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This study focused on the effects of dietary selenium deficiency on structural integrity of the head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp (mean weight 226.48 ± 0.68 g) were randomly divided into six groups and fed six separate diets with graded dietary levels of selenium (0.025-1.049 mg/kg diet) for 80 days. Results showed that selenium deficiency (1) caused oxidative damage in part by reducing the activities of antioxidant enzymes (such as SOD, CAT, GPx, GST and GR) and glutathione (GSH) content, down-regulating the transcript abundances of antioxidant enzymes (except GSTp1) partly related to Kelch-like-ECH-associated protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2) signalling; (2) aggravated apoptosis in part by up-regulating the mRNA levels of caspase-2, -3, -7, -8 and -9, which were partially related to p38MAPK/FasL/caspase-8 signalling and JNK/(BAX, Bcl-2, Mcl-1b, IAP)/(Apaf1, caspase-9) signalling; (3) damaged the tight junctions in part by down-regulating the mRNA levels of ZO-1 (except spleen), ZO-2 (except spleen), claudin-c, -f, -7, -11 and claudin-15, and up-regulating the mRNA levels of claudin-12, which were partially related to myosin light chain kinase (MLCK) signalling. Interesting, selenium deficiency failed to affect the expression of GSTp1, Keap1a, occludin, claudin-b, claudin-3c, ZO-1 (spleen only) and ZO-2 (spleen only) in the head kidney, spleen and skin of grass carp. Finally, based on the activities of glutathione peroxidase (GPx) and reactive oxygen species (ROS) content in the head kidney, spleen and skin, the dietary selenium requirements for young grass carp were estimated to be 0.558-0.588 mg/kg diet., (Copyright © 2018. Published by Elsevier Ltd.)

The aim of this study was to assess the effects of dietary pyridoxine (PN) deficiency on intestinal antioxidant capacity, cell apoptosis and intercellular tight junction in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (231.85 ± 0.63 g) were fed six diets containing graded levels of PN (0.12-7.48 mg/kg diet) for 10 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results showed that compared with the optimal PN level, PN deficiency (1) increased the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC), decreased the activities and mRNA levels of antioxidant enzymes such as copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) (P < .05); (2) up-regulated the mRNA levels of cysteinyl aspartic acid-protease-3 (caspase-3), caspase-7, caspase-8, caspase-9, Bcl-2 associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1) and Fas ligand (FasL), and down-regulated the mRNA levels of inhibitor of apoptosis proteins (IAP), B-cell lymphoma protein-2 (Bcl-2) and myeloid cell leukaemia-1 (Mcl-1) (P < .05); (3) down-regulated the mRNA levels of ZO-1, occludin [only in middle intestine (MI)], claudin-b, claudin-c, claudin-f, claudin-3c, claudin-7a, claudin-7b and claudin-11, and up-regulated the mRNA levels of claudin-12 and claudin-15a (P < .05), which might be partly linked to Kelch-like-ECH-associated protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2), p38 mitogen-activated protein kinase (p38MAPK) and myosin light chain kinase (MLCK) signalling in the intestines of fish. However, the activities and mRNA levels of MnSOD, the mRNA levels of Keap1b, c-Jun N-terminal protein kinase (JNK) and claudin-15b in three intestinal segments, and the mRNA levels of occludin in the proximal intestine (PI) and distal intestine (DI) were not affected by graded levels of PN. These data indicate that PN deficiency could disturb the intestinal physical barrier function of fish. Additionally, based on the quadratic regression analysis for MDA content and GST activity, the dietary PN requirements for young grass carp were estimated as 4.85 and 5.02 mg/kg diet, respectively., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, fish were fed six diets containing different levels of pyridoxine (PN) for 10 weeks, and then infected with Aeromonas hydrophila for 2 weeks. The results showed that dietary supplementation with PN had a powerful effect on the inhibition of skin haemorrhage and lesion morbidity, and the enhancement of immunity after fish were infected with A. hydrophila. For example, PN increased the levels of immunoglobulin M (IgM), acid phosphatase (ACP), complement 3 (C3), lysozyme (LZ), C4, antimicrobial peptides, target of rapamycin (TOR), anti‐inflammatory cytokines, ribosomal protein S6 kinases 1 (S6K1) and inhibitor of κBα (IκBα), while decreasing the levels of proinflammatory cytokines [except interleukin‐12 p40 (IL‐12p40)], eIF4E‐binding proteins (4E‐BP), IκB kinase β (IKKβ) and nuclear factor κB p65 (NF‐κBp65) in grass carp skin. Additionally, dietary supplementation with PN increased the levels of tight junction proteins (not claudin‐15 and claudin‐12), antioxidant enzymes [except manganese superoxide dismutase (MnSOD) activity and gene expression, glutathione peroxidase 4a (GPx4a) and GPx4b gene expression], NF‐E2‐related Factor 2 (Nrf2), myeloid cell leukaemia‐1 (Mcl‐1) and inhibitor of apoptosis proteins (IAP), while decreasing the levels of malondialdehyde (MDA), reactive oxygen species (ROS), protein carbonyl (PC), Kelch‐like ECH‐associating protein 1a (Keap1a) myosin light chain kinase (MLCK), cysteinyl aspartic acid protease‐9 (caspase‐9), caspase‐8, caspase‐7, caspase‐3, Bcl‐2‐associated X protein (Bax), p38 mitogen‐activated protein kinase (p38MAPK) and apoptotic protease‐activating factor 1 (Apaf‐1) in grass carp skin. In summary, dietary PN can enhance the immunity of fish skin via elevated immune substances, an eased inflammatory response and an improved barrier integrity. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA), a notorious pollutant widely present worldwide, seriously pollutes aquafeeds. This paper aims to explore the toxicity effects of OTA by the way of diet on the skin barrier in grass carp (Ctenopharyngodon idella). Results were shown as follows in the skin: (1) OTA increased the mRNA abundances of uptake transporter proteins (e.g., OAT3) and decreased efflux transporter proteins (e.g., ABCG2), which caused the accumulation of OTA in the skin of grass carp. (2) OTA upregulated the gene expression related to ROS production by enhancing the NOX (1, 2, 4) signaling pathway and decreased the ability to ROS elimination with downregulation of GPx1 (a,b), Trx by inhibiting the PGC1-α/Nrf2 signaling pathway, which caused oxidative damage to the skin. (3) OTA exacerbated apoptosis in the skin by upregulating the expression of apoptosis-related proteins mediated by ways of endoplasmic reticulum stress and mitochondrial apoptosis. Moreover, OTA down-regulated the mRNA and protein abundances of tight junction-related proteins by inhibiting the MLCK signaling pathway, which in turn disrupted the tight junctions. (4) OTA reduced the number of mucous cup cells and decreased f LZ activities and IgM contents, and finally down-regulated the mRNA abundances of mucin (2, 3), LEAP-2 (A, B), and β-defensin (1, 2, 3), which in turn resulted in impairing skin chemical barrier. Moreover, based on the antimicrobial-related indexes (LZ activities and IgM contents), the OTA-safe upper doses were 814.827 and 813.601 μg/kg. [Display omitted] • Ochratoxin A (OTA) residues destroys the skin histological structure. • OTA damaged fish skin physical barrier through causing tight junction failure. • OTA damaged fish skin chemical barrier through causing reducing mucous cup cells. • The safe upper limit dose of skin barrier function damaged by OTA were 814.83–813.60 μg/kg diet. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) contamination widely occurs in various feed ingredients and food crops, potentially posing a serious health threat to animals. In this research, 1260 juvenile grass carp were separately fed with seven distinct experimental diets (0, 406, 795, 1209, 1612, 2003 and 2406 µg of OTA/kg of diet) for 60 consecutive days to evaluate OTA's toxic effect on the intestinal apical junctional complex (including the tight junction (TJ) and the adherents junction (AJ)) and the underlying action mechanisms. Our experiment firstly confirmed that OTA caused fish growth retardation and disrupted the intestinal structural integrity. The detailed results show that OTA (1) depressed the feed efficiency, percentage weight gain and specific growth rate; (2) accumulated in the intestine; (3) caused oxidative damage and increased intestinal permeability; and (4) induced the RhoA/ROCK signaling pathway, destroying intestinal apical junctional complexes. Notably, OTA intervention did not result in changes in the gene expression of claudin-3c (in the proximal intestine (PI)), claudin-b and ZO-2b (in the mid intestine (MI) and distal intestine (DI)) in the fish intestine. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEA) is a prevalent mycotoxin with high toxicity in animals. In order to study its effect on juvenile grass carp (Ctenopharyngodon idella), six diets supplemented with different levels of ZEA (0, 535, 1041, 1548, 2002, and 2507 μg/kg diet) for 10 weeks were studied to assess its toxicity on intestinal structural integrity and potential mechanisms of action. Our report firstly proved that ZEA led to growth retardation and body deformity, and impaired the intestinal structural integrity of juvenile grass carp, as revealed by the following findings: (1) ZEA accumulated in the intestine and caused histopathological lesions; (2) ZEA resulted in oxidative injury, apoptosis, and breached tight junctions in the fish intestine, which were probably associated with Nuclear factor-erythroid 2-related factor 2 (Nrf2), p38 mitogen activated protein kinases (p38MAPK), and myosin light chain kinase (MLCK) signaling pathways, respectively. ZEA had no influence on the antioxidant gene levels of Kelch-like ECH associating protein 1 (Keap1)b (rather than Keap1a), glutathione-S-transferase (GST)P1, GSTP2 (not in the distal intestine (DI)), tight junctions occludin, claudin-c (not in the proximal intestine (PI)), or claudin-3c (not in the mid intestine (MI) or DI). [ABSTRACT FROM AUTHOR]

The current study explored the protective effect of leucine on antioxidant status, apoptosis and tight junction damage in the gill of grass carp ( Ctenopharyngodon idella Val.). The trial was conducted by feeding grass carp with six graded level of leucine (7.1, 8.9, 11.0, 13.3, 15.2 and 17.1 g kg −1 diet) for 8 weeks. The fish were fed to apparent satiation 4 times per day. The results indicated that compared with the leucine deficiency group, 8.9–11.3 g leucine kg −1 diet supplementations decreased protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the improvement of antioxidant status in the gill by increasing hydroxyl radical capacity and anti-superoxide radicals, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT), glutathione-S-transferase (GST) and glutathione reductase (GR), that referring to the up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression. Moreover, leucine deficiency induced DNA fragmentation via the up-regulation of caspase-3, caspase-8 and caspase-9 expressions and down-regulation of target of rapamycin and ribosomal S6 protein kinase 1 expressions. Furthermore, leucine deficiency increased interleukin-1β (IL-1β), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) mRNA expression and decreased IL-10 and transforming growth factor β (TGF-β), which was partly related to nuclear factor κB (NF-κB) and its inhibitor (IκB). In contrast, the relative mRNA expression of IL-1, IL-8 and TNF-α was down-regulated with 8.9–11.3 g leucine kg −1 diet supplementations. Finally, the relative mRNA expression of tight junction protein, including occludin, zonula occludens-1, claudin b, claudin 3 and claudin 12 was up-regulated with leucine diet supplementations. Our results indicate that leucine protected the fish gill structural integrity partially because of the inhibition of apoptosis, the improvement of antioxidant status, the regulation of tight junction protein and related signalling molecules mRNA expressions in the fish gill. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is a prevalent contaminant in fish feed, posing a threat to aquatic health. Curcumin (Cur), a widely used feed additive renowned for its antioxidant and anti-inflammatory functions, has emerged as a potential solution. Gills are one of the mucosal immune organs in fish. Adding a natural product to feed is one of the most popular strategies to alleviate mycotoxin. At present, it is unknown whether dietary Cur can alleviate the damage to the fish gill barrier caused by ochratoxin A in the diet. Here, we explored the mitigation effects of Cur on the physical, chemical, and immune barriers of gills in grass carp damaged by OTA. This study employed 720 juvenile grass carp (initial average body weight: 11.06 ± 0.05 g) were randomly assigned into 4 groups: the control group (devoid of OTA and Cur), the 1.2 mg/kg OTA group, the 400 mg/kg Cur group, with three replicates per treatment (180 fish) over a 60-day period. Results demonstrated that Cur supplementation could significantly counter OTA-induced damage to the gill barrier through distinct mechanisms: (1) Inhibition of OTA absorption while promoting efflux transporters mRNA expression, thereby diminishing OTA residuals. (2) Enhancement of the physical barrier by fortifying antioxidant defenses, reducing apoptosis, and strengthening tight junctions. (3) Improvement of the chemical barrier by elevating goblet cells, immune components, and antimicrobial peptides. (4) Enhanced the immune barrier by suppressing toll-like receptor 4 and nuclear factor-κB signaling pathways, leading to reduced expression of pro-inflammatory factors and increased T cell differentiation-related genes. In conclusion, Cur emerges as an effective agent in alleviating OTA-induced gill immune function, enhancing physical, chemical, and immune barriers. This study provides a valuable theoretical foundation and practical significance for employing Cur to mitigate the mucosal immune toxicity of OTA in fish. • Curcumin alleviates OTA-induced gill histological injury by reducing OTA residues. • Curcumin alleviates the gills physical, chemical and immune barrier of OTA damage. • There is a potential regulatory network affected by Cur: Nrf2/RhoA/MAPK/Mucin/ NFκB. [ABSTRACT FROM AUTHOR]

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 pyridoxine deficiency impaired immune function of fish gill. • Dietary pyridoxine deficiency impaired apoptosis, antioxidant system and intercellular tight junction of fish gill. • Dietary pyridoxine regulated the signalling of NF-κB, TOR, p38MAPK, Nrf2 and MLCK in fish. [ABSTRACT FROM AUTHOR]

This research was directed to examine the influences of dietary silymarin supplementation in growth, intestinal mucosal permeability, histology and apical junctional complex (AJC) (including tight junction (TJ) and adherents junction (AJ)), as well as the appropriate level of silymarin supplementation to juvenile grass carp (Ctenopharyngodon idella). Fish was fed at 6 graded levels of silymarin (0, 20, 40, 60, 80 and 100 mg/kg) for 70 days. Our results showed that, dietary appropriate silymarin supplementation (1) enhanced growth performance and promoted intestinal growth of fish; (2) reduced intestinal mucosa permeability and improved intestinal apparent morphology of fish; (3) ameliorated intestinal AJC partly related to the enhancement of barrier-forming TJ-related proteins (TJs) and AJ-related proteins (AJs) mRNA levels and the reduction of pore-forming TJs mRNA levels through suppressing RhoA/ROCK signalling pathway (RhoA/ROCK/MLCK/NMII) in fish intestine. Interestingly, silymarin supplementation in diet did not affect the mRNA levels of ZO-2b (not in DI), claudin-7a, −7b, and -15b in fish intestine. Ultimately, based on the growth indicator PWG and intestinal mucosa permeability index serum D-lactate, the appropriate levels of silymarin supplementation to juvenile grass carp (24–223 g) were assessed to be 57.63 and 57.50 mg/kg diet, respectively. • Silymarin enhanced the growth performance of fish. • Silymarin reduced intestinal mucosal permeability and ameliorated intestinal histology of fish. • Silymarin improved intestinal AJC by modulation of RhoA/ROCK signalling pathway of fish. [ABSTRACT FROM AUTHOR]

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. • Deoxynivalenol (DON) caused oxidative damage and declining antioxidant capacity in the fish gill. • DON aggravated apoptosis in the fish gill. • DON disrupted tight junctions in the fish gill. • DON regulated Nrf2, JNK and MLCK signalling. • DON did not affect Keap1b, claudin-b, claudin-3c and claudin-15b in fish gill. [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) is one of the most common mycotoxin contaminants of animal feed worldwide and brings significant threats to the animal production. However, studies concerning the effect of DON on fish intestine are scarce. This study explored the effects of DON on intestinal physical barrier in juvenile grass carp ( Ctenopharyngodon idella ). A total of 1440 juvenile grass carp (12.17 ± 0.01 g) were fed six diets containing graded levels of DON (27, 318, 636, 922, 1243 and 1515 μg/kg diet) for 60 days. This study for the first time documented that DON caused body malformation in fish, and histopathological lesions, oxidative damage, declining antioxidant capacity, cell apoptosis and destruction of tight junctions in the intestine of fish. The results indicated that compared with control group (27 μg/kg diet), DON: (1) increased the reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) content, and up-regulated the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1: Keap1a but not Keap1b), whereas decreased glutathione (GSH) content and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes (except GSTR in MI) and NF-E2-related factor 2 (Nrf2), as well as the protein levels of Nrf2 in fish intestine. (2) up-regulated cysteinyl aspartic acid-protease (caspase) -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl2-associated X protein (Bax), Fas ligand (FasL) and c-Jun N-terminal protein kinase (JNK) mRNA levels, whereas down-regulated B-cell lymphoma-2 (bcl-2) and myeloid cell leukemia-1 (Mcl-1) mRNA levels in fish intestine. (3) down-regulated the mRNA levels of ZO-1, ZO-2b, occludin, claudin-c, -f, -7a, -7b, -11 (except claudin-b and claudin-3c), whereas up-regulated the mRNA levels of claudin-12, -15a (not -15b) and myosin light chain kinase (MLCK) in fish intestine. All above data indicated that DON caused the oxidative damage, apoptosis and the destruction of tight junctions via Nrf2, JNK and MLCK signaling in the intestine of fish, respectively. Finally, based on PWG, FE, PC and MDA, the safe dose of DON for grass carp were all estimated to be 318 μg/kg diet. [ABSTRACT FROM AUTHOR]

The present study explored the possible preventive effects of dietary glutamate (Glu) on LPS-induced oxidative damage, mRNA expression changes of tight junction (TJ) and defensin proteins, inflammatory and apoptosis response signaling molecules in fish intestine. Young Jian carp were fed five diets supplemental graded levels of Glu (0, 4, 8, 16 and 32 g kg −1 diet) for 63 days. The results indicated that Glu supplementation depressed LPS induced the production of reactive oxygen species (ROS) and severe oxidative damage (lipid peroxidation and protein oxidation) in fish intestine, which was partially due to the increased glutathione (GSH) content and antioxidant enzyme activities including superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione- S -transferase (GST), and glutathione reductase (GR) ( P < 0.05). Further investigations indicated that Glu supplementation caused elevation of those antioxidant enzyme activities are related to the up-regulation of corresponding antioxidant enzymes and the related signaling factor Nrf2 mRNA levels ( P < 0.05). Meanwhile, Glu pre-treatment significantly suppressed LPS-induced COX-2 and inflammatory cytokines mRNA expression and down-regulated NF-κB p65 and MAPK p38 transcription. Furthermore, pre-treatment with Glu prevented LPS induced apoptosis-related gene expression (caspase 3 and 9, P < 0.05). Lastly, Glu supplementation also attenuated LPS induced intestinal barrier function-related gene TJ proteins (ZO-1, occludin1, claudin2, 3, and 7), β-defensin1 and 3 mRNA expressions decreasing ( P < 0.05). Taken together, the present results showed Glu could attenuate LPS induced the oxidative damage by Nrf2 signal pathway and depress LPS induced inflammation response (cytokines, COX-2, NF-κB p65, and MAPK p38), apoptosis (caspase3 and 9), and barrier function (ZO-1, occludin1, claudin2, 3 and 7, and β-defensin 1 and 3) - related gene expression changes of fish intestine. [ABSTRACT FROM AUTHOR]

The current study explores the hypothesis that low or excess levels of lipids decrease the enteritis resistance ability through impairing the intestinal physical and immune barrier function of young grass carp (Ctenopharyngodon idella) . A total of 540 young grass carp with an average initial weight of 261.41 ± 0.53 g were fed diets containing six graded levels of lipids at 5.9, 21.4, 36.0, 50.2, 66.6 and 80.1 g/kg for 8 weeks. After that, a challenge trial was conducted by injection of Aeromonas hydrophila over 2 weeks. The results show that compared with optimal lipids level, low or excess levels of lipids impair fish immune barrier function through declining humoral compounds and down-regulating the mRNA levels of interleukin 4/13A (IL-4(13)A) [not in the proximal intestine (PI)], IL-4(13)B, IL-6, IL-10, transforming growth factor β1 (TGF-β1), inhibitor of κBα (IκBα), TOR and ribosomal p70S6 kinase (S6K1) ( P < 0.05), and up-regulating tumor necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), IL-1β, IL-8, IL-12 p40 (not p35), nuclear factor κB p65 (NF-κB p65), IκB kinase α (IKKα), IKKβ, IKKγ, and eIF4E-binding protein (4EBP) in the intestine of young grass carp ( P < 0.05). In addition, low or excess levels of lipids also decrease young grass carp physical barrier function through down-regulating the mRNA levels of zonula occludens (ZO-1), ZO-2b [only in the distal intestine (DI)], Claudin b, c, 3, 12, 15a, 15b (only in the DI), 7b (not 7a) and Occludin by MAPKK 6/p38 MAPK (not JNK)/MLCK signaling molecules, down-regulating B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) (not in the DI), and decreasing antioxidant ability via Kelch-like ECH-associating protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) signaling molecules in the intestine. Based on the quadratic regression analysis for the enteritis resistance ability, LA activities and GSH content in the MI were established to be 54.5, 49.91 and 47.83 g lipid/kg diets, respectively. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of dietary myo -inositol on the growth and intestinal physical barrier functions of young grass carp ( Ctenopharyngodon idella ). A total of 540 young grass carp (221.83 ± 0.84 g) were fed six diets containing graded levels of myo -inositol (27.0, 137.9, 286.8, 438.6, 587.7 and 737.3 mg/kg) for 10 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila for 14 days. The results indicated that compared with optimal myo -inositol levels, myo -inositol deficiency (27.0 mg/kg diet): (1) decreased glutathione (GSH) contents and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes [not glutathione-S-transferase ( gst ) p1 and gstp2 ] and NF-E2-related factor 2 ( nrf2 ), whereas up-regulated the reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents, and the mRNA levels of Kelch-like-ECH-associated protein 1 ( keap1 ) in three intestinal segments of young grass carp ( P < 0.05). (2) Up-regulated cysteinyl aspartic acid-protease ( caspase )- 2 , -3 , -7 , -8 , -9 , apoptotic protease activating factor-1 ( apaf-1 ), Bcl2-associated X protein ( bax ), fas ligand ( fasl ), gen-activated protein kinase ( p38mapk ) and c-Jun N-terminal protein kinase ( jnk ) mRNA levels, whereas down-regulated B-cell lymphoma-2 ( bcl-2 ), inhibitor of apoptosis proteins ( iap ) and myeloid cell leukemia-1 ( mcl-1 ) mRNA levels in three intestinal segments of young grass carp ( P < 0.05). (3) Down-regulated mRNA levels of cell cycle proteins cyclin b , cyclin d , cyclin e and E2F transcription factor 4 ( e2f4 ) in three intestinal segments of young grass carp ( P < 0.05). (4) Down-regulated the mRNA levels of zonula occludens ( zo ) 1 , zo-2 , occludin , claudin-b , -c , -f , -3c , -7a , -7b as well as -11 , and up-regulated the mRNA levels of claudin-12 , -15a (not -15b ) and myosin light chain kinase ( mlck ) in three intestinal segments of young grass carp ( P < 0.05). All above data indicated that dietary myo -inositol deficiency could damage physical barrier function in three intestinal segments of fish. Finally, the myo -inositol requirements based on the percent weight gain (PWG), reactive oxygen species (ROS) contents in the proximal intestine (PI), relative mRNA levels of caspase-2 (PI), cyclin b (MI) as well as claudin-b (PI) were estimated to be 276.7, 304.1, 327.9, 416.7 and 313.2 mg/kg diet, respectively. [ABSTRACT FROM AUTHOR]

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp ( Ctenopharyngodon idella ) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill. [ABSTRACT FROM AUTHOR]

This study explored the possible preventive effects of dietary phenylalanine (Phe) on antioxidant responses, apoptosis and tight junction protein transcription in the gills of young grass carp ( Ctenopharyngodon idella ). Fish were fed six different experimental diets containing graded levels of Phe (3.4–16.8 g kg −1 ) for 8 weeks. The results showed that Phe deficiency induced protein oxidation and lipid peroxidation by decreasing the glutathione content and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) in fish gill ( P < 0.05). These results may be ascribed to the downregulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), and the upregulation of Kelch-like-ECH-associated protein 1 a (Keap1a) expression in grass carp gills ( P < 0.05). Additionally, Phe deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 mRNA expression ( P < 0.05). These results may be ascribed to the improvement in reactive oxygen species (ROS) levels in the fish gills ( P < 0.05). Furthermore, the results indicated that Phe deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gills ( P < 0.05). These effects were partly due to the downregulation of interleukin 10 (IL-10), transforming growth factor β (TGF-β) and inhibitor factor κBα (iκBα) and the upregulation of relative mRNA expression of interleukin 1β (IL-1β), interleukin 8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear transcription factor-κB p65 (NF-κB p65) ( P < 0.05). Taken together, the results showed that Phe deficiency impaired the structural integrity of fish gills by regulating the expression of tight junction proteins, cytokines, antioxidant enzymes, NF-κB p65, iκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gills. [ABSTRACT FROM AUTHOR]

This study investigated the effects of choline on intestinal mucosal immune and the possible mechanisms in fish by feeding juvenile Jian carp ( Cyprinus carpio var. Jian) with graded levels of dietary choline (165–1820 mg/kg diet) for 65 days. The results firstly showed that choline deficiency induced inflammatory infiltration in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of fish. Meanwhile, compared with the optimal choline group, choline deficiency decreased the activities of lysozyme and acid phosphatase, contents of complement 3 and IgM in the intestine, downregulated the mRNA levels of antimicrobial peptides (liver-expressed antimicrobial peptide (LEAP) 2A and defensin-3 in the PI and MI, LEAP-2B and hepcidin in the PI, MI and DI), anti-inflammatory cytokines (interleukin (IL) 10 and transforming growth factor β2 in the PI, MI and DI), and signaling molecule IκB in the PI, MI and DI; while upregulated the mRNA levels of pro-inflammatory cytokines (IL-6a and tumor necrosis factor α in the MI and DI, interferon γ2b in the PI and MI, IL-1β and IL-6b in the PI, MI and DI), and signaling molecules (Toll-like receptor 4 in the MI, myeloid differentiation primary response 88 in the PI and MI, Janus kinase 3 and tyrosine kinase 2 in the MI and DI, nuclear factor kappa B (NF-κB), signal transducers and activators of transcription (STAT) 4 and STAT5 in the PI, MI and DI) of juvenile Jian carp, further indicating that choline deficiency caused inflammation and immunity depression in the intestine of fish. But choline deficiency decreased the PI IL-6a mRNA level, and increased the DI LEAP-2A and defensin-3 mRNA levels with unknown reasons. Furthermore, dietary choline deficiency downregulated mRNA levels of tight junction (TJ) proteins (claudin 3c in the PI and MI, claudin 7, claudin 11 and occludin in the PI, MI and DI) and signaling molecule mitogen-activated protein kinases p38 in the PI, MI and DI of juvenile Jian carp, whereas upregulated the mRNA levels of claudin 3b in the MI and DI, and claudin 3c in the DI. Moreover, the excessive choline exhibited negative effects on intestinal immunity and TJ proteins that were similar to the choline deficiency. In summary, dietary choline deficiency or excess caused the depression of intestinal mucosal immune by inducing inflammation and dysfunction of the intestinal physical barrier, and regulating related signaling molecules of fish. [ABSTRACT FROM AUTHOR]

This study firstly explored the possible effects of dietary histidine on structural integrity and the related signaling factor gene expression in the gills of fish. Young grass carp ( Ctenopharyngodon idella ) were fed with six diets containing gradual levels of histidine for 8 weeks. The results firstly demonstrated that histidine deficiency caused increases in reactive oxygen species (ROS) contents, and severe oxidative damage (lipid peroxidation and protein oxidation) in the gills of fish, which was partially due to the decreased glutathione (GSH) content and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione- S -transferase (GST) and glutathione reductase (GR)]. Further investigations indicated that histidine deficiency caused depressions of those antioxidant enzyme activities are related to the down-regulation of corresponding antioxidant enzyme genes and the related signaling factor Nrf2 mRNA levels. Meanwhile, histidine deficiency induced DNA fragmentation via up-regulation of caspase-3, caspase-8 and caspase-9 expressions that referring to the down-regulation of TOR and S6K mRNA levels. Furthermore, His deficiency down-regulated claudin-b, claudin-c, claudin-3, claudin-12, claudin-15, occludin and ZO-1 transcription in fish gills. These effects were partially related to the up-regulation of pro-inflammatory cytokines, interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and related signaling factor nuclear factor κB P65 (NF-κB P65) mRNA levels, and the down-regulation of anti-inflammatory cytokines, interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and related signaling factor IκBα mRNA levels. Excessive histidine exhibited negative effects that were similar to histidine deficiency, whereas the optimal histidine levels reversed those negative effects. Taken together, our results showed that histidine deficiency or excess impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, apoptosis, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gills. [ABSTRACT FROM AUTHOR]

To investigate the effects of dietary niacin on gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression, young grass carp ( Ctenopharyngodon idella ) were fed six diets containing graded levels of niacin (3.95–55.01 mg/kg diet) for 8 weeks. The study indicated that niacin deficiency decreased lysozyme and acid phosphatase activities, and complement 3 content, and caused oxidative damage that might be partly due to the decreased copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in fish gills ( P < 0.05). Moreover, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1), tight junction proteins (Occludin, zonula occludens 1, Claudin-15 and -3), signaling molecules (inhibitor of κBα (IκBα), target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and NF-E2-related factor 2 (Nrf2)) and antioxidant enzymes were significantly decreased ( P < 0.05) in niacin-deficient diet group. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), signaling molecules (nuclear factor kappa B p65, IκB kinase α, IκB kinase β, IκB kinase γ, Kelch-like-ECH-associated protein 1b, myosin light chain kinase and p38 mitogen-activated protein kinase (p38 MAPK) were significantly increased ( P < 0.05) in fish gills fed niacin-deficient diet. Interestingly, the varying niacin levels of 3.95–55.01 mg/kg diet had no effect on the mRNA level of Kelch-like-ECH-associated protein 1a, Claudin-c and −12 in fish gills ( P > 0.05). In conclusion, niacin deficiency decreased gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling molecules of fish. [ABSTRACT FROM AUTHOR]

In this study, we investigate the effects of dietary thiamin deficiency on immune responses, tight junctions, antioxidant capacity and related signaling molecules in the gills of young grass carp ( Ctenopharyngodon idella ). Fish were fed diets that contained 0.12–2.04 mg thiamin kg −1 for 8 weeks. We found that dietary thiamin deficiency resulted in reduced complement 3 content, lysozyme and acid phosphatase activities, mRNA levels of hepcidin, liver-expressed antimicrobial peptides 2, transforming growth factor (TGF)-β1, interleukin (IL)-10, inhibitor protein-κBα (IκBα), ribosomal S6 protein kinase 1 and target of rapamycin (TOR) and increased expression of interferon-γ2, tumor necrosis factor-α, TGF-β2, IL-1β, IL-8, IκB kinases (IKKβ and IKKγ) and nuclear factor-κB p65 (NF-κB p65). Our findings showed that thiamin deficiency reduced the immune status of fish gills. Furthermore, thiamin deficiency resulted in reduced mRNA transcript levels of claudin b, claudin 3, claudin 12, zonula occludens 1 (ZO-1) and occludin and increased mRNA transcript levels of claudin 15a, myosin light-chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) in fish gill tissues. These data suggested that thiamin deficiency disrupted tight junction-mediated fish gill barrier function. Additionally, reactive oxygen species, malondialdehyde and protein carbonyl levels and both the activities and expression levels of Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferases and glutathione reductase, as well as NF-E2-related factor 2 gene expression in fish gills, were lower in fish fed a thiamin-deficient diet. By contrast, thiamin deficiency increased levels of Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b mRNA transcript expression in fish gills. Taken together, our findings indicated that thiamin deficiency impaired fish gill health by effects on the expression of genes encoding cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, MLCK and Nrf2. [ABSTRACT FROM AUTHOR]

This study investigated the effect of dietary arginine on the immune response, antioxidant status and tight junction mRNA expression in the intestine of juvenile Jian carp ( Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with an average initial weight of 6.33 ± 0.03 g were fed graded levels of arginine (9.8–24.5 g kg −1 diet) for nine weeks. The study showed that arginine deficiency up-regulated interleukin 1, interleukin 8 and transforming growth factor-β and down-regulated tumour necrosis factor α gene expression ( P < 0.05). Additionally, arginine deficiency increased malondialdehyde (MDA), protein carbonyl (PC) and glutathione contents and decreased the activities of copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) and glutathione-S-transferase (GST) ( P < 0.05). Meanwhile, arginine deficiency significantly increased claudin 7, occludin, protein kinase C, NF-E2-related factor 2 and Kelch-like-ECH- associated protein 1 mRNA expression and decreased SOD1, CAT and GR mRNA expression ( P < 0.05). All of these results indicated that arginine deficiency impaired intestinal immune function via the regulation of mRNA expression of cytokines, tight junction proteins, antioxidant enzymes, Nrf2/Keap1 and PKC in fish intestine. [ABSTRACT FROM AUTHOR]

This investigation used the same growth trial as the previous study, which showed that folic acid deficiency retarded growth in young grass carp (the percent weight gain of Groups 1–6 were 102.32 ± 3.41%, 137.25 ± 10.48%, 179.78 ± 3.95%, 164.33 ± 3.21%, 143.35 ± 8.12% and 115.28 ± 2.66%) [1]. In the present study, we investigated the effects of dietary folic acid on the immune response, antioxidant status and tight junctions in the intestine of young grass carp ( Ctenopharyngodon idella ). A total of 540 young grass carp were fed diets containing graded levels of folic acid at 0.10, 0.47, 1.03, 1.48, 1.88 and 3.12 mg kg −1 diet for 8 weeks. The results indicated that acid phosphatase and lysozyme activities, and the complement component 3 content in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) were decreased with folic acid deficiency (0.1 mg kg −1 ) ( P < 0.05). Folic acid deficiency (0.1 mg kg −1 ) up-regulated interleukin 1β, interleukin 8, tumor necrosis factor α, nuclear factor κB p65 (NF-κB p65), IκB kinase α (IKK-α), IKK-β and IKK-γ gene expression, meanwhile down-regulated interleukin 10, transforming growth factor β, IκB and target of rapamycin gene expression in the PI, MI and DI ( P < 0.05). These data suggested that folic acid deficiency decreased fish intestinal innate immune function may be partly contributed to the regulation of NF-κB p65 pathway. Moreover, the activities and corresponding gene expression of glutathione content, Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione s-transferases and glutathione reductase in fish intestine were depressed by deficient folic acid diet (0.1 mg kg −1 ) ( P < 0.05). Furthermore, folic acid deficiency (0.1 mg kg −1 ) down-regulated NF-E2-related factor 2 (Nrf2) gene expression, up-regulated Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b gene expression in fish intestine ( P < 0.05). These data indicated that deficient folic acid diet damaged fish intestinal antioxidant capacity partly by regulating Nrf2/Keap1 pathway. Additionally, folic acid deficiency (0.1 mg kg −1 ) down-regulated claudin-b, claudin-c, claudin-3, occludin and zonula occludens 1 gene expression; whereas folic acid deficiency (0.1 mg kg −1 ) up-regulated claudin-12, claudin-15, myosin light chain kinase (MLCK) and p38 mitogen activated protein kinase (p38 MAPK) gene expression in the PI, MI and DI ( P < 0.05), suggesting that folic acid deficiency may damage fish intestinal tight junctions associated with the mediation of MLCK and p38 MAPK gene expression. In conclusion, folic acid deficiency (0.1 mg kg −1 ) impaired fish intestinal immunity, antioxidant capacity and tight junctions. [ABSTRACT FROM AUTHOR]

This study investigated the effects of riboflavin on intestinal immunity, tight junctions and antioxidant status of young grass carp ( Ctenopharyngodon idella ). Fish were fed diets containing graded levels of riboflavin (0.63–10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme, acid phosphatase, copper/zinc superoxide dismutase, glutathione reductase and glutathione peroxidase activities, and contents of complement component 3 and reduced glutathione in the intestine of fish ( P < 0.05). Meanwhile, riboflavin deficiency increased reactive oxygen species, malondialdehyde and protein carbonyl contents and catalase activity ( P < 0.05) in the intestine of fish. Furthermore, real-time polymerase chain reaction analysis was used to investigate mRNA expression patterns and found that the mRNA levels of interleukin 10 and transforming growth factor β1, Occludin, zonula occludens 1, Claudin-b and Claudin-c, inhibitor protein κBα, target of rapamycin, ribosomal S6 protein kinase 1 and NF-E2-related factor 2, copper/zinc superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased ( P < 0.05) in the intestine of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of tumor necrosis factor α, interleukin 1β, interleukin 8, nuclear factor kappa B p65, Ikappa B kinase β, Ikappa B kinase γ, Kelch-like-ECH-associated protein 1b, p38 mitogen-activated protein kinase, myosin light chain kinase and Claudin-12 were increased ( P < 0.05) in the intestine of fish fed riboflavin-deficient diet. In conclusion, riboflavin deficiency decreased immunity and structural integrity of fish intestine. The optimum riboflavin level for intestinal acid phosphatase activity of young grass carp was estimated to be 6.65 mg/kg diet. [ABSTRACT FROM AUTHOR]

Intestinal mucosal immune components and mRNA levels of inflammatory cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules in young grass carp ( Ctenopharyngodon idellus ) under dietary manganese (Mn) deficiency or excess were investigated. Fish were fed the diets containing graded levels of Mn [3.65–27.86 mg Mn kg −1 diet] for 8 weeks. The results demonstrated that Mn deficiency significantly decreased the lysozyme and acid phosphatase (ACP) activities, up-regulated tumour necrosis factor α (TNF-α), interleukin 8 and the signalling factor nuclear factor-κB p65, and down-regulated interleukin 10 (IL-10), transforming growth factor β1, inhibitor of signalling factors κB-α and target of rapamycin mRNA levels in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI). However, Mn deficiency did not change the C3 content in the PI, whereas it decreased the C3 contents in the MI and DI. Additionally, Mn depletion also resulted in significantly low mRNA levels for tight junction proteins (claudin-b, claudin-c, claudin-15, occludin and zonula occludens-1), antioxidant enzymes (MnSOD, GPx and CAT) and NF-E2-related factor-2 in the intestines of fish. Excessive Mn exhibited toxic effects similar to Mn deficiency, where optimal Mn contents reversed those indicators. In conclusion, Mn deficiency or excess causes the depression of intestinal immunity, induction of inflammation and dysfunction of the intestinal physical barrier relating to NF-κB, TOR and Nrf2 signalling in grass carp. Furthermore, quadratic regression analysis at 95% maximum response of lysozyme and acid phosphatase activities in the distal intestine of young grass carp revealed the optimum dietary Mn levels to be 8.90 and 8.99 mg kg −1 diet, respectively. [ABSTRACT FROM AUTHOR]

This study is for the first time to explore the possible effects of dietary tryptophan (Trp) on structural integrity and the related signalling factor gene expression in the gill of young grass carp ( Ctenopharyngodon idella ). Fish were fed with six different experimental diets containing graded levels of Trp at 0.7 (control), 1.7, 3.1, 4.0, 5.2 and 6.1 g kg −1 diet for 8 weeks. The results firstly demonstrated that Trp deficiency or excess caused increases in reactive oxygen species (ROS) contents, and severe oxidative damage (lipid peroxidation and protein oxidation) in the gill of fish, and those negative effects could be reversed by optimal Trp levels. Secondly, compared with the optimal Trp levels, Trp deficiency could cause decreases in the mRNA levels of the barrier functional proteins (occludin, zonula occludens-1, claudin-c, and -3) and increases in the mRNA levels of the pore-formation proteins (claudin-12 and -15) mRNA levels in the gill of fish, and those were reversed by the optimal levels of Trp. The negative effects of Trp deficiency on those tight junction protein gene expression might be partly related to the increases in the mRNA levels of pro-inflammatory cytokines and related signalling factors (tumor necrosis factor α, interleukin 8, interleukin 1β and transcription factor-κB) and decreases in the mRNA levels of anti-inflammatory cytokines and related signalling factors [interleukin 10, transforming growth factor-β1, nuclear inhibitor factor κBα (iκBα), target of rapamyc and ribosome protein S6 kinase 1 (S6K1)] in the gill of fish. In addition, optimal dietary Trp protected the gill of fish against its deficiency-caused increases in the mRNA levels of the apoptosis signalling (caspase-3, caspase-8, caspase-9) and decreases in anti-superoxide radicals capacity, anti-hydroxyl radical capacity, glutathione contents and the activities of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) in the gill of fish. Additionally, compared with the Trp deficiency, optimal Trp up-regulated the mRNA levels of SOD, CAT, GPx, GR and GST, which might be partly ascribed to the up-regulation of the NF-E2-related factor 2 (Nrf2) mRNA levels and the down-regulation of Kelch-like-ECH-associated protein 1 (Keap1) mRNA levels in the gill of fish. Interestingly, excessive Trp caused similar results with its deficiency. Collectively, Trp deficiency or excess could cause antioxidant system disruption and change tight junction protein transcription abundances, which were partly related to the signalling factors, NF-κB p65, TOR, caspase-(3,8,9) and Nrf2, in fish gill, those could be blocked by the optimal Trp levels. [ABSTRACT FROM AUTHOR]

This study investigated the effects of dietary valine on tight junction protein transcription, antioxidant status and apoptosis on grass carp gills ( Ctenopharyngodon idella ). Fish were fed six different experimental diets containing graded levels of valine (4.3, 8.0, 10.6, 13.1, 16.7, 19.1 g/kg). The results indicated that valine deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gill ( P < 0.05). These effects were partly due to the down-regulation of interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and IκB α and the up-regulation of relative mRNA expression of interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and nuclear factor κB P65 (NF-κB P65) (P < 0.05). However, valine deficiency and valine supplementation did not have a significant effect on Claudin c and Claudin 12 expression in grass carp gills ( P > 0.05). Valine deficiency also disrupted antioxidant status in the gill by decreasing anti-superoxide radicals and hydroxyl radical capacity, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) ( P < 0.05). These results may be ascribed to the down-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the up-regulation of Kelch-like-ECH-associated protein 1 (Keap1) ( P < 0.05). Additionally, valine deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 expressions ( P < 0.05). These results may be ascribed to the improvement in ROS levels in the fish gill ( P < 0.05). Taken together, the results showed that valine deficiency impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gill. [ABSTRACT FROM AUTHOR]

This study investigated the effects of dietary riboflavin on the growth, gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression of young grass carp ( Ctenopharyngodon idella ). Fish were fed six diets containing graded levels of riboflavin (0.63–10.04 mg/kg diet) for 8 weeks. The study indicated that riboflavin deficiency decreased lysozyme and acid phosphatase activities, and complement component 3 content in the gills of fish ( P < 0.05). Moreover, riboflavin deficiency caused oxidative damage, which might be partly due to decrease copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in the gills of fish ( P < 0.05). Furthermore, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1), tight junction proteins (Occludin, zonula occludens 1, Claudin-c and Claudin-3), signaling molecules (inhibitor of κBα, target of rapamycin and NF-E2-related factor 2) and antioxidant enzymes (copper, zinc superoxide dismutase and glutathione reductase) were significantly decreased ( P < 0.05) in the gills of fish fed riboflavin-deficient diet. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), signaling molecules (nuclear factor kappa B p65, IκB kinase β, IκB kinase γ, Kelch-like-ECH-associated protein 1b and myosin light chain kinase) and tight junction protein Claudin-12 were significantly increased ( P < 0.05) in the gills of fish fed riboflavin-deficient diet. In addition, this study indicated for the first time that young fish fed a riboflavin-deficient diet exhibited anorexia and poor growth. In conclusion, riboflavin deficiency decreased growth and gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling molecules of fish. Based on percent weight gain, gill lysozyme activity and reduced glutathione content, the dietary riboflavin requirements for young grass carp (275–722 g) were estimated to be 5.85, 7.39 and 6.34 mg/kg diet, respectively. [ABSTRACT FROM AUTHOR]

The present work evaluated the effects of dietary phenylalanine (Phe) on the intestinal immune response, tight junction proteins transcript abundance, and the gene expression of immune- and antioxidant-related signalling molecules in the intestine. In addition, the dietary Phe (and Phe + Tyr) requirement of young grass carp ( Ctenopharyngodon idella ) was also estimated. Fish were fed fish meal–casein–gelatin based diets (302.3 g crude protein kg −1 ) containing 3.4 (basal diet), 6.1, 9.1, 11.5, 14.0 and 16.8 g Phe kg −1 with a fixed amount of 10.7 g tyrosine kg −1 for 8 weeks. The results showed that Phe deficiency or excess Phe reduced the lysozyme and acid phosphatase activities and complement C 3 content in the intestine ( P < 0.05). Moreover, zonula occludens-1 ( ZO-1 ), occludin and claudin c mRNA levels were highest in the fish fed the diet containing 11.5 g Phe kg −1 ( P < 0.05). However, claudin 12 and claudin b mRNA levels were not significantly affected by dietary Phe ( P > 0.05). Gene expression of interleukin-10 ( IL-10 ), transforming growth factor-β1 ( TGF-β1 ), target of rapamycin ( TOR ) and inhibitor of nuclear factor κBα ( IκBα ) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) increased as dietary Phe increased up to 6.1, 9.1, 11.5 and 14.0 g kg −1 , respectively ( P < 0.05). However, interleukin-8 ( IL-8 ), tumour necrosis factor-α ( TNF-α ) and nuclear factor-κB p65 ( NF-κB p65 ) mRNA levels showed opposite tendencies. In addition, the mRNA level of superoxide dismutase (SOD) was significantly lower in the intestinal tissue of the group fed a diet with Phe levels of 16.8 g kg −1 than in those of other groups ( P < 0.05). The expression of NF-E2-related factor 2 ( Nrf2 ) gene was increased as dietary Phe increased up to 9.1 g kg −1 ( P < 0.05). In conclusion, Phe improved intestinal immune status, and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65 , IκBα , TOR , and Nrf2 in the fish intestine. Based on the quadratic regression analysis of lysozyme activity at a 95% maximum, the dietary Phe requirement of young grass carp (256–629 g) was estimated to be 8.31 g kg −1 , corresponding to 2.75 g 100 g −1 protein. [ABSTRACT FROM AUTHOR]

The role of leucine (Leu) in the regulation of the intestinal immune status, immune-related signalling molecules and tight junction (TJ) protein transcript abundance in the intestine of grass carp ( Ctenopharyngodon idella ) was investigated. Six iso-nitrogenous diets that contained graded levels of Leu (7.1–17.1 g Leu kg − 1 diets) were fed to the fish for 8 weeks. Compared with the control group, appropriate Leu supplementation increased ( P < 0.05) the following: (1) the lysozyme activity, acid phosphatase activity and complement component 3 (C3) content in all intestinal segments; (2) the mRNA levels of interleukin 10, inhibitor factor κBα (IκBα) and target of rapamycin (TOR) in the mid and distal intestine as well as transforming growth factor β1 in all intestinal segments; and (3) the transcript levels for claudin b, claudin 3, claudin 15, occludin and zonula occludens-1 (ZO-1) in the intestine of young grass carp. At the same time, appropriate Leu supplementation down-regulated the mRNA levels of tumour necrosis factor α, interleukin 8 and nuclear factor κB p65 (NF-κB p65) in the mid and distal intestine of young grass carp ( P < 0.05). Interestingly, the transcript levels for claudin c and claudin 12 showed no significant differences among the groups in the intestine of young grass carp. In conclusion, the positive effect of Leu on intestinal health is associated with the improvement of the intestinal immune status and the regulation of immune-related signalling molecules and tight junction transcripts of fish. [ABSTRACT FROM AUTHOR]

This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp ( Ctenopharyngodon idella ). Six iso-nitrogenous diets containing graded levels of valine (4.3–19.1 g kg − 1 diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet ( P < 0.05). In addition, valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine ( P < 0.05), down-regulated mRNA levels of interleukin 10, transforming growth factor β1, IκBα and target of rapamycin (TOR) ( P < 0.05), and up-regulated tumor necrosis factor α, interleukin 8 and nuclear factor κB P65 (NF-κB P65) gene expression ( P < 0.05). Additionally, valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 ( P < 0.05), and improved Claudin 15 expression in the fish intestine ( P < 0.05). However, valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp ( P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268–679 g) were established to be 14.47 g kg −1 diet (4.82 g 100 g −1 CP) or 14.00 g kg −1 diet (4.77 g 100 g −1 CP), respectively. [ABSTRACT FROM AUTHOR]

Low-molecular-weight vitamin biotin is an essential nutrient substance for all animal normal growth. Previous exploration has documented that dietary biotin deficiency led to fish growth retardation and a decrease in immunity, whereas its detrimental impacts on fish other physiological status were still not revealed yet. Herein, further exploration was performed to excavate the latent influence of dietary biotin deficiency on antioxidant status, apoptosis and tight junction protein of immune organs (the skin, spleen and head kidney). In this research, grass carp (Ctenopharyngodon idella) (117.11 ± 0.48 g) as a model were fed six graded levels of biotin diets (0.012, 0.110, 0.214, 0.311, 0.427 and 0.518 mg/kg diet) in triplicate for 70 days, followed by a challenge with A. hydrophila for 6 days. Our data from above three immune organs manifested that biotin deficiency (0.012 mg/kg diet): (1) markedly increased oxidative damage degree as evidenced by the obtained highest value in all oxidative damage indicators and exhibited the lowest significant value in almost all antioxidant enzymes activities and corresponding gene expressions, where this action may be in part correlated with the modulation of Nrf2 signaling (P < 0.05); (2) signally aggravated cell apoptosis by altering the expression profiles of mitochondria and death receptor pathways involved genes (such as caspase-3 , 8 and FasL), where this mechanism may be partly connected with p38MAPK signaling (P < 0.05); (3) dramatically damaged tight junctions by regulating the expression profiles of a series of complexes (such as occludin and claudins), where the reason may be in part correlated with myosin light chain kinase (MLCK) signaling (P < 0.05). Differently, the transcriptional levels of MnSOD , GPx4 , JNK and ZO-2 (only in spleen) were not regulated by dietary biotin in above three immune organs (P > 0.05). Furthermore, a broken line analysis showed that dietary biotin requirement levels for on-growing grass carp is 0.245–0.354 mg/kg diet for parameters (ROS content, caspase3 and claudin-3c expression levels). • This study provided new insights into the role of biotin in enhancing fish growth. • Biotin deficiency aggravated cell apoptosis related to P38MAPK signaling in fish immune organs. • Biotin deficiency decreased antioxidant capacity related to Nrf2 signaling in fish immune organs. • Biotin deficiency damaged tight junction capacity related to MLCK signaling in fish immune organs. [ABSTRACT FROM AUTHOR]

The immune function operation of fish immune organs depends tightly on the immune organs' structural integrity which is mainly regulated by various biological respects such as oxidative status, apoptosis and tight junctions. Previous explorations have documented that dietary (2-carboxyethyl) dimethylsulfonium Bromide (Br-DMPT) intervention boosted fish growth in association with the strengthened immune function of the head kidney, spleen and skin. Here, further exploration was performed to better decipher the primary action mechanisms by which Br-DMPT maintains the immune organs' integrity of fish. In this research, grass carp (Ctenopharyngodon idella) (216.49 ± 0.29 g) as a model were fed five graded levels of Br-DMPT diets (0-520.0 mg/kg) in triplicate for 60 days, followed by a challenge with Aeromonas hydrophila for 2 weeks. Subsequently, immune organ samples were selectively collected to investigate oxidative status, apoptosis and tight junctions associated parameters. Our data from above three immune organs manifested that compared to the control group, 260.0 mg/kg Br-DMPT diet group: (1) markedly decreased oxidative damage degree as evidenced by the obtained lowest value in all oxidative damage indicators and exhibited the highest significant value in almost all antioxidant enzymes activities and corresponding gene expressions, where this action may be in part correlated with the modulation of Nrf2 signalling (P < 0.05); (2) signally alleviated cell apoptosis by altering the expression profiles of mitochondria and death receptor pathways involved genes (such as caspase-3 , 8 and FasL), where this mechanism may be partly connected with p38MAPK and JNK signalling (P < 0.05); (3) dramatically elevated tight junctions by regulating the expression profiles of a series of complexes (such as occludin and claudins), where the reason may be in part correlated with myosin light chain kinase (MLCK) signalling (P < 0.05). Noticeably, no statistical distinction was examined between all Br-DMPT diet groups with respect to the gene expressions of GPx4a , GPx4b , GSTP2 , Bcl-2 , claudin-b and claudin-7 in above diverse immune organs (P > 0.05). Overall, for the first time, our present results confirm that the enhanced structural integrity of immune organs in Br-DMPT- supplemented fish appears to partly be associated with the increased antioxidant status and the inhibited cell apoptosis, as well as the elevated tight junctions, which provides groundbreaking insight into the undiscovered role of Br-DMPT in accelerating fish growth and increases Br-DMPT more potential to be popularly applied in future fishery production. • This study provided new insights into the role of Br-DMPT in enhancing fish growth. • Br-DMPT inhibited cell apoptosis related to JNK and P38MAPK signaling in fish immune organs. • Br-DMPT improved antioxidant capacity related to Nrf2 signaling in fish immune organs. • Br-DMPT enhanced tight junction capacity related to MLCK signaling in fish immune organs. [ABSTRACT FROM AUTHOR]

Glycinin is a major anti-nutritional factor of soybean. Glycinin inhibits growth and impairs intestinal health in grass carp and Jian carp, but the underlying mechanisms are largely unknown. This study used grass carp to examine whether the negative influences of dietary glycinin are related to intestinal apoptosis signalling and tight junctions (TJs). The results showed that 8% dietary glycinin caused higher diamine oxidase activities and D-lactate content in the serum than did the control, indicating impairment of intestinal structural integrity. For the apoptosis indices, in the midgut and hindgut, glycinin aggravated the DNA fragmentation phenomenon, promoted caspase-3, −8, and − 9 activities, and increased cytochrome c protein abundance in the cytoplasm (apart from mitochondria) compared to those the control. In the midgut, glycinin increased the mRNA levels of pro-apoptotic molecules, including caspase-3, tumour necrosis factor-α (TNF-α), B-cell lymphoma 2 (Bcl-2)-associated X (Bax), and apoptotic protease activating factor 1 (Apaf-1), and decreased the mRNA levels of the anti-apoptotic molecule Bcl-2 compared to those in the control. In the hindgut, glycinin increased caspase-3, −8, and − 9, TNF-α, Fas-ligand (FasL), Bax, and Apaf-1 mRNA levels and decreased Bcl-2, myeloid cell leukaemia 1 (Mcl-1), and inhibitors of apoptosis proteins (IAP) mRNA levels compared to those in the control. For TJs, compared to that in the control, glycinin reduced the mRNA abundance of barrier-forming TJs—including claudin-c in the foregut; ZO-1, ZO-2b, occludin, and claudin-7a in the midgut; and ZO-1, ZO-2b, occludin, claudin-3c, claudin-7a, claudin-7b, and claudin-11 in the hindgut. In contrast, glycinin increased the mRNA abundance of pore-forming TJs, including claudin-15a and claudin-15b, in the midgut and hindgut. As an important nuclear factor of TJ transcription, hepatic nuclear factor 4 alpha (HNF-4α) protein abundance was reduced by glycinin in the midgut and hindgut. Glutamine alleviated apoptosis in the midgut. Glycinin aggravated apoptosis associated with both extrinsic and intrinsic signalling and disrupted TJ related to HNF-4α signalling in the midgut and hindgut, ultimately disrupting the intestinal structural integrity of grass carp. • Glycinin disrupted intestinal structural integrity of grass carp. • Glycinin triggered extrinsic and intrinsic apoptosis in the midgut and hindgut of grass carp. • Glycinin decreased transcription abundances of TJ proteins partially related to HNF-4α signalling. [ABSTRACT FROM AUTHOR]

Erucic acid (EA), an anti-nutritional factor naturally present in rapeseed meal, could inhibit animal growth. While most studies about the detrimental effect of EA have been restricted mainly to terrestrial animal, and the information on aquatic animal is lacking. This study focused on investigating the impact of EA on growth performance, intestinal oxidative damage, cell apoptosis and dysfunction of cell-cell tight junction of on-growing grass carp (Ctenopharyngodon idella) (129.17 ± 0.19 g) which were offered with six diets containing graded levels of EA (0.00, 0.29, 0.60, 0.88, 1.21 and 1.50% diet) for 60 days. We were the first to find that EA inhibited growth and disrupted intestinal structural integrity of on-growing grass carp involved in: (1) reducing feed utilization and decreasing intestinal growth of fish; (2) causing intestinal hyperemia and hyperplasia of intestine villi of fish; (3) inducing oxidative damage, cell apoptosis and dysfunction of cell-cell tight junction associated with Nrf2, p38MAPK and MLCK signaling pathways in the intestine of fish, respectively. Interestingly, EA did not affect the activity and mRNA level of CuZnSOD and the mRNA levels of GSTP2 , Keap1b , JNK , Bcl-2 (only in PI and DI), caspase-3 (only in DI), claudin-12 and -15b of fish intestine. Finally, broken-line regression analysis demonstrated that the maximum tolerance levels of EA for on-growing grass carp (129.17–471.18 g) for 60 days based on PWG (percent weight gain), MDA in PI (proximal intestine), ROS in MI (mid intestine) and PC in DI (distal intestine) were estimated to be 0.64%, 0.48%, 0.48% and 0.53% diet, respectively. • EA decreased the growth performance of fish. • The residues of EA could be detected in three intestinal segments of fish. • EA caused oxidative damage, cell apoptosis and dysfunction of cell-cell tight junction to disrupt the structural integrity of fish intestine. • EA disrupted intestinal structural integrity associated with Nrf2, p38MAPK and MLCK signaling pathways of fish intestine. [ABSTRACT FROM AUTHOR]