From growth promotion to intestinal inflammation alleviation: Unraveling the potential role of Lactobacillus rhamnosus GCC-3 in juvenile grass carp (Ctenopharyngodon idella).

Lactobacillus rhamnosus is a probiotic, which not only promotes the growth of animals, but also has anti-inflammatory effects. However, the mechanism by which Lactobacillus rhamnosus regulates intestinal immunity is not well comprehended. Hence, the study aimed to research how Lactobacillus rhamnosus affects the intestinal immunity using juvenile grass carp (Ctenopharyngodon idella) as a model. We selected 1800 juvenile grass carp for testing. They were divided into six treatments and fed with six gradients of Lactobacillus rhamnosus GCC-3 (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 g/kg) for 70 days. Enteritis was subsequently induced with dextroside sodium sulfate. Results indicated that dietary Lactobacillus rhamnosus GCC-3 addition improved growth performance. Meanwhile, appropriate levels of Lactobacillus rhamnosus GCC-3 alleviated excessive inflammatory response by down-regulating the expression of TLR4 and NOD receptors, up-regulating the expression of TOR, and then down-regulating the expression of NF-κB. Additionally, appropriate Lactobacillus rhamnosus GCC-3 improved intestinal immunity by reducing pyroptosis triggered by NLRP3 inflammasome and mediated by GSDME. Furthermore, 16 S rRNA sequencing showing appropriate levels of Lactobacillus rhamnosus GCC-3 increased Lactobacillus and Bifidobacterium abundance and decreased Aeromonas abundance. These results suggest that Lactobacillus rhamnosus GCC-3 can alleviate intestinal inflammation through down-regulating NF-κB and up-regulating TOR signaling pathways, as well as by inhibiting pyroptosis.

Exploring the novel benefits of leucine: Protecting nitrite-induced liver damage in sub-adult grass carp (Ctenopharyngodon idella) through regulating mitochondria quality control.

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.

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

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

Novel insights on toxicology of ochratoxin A contaminated diets to skin: Residues, mucus disruption and barrier impairment in teleost model grass carp (Ctenopharyngodon idella).

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

Vitamin D Promotes Mucosal Barrier System of Fish Skin Infected with Aeromonas hydrophila through Multiple Modulation of Physical and Immune Protective Capacity.

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.

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

BACKGROUND: Deterioration of flesh quality has bad effects on consumer satisfaction. Therefore, effects of safe mannan-oligosaccharides (MOS) on flesh quality of grass carp (Ctenopharyngodon idella) muscle were studied. A total of 540 healthy fish (215.85 ± 0.30 g) were randomly divided into six groups and fed six separate diets with graded levels of MOS (0, 200, 400, 600, 800 and 1000 mg kg-1 ) for 60 days. This study aimed at investigating the benefits of dietary MOS on flesh quality (fatty acids, amino acids and physicochemical properties) and the protection mechanism regarding antioxidant status. RESULTS: Optimal MOS could improve tenderness (27.4%), pH (5.5%) while decreasing cooking loss (16.6%) to enhance flesh quality. Meanwhile, optimal MOS improved flavor inosine 5'-monophosphate (IMP) of 11.8%, sweetness and umami-associated amino acid, healthy unsaturated fatty acid (UFA) of 14.9% and n-3 polyunsaturated fatty acids (n-3 PUFAs) especially C20:5n-3 (15.8%) and C22:6n-3 (38.3%). Furthermore, the mechanism that MOS affected pH, tenderness and cooking loss could be partly explained by the reduced lactate, cathepsin and oxidation, respectively. The enhanced flesh quality was also associated with enhanced antioxidant ability concerning improving antioxidant enzymes activities and the corresponding transcriptional levels, which were regulated through NF-E2-related factor 2 (Nrf2) and target of rapamycin (TOR) signaling. Based on pH24h , cooking loss, shear force and DHA (docosahexaenoic acid, C22:6n-3), optimal MOS levels for grass carp were estimated to be 442.75, 539.53, 594.73 and 539.53 mg kg-1 , respectively. CONCLUSION: Dietary MOS is a promising alternative strategy to improve flesh quality of fish muscle. © 2022 Society of Chemical Industry.

Effects of dietary tryptophan on muscle growth, protein synthesis and antioxidant capacity in hybrid catfish Pelteobagrus vachelli♀ × Leiocassis longirostris♂.

The present study evaluated effects of dietary supplementation with tryptophan (Trp) on muscle growth, protein synthesis and antioxidant capacity in hybrid catfish Pelteobagrus vachelli♀ × Leiocassis longirostris♂. Fish were fed six different diets containing 2·6 (control), 3·1, 3·7, 4·2, 4·7 and 5·6 g Trp/kg diet for 56 d, respectively. Results showed that dietary Trp significantly (1) improved muscle protein content, fibre density and frequency of fibre diameter; (2) up-regulated the mRNA levels of PCNA, myf5, MyoD1, MyoG, MRF4, IGF-I, IGF-II, IGF-IR, PIK3Ca, TOR, 4EBP1 and S6K1; (3) increased phosphorylation levels of AKT, TOR and S6K1; (4) decreased contents of MDA and PC, and increased activities of CAT, GST, GR, ASA and AHR; (5) up-regulated mRNA levels of CuZnSOD, CAT, GST, GPx, GCLC and Nrf2, and decreased Keap1 mRNA level; (6) increased nuclear Nrf2 protein level and the intranuclear antioxidant response element-binding ability, and reduced Keap1 protein level. These results indicated that dietary Trp improved muscle growth, protein synthesis as well as antioxidant capacity, which might be partly related to myogenic regulatory factors, IGF/PIK3Ca/AKT/TOR and Keap1/Nrf2 signalling pathways. Finally, based on the quadratic regression analysis of muscle protein and MDA contents, the optimal Trp requirements of hybrid catfish (21·82-39·64 g) were estimated to be 3·94 and 3·93 g Trp/kg diet (9·57 and 9·54 g/kg of dietary protein), respectively.

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

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

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

In the current research, a 60-d experiment was conducted with the purpose of exploring the impacts of methionine (Met) on growth performance, muscle nutritive deposition, muscle fibre growth and type I collagen synthesis as well as the related signalling pathway. Six diets (iso-nitrogenous) differing in Met concentrations (2·54, 4·85, 7·43, 10·12, 12·40 and 15·11 g/kg diets) were fed to 540 grass carp (178·47 (SD 0·36) g). Results showed (P < 0·05) that compared with Met deficiency, optimal level of dietary Met (1) increased feed intake, feed efficiency, specific growth rate and percentage weight gain (PWG); (2) increased fish muscle protein, lipid and free amino acid contents and improved fish muscle fatty acid profile as well as increased protein content in part associated with the target of rapamycin complex 1 (TORC1)/S6K1 signalling pathway; (3) increased the frequency distribution of muscle fibre with >50 µm of diameter; (4) increased type I collagen synthesis partly related to the transforming growth factor-ß1/Smads and CK2/TORC1 signalling pathways. In conclusion, dietary Met improved muscle growth, which might be due to the regulation of muscle nutritive deposition, muscle fibre growth and type I collagen synthesis-related signal molecules. Finally, according to PWG and muscle collagen content, the Met requirements for on-growing grass carp (178-626 g) were estimated to be 9·56 g/kg diet (33·26 g/kg protein of diet) and 9·28 g/kg diet (32·29 g/kg of dietary protein), respectively.

Isoleucine improved growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris.

This study was performed to determine effects of dietary isoleucine (Ile) on growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris. Six hundred and thirty fish (33.11 ± 0.09 g) were randomly divided into seven experimental groups with three replicates each, and respectively fed seven diets with 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 g Ile kg-1 diets for 8 weeks. The results showed improvement of growth performance, feed intake, feed utilization, relative gut length (RGL), and intestinal fold height and width by dietary Ile (P < 0.05). Meanwhile, dietary Ile (12.5 g kg-1 diet) improved the activities of lysozyme (LZM), acid phosphatase, alkaline phosphatase and the contents of complement 3 (C3), C4, and immunoglobulin M (IgM) (P < 0.05). The c-type-lectin, c-LZM, g-LZM, and hepcidin mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0-20.0 g Ile kg-1 diet (P < 0.05). Dietary Ile (10.0-12.5 g Ile kg-1 diet) increased intestinal ß-defensin mRNA expression partially in association with Sirt1/ERK/90RSK signaling pathway. Dietary Ile (12.5-15.0 g Ile kg-1 diet) decreased oxidative damage and improved antioxidant ability by increasing activities and expressions of superoxide dismutase, glutathione peroxidase, and glutathione reductase, glutathione-S-transferase (P < 0.05). The occludin, ZO-1, ZO-2, claudin3, and claudin 7 mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0 and 12.5 g Ile kg-1 diet (P < 0.05), whereas the myosin light chain kinase gene expression was decreased in fish fed diets with 7.5-17.5 g Ile kg-1 diet. Dietary Ile (10-12.5 g Ile kg-1 diet) decreased apoptotic responses by reducing the expression of caspase3 and caspase 9 via the AKT/TOR signaling pathway. Based on the quadratic regression analysis of PWG, the dietary Ile requirement of hybrid catfish was estimated to be 12.43 g Ile kg-1 diet, corresponding to 32.05 g Ile kg-1 dietary protein. Collectively, dietary Ile improved growth performance and immunological and physical barrier function of intestine in hybrid catfish.

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

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

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

The present study investigated the effects of condensed tannins (CT) on intestinal immune function in on-growing grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp were fed six diets containing different levels of CT (0, 10·00, 20·00, 30·00, 40·00 and 50·00 g/kg diet) for 70 d and then challenged with Aeromonas hydrophila for 14 d. The results showed that, compared with the control group, dietary CT (1) induced intestinal histopathological lesions and aggravated enteritis; (2) decreased lysozyme and acid phosphatase activities, complement 3 (C3), C4 and IgM contents and down-regulated the Hepcidin, liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, Mucin2 and ß-defensin-1 mRNA levels in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) (P < 0·05); (3) down-regulated the mRNA levels of anti-inflammatory cytokines transforming growth factor (TGF)-ß1, TGF-ß2 (not in MI and DI), IL-4/13A (not IL-4/13B), IL-10 and IL-11 partly correlated with target of rapamycin (TOR) signalling; and (4) up-regulated the mRNA levels of pro-inflammatory cytokines interferon-γ2, IL-1ß, IL-6, IL-8 (not in PI), IL-12p35, IL-12p40, IL-15 and IL-17D partly related to NF-κB signalling in the intestine of on-growing grass carp. Overall, the results indicated that CT could impair the intestinal immune function, and its potential regulation mechanisms were partly associated with the TOR and NF-κB signalling pathways. Finally, based on the percentage weight gain and enteritis morbidity, the maximum allowable levels of CT for on-growing grass carp (232·22-890·11 g) were estimated to be 18·6 and 17·4 g/kg diet, respectively.

Effect of dietary threonine on growth performance and muscle growth, protein synthesis and antioxidant-related signalling pathways of hybrid catfish Pelteobagrus vachelli♀ × Leiocassis longirostris♂.

The experiment was conducted to investigate the effects of dietary threonine (Thr) on growth performance and muscle growth, protein synthesis and antioxidant-related signalling pathways of hybrid catfish Pelteobagrus vachelli♀ × Leiocassis longirostris♂. A total of 1200 fish (14·19 (se 0·13) g) were randomly distributed into six groups with four replicates each, fed six diets with graded level of Thr (9·5, 11·5, 13·5, 15·4, 17·4 and 19·3 g/kg diets) for 56 d. Results showed (P < 0·05) that dietary Thr (1) increased percentage weight gain, specific growth rate, feed efficiency and protein efficiency ratio; (2) up-regulated growth hormone (GH), insulin-like growth factor 1 (IGF-1), proliferating cell nuclear antigen, myogenic regulation factors (MyoD, Myf5, MyoG and Mrf4) and myosin heavy chain (MyHC) mRNA levels; (3) increased muscle protein content via regulating the protein kinase B/target of rapamycin signalling pathway and (4) decreased malondialdehyde and protein carbonyl contents, increased catalase, glutathione-S-transferase, glutathione reductase and GSH activities, up-regulated mRNA levels of antioxidant enzymes related to NFE2-related factor 2 and γ-glutamylcysteine ligase catalytic subunit. These results suggest that Thr has a potential role to improve muscle growth and protein synthesis, which might be due to the regulation of GH-IGF system, muscle growth-related gene, antioxidative capacity and protein synthesis-related signalling pathways. Based on the quadratic regression analysis of specific growth rate, the Thr requirement of hybrid catfish (14·19-25·77 g) was estimated to be 13·77 g/kg of the diet (33·40 g/kg of dietary protein).

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

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

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

The present study aimed to explore the effects of phytic acid (PA) on the antimicrobial activity and inflammatory response in three immune organs (head kidney, spleen and skin) of on-growing grass carp (Ctenopharyngodon idella). To achieve this goal, we first conducted a 60-day growth trial by feeding fish with graded levels of PA (0, 0.8, 1.6, 2.4, 3.2 and 4.0%). Then, the fish were challenged with Aeromonas hydrophila for 6 days. Compared with the control group, the following results were obtained regarding supplementation with certain levels of PA in the diet. (1) There was an increase in skin haemorrhage and lesion morbidity in fish. (2) There was a decrease in activities or contents of immune factors, including lysozyme (LZ), complement 3 (C3), C4 and immunoglobulin M (IgM), and there was downregulation of gene expression levels of hepcidin, liver-expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, and ß-defensin-1 in immune organs. (3) There was upregulation in the gene expression of the following pro-inflammatory cytokines: tumour necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) (except in the spleen), interferon γ2 (IFN-γ2), IL-6 (except in the spleen), IL-8, IL-12p40, IL-15 and IL-17D. These changes were partly related to the nuclear factor kappa B (NF-κB) signalling pathway, but downregulation of mRNA levels of anti-inflammatory cytokines (transforming growth factor ß1 (TGF-ß1), TGF-ß2, IL-413/A, IL-413/B, IL-10 (except in the skin) and IL-11) occurred in a manner partially related to the target of rapamycin (TOR) signalling pathway. Finally, based on the broken-line analysis of skin haemorrhage and lesion morbidity and IgM content in the head kidney, the maximum tolerance levels of PA for on-growing grass carp (120.56-452.00 g) were estimated to be 1.79 and 1.31% of the diet, respectively.

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

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

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

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

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

Glycinin is a major protein and antinutritional factor of soybean. However, how dietary glycinin affect intestinal immune function of fish were largely unknown. In this study, we used juvenile grass carp as a model to investigate the impacts of glycinin on intestinal immune function of fish and involved mechanisms. We set three treatments including control, glycinin and glycinin + glutamine in this trial. For immune components, results revealed that compared with control group, glycinin group had lower acid phosphatase activities in the foregut, midgut and hindgut, lower C3 and C4 content, and lower mRNA abundances of IgM, IgZ, hepcidin, LEAP-2A, LEAP-2B and ß-defensin-1 in the midgut and hindgut rather than foregut of grass carp. For pro-inflammatory cytokines and relevant signaling, glycinin elevated mRNA abundances of IL-1ß, IL-8, IL-12p35, IL-12p40 and IL-17D in the midgut and IL-1ß, IFN-γ2, IL-6, IL-8, IL-12p35, IL-12p40 and IL-17D in the hindgut, and increased protein abundances of PKC-ζ and nuclear NF-κB p65 in the midgut and hindgut in comparison to control. For anti-inflammatory cytokines and relevant signaling, glycinin reduced mRNA abundances of TGF-ß1, TGF-ß2, IL-4/13B (rather than IL-4/13A), IL-10 and IL-11 in the midgut and hindgut, and reduced p-mTOR (Ser 2448), p-S6K1 (Thr 389) and p-4EBP1 (Thr 37/46) protein abundances in the midgut and hindgut rather than foregut. Co-administration of glutamine with glycinin could partially enhance intestinal function and reduce intestinal inflammation compared with glycinin treatment. Concluded, glycinin decreased intestinal immune components and caused intestinal inflammation associated with PKC-ζ/NF-κB and mTORC1 signaling.

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

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

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

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