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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.)

This study evaluated the effect of dietary sodium butyrate (SB) supplementation on the intestinal physical barrier function of young grass carp (Ctenopharyngodon idella). The fish were fed one powdery sodium butyrate (PSB) diet (1000.0 mg kg -1 diet) and five graded levels of microencapsulated sodium butyrate (MSB) diets: 0.0 (control), 500.0, 1000.0, 1500.0 and 2000.0 mg kg -1 diet for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila to explore the effect of SB supplementation on intestinal physical barrier function and the potential mechanisms in fish. The results showed that optimal SB supplementation: (1) down-regulated the cysteine-aspartic protease-2 (caspase-2), caspase-3 (rather than PI), caspase-7, caspase-8 (rather than PI), caspase-9, fatty acid synthetase ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell lymphoma 2 associated X protein (Bax) and c-Jun Nterminal protein kinase (JNK) mRNA levels, up-regulated the B-cell lymphoma protein-2 (Bcl-2) (rather than PI), inhibitor of apoptosis proteins (IAP) and myeloid cell leukemia-1 (Mcl-1) mRNA levels in the intestine (P < 0.05), inhibited the intestinal cell apoptosis, maintained the intestine cell structure integrity; (2) increased NF-E2-related factor 2 (Nrf2) mRNA levels and nucleus protein levels, and down-regulated kelch-like-ECH-associated protein (Keap1b) (rather than Keap1a) mRNA levels in the intestine, up-regulated copper/zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase 1a (GPx1a), GPx1b, GPx4a, GPx4b, glutathione S-transferases R (GSTR), GSTP1, GSTP2, GSTO1, GSTO2 and glutathione reductase (GR) mRNA levels in the intestine, increased the corresponding antioxidant enzymes activity (P < 0.05), thus enhancing the ability of scavenging free radicals and decreasing the reactive oxygen species (ROS) content, decreasing the lipid and protein peroxidation, as well as alleviating oxidative damage; (3) down-regulated the molecule myosin light-chain kinase (MLCK) mRNA levels in the intestine, and up-regulated the occludin, zonula occludens-1 (ZO-1), ZO-2, claudin-b, claudin-c, claudin-f, claudin-3c (rather than PI), claudin-7a, claudin-7b and claudin-11 mRNA levels, down-regulated claudin-12, claudin-15a and claudin-15b mRNA levels (P < 0.05), thus maintaining the structural integrity between cells. This study suggests that SB supplementation could improve fish intestinal physical barrier function. Furthermore, according to the positive effect, MSB was superior to PSB on improving intestinal physical barrier function of fish. Finally, based on protein carbonyl content in the PI, the optimal SB supplementation (MSB as SB source) for young grass carp was estimated to be 338.8 mg kg -1 diet., (Copyright © 2017. Published by Elsevier Ltd.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2 mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively., (Copyright © 2016. Published by Elsevier Ltd.)

This study explored the possible preventive effects of dietary arginine on copper (Cu)-induced tight junction mRNA expression changes, apoptosis and antioxidant responses in the gills of young grass carp (Ctenopharyngodon idella). The results indicated that exposure to 0.7 mg/L (11.01 μmol/L) Cu for 96 h induced the production of reactive oxygen species (ROS), thereby increasing protein oxidation, lipid peroxidation and DNA damage in the gills of fish. However, these oxidative effects were prevented by arginine supplementation. Arginine also prevented the toxic effects of Cu on the activities of copper/zinc superoxide dismutase (SOD1), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) and the glutathione (GSH) content (P<0.05). However, Cu induced an adaptive increase in the activity of catalase (CAT), and arginine supplementation further increased CAT activity (P<0.05). Moreover, Cu induced increases in the relative mRNA expressions of SOD1, CAT, GPx, GST, caspase-3, caspase-9, NF-E2-related factor 2 (Nrf2), Kelch-like-ECH-associated protein 1a (Keap1a), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), transforming growth factor-β (TGF-β) and nuclear transcription factor-κB p65 (NF-κB p65) in the gills of grass carp (P<0.05). In contrast, the relative mRNA expression levels of occludin, zonula occludens-1 (ZO-1), claudin b, claudin 3, claudin 12, target of rapamycin (TOR) and inhibitor factor κBα (IκBα) in the gills were decreased by Cu (P<0.05). However, pre-treatment of fish with arginine prevented Cu-induced relative mRNA expression decrease. Interestingly, Cu exposure resulted in increases in claudin 15a mRNA expression (P<0.05) but could not induce claudin c, caspase-8 and interleukin-10 (IL-10) mRNA expression changes in the gill of fish (P>0.05). These results indicated that Cu exposure induced apoptosis and antioxidant system and tight junction mRNA changes in the fish gills, which could be completely blocked by dietary arginine pre-supplementation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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

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

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

Health benefits, flavour quality indicators and physical properties were analysed after feeding grass carp graded concentrations of soybean isoflavones (SIF) (0, 25, 50, 75, 100 and 125 mg/kg) for 60 days. The results demonstrated that optimal dietary SIF supplementation improved the protein and total PUFA content, especially healthcare n-3 PUFA (C18: 3n-3, EPA and DHA), and increased the flavour-related free amino acid [especially umami amino acid] and 5'-inosine monophosphate content, improving the health benefits and flavour quality indicators in the muscle of grass carp. In addition, optimal dietary SIF supplementation (25 or 50 mg SIF/kg diet) enhanced some physical properties [water-holding capacity and tenderness] and increased the collagen content; however, it reduced cathepsin activity and apoptosis. SIF supplementation enhanced the glutathione content and the activity of antioxidant enzymes (except CuZnSOD) by regulating their gene expression. The gene expression could be regulated by NF-E2-related factor 2 (Nrf2) signalling in the muscle of grass carp. We demonstrated that optimal dietary SIF supplementation elevated the health benefits, flavour quality indicators and physical properties of fish muscle. [ABSTRACT FROM AUTHOR]

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

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]

Leucine is an essential amino acid for fish. The ability of leucine to resist stress in fish has not been reported. Nitrite is a common pollutant in the aquatic environment. Therefore, we investigated the effects of dietary leucine on growth performance and nitrite-induced liver damage, mitochondrial dysfunction, autophagy, and apoptosis for sub-adult grass carp. A total of 450 grass carp (615.91 ± 1.15 g) were selected and randomly placed into 18 net cages. The leucine contents of the six diets were 2.91, 5.90, 8.92, 11.91, 14.93, and 17.92 g/kg, respectively. After a 9-week feeding trial, the nitrite exposure experiment was set up for 96 h. These results indicated that dietary leucine significantly promoted FW, WG, PWG, and SGR of sub-adult grass carp (P < 0.05). Appropriate levels of dietary leucine (11.91–17.92 g/kg) decreased the activities of serum parameters (glucose, cortisol, and methemoglobin contents, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase), the contents of reactive oxygen species (ROS), nitric oxide (NO) and peroxynitrite (ONOO−). In addition, appropriate levels of dietary leucine (11.91–17.92 g/kg) increased the mRNA levels of mitochondrial biogenesis genes (PGC-1α, Nrf1/2, TFAM), fusion-related genes (Opa1, Mfn1/2) (P < 0.05), and decreased the mRNA levels of caspase 3, caspase 8, caspase 9, fission-related gene (Drp1), mitophagy-related genes (Pink1, Parkin) and autophagy-related genes (Beclin1, Ulk1, Atg5, Atg7, Atg12) (P < 0.05). Appropriate levels of dietary leucine (8.92–17.92 g/kg) also increased the protein levels of AMP-activated protein kinase (AMPK), prostacyclin (p62) and decreased the protein levels of protein light chain 3 (LC3), E3 ubiquitin ligase (Parkin), and Cytochrome c (Cytc). Appropriate levels of leucine (8.92–17.92 g/kg) could promote growth performance and alleviate nitrite-induced mitochondrial dysfunction, autophagy, apoptosis for sub-adult grass carp. Based on quadratic regression analysis of PWG and serum GPT activity, dietary leucine requirements of sub-adult grass carp were recommended to be 12.47 g/kg diet and 12.55 g/kg diet, respectively. [Display omitted] • Leucine could alleviate nitrite-induced liver damage of grass carp. • Leucine alleviated liver damage probably via reducing nitrite-induced autophagy, apoptosis, and mitochondrial dysfunction. • Based on PWG and serum GPT, leucine requirement was determined to be 12.47 g/kg and 12.55 g/kg diet for sub-adult grass carp. [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]

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. • Vitamin A deficiency impaired immunity in the head kidney and spleen of fish. • Vitamin A deficiency disrupted the structural integrity of systemic immune organs in fish. • Vitamin A regulated p38MAPK, NF-κB, TOR, Nrf2 and MLCK signaling pathways. [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]

Abstract A 60 days feeding trial was conducted to investigate the effects of dietary nucleotides levels on growth performance, proximate composition, physicochemical properties, amino acids constituents, fatty acids composition, apoptosis and antioxidant status related mechanism of grass carp (Ctenopharyngodon idella) (initial weight: 200.00 ± 1.00 g). Six graded levels of dietary nucleotides were designed as 0, 200.0, 400.0, 600.0, 800.0 and 1000.0 mg/kg, respectively. The results showed that, compared with control group, dietary nucleotides supplementation increased the growth of grass carp with higher final body weight, percentage weight gain and specific growth rate, decreased moisture but increased protein and lipid in muscle. However, dietary nucleotides had no effect on ash, calcium and phosphorus in fish fillets (P >.05). Dietary nucleotides supplementation increased the content of umami and sweetness-associated amino acids, flavor nucleotide (inosine mono-phosphate), healthcare n-3 PUFAs (EPA and DHA) and the ratio of n-3/n6 in fish muscle. Meanwhile, dietary nucleotides increased water holding capacity, tenderness and pH to enhance flesh quality, which were partly related to inhibited apoptosis with regulation of apoptosis-related gene expression, decreased hydroxyproline with increase of cathepsins activities and decreased lactate in muscle, respectively. In addition, dietary nucleotides enhanced flesh quality of grass carp fillets concerning increasing antioxidant enzymes activities and its gene expression, which were modulated by signaling molecules (TOR and Nrf2) gene and protein expression in grass carp muscle. In summary, dietary nucleotides supplementation increased growth performance, flavor characteristics, healthy fatty acid contents, physicochemical properties (water holding capacity, tenderness and pH), anti-apoptotic ability and antioxidant capacity in fish. This study demonstrated that nucleotides was a promising feed additives to enhance flesh quality in fish. Highlights • Nucleotides enhance muscle flavor quality and health benefits in fish. • Nucleotides are effective in enhancing flesh quality related parameters. • Nucleotides-enhanced WHC, tenderness and pH is related to decreasing apoptosis, collagen and lactate content in muscle. • Inhibition of oxidation is due to increases of antioxidant capacity regulated by Nrf2 and TOR signaling. • The nucleotides supplementation for fish growth and flesh quality were evaluated. [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 aim of this study was to investigate the effect of graded levels of vitamin E (0–225 mg/kg) on gill immune response and physical barriers, and relative mRNA levels of signalling molecules in the gill of grass carp ( Ctenopharyngodon idella ) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin E supplementation, vitamin E deficiency (1) increased the rate of gill rot morbidity and aggravated gill histopathological injuries following F. columnare infection ( P < 0.05); (2) decreased LA and ACP activities, complement component 3, complement component 4 and IgM contents and down-regulated antimicrobial peptides (liver-expressed antimicrobial peptide-2A, -2B, hepcidin, β-defensin) and IgT mRNA levels ( P < 0.05); (3) down-regulated the mRNA levels of anti-inflammatory cytokines and the signalling molecules inhibitory protein κBα, the target of rapamycin and ribosome protein S6 kinase 1 ( P < 0.05) and up-regulated the mRNA levels of pro-inflammatory cytokines and the signalling molecules NF-κB p65, IκB kinase α (IKKα), IKKβ and eIF4E-binding protein 1 ( P < 0.05); (4) decreased activities of anti-superoxide anion, Cu/ZnSOD, MnSOD, GPx, GST and GR, and down-regulated mRNA levels of those antioxidant enzymes and signalling molecule NF-E2-related factor 2 in the gills of fish ( P < 0.05); (5) down-regulated the relative mRNA levels of B cell lymphoma-2 protein, and up-regulated the relative mRNA levels of caspase-2, -3, -7, -9, Bcl-2 associated X protein and apoptotic protease activating facter-1 ( P < 0.05); (6) down-regulated the relative mRNA levels of tight junction proteins (occludin, zonula occludens-1, ZO-2, claudin-3, -b, -c and -11a) and up-regulated signalling molecule myosin light chain kinase mRNA levels ( P < 0.05). In conclusion, vitamin E deficiency disrupted gill immune barriers and physical barrier function via impairing gill immunity and antioxidant capacity, inducing apoptosis and changing tight junction protein transcription abundances and the related signalling molecules in the gills of fish. The optimal vitamin E requirements for against gill rot morbidity of grass carp (266–1026 g) were estimated to be 139.56 mg/kg diet. Meanwhile, based on immune indicator (ACP activity) and antioxidant indicator (MDA content), the optimal vitamin E requirements for young grass carp were estimated to be 130.67 and128.53 mg/kg diet, respectively. [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]

This study is for the first time to explore the possible effects of dietary manganese (Mn) on structural integrity and the related signaling in the gills of fish. Grass carp ( Ctenopharyngodon idella ) were fed with six diets containing graded levels of Mn [3.65–27.86 mg Mn/kg diet] for 8 weeks. The results firstly demonstrated that Mn deficiency aggravated inflammation indicated by up-regulation of pro-inflammatory cytokines (tumour necrosis factor α, interleukin 8, and interleukin 1β mRNA levels) and down-regulation of anti-inflammatory cytokines (interleukin 10, transforming growth factor-β1) mRNA levels, which might be partially related to the up-regulation of nuclear factor kappa B (NF-κB p65) and down-regulation of nuclear inhibitor factor κBα (iκBα) mRNA levels in the gills of fish. Meanwhile, Mn deficiency caused DNA fragmentation, which might be partially associated with the up-regulation of the apoptosis signaling (caspase-3, caspase-8 and caspase-9) in the gills of fish. Furthermore, Mn deficiency-caused apoptosis might be partly related to the increases of oxidative damage that indicated by increases of lipid peroxidation and protein oxidation, and decreases of antioxidant enzyme activities [included Mn superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione- S -transferase (GST)]. However, Mn deficiency only down-regulated MnSOD and GST mRNA levels, which might be partially related to the up-regulation of NF-E2-related factor-2 (Nrf2) inhibitor (Keap1), and only down-regulated the gene expression of claudin-b and claudin-15 to disrupt the TJ in the gills of fish. Excessive Mn led to negative effects on partial parameters studied in the gills of fish. The optimal levels of Mn based on protecting against ROS, MDA and PC in the gills of grass carp were 17.04, 16.86 and 21.20 mg/kg diet, respectively. Collectively, Mn deficiency or excess could cause inflammation, apoptosis, antioxidant system disruption and change tight junction protein (claudin-b and claudin-15) transcription abundances, which might be partially related to the NF-κB p65, caspase-(3,8,9) and Nrf2 signaling, in the gills of fish. [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]

An 8-week feeding trial was conducted to determine the effect of dietary glutamine (Gln) on the erythrocyte function in juvenile Jian carp. The results indicated that dietary Gln caused significant increases in haematocrit, erythrocyte count, haemoglobin concentration, mean corpuscular volume and mean corpuscular haemoglobin content in carp blood. Dietary Gln caused significant decreases in the levels of superoxide anion, hydrogen peroxide, met-haemoglobin, malonaldehyde and protein carbonyl and increases in the level of reduced glutathione and the activities of Na + , K + -ATPase and lactate dehydrogenase in carp erythrocytes. These results demonstrated that dietary Gln could improve the function of erythrocytes in fish. Meanwhile, the present study explored the effect of dietary Gln on hydroxyl radical ( OH)-induced apoptosis in carp erythrocytes. We found that dietary Gln prevented the OH-induced apoptosis by inhibiting the generation of reactive oxygen species and the oxidation of cellular components, and restoring the non-enzymatic and enzymatic antioxidant activities in fish erythrocytes. These results revealed that the effects of Gln on the function may be closely associated with apoptosis in fish erythrocytes. Moreover, our data suggested that dietary Gln raised the levels of alanine (Ala), citrulline (Cit) and proline (Pro) in plasma of carp. We then examined the protective effects of Ala, Cit and Pro at the physiological concentrations in OH-induced carp erythrocytes. The results indicated that Ala, Cit or Pro at the physiological concentrations suppressed apoptosis and the oxidation of cellular components, and restored the GSH antioxidant system in the OH-induced carp erythrocytes. Moreover, the combination of Ala, Cit and Pro at the physiological concentrations produces a greater protective effect than their individual effects in fish erythrocytes. These results displayed that the effects of Gln on erythrocytes are at least partly dependent on that of its metabolites in fish. Ala, Cit, Pro and their combination could serve as an antioxidant or apoptosis-inhibitor in fish. [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 investigated the effects of dietary vitamin E on growth, disease resistance and the immunity and structural integrity of head kidney, spleen and skin in grass carp ( Ctenopharyngodon idella ). The fish were fed six diets containing graded levels of vitamin E (0, 45, 90, 135, 180 and 225 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila . The results showed that compared with optimal vitamin E supplementation, vitamin E deficiency caused depressed growth, poor survival rates and increased skin lesion morbidity in grass carp. Meanwhile, vitamin E deficiency decreased lysozyme and acid phosphatase activities, complement component 3 and complement component 4 contents in the head kidney, spleen and skin of grass carp ( P < 0.05). Moreover, vitamin E deficiency down-regulated antimicrobial peptides (Hepcidin, liver-expressed antimicrobial peptide-2A, -2B, β-defensin), IL-10, TGFβ1, IκBα, TOR and S6K1 mRNA levels ( P < 0.05) and up-regulated IL-1β, IL-6, IL-8, IFN-γ2 and TNFα, NF-κB p65, IKKα, IKKβ and 4EBP1 (not in the head kidney) mRNA levels ( P < 0.05). In addition, vitamin E deficiency caused oxidative damage, decreased superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) activities, and down-regulated the mRNA levels of antioxidant enzymes and signaling molecules Nrf2 ( P < 0.05). Vitamin E deficiency also induced apoptosis by up-regulating capase-2, -3, -7, and -8 mRNA levels in the head kidney, spleen and skin of grass carp. In conclusion, this study indicated that dietary vitamin E deficiency depressed fish growth, impaired the immune function and disturbed the structural integrity of the head kidney, spleen and skin in grass carp, but optimal vitamin E supplementation can reverse those negative effects in fish. The optimal vitamin E requirements for young grass carp (266.39–1026.63 g) to achieve optimal growth performance and disease resistance based on the percent weight gain (PWG) and skin lesion morbidity were estimated to be 116.2 and 130.9 mg/kg diet, respectively. Meanwhile, based on immune indicator (LA activity in the head kidney) and antioxidant indicator (protection of spleen against MDA), the optimal vitamin E requirements for young grass carp were estimated to be 123.8 and 136.4 mg/kg diet, respectively. [ABSTRACT FROM AUTHOR]

This research discovered the impacts of dietary iron on intestine health of on-growing grass carp (Ctenopharyngodon idella). Six hundred and thirty grass carp (242.32 ± 0.58 g) were fed different diets containing 12.15, 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg iron/kg, where the iron source first six groups was ferrous fumarate, and the last group was ferrous sulfate. Feeding trial lasted for sixty days. Thereafter, a fourteen days' challenge test was carried out through Aeromonas hydrophila infection. Previous study showed that the optimum iron level of on-growing grass carp based on per weight gain was estimated to be 75.65 mg/kg. Results from the present study displayed that in contrast to optimum iron level, deficient iron downregulated gene expression of tight junction complexes, Apoptosis regulator Bcl-2 (Bcl-2), MCL1 apoptosis regulator BCL2 family member (Mcl-1), inhibitor of apoptosis protein (IAP) and NF-E2-related factor 2 (Nrf2). It also reduced activities and gene expression of antioxidant enzymes, and upregulated gene expression of apoptosis-related cysteine peptidase CASP (caspase) -2, -7, -8, -9, apoptotic protease activating factor 1 (Apaf-1), Apoptosis regulator Bax (Bax) (in MI and DI), Fas ligand (FasL), p38 mitogen activated protein kinase (p38MAPK), Kelch like ECH associated protein 1a (Keap1a), Keap1b, claudin-12 and myosin light chain kinase (MLCK). In addition, deficient iron raised malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) generation in intestine of grass carp (P < 0.05). These data demonstrated that deficient iron is harmful to intestinal physical barrier of fish. Besides, the efficiency of ferrous fumarate on intestinal physical barrier function was better than ferrous sulfate in fish. At last, based on MDA contents in MI and DI, the iron requirements were 83.40 and 80.20 mg/kg diet (ferrous fumarate as sources), respectively. • Iron deficiency impaired physical barrier function in intestine of fish. • Iron deficiency disturbed tight junctions, impairing intercellular integrity in intestine of fish. • Iron deficiency aggravated apoptosis and induce oxidative damage, impairing cellular integrity in intestine of fish. • Iron regulated MLCK, JNK, p38MAPK and Nrf2 signaling in intestine of fish. [ABSTRACT FROM AUTHOR]

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp ( Ctenopharyngodon idella ) under infection of Flavobacterium columnare . The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents ( P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels ( P < 0.05), and glutathione and vitamin C contents ( P < 0.05), down-regulated NF-E2-related factor 2 mRNA level ( P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level ( P < 0.05) in the gill of grass carp under infection of F. columnare , suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels ( P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level ( P < 0.05) in the gill of grass carp under infection of F. columnare , suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels ( P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels ( P < 0.05) in the gill of grass carp under infection of F. columnare , suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents ( P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, β-defensin mRNA levels ( P < 0.05) in the gill of grass carp under infection of F. columnare , suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) ( P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels ( P < 0.05) in the gill of grass carp under infection of F. columnare , suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264–1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264–1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively. [ABSTRACT FROM AUTHOR]

The effects of dietary protein levels on the disease resistance, gill immune function and physical barrier function of grass carp ( Ctenopharyngodon idella ) were investigated in this study. A total of 540 grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg −1 diet) for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that optimal levels of dietary protein had the following effects: (1) the production of antibacterial components increased, and anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels were up-regulated, whereas mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, IκB kinase (IKK) α, IKKβ, IKKγ, eIF4E-binding proteins (4E-BP) 1 and 4E-BP2 were down-regulated in the gills of grass carp ( P < 0.05), indicating that fish gill immune function was enhanced at an optimal level of dietary protein; (2) the activities and mRNA levels of antioxidant enzymes and glutathione content increased, the contents of reactive oxygen species, malondialdehyde and protein carbonyl (PC) decreased, and NF-E2-related factor 2, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and tight junction complexes mRNA levels were up-regulated, whereas Kelch-like-ECH-associated protein (Keap) 1a, Keap1b, cysteinyl aspartic acid-protease 3, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, c-Jun N-terminal protein kinase, myosin light chain kinase and p38 mitogen-activated protein kinase mRNA levels were down-regulated in the gills of grass carp ( P < 0.05), indicating that the fish gill physical barrier function improved at an optimal level of dietary protein. Finally, based on the gill rot morbidity, ACP activity and PC content, the optimal levels of dietary protein for grass carp were estimated to be 286.65 g kg −1 diet (253.73 g digestible protein kg −1 diet), 290.46 g kg −1 diet (257.76 g digestible protein kg −1 diet) and 296.25 g kg −1 diet (260.69 g digestible protein kg −1 diet), respectively. [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]

This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp ( Ctenopharyngodon idella ). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg −1 diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp ( P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp ( P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis morbidity of young grass carp were estimated to be 286.82 g kg −1 diet (250.66 g digestible protein kg −1 diet) and 292.10 g kg −1 diet (255.47 g digestible protein kg −1 diet), respectively. [ABSTRACT FROM AUTHOR]

Our study explored the effect of dietary lipids on growth and immunity and structure (head kidney, spleen and skin) 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 5.9–80.1 g/kg diet for 8 weeks. After that, a challenge trial was conducted by injection of Aeromonas hydrophila over 2 weeks. The results indicated that compared with optimal lipids supplementation, low and excess levels of lipids down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα) and ribosomal p70S6 kinase (S6K1), and up-regulated pro-inflammatory cytokines, nuclear factor κB p65 (NF-κB p65), NF-κB c-Rel (not p52), IκB kinase α (IKKα), IKKβ, IKKγ, and eIF4E-binding protein (4EBP) mRNA levels in the head kidney and spleen of young grass carp ( P < 0.05). Low or excess levels of lipids also increased reactive oxygen species (ROS) production and malondialdehyde (MDA) and protein carbonyl (PC) contents, reduced the activities of antioxidant enzymes ( P < 0.05), down-regulate the relative mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2), and up-regulated the expression levels of Kelch-like ECH-associating protein 1a (Keap1a) and Keap1b in the head kidney and spleen. In addition, low or excess levels of lipids down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) in the head kidney and spleen, whereas up-regulated the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), caspase 3, 7, 8 and 9 mRNA levels in the head kidney and spleen and Fas ligand (FasL) mRNA levels in the spleen of young grass carp, suggesting that low or excess levels of lipids could decrease the head kidney and spleen immune function, induce oxidative damage and apoptosis and impair antioxidant system of young grass carp. At last, low or excess levels of lipids also impaired the immune function and structure in the skin of young grass carp. Based on the quadratic regression analysis for PWG, skin haemorrhage and lesions morbidity and IgM content, the dietary lipids requirements for young grass carp were estimated to be 43.7, 60.2, 55.0 and 52.1 g/kg 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]

Ochratoxin A (OTA) is a common hazardous food contaminant that seriously endangers human and animal health. However, limited study is focused on aquatic animal. This research investigated the multiple biotoxicity of OTA on spleen (SP) and head kidney (HK) in grass carp and its related mechanism. Our data showed that, dietary supplemented with OTA above 1209 μg/kg caused histopathological damages by decreasing the number of lymphocytes and necrotizing renal parenchymal cells. Meanwhile, OTA caused oxidative damage and reduced the isoforms mRNAs transcripts of antioxidant enzymes (e.g., GPX1, GPX4, GSTO) partly due to suppressing NF-E2-related factor 2 (Nrf2). OTA triggered apoptosis through mitochondria and death receptor pathway potentially by p38 mitogen-activated protein kinase (p38MAPK) activation. Besides, OTA exacerbated inflammation by down-regulation of anti-inflammatory factor (e.g., IL-10, IL-4) and up-regulations of pro-inflammatory factors (e.g., TNF-α, IL-6), which could be ascribed to signaling meditation of Janus kinase / signal transducer and activator of transcription (JAK/STAT). Additionally, the safe upper limits of OTA were estimated to be 677.6 and 695.08 μg/kg based on the immune-related indexes (C3 contents in the SP and LZ activities in the HK, respectively). Our study has provided a wide insight for toxicological assessment of feed pollutant in aquatic animals. [Display omitted] • Dietary exposure to OTA destroyed the spleen and head kidney histological structure. • OTA exposure caused oxidative damage, apoptosis, and immunosuppression in the spleen and head kidney. • The safe upper limit dose of biotoxicity induced by OTA were 677.6 and 695.08 μg/kg diet. [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]

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 gills are some of the most metabolically active organs in fish and are involved in gas exchange, osmotic regulation, acid-base balance, and ammonia nitrogen waste excretion. Here, we explored the actions of graded levels of dietary myo -inositol [27.0 (un-supplemented), 137.9, 286.8, 438.6, 587.7, and 737.3 mg/kg diet)] on the physical barrier functioning and signaling in the gills of grass carp (Ctenopharyngodon idella) for 10-weeks, and then infected by Flavobacterium columnare. It was found that optimal myo -inositol levels (approximately 137.9–438.6 mg myo -inositol /kg diet): (1) decreased the oxidative products such as malondialdehyde (MDA) and protein carbonyl (PC), while increased the activities and mRNA levels of SOD (CuZnSOD and MnSOD), CAT, GPx (GPx1a, GPx1b, GPx4a and GPx4b), GST (GSTO1, GSTO2, GSTP1 and GSTP2) and GR enzymes, possibly related to the Nrf2/Keap1 signaling pathway, suggesting that myo -inositol inhibited oxidative injury in fish gills; (2) inhibited cell apoptosis related to the down-regulation of caspases-2, −3, −7, −8, and-9 mRNA levels, possibly linked to the inhibition of the JNK/FasL/caspase 8 and p38MAPK/(Apaf-1, Bax, Bcl-2, Mcl-1, IAP)/caspase 9/caspase 3 signaling pathways; (3) promoted cell proliferation by up-regulating cyclins B, D, and E mRNA levels, possibly related to the E2F4 signaling; (4) maintained intercellular structural integrity by up-regulating tight junctional complexes (ZO-1, ZO-2, occludin, and claudins b, −c, f, −3c, −7a, −7b, and − 11), and down-regulating pore-forming TJs (claudins-12,-15a, and-15b) mRNA levels, possibly associated with the down-regulation of MLCK signaling. Based on the inhibition of gill rot and MDA content, the optimal myo -inositol intake was calculated as 285.8 and 305.4 mg/kg diet for grass carp (initial 221 g-final 792 g), respectively. [Display omitted] • Compared with myo -inositol deficiency, optimal myo -inositol supplementation. • Enhanced antioxidant capacity in the gills of fish. • Inhibited cell apoptosis in the gills of fish. • Promoted cell proliferation of fish gills. • Maintained the cell-cell integration of the gills in fish. [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]

Abstract: The present study explored the protective effects of glutamine (Gln), alanine (Ala), citrulline (Cit) and proline (Pro) on hydroxyl radical (ed apoptosis in isolated carp erythrocytes. Hydroxyl radicals were generated by ferrous ion (Fe2+)-mediated decomposition of hydrogen peroxide (H2O2) (Fenton reaction). In order to select an optimal tration to induce apoptosis, cultures were treated with different concentrations of FeSO4/H2O2 (0μM/0μM–50μM/25μM). The results showed that exposure to FeSO4/H2O2 (0μM/0μM–40μM/20μM) increased apoptosis in a dose-dependent manner. Moreover, apoptosis was at its highest level at 40μM FeSO4/20μM H2O2. We then examined the cytoprotective effects of Gln, Ala, Cit, Pro or the combination of Ala, Cit and Pro under conditions of apoptosis. Carp erythrocytes were treated with the substances listed above in the presence of 40μM FeSO4/20μM H2O2 for 9h. The controls were grown in Gln, Ala, Cit, Pro-free culture medium. The results showed that Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively protected against annexin binding, decrease of forward scatter and DNA fragmentation in carp erythrocytes induced by ermore, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively blocked ated erythrocyte hemolysis, reduced the increase of superoxide anion and H2O2 concentrations, inhibited the formation of malondialdehyde, protein carbonyls and met-hemoglobin, and prevented the decrease of superoxide dismutase, catalase and glutathione peroxidase activities and glutathione content in carp erythrocytes induced by dition, the results suggest that the combination of Ala, Cit and Pro produces a greater anti-apoptotic and anti-oxidative effect than their individual effects at the same concentrations. Taken together, the results showed that s apoptosis and oxidative damage in carp erythrocytes. In addition to inhibiting apoptosis, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro protected carp erythrocytes against oxidative damage induced by may be a major factor in the protection of erythrocytes from apoptosis. [Copyright &y& Elsevier]

The purpose of this study was to confirm the efficacy of xylooligosaccharides (XOS) on the growth performance and intestinal apoptosis of on-growing grass carp Ctenopharyngodon idella (167.46 ± 0.61 g). Fish were separated into six groups, which were given six different types of diet (supplemented with 0.00%, 0.002%, 0.004%, 0.006%, 0.008% and 0.010% of XOS) for 60 days. Grass carp were challenged with Aeromonas hydrophila (6 days) after the growth experiment. To our knowledge, the present study is the first to report that appropriate XOS supplementation: (1) improved growth performance in fish and (2) attenuated apoptosis via the internal mitochondrial signalling pathways (JNK and p38MAPK/Cytochrome-c/caspase-9) in the proximal intestine (PI) and mid intestine (MI) and the external death receptor signalling pathways (p38MAPK/FasL/caspase-8) in the distal intestine (DI). Interestingly, XOS supplementation had no effect on the mRNA levels of factor-related apoptosis ligand (FasL) or caspase-8 in the PI and MI, Bcl-2 in the PI and JNK , caspase-9, Bax or Apaf-1 in the DI. Finally, appropriate levels of XOS supplementation based on the percentage of weight gain (PWG), feed intake (FI), and caspase-3, -8, and -9 activities in the MI were estimated to be 51.81, 51.61, 57.15, 57.90, and 55.36 mg/kg diet, respectively. • Xylooligosaccharides (XOS) promoted growth performance of fish. • XOS inhibited apoptosis by the caspase-dependent transduction pathway in fish intestines. • XOS attenuated apoptosis via the internal signalling in the proximal intestine and the mid intestine. • XOS attenuated apoptosis via the external signalling in the distal intestine. [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]