grass carp (Ctenopharyngodon idella) were divided into six groups, feeding graded levels of VA (0, 600, 1200, 1800, 2800 and 3800 IU/kg diet) for 70 days. Results showed that dietary VA deficiency depressed antibacterial ability and aggravated inflammatory response partially linked to nuclear factor κB p65 (NF-κB p65) and target of rapamycin (TOR) signaling pathways in the head kidney and spleen of fish. Meanwhile, VA deficiency caused oxidative damage, apoptosis and disruption of tight junctions (TJs), which were partially attributed to the down-regulation of NF-E2-related factor 2 (Nrf2) signaling mediated antioxidant ability, the up-regulation of p38 mitogen-activated protein kinase (p38MAPK) signaling mediated apoptosis and myosin light chain kinase (MLCK) signaling mediated disruption of tight junctions (TJs). Taken together, current study firstly demonstrated that VA deficiency decreased the immune function and damaged the structural integrity of the head kidney and spleen in fish.

Mannan oligosaccharides supplementation enhanced head-kidney and spleen immune function in on-growing grass carp (Ctenopharyngodon idella).

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.

Soybean glycinin impaired immune function and caused inflammation associated with PKC-ζ/NF-κb and mTORC1 signaling in the intestine of juvenile grass carp (Ctenopharyngodon idella).

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 ß-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-1ß, IL-8, IL-12p35, IL-12p40 and IL-17D in the midgut and IL-1ß, 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-ß1, TGF-ß2, 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.

Protective effects and potential mechanisms of (2-Carboxyethyl) dimethylsulfonium Bromide (Br-DMPT) on gill health status of on-growing grass carp (Ctenopharyngodon idella) after infection with Flavobacterium columnare.

In this study, the protective effects and potential mechanisms of (2-Carboxyethyl) dimethylsulfonium Bromide (Br-DMPT) were evaluated in relation to the gill health status of on-growing young grass carp (Ctenopharyngodon idella). A total of 450 grass carp (216.49 ± 0.29 g) were randomly distributed into five treatments of three replicates each (30 fish per replicate) and were fed diets supplemented with gradational Br-DMPT (0-520.0 mg/kg levels) for 60 days. Subsequently, the fish were challenged with Flavobacterium columnare for 3 days, and the gills were sampled to evaluate antioxidant status and immune responses evaluation. Our results showed that, when compared to the control group, dietary supplementation with appropriate Br-DMPT levels resulted in the following: (1) decreased gill rot morbidity and improved gill histological symptoms after exposure to F. columnare (P < 0.05); (2) improved activities and gene expression levels (except GSTP2 gene) of antioxidant enzymes and decreased oxidative damage parameter values (reactive oxygen species, malondialdehyde and protein carbonyl) (P < 0.05), which may be partially associated with the nuclear factor-erythroid 2-related factor 2 (Nrf2) signalling pathway (P < 0.05); (3) increased lysozyme (LZ) and acid phosphatase (ACP) activities and complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and upregulated genes expressions of antibacterial peptides (liver-expressed antimicrobial peptide-2A, -2B, hepcidin, ß-defensin and mucin2) (P < 0.05); (4) upregulated gene expressions of anti-inflammatory cytokines (except IL--4/13B) that may be partially to the TOR/(S6K1, 4E-BP1) signalling pathway, and downregulated gene expressions of pro-inflammatory cytokines (except IL-12P35) may be partially to the IKK ß, γ/IκBα/NF-kB) signalling pathway (P < 0.05). Taken together, our results indicate that dietary supplementation with appropriate amounts of Br-DMPT may effectively protect on-growing grass carp from F. columnare by strengthening gill antioxidant capacity and immunity. Furthermore, based on measures of combatting gill rot, antioxidant indices (MDA) and immune indices (LZ), the dietary Br-DMPT supplementation levels for on-growing grass carp are recommended to be 291.14, 303.38 and 312.01 mg/kg diet, respectively.

Enzyme-treated soy protein supplementation in low protein diet enhanced immune function of immune organs in on-growing grass carp.

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 ß (IKKß)/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.

(2-Carboxyethyl) dimethylsulfonium bromide supplementation in non-fish meal diets for on-growing grass carp (Ctenopharyngodon idella): Beneficial effects on immune function of the immune organs via modulation of NF-κB and TOR signalling pathway.

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). (2) significantly improved the innate and adaptive immune components (lysozyme, complement 3, immunoglobulin M and antibacterial peptides et al.) (P < 0.05). (3) increased the gene expressions of main anti-inflammatory cytokines partially by referring to TOR signalling pathway, and decreased the gene expressions of main pro-inflammatory cytokines partially by referring to NF-kB signalling pathway (P < 0.05). Strikingly, no statistical difference could be found in the most of above immune parameters between 260.0 mg/kg Br-DMPT diet group and FM diet group (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.

Dietary biotin deficiency decreased growth performance and impaired the immune function of the head kidney, spleen and skin in on-growing grass carp (Ctenopharyngodon idella).

The aim of this study was to investigate the effects of dietary biotin deficiency on the growth performance and immune function of the head kidney, spleen and skin in on-growing grass carp (Ctenopharyngodon idella). A total of 540 on-growing grass carp (117.11 ± 0.48 g) were fed six diets containing increasing levels of biotin (0.012, 0.110, 0.214, 0.311, 0.427 and 0.518 mg/kg diet) for 70 days. Subsequently, a challenge experiment was performed by infecting them with Aeromonas hydrophila for six days. Our results showed that compared with the appropriate biotin level, (1) biotin deficiency (0.012 mg/kg diet) reduced the activities of lysozyme (LZ) and acid phosphatase (ACP), decreased the contents of complement 3 (C3), C4 and immunoglobulin M (IgM), as well as reduced the mRNA levels of antimicrobial peptides in the head kidney, spleen and skin of on-growing grass carp; (2) biotin deficiency reduced the mRNA levels of anti-microbial substances: liver-expressed antimicrobial peptide (LEAP) -2A, LEAP-2B, hepcidin, ß-defensin-1 and mucin 2 in the head kidney, spleen and skin of on-growing grass carp; (3) biotin deficiency increased the mRNA levels of pro-inflammatory cytokines interleukin 1ß (IL-1ß), IL-6, IL-8, IL-12p40, IL-15, IL-17D, tumour necrosis factor α (TNF-α) and interferon γ2 (IFN-γ2) partially in association with nuclear factor-kappa B (NF-κB) signalling and reduced anti-inflammatory IL-4/13A, IL-10, IL-11 and transforming growth factor ß1 (TGF-ß1) mRNA levels partially in association with target of rapamycin (TOR) signalling in the head kidney, spleen and skin of on-growing grass carp. Interestingly, biotin deficiency had no effect on the expression of IL-12p35, IL-4/13B, TGF-ß2, 4E-BP1 (skin only) or IKKα in the head kidney, spleen and skin of on-growing grass carp. In conclusion, the results indicated that biotin deficiency impaired the immune function of the head kidney, spleen and skin in fish. Finally, based on the percent weight gain (PWG), the ability to prevent skin haemorrhages and lesions, the LZ activity in the head kidney and the C4 content in the spleen, the optimal dietary biotin levels for on-growing grass carp (117-534 g) were estimated as 0.210, 0.230, 0.245 and 0.238 mg/kg diet, respectively.

Novel insights into the intestinal immune regulatory effects of (2-Carboxyethyl) dimethylsulfonium Bromide (Br-DMPT) in on-growing grass carp (Ctenopharyngodon idella).

The present study evaluated the effects of (2-Carboxyethyl)dimethylsulfonium Bromide (Br-DMPT) supplementation on the intestinal immune function and potential mechanisms of on-growing grass carp (Ctenopharyngodon idella) by feeding fish (initial weight 216.49 ± 0.29 g) five diets with gradational Br-DMPT (0-520 mg/kg diet) concentrations for 60 days and then infecting them with Aeromonas hydrophila for 14 days. Our results firstly indicated that compared with the control group, appropriate Br-DMPT supplementation increased the number of beneficial bacteria Lactobacillus and Bifidobacterium and enteritis resistance, decreased the number of detrimental bacteria Aeromonas and E. coli, and relieved the intestinal histopathological symptoms of fish. In addition, compared with the control group, appropriate Br-DMPT supplementation (1) increased lysozyme (LZ) and acid phosphatase (ACP) activities, as well as complement 3 (C3), C4 and immunoglobulin M (IgM) content; (2) upregulated the mRNA levels of anti-microbial substance: liver expressed anti-microbial peptide (LEAP) -2A, LEAP-2B, hepcidin, ß-defensin-1 and Mucin2; (3) partially downregulated the mRNA levels of pro-inflammatory cytokines [interleukin 1ß (IL-1ß), IL-6, IL-8, IL-12p40, IL-15, IL-17D, tumour necrosis factor α (TNF-α) and interferon γ2 (IFN-γ2)] by inhibiting [IKKß/IκBα/(NF-κBp65 and c-Rel)] signalling; and (4) partially upregulated the mRNA levels of anti-inflammatory cytokines [IL-4/13A, IL-10, IL-11, transforming growth factor (TGF)-ß1] by activating [TOR/(S6K1 and 4E-BP)] signalling. The aforementioned results indicated that appropriate amount of Br-DMPT exerted a positive effect on the regulation of intestinal immune function in fish. Finally, based on enteritis morbidity, the IgM content and the lysozyme activity in the PI, the appropriate levels of Br-DMPT supplementation for on-growing grass carp were established as 295.43, 301.73 and 320.36 mg/kg diet, respectively.

The dynamic process of dietary soybean ß-conglycinin in digestion, absorption, and metabolism among different intestinal segments in grass carp (Ctenopharyngodon idella).

The present study aimed to investigate the dynamic process of soybean ß-conglycinin in digestion, absorption, and metabolism in the intestine of grass carp (Ctenopharyngodon idella). Fish fed with 80 g ß-conglycinin/kg diet for 7 weeks, the intestinal digestive enzyme was extracted to hydrolyze ß-conglycinin in vitro, the free amino acid and its metabolism product contents in intestinal segments were analyzed. The present study first found that ß-conglycinin cannot be thoroughly digested by fish intestine digestive enzyme and produces new products (about 60- and 55-kDa polypeptides). The indigestible ß-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 ß-conglycinin in amino acid transporters and tight junction-regulated paracellular pathway. Finally, dietary ß-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 ß-conglycinin in digestion, absorption, and metabolism, which further supported our previous study that dietary ß-conglycinin suppressed fish growth and caused intestine injure.

Cinnamaldehyde improves the growth performance and digestion and absorption capacity in grass carp (Ctenopharyngodon idella).

The present study evaluated the effect of cinnamaldehyde (CIN) on the growth performance and digestion and absorption capacity of grass carp (Ctenopharyngodon idella). Fish were fed five diets including graded levels of CIN for 60 days. The results indicated that (1) appropriate CIN supplementation increased the growth performance and promoted the intestine growth of grass carp; (2) dietary appropriate CIN supplementation increased the digestion and absorption capacity by increasing the activities of intestinal and hepatopancreas digestive enzymes (lipase, chymotrypsin, trypsin, and amylase) and intestinal brush border enzymes (creatine kinase (CK), Na+/K+-ATPase, γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (AKP)); (3) dietary CIN increased the absorption capacity which may be associated with the upregulated messenger RNA (mRNA) abundances of their amino acid transporters (AATs) in the intestine, which might be associated with activating the target of rapamycin (TOR) signaling pathway. The best CIN supplementation in the diets of grass carp was estimated to be 76.40 mg kg-1 diet based on the best percent weight gain (PWG). In general, CIN increased the digestion and absorption capacity of grass carp and raised the mRNA abundances of AATs which may be partly related to activation of the TOR signaling pathway.

Soybean ß-conglycinin caused intestinal inflammation and oxidative damage in association with NF-κB, TOR and Nrf2 in juvenile grass carp (Ctenopharyngodon idella): varying among different intestinal segments.

The current study aimed to investigate the effects and mechanisms of dietary soybean ß-conglycinin in immune function and oxidative damage among different intestinal segments of juvenile grass carp (Ctenopharyngodon idella). 240 fish (13.77 ±â€¯0.10 g) were fed control or 8% ß-conglycinin diet for 7 weeks. Dietary ß-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 ß-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 ß-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 ß-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 ß-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 ß-conglycinin only increased PC content in association with the low activities of CAT, GPx and glutathione peroxidase (GR). Unexpectedly, in the proximal intestine, dietary ß-conglycinin did not significantly change MDA and PC contents while decreased antioxidant enzyme activities. Furtherly, dietary ß-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 ß-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.

Dietary iron deficiency impaired intestinal immune function of on-growing grass carp under the infection of Aeromonas hydrophila: Regulation of NF-κB and TOR signaling.

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, ß-defensin-1, anti-inflammatory cytokines transforming growth factor ß1 (TGF-ß1), TGF-ß2, 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-1ß, interferon γ2 (IFN-γ2), IL-8, IL-12p35, IL-12p40 and IL-17D, nuclear factor kappa B (NF-κB) p65, IκB kinases α (IKKα), IKKß 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.