Novel Insight Into Nutritional Regulation in Enhancement of Immune Status and Mediation of Inflammation Dynamics Integrated Study In Vivo and In Vitro of Teleost Grass Carp (Ctenopharyngodon idella): Administration of Threonine.

This study aims to investigate the effects of threonine (Thr) on immunoregulation in vivo and in vitro of teleost grass carp (Ctenopharyngodon idella). Juveniles (9.53 ± 0.02 g) were reared for 8 weeks with respective Thr diet (3.99, 7.70, 10.72, 14.10, 17.96, and 21.66 g/kg) and then challenged with Aeromonas hydrophila for in vivo study. Macrophages isolated from head kidney were treated in vitro for 48 h with L-Thr (0, 0.5, 1.0, 2.0, 4.0, and 8.0 mM) after 6 h of lipopolysaccharide induction. The results showed that, compared with Thr deficiency (3.99 g/kg), the optimal dietary Thr (14.10g/kg) affected the immunocyte activation in the head kidney (HK) and spleen (SP) by downregulating the mRNA expressions of MHC-II and upregulating CD4 (not CD8), and it mediated the innate immune by enhancing the activities of lysozyme (LZ), acid phosphatase content of complement 3 (C3) and C4, increasing the mRNA abundances of hepcidin, liver expressed antimicrobial peptide-2A (LEAP-2A), LEAP-2B, ß-defensin1, downregulating tumor necrosis factor α (TNF-α), IL-6, IL-1ß, IL-12p35, IL-12p40, IL-17AF1, and IL-17D partly by attenuating RORγ1 transcriptional factor and nuclear factor kappa B p65 (NF-κBp65) signaling cascades [IKKß/IκBα/NF-κBp65] and upregulating transforming growth factor ß1 (TGF-ß1), IL-4/13A, -4/13B, IL-10, and IL-22 partly by GATA-3. Besides these, the optimal dietary Thr regulated the adaptive immune by upregulating the mRNAs of immunoglobulin M (IgM) and IgZ (not IgD). Moreover, 2 mM Thr downregulated in vitro the mRNA abundances of colony stimulating factor-1, inducible nitric oxide synthase, mannose receptor 1, matrix metalloproteinase2 (MMP-2), and MMP-9 significantly (P < 0.05), indicating that Thr could attenuate the M1-type macrophages' activation. Moreover, L-Thr downregulated the mRNA transcripts of TNF-α, IL-6, and IL-1ß associated with impairing the SOCS1/STAT1 signaling and upregulated IL-10 and TGF-ß1 partly by accentuating the SOCS3/STAT3 pathway. The above-mentioned observations suggested that Thr improved the immune status in the immune organs of fish by enhancing the immune defense and mediating the inflammation process. Finally, based on the immune indices of LZ activity in HK and C3 content in SP, the optimal Thr for immune enhancement in juvenile grass carp (9.53-53.43 g) was determined to be 15.70 g/kg diet (4.85 g/100 g protein) and 14.49 g/kg diet (4.47 g/100 g protein), respectively.

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

Iron is an important mineral element for fish. In this study, we investigated the influences of dietary iron deficiency on intestinal immune function as well as underlying signaling of on-growing grass carp (Ctenopharyngodon idella). Fish were fed with six graded level of dietary iron for sixty days, and a fourteen days' challenge test under infection of Aeromonas hydrophila thereafter. Results showed that compared with optimal iron level, iron deficiency increased enteritis morbidity, decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) concentrations and down-regulated mRNA levels of hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, Mucin2, ß-defensin-1, anti-inflammatory cytokines transforming growth factor ß1 (TGF-ß1), TGF-ß2, interleukin 4/13A (IL-4/13A), IL-4/13B, IL-10, IL-11 and IL-15, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated mRNA levels of pro-inflammatory cytokines IL-1ß, interferon γ2 (IFN-γ2), IL-8, IL-12p35, IL-12p40 and IL-17D, nuclear factor kappa B (NF-κB) p65, IκB kinases α (IKKα), IKKß and eIF4E-binding protein (4E-BP) in intestine of on-growing grass carp, indicating that iron deficiency impaired intestinal immune function of fish under infection of A. hydrophila. Besides, iron excess also increased enteritis morbidity and impaired immune function of fish under infection of A. hydrophila. In addition, the effect of ferrous fumarate on intestinal immune function of on-growing grass carp is more efficient than ferrous sulfate. Finally, based on ability against enteritis, LZ activities in mid intestine and distal intestine, we recommended adding 83.37, 86.71 and 85.39 mg iron/kg into diet, respectively.

Soybean glycinin decreased growth performance, impaired intestinal health, and amino acid absorption capacity of juvenile grass carp (Ctenopharyngodon idella).

The present study evaluated the influence of dietary soybean glycinin on growth performance, intestinal morphology, free intestinal amino acid (AA) content, and intestinal AA transporter (AAT) mRNA levels in juvenile grass carp (Ctenopharyngodon idella). Results were displayed as follows: (1) 8% dietary glycinin decreased growth performance, inhibited intestinal growth, and caused intestinal histology damage of grass carp; (2) dietary glycinin decreased the content of free neutral AAs including Val, Ser, Tyr, Ala, Pro, and Gln in all intestinal segments, and Thr, Ile, Leu, Phe, and Gly in the MI and DI while downregulated the mRNA levels of corresponding transporters including SLC38A2, SLC6A19b, and SLC6A14 in all intestinal segments, and SLC7A5, SLC7A8, and SLC1A5 in the MI and DI. Dietary glycinin decreased the content of free basic AAs including Arg in the MI and DI and His in all intestinal segments while downregulated cationic AAT SLC7A1 mRNA levels in the MI and DI. Dietary glycinin decreased the content of free acidic AAs including Glu in all intestinal segments and Asp in the MI and DI while decreased mRNA levels of corresponding transporters including SLC1A2a in all intestinal segments and SLC1A3 in the MI and DI; (3) the digestion trial showed that basic subunits of glycinin was hard to digest in the intestine of grass carp; (4) co-administration of glutamine with glycinin partially alleviated the negative effects. Overall, glycinin decreased intestinal AA absorption capacity partly contributed by decreased AATs' mRNA levels and the indigestibility of glycinin.

Impact and consequences of dietary riboflavin deficiency treatment on flesh quality loss in on-growing grass carp (Ctenopharyngodon idella).

Fish is among the cheapest and most promising sources of animal protein. The main edible portion of fish is muscle. This study explored the impact of dietary riboflavin on fish flesh quality and showed the possible role of muscle antioxidant defense in flesh quality in relation to dietary riboflavin. On-growing grass carp (initial average weight of 275.82 ± 0.57 g) were fed diets containing graded levels of riboflavin (0.63, 1.95, 3.98, 6.02, 7.96, and 10.04 mg kg-1 diet) for eight weeks. The results indicated that compared with the optimal riboflavin levels (3.98 and/or 6.02 mg riboflavin per kg diet), riboflavin deficiency treatment (0.63 mg riboflavin per kg diet) significantly reduced the muscle nutrients, including the protein, lipid, flavor amino acid, and total essential amino acid contents. Furthermore, the muscle shear force, pH value, and hydroxyproline concentration were reduced, while the muscle cooking loss and lactic acid content increased (P < 0.05). Compared with optimal riboflavin levels, the riboflavin deficiency treatment increased the reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl contents, while riboflavin treatments of 3.98-10.04 mg riboflavin per kg diet showed the lowest ROS and MDA contents (P < 0.05). Compared with the optimal riboflavin levels, the riboflavin deficiency treatment decreased the activities of copper/zinc superoxide dismutase (CuZnSOD), glutathione reductase (GR), catalase (CAT), and glutathione peroxidase (GPx), and reduced the glutathione (GSH) content (P < 0.05). Furthermore, the relative mRNA levels of antioxidant enzymes, including CuZnSOD, CAT, GR and GPx, and antioxidant-related signaling molecules, including NF-E2-related factor 2 (Nrf2) and casein kinase 2, were down-regulated, while those of Kelch-like ECH-associated protein 1b were up-regulated (P < 0.05). Collectively, the present study indicates that riboflavin deficiency treatment reduces the flesh quality, partly due to inhibition of the antioxidant defense through the Nrf2 signaling pathway, while optimal riboflavin levels reverse these negative effects.

Vitamin A deficiency impairs intestinal physical barrier function of fish.

The present study was the first to investigate the effects of dietary vitamin A (VA) on the intestinal physical barrier function associated with oxidation, antioxidant system, apoptosis and cell-cellular tight junction (TJ) in the proximal (PI), mid (MI) and distal (DI) intestines of young grass carp (Ctenopharyngodon idella). Fish were fed graded levels of dietary VA for 10 weeks, and then a challenge test using an injection of Aeromonas hydrophila was conducted for 14 days. Results indicated that dietary VA deficiency caused oxidative damage to fish intestine partly by the reduced non-enzymatic antioxidant components glutathione (GSH) and VA contents as well as reduced antioxidant enzyme activities [not including manganese superoxide dismutase (MnSOD)]. Further results observed that the decreased antioxidant enzyme activities by VA deficiency were partly related to the down-regulation of their corresponding mRNA levels which were regulated by the down-regulation of NF-E2-related factor 2 (Nrf2) mRNA levels and up-regulation of kelch-like-ECH-associated protein (Keap1a) (rather than Keap1b) mRNA levels in three intestinal segments of fish. Meanwhile, VA deficiency up-regulated the mRNA levels of the apoptosis signalling [caspase-3, caspase-8, caspase-9 (rather than caspase-7)] associated with the inhibition of the target of rapamycin (TOR) signalling pathway in three intestinal segments of fish. Additionally, VA deficiency decreased the mRNA levels of TJ complexes [claudin-b, claudin-c, claudin-3, claudin-12, claudin-15a, occludin and zonula occludens-1 (ZO-1) in the PI, MI and DI, as well as claudin-7 and claudin-11a in the MI and DI] linked to the up-regulation of myosin light chain kinase (MLCK) signalling. These results suggested that VA deficiency impaired structural integrity in three intestinal segments of fish. Meanwhile, excessive VA also showed similar negative effects on these indexes. Taken together, the current study firstly demonstrated that VA deficiency impaired physical barrier functions associated with impaired antioxidant capacity, aggravated cell apoptosis and disrupted TJ complexes in the PI, MI and DI, but different segments performed different actions in fish. Based on protecting fish against protein oxidation, the optimal VA levels for grass carp were estimated to be 2622 IU/kg diet.

Soybean isoflavones improve the health benefits, flavour quality indicators and physical properties of grass carp (Ctenopharygodon idella).

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.

Impairing of gill health through decreasing immune function and structural integrity of grass carp (Ctenopharyngodon idella) fed graded levels dietary lipids after challenged with Flavobacterium columnare.

The current study conducted to investigate the hypothesis that low or excess levels of lipids increased the gill rot morbidity through impairing the immune function and structural integrity in the gill of grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ±â€¯0.53 g were fed diets containing six graded levels of lipids at 0.59%, 2.14%, 3.60%, 5.02%, 6.66% and 8.01% diets for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that compared with optimal lipids supplementation (2.14%-8.01% lipids diets), low or excess levels of lipids impaired fish immune function through declining the activities of humoral compounds, down-regulated the mRNA levels of 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) (not p52), IκB kinase α (IKKα) (not IKKß), IKKγ and eIF4E-binding protein (4EBP) in the gill of young grass carp. In addition, low or excess levels of lipids decreased young grass carp physical barrier function through down-regulating the mRNA levels of ZO-1 (rather than ZO-2b), Claudin b, c, 3, 12, 15a, 15b, 7b, 7a and Occludin through MAPKK 6/p38 MAPK/MLCK signaling molecules, decreasing antioxidant ability via Kelch-like ECH-associating protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2) signaling molecules, and down-regulating the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) in the gill of grass carp. Based on the quadratic regression analysis for the gill rot morbidity, C3 and MDA contents, the dietary lipids requirements for young grass carp have been estimated to be 5.60%, 6.01% and 4.58% diets.

Low or excess levels of dietary cholesterol impaired immunity and aggravated inflammation response in young grass carp (Ctenopharyngodon idella).

The present study explored the effect of cholesterol on the immunity and inflammation response in the immune organs (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) fed graded levels of dietary cholesterol (0.041-1.526%) for 60 days and then infected with Aeromonas hydrophila for 14 days. The results showed that low levels of cholesterol (1) depressed the innate immune components [lysozyme (LZ), acid phosphatase (ACP), complements and antimicrobial peptides] and adaptive immune component [immunoglobulin M (IgM)], (2) up-regulated the mRNA levels of pro-inflammatory cytokines [interleukin 1ß (IL-1ß), IL-6, IL-8, IL-12p35, IL-12p40, IL-15, IL-17D, tumor necrosis factor α (TNF-α) and interferon γ2 (IFN-γ2)], partly due to the activated nuclear factor kappa B (NF-κB) signalling, and (3) down-regulated the mRNA levels of anti-inflammatory cytokines [IL-4/13B, IL-10, IL-11, transforming growth factor (TGF)-ß1 and TGF-ß2], partly due to the suppression of target of rapamycin (TOR) signalling in the immune organs of young grass carp. Interestingly, dietary cholesterol had no influences on the IκB kinase α (IKKα) and IL-4/13A mRNA levels in the head kidney, spleen and skin, the IL-1ß and IL-12p40 mRNA levels in the spleen and skin, or the ß-defensin-1 mRNA level in the skin of young grass carp. Additionally, low levels of cholesterol increased the skin haemorrhage and lesion morbidity. In summary, low levels of cholesterol impaired immunity by depressing the innate and adaptive immune components, and low levels of cholesterol aggravated the inflammation response via up-regulating the expression of pro-inflammatory cytokines as well as down-regulating the expression of anti-inflammatory cytokines partly through the modulation of NF-κB and TOR signalling in the immune organs of fish. Similar to the low level of cholesterol, the excess level of dietary cholesterol impaired immunity and aggravated inflammation response in the immune organs of fish. Finally, based on the percent weight gain (PWG), the ability against skin haemorrhage and lesions as well as the LZ activity in the head kidney and the ACP activity in the spleen, the optimal dietary cholesterol levels for young grass carp were estimated as 0.721, 0.826, 0.802 and 0.772% diet, respectively.

Dietary myo-inositol deficiency decreased intestinal immune function related to NF-κB and TOR signaling in the intestine of young grass carp (Ctenopharyngodon idella).

In this study, we investigated the effects of dietary myo-inositol on the intestinal immune barrier function and related signaling pathway in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (221.33 ±â€¯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. The results indicated that compared with the optimal dietary myo-inositol level, myo-inositol deficiency (27.0 mg/kg diet): (1) decreased lysozyme (LZ) and acid phosphatase (ACP) activities, as well as complement 3 (C3), C4 and immunoglobulin M (IgM) contents in the proximal intestine (PI), middle intestine (MI) and distal intestine (DI) of young grass carp (P < 0.05). (2) down-regulated the mRNA levels of anti-microbial substance: liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, ß-defensin-1 and mucin2 in the PI, MI and DI of young grass carp (P < 0.05). (3) up-regulated pro-inflammatory cytokines [IL-1ß (not in DI), TNF-α and IL-8], nuclear factor kappa B P65 (not NF-κB P52), c-Rel, IκB kinaseα (IKKα), IKKß and IKKγ mRNA levels in the PI, MI and DI of young grass carp (P < 0.05); and down-regulated pro-inflammatory cytokines IL-15 (not in DI) and inhibitor of κBα (IκBα) mRNA levels (P < 0.05). (4) down-regulated the mRNA levels of anti-inflammatory cytokines [IL-10 (not in DI), IL-11, IL-4/13B (not IL-4/13A), TGF-ß1 and TGF-ß2], target of rapamycin (TOR), eIF4E-binding proteins 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6k1) in the PI, MI and DI of young grass carp (P < 0.05). All data indicated that myo-inositol deficiency could decrease fish intestine immunity and cause inflammation under infection of A. hydrophila. Finally, the optimal dietary myo-inositol levels for the ACP and LZ activities in the DI were estimated to be 415.1 and 296.9 mg/kg diet, respectively.

Impaired intestinal immune barrier and physical barrier function by phosphorus deficiency: Regulation of TOR, NF-κB, MLCK, JNK and Nrf2 signalling in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

In aquaculture, the occurrence of enteritis has increased and dietary nutrition is considered as one of the major strategies to solve this problem. In the present study, we assume that dietary phosphorus might enhance intestinal immune barrier and physical barrier function to reduce the occurrence of enteritis in fish. To test this assumption, a total of 540 grass carp (Ctenopharyngodon idella) were investigated by feeding graded levels of available phosphorus (0.95-8.75 g/kg diet) and then infection with Aeromonas hydrophila. The results firstly showed that phosphorus deficiency decreased the ability to combat enteritis, which might be related to the impairment of intestinal immune barrier and physical barrier function. Compared with optimal phosphorus level, phosphorus deficiency decreased fish intestinal antimicrobial substances activities or contents and down-regulated antimicrobial peptides mRNA levels leading to the impairment of intestinal immune response. Phosphorus deficiency down-regulated fish intestinal anti-inflammatory cytokines mRNA levels and up-regulated the mRNA levels of pro-inflammatory cytokines [except IL-1ß and IL-12p35 in distal intestine (DI) and IL-12p40] causing aggravated of intestinal inflammatory responses, which might be related to the signalling molecules target of rapamycin and nuclear factor kappa B. In addition, phosphorus deficiency disturbed fish intestinal tight junction function and induced cell apoptosis as well as oxidative damage leading to impaired of fish intestinal physical barrier function, which might be partially associated with the signalling molecules myosin light chain kinase, c-Jun N-terminal protein kinase and NF-E2-related factor 2, respectively. Finally, based on the ability to combat enteritis, dietary available phosphorus requirement for grass carp (254.56-898.23 g) was estimated to be 4.68 g/kg diet.

The impaired immune function and structural integrity by dietary iron deficiency or excess in gill of fish after infection with Flavobacterium columnare: Regulation of NF-κB, TOR, JNK, p38MAPK, Nrf2 and MLCK signalling.

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ±â€¯0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKß, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively.

Methionine hydroxy analogue supplementation modulates gill immunological and barrier health status of grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of methionine hydroxy analogue (MHA) on the physical barrier and immune defence in the gill of young grass carp (Ctenopharyngodon idella). A total 630 young grass carp with an average initial weight of 259.70 ±â€¯0.47 g were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one DL-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. After feeding trial, 15 fish from each treatment were challenged with Flavobacterium columnare. Compared to the basal diet, optimal MHA improved cellular structure integrity of gill via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < .05). Simultaneously, optimal MHA supplementation improved cellular structure integrity of gill via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < .05). Besides, optimal MHA improved intercellular structure integrity of immune organs via up-regulating the mRNA levels of intercellular tight junctions-related genes, which might be owing to the down-regulation of myosin light chain kinase (MLCK) mRNA levels (P < .05). Summarily, MHA could improve the physical barrier of fish gill. In addition, optimal MHA supplementation increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M contents and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin and ß-defensin, suggesting that MHA could enhance antimicrobial ability of fish gill. Meanwhile, optimal MHA supplementation enhanced the immune defence of gill via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase ß, p38 mitogen activated protein kinase, eIF4E-binding protein1 (4E-BP1) and 4E-BP2 mRNA levels and up-regulation of inhibitor of κBα, ribosomal protein S6 kinase 1 and target of rapamycin mRNA levels (P < .05). In conclusion, the positive effect of MHA on gill health is associated with the improvement of the defence against apoptosis, antioxidant status, tight junctions and immune defence of fish gill. Meanwhile, MHA was superior to DLM on improving the physical barrier of fish gill. For the direction to healthy breeding of young grass carp, the optimal MHA supplementation levels on the premise of 4.01 g/kg methionine basal were estimated by quadratic regression curve, such as 5.49, 6.17 and 6.02 g/kg diet bases on the defence against gill-rot, malondialdehyde content and LZ activity in the gill, respectively.

Sodium butyrate enhanced physical barrier function referring to Nrf2, JNK and MLCK signaling pathways in the intestine of young grass carp (Ctenopharyngodon idella).

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.

Dietary threonine deficiency depressed the disease resistance, immune and physical barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study was conducted to investigate the effects of dietary threonine on the disease resistance, gill immune and physical barriers function of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juveniles were fed six iso-nitrogenous diets containing graded levels of threonine (3.99-21.66 g kg-1 diet) for 8 weeks, and then challenged with Flavobacterium columnare. Results showed that threonine deficiency (3.99 g kg-1 diet): (1) increased the gill rot morbidity after exposure to F. columnare; (2) attenuated the gill immune barrier function by decreasing antimicrobial substances production, up-regulating the mRNA levels of pro-inflammatory cytokines (except IL-12p40), and down-regulating the anti-inflammatory cytokines partly due to the modulation of NF-κB and TOR signaling. (3) disrupt the gill tight junction complexes by down-regulating TJs (claudin-3, -b, -c, 12, occludin, ZO-1 and ZO-2) and up-regulating TJs (claudin-7a, -7b) as well as related signaling molecule myosin light chain kinase mRNA levels (P < 0.05). (4) exacerbated the gill apoptosis by up-regulating cysteinyl aspartic acid-protease-3, 8, 9, c-Jun N-terminal kinases and mediating apoptosis related factors mRNA levels (P < 0.05); (5) exacerbated oxidative injury with increased reactive oxygen species, malondialdehyde and protein carbonyl contents (P < 0.05), decreased the antioxidant related enzymes activities and corresponding mRNA levels (except glutathione peroxidase-1b and glutathione-S-transferase-omega 2) as well as glutathione contents (P < 0.05) partly ascribe to the abridgement of NF-E2-related factor 2 signaling [Nrf2/Keap1a (not Keap1b)] in fish gill. Overall, threonine deficiency depressed the disease resistance, and impaired immune and physical barriers in fish gill. Finally, based on the gill rot morbidity and biochemical indices (immune indices LA activity and antioxidant indices MDA content), threonine requirements for juvenile grass carp (9.53-53.43 g) were estimated to be 15.32 g kg-1 diet (4.73 g 100 g-1 protein), 15.52 g kg-1 diet (4.79 g 100 g-1 protein), 15.46 g kg-1 diet (4.77 g 100 g-1 protein), respectively.

Dietary pyridoxine deficiency reduced growth performance and impaired intestinal immune function associated with TOR and NF-κB signalling of young grass carp (Ctenopharyngodon idella).

The objective of this study was to evaluate the effects of dietary pyridoxine (PN) deficiency on growth performance, intestinal immune function and the potential regulation mechanisms in young grass carp (Ctenopharyngodon idella). Fish were fed six diets containing graded levels of PN (0.12-7.48 mg/kg) for 70 days. After that, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results showed that compared with the optimal PN level, PN deficiency: (1) reduced the production of innate immune components such as lysozyme (LZ), acid phosphatase (ACP), complements and antimicrobial peptides and adaptive immune components such as immunoglobulins in three intestinal segments of young grass carp (P < 0.05); (2) down-regulated the mRNA levels of anti-inflammatory cytokines such as transforming growth factor ß (TGF-ß), interleukin 4/13A (IL-4/13A) (rather than IL-4/13B), IL-10 and IL-11 partly relating to target of rapamycin (TOR) signalling [TOR/ribosomal protein S6 kinases 1 (S6K1) and eIF4E-binding proteins (4E-BP)] in three intestinal segments of young grass carp; (3) up-regulated the mRNA levels of pro-inflammatory cytokines such as tumour necrosis factor α (TNF-α) [not in the proximal intestine (PI) and distal intestine (DI)], IL-1ß, IL-6, IL-8, IL-12p35, IL-12p40, IL-15 and IL-17D [(rather than interferon γ2 (IFN-γ2)] partly relating to nuclear factor kappa B (NF-κB) signalling [IκB kinase ß (IKKß) and IKKγ/inhibitor of κBα (IκBα)/NF-κB (p65 and c-Rel)] in three intestinal segments of young grass carp. These results suggest that PN deficiency could impair the intestinal immune function, and the potential regulation mechanisms were partly associated with TOR and NF-κB signalling pathways. In addition, based on percent weight gain (PWG), the ability against enteritis and LZ activity, the dietary PN requirements for young grass carp were estimated to be 4.43, 4.75 and 5.07 mg/kg diet, respectively.

Towards the modulation of oxidative damage, apoptosis and tight junction protein in response to dietary leucine deficiency: A likely cause of ROS-induced gill structural integrity impairment.

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-1ß (IL-1ß), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) mRNA expression and decreased IL-10 and transforming growth factor ß (TGF-ß), 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.

Impairment of gill structural integrity by manganese deficiency or excess related to induction of oxidative damage, apoptosis and dysfunction of the physical barrier as regulated by NF-κB, caspase and Nrf2 signaling in fish.

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 1ß mRNA levels) and down-regulation of anti-inflammatory cytokines (interleukin 10, transforming growth factor-ß1) 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.

Threonine deficiency decreased intestinal immunity and aggravated inflammation associated with NF-κB and target of rapamycin signalling pathways in juvenile grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

This study aimed to investigate the impacts of dietary threonine on intestinal immunity and inflammation in juvenile grass carp. Six iso-nitrogenous semi-purified diets containing graded levels of threonine (3·99-21·66 g threonine/kg) were formulated and fed to fishes for 8 weeks, and then challenged with Aeromonas hydrophila for 14 d. Results showed that, compared with optimum threonine supplementation, threonine deficiency (1) decreased the ability of fish against enteritis, intestinal lysozyme activities (except in the distal intestine), acid phosphatase activities, complement 3 (C3) and C4 contents and IgM contents (except in the proximal intestine (PI)), and it down-regulated the transcript abundances of liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, hepcidin, IgZ, IgM and ß-defensin1 (except in the PI) (P<0·05); (2) could up-regulate intestinal pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IL-17D mRNA levels partly related to NF-κB signalling; (3) could down-regulate intestinal anti-inflammatory cytokine transforming growth factor (TGF)-ß1, TGF-ß2, IL-4/13A (not IL-4/13B) and IL-10 mRNA levels partly by target of rapamycin signalling. Finally, on the basis of the specific growth rate, against the enteritis morbidity and IgM contents, the optimum threonine requirements were estimated to be 14·53 g threonine/kg diet (4·48 g threonine/100 g protein), 15.05 g threonine/kg diet (4·64 g threonine/100 g protein) and 15·17 g threonine/kg diet (4·68 g threonine/100 g protein), respectively.

Dietary protein levels regulated antibacterial activity, inflammatory response and structural integrity in the head kidney, spleen and skin of grass carp (Ctenopharyngodon idella) after challenged with Aeromonas hydrophila.

This study investigated the effects of dietary protein levels on disease resistance, immune function and structural integrity in the head kidney, spleen and skin of grass carp (Ctenopharyngodon idella). 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 Aeromonas hydrophila for 14 days. The results indicated that optimal levels of dietary protein: (1) (1) increased the lysozyme (LA) and acid phosphatase (ACP) activities and the complement 3 (C3) and C4 contents, up-regulated antimicrobial peptides, 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, IKKß, IKKγ, eIF4E-binding proteins (4E-BP) 1 and 4E-BP2 mRNA levels in the head kidney, spleen and skin of grass carp (P < 0.05), suggesting that optimal level of dietary protein could enhance immune function in the head kidney, spleen and skin of fish; (2) increased the activities and mRNA levels of antioxidant enzymes, enhanced the glutathione content, decreased reactive oxygen species, malondialdehyde (MDA) and protein carbonyl contents, and up-regulated the mRNA levels of NF-E2-related factor 2, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and tight junction complexes, whereas down-regulated Kelch-like-ECH-associated protein (Keap) 1b, cysteinyl aspartic acid-protease 3, 8, 9, Fas ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein and myosin light chain kinase mRNA levels in the head kidney, spleen and skin of grass carp (P < 0.05), indicating that optimal level of dietary protein could improve structural integrity in the head kidney, spleen and skin of fish. Finally, based on the skin hemorrhage and lesion morbidity, LA activity and MDA content, the optimal levels of dietary protein for grass carp (264 g-787 g) were estimated to be 241.45 g kg-1 diet (217.68 g digestible protein kg-1 diet), 301.68 g kg-1 diet (265.48 g digestible protein kg-1 diet) and 307.84 g kg-1 diet (272.71 g digestible protein kg-1 diet), respectively.

Decreased enteritis resistance ability by dietary low or excess levels of lipids through impairing the intestinal physical and immune barriers function of young grass carp (Ctenopharyngodon idella).

The current study explores the hypothesis that low or excess levels of lipids decrease the enteritis resistance ability through impairing the intestinal physical and immune barrier function of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ± 0.53 g were fed diets containing six graded levels of lipids at 5.9, 21.4, 36.0, 50.2, 66.6 and 80.1 g/kg for 8 weeks. After that, a challenge trial was conducted by injection of Aeromonas hydrophila over 2 weeks. The results show that compared with optimal lipids level, low or excess levels of lipids impair fish immune barrier function through declining humoral compounds and down-regulating the mRNA levels of interleukin 4/13A (IL-4(13)A) [not in the proximal intestine (PI)], IL-4(13)B, IL-6, IL-10, transforming growth factor ß1 (TGF-ß1), inhibitor of κBα (IκBα), TOR and ribosomal p70S6 kinase (S6K1) (P < 0.05), and up-regulating tumor necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), IL-1ß, IL-8, IL-12 p40 (not p35), nuclear factor κB p65 (NF-κB p65), IκB kinase α (IKKα), IKKß, IKKγ, and eIF4E-binding protein (4EBP) in the intestine of young grass carp (P < 0.05). In addition, low or excess levels of lipids also decrease young grass carp physical barrier function through down-regulating the mRNA levels of zonula occludens (ZO-1), ZO-2b [only in the distal intestine (DI)], Claudin b, c, 3, 12, 15a, 15b (only in the DI), 7b (not 7a) and Occludin by MAPKK 6/p38 MAPK (not JNK)/MLCK signaling molecules, down-regulating B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) (not in the DI), and decreasing antioxidant ability via Kelch-like ECH-associating protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) signaling molecules in the intestine. Based on the quadratic regression analysis for the enteritis resistance ability, LA activities and GSH content in the MI were established to be 54.5, 49.91 and 47.83 g lipid/kg diets, respectively.