Effect of phosphorus on growth performance, intestinal tight junctions, Nrf2 signaling pathway and immune response of juvenile mirror carp (Cyprinus carpio) fed different α-ketoglutarate levels.

Previous research has shown that dietary α-ketoglutarate (AKG) supplementation can promote growth performance, phosphorus metabolism, and skeletal development of juvenile mirror carp (Cyprinus carpio) fed low phosphorous diets. In the current study, we tested the hypothesis that 1% AKG dietary supplementation reduces the dietary phosphorus requirements of juvenile mirror carp. A total of 12 experimental isoproteic and isolipidic diets containing available phosphorus levels of 0.21%, 0.38%, 0.55%, 0.72%, 0.89%, and 1.07% dry matter with either 0 or 1% AKG supplementation were used in the study. A total of 1080 juvenile fish of similar initial weight (0.90 ± 0.03 g) were selected and randomly assigned to 36 tanks. There were three replicates for each experimental group, with a density of 30 fish per tank. Fish were fed to satiation for 8 weeks. The results indicated that fish fed the diet supplemented with 1% AKG showed a significant increase in final body weight (FBW), weight gain rate (WGR), feed intake (FI) and phosphorus intake (PI) compared to the diet without AKG (P < 0.05). FBW and WGR increased significantly with increasing available phosphorus levels from 0.21% to 0.89% (P < 0.05). The mRNA expression of ZO-1, claudin 11, and occludin was significantly increased by dietary AKG and phosphorus (P < 0.05). The mRNA expression of Nrf2, GPx1a, and CAT in the Nrf2 signaling pathway was significantly increased by dietary AKG and phosphorus (P < 0.05). The expression levels of IL-10 and TGF-ß2 were significantly increased by dietary AKG and phosphorus, but the expression levels of IL-1ß, IL-8, IL-10, TNF-a and NF-κB were significantly decreased with dietary AKG and phosphorus supplementation (P < 0.05). Based on second-order polynomial regression analysis of WGR against dietary phosphorus levels, the optimal dietary phosphorus level was found to be 0.79% of dry feed for juvenile mirror carp fed a diet with 1% AKG supplementation and 0.93% of dry feed without AKG supplementation. This study confirmed that AKG supplementation can reduce the phosphorus requirements of juvenile mirror carp by promoting growth performance, intestinal tight junctions, Nrf2 signaling pathways and immune response.

Effect of dietary Ficus carica polysaccharides on the growth performance, innate immune response and survival of crucian carp against Aeromonas hydrophila infection.

Ficus carica polysaccharides (FCPS), one of the most effective and important compo-nents in Ficus carica L., had been considered to be a beneficial immunostimulant and may be used in immunotherapy for animals and human. However, studies were little about the effect of FCPS used as immunomodulatory and the suitable dosage in fish. The present study investigated the effect of four different dietary levels of FCPS (0.1%, 0.2%, 0.4%, 0.8%) on the growth performance, innate immune responses and survival of crucian carp against Aeromonas hydrophila infection. The results showed that compared with control group, dietary FCPS had positive effects the growth performance (final weight, feed conversion ratio and survival rate) of crucian carp. FCPS induced significant higher (p < 0.05) leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, com-plement C3, SOD activity and total protein level in the serum of crucian carp. Moreover, innate immune response of fish in FCPS groups increased first and then decreased with increasing dietary FCPS from 0.1% to 0.8%, and reached up to the peak in 0.4% dietary FCPS groups. Besides, the cumulative mortalities in FCPS groups were remarkably lower than that of control group when challenged with A. hydrophila, the relative percent survivals were 22.67%, 55.56%, 62.22% and 17.78% in 0.1% group, 0.2% group, 0.4% group and 0.8% group, respectively. These results suggested that dietary FCPS could improve the growth performance, innate immune response and disease resistance against A. hydrophila in fish, and the suitable dietary dose of FCPS was 0.4% in crucian carp.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1ß (IL-1ß) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

Effect of Pandanus tectorius extract as food additive on oxidative stress, immune status, and disease resistance in Cyprinus carpio.

The present study investigated the effect of Pandanus tectorius (PT) extract on Cyprinus carpio growth, antioxidant capacity, immunological and oxidative stress, immune-related gene expression, and resistance against pathogen challenge. Fish (average weight: 12.18 ± 0.32 g) were fed with diets containing various concentration (g kg-1) of PT extract: 0 g (basal diet), 5 g [PT5], 10 g [PT10], 20 g [PT20], and 30 g [PT30] for 8 weeks. Our results revealed an increase (p < 0.05) in the final weight gain only in the PT20 (63.12 ± 1.4 g) group. Activity of liver antioxidant parameters such as catalase (CAT) (27.67 ± 1.12 U mg protein-1), superoxide dismutase (SOD) (13.17 ± 0.428 U mg protein-1), and glutathione peroxidase (GPx) (47.14 ± 1.47 U mg protein-1) was highest in the PT20 group. Among the serum immunological parameters examined, lysozyme (37.45 ± 0.67 U mL-1), alternative complement pathway (133.22 ± 1.92 U mL-1), and phagocytic activities (25.68 ± 0.93%) were high (p < 0.05) in PT20, whereas there was no significant effect on serum immunoglobulin or total protein levels, compared to the control. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and malondialdehyde (MDA) levels were found to be lower (p < 0.05) in the PT20 and PT30 groups than those in the control. However, myeloperoxidase (MPO) levels were significantly higher (p < 0.05) in the PT20 and PT30 groups. Gene expression analysis revealed that mRNA expressions of antioxidant genes (SOD, CAT, nuclear factor erythroid 2-related factor 2) and anti-inflammatory cytokine (IL-10) genes were significantly upregulated in the PT10 - PT30 groups, except for the SOD gene in PT10. Conversely, the signalling molecule NF-κBp65 was downregulated in PT20 and PT30. Expression of Toll-like receptor 22 (TLR22) and hepcidin was significantly upregulated in PT20 and PT30, respectively. Fish in the PT20 group exhibited highest relative post-challenge survival (70.37%) against Aeromonas hydrophila challenge. The results of the present study suggest that dietary supplementation of P. tectorius extract at 20 g kg-1 can significantly improve weight gain, serum antioxidant parameters, strengthen immunity, and increase the disease resistance of C. carpio. Therefore, P. tectorius extract could be exploited for its use as a food additive in aquaculture.

Remarkable positive effects of figwort (Scrophularia striata) on improving growth performance, and immunohematological parameters of fish.

This study was conducted to investigate the effect of figwort on the growth and immunohematological parameters of common carp (14.20 ± 0.53 g). Four experimental diets were developed to feed fish for eight weeks: control, Figw10 (10 g/kg figwort), Figw20 (20 g/kg figwort), and Figw30 (30 g/kg figwort). The results showed that fish fed dietary Figw10 gained more weight (38.25 g) than control (P < 0.05). Regarding immunohematological parameters, fish fed dietary Figw30 had a higher level of white blood cells (31.2 103/mm3), hematocrit (35.82%), blood performance (14.63), total protein (1.96 g/dL), albumin (0.79 g/dL), globulin (1.17 g/dL), lymphocyte (70.53%), monocyte (3.03%), alternative hemolytic complement activity (ACH50) (147.76 u/mL), lysozyme (62.19 u/mL), and bactericidal activities (135.24) than the control group (P < 0.05). After 14 days of the challenge with Aeromonas hydrophila, the Figw30 treatment had the highest survival ratio (61.76%) compared to the control with 26.46%. Further, after the challenge, fish fed dietary Figw30 had a higher value of immunoglobulin M (42.00 µg/mL), antibody titer (19.23), complement component 3 (296.39 µg/mL), and complement component 4 (97.91 µg/mL) when compared with those fed control diet (P < 0.05). In conclusion, the optimum dosage for providing the best immune response was 30 g/kg in diet.

Evaluation of the effects of Astragalus polysaccharides as immunostimulants on the immune response of crucian carp and against SVCV in vitro and in vivo.

This experiment was conducted to evaluate the immunomodulatory effect and antiviral activity of Astragalus polysaccharides (APS) in crucian carp and epithelioma papulosum cyprinid (EPC) cells. Two diets containing 0 and 2 g/kg, APS were fed crucian carp for 56 days. The results showed that supplementation with APS significantly upregulated the immune-related indices including the levels of IgM, the activities of LZM, AKP and ACP, and the contents of C3 and C4. At the same time, compared with the CK group, adding APS to the feed significantly upregulated the expression of IL-8, IL-10, IL-1ß, IFN-α, IFN-γ, MyD88, TGF-ß and TNF-α in the spleen, kidney, liver and intestine of crucian carp. In addition, when the crucian carp were injected with SVCV, the survival rates of fish in the APS group and the control group were 48.87% and 13.76%, respectively. These results indicated that dietary APS could improve the resistance of crucian carp against SVCV infection. APS also significantly decreased viral titer and inhibited apoptosis induced by SVCV in EPC cells. These results indicated that APS could stimulate the immune response of crucian carp and improve the abilities of crucian carp and EPC cells to resist SVCV infection.

Evaluation of Alpha-Ketoglutarate Supplementation on the Improvement of Intestinal Antioxidant Capacity and Immune Response in Songpu Mirror Carp (Cyprinus carpio) After Infection With Aeromonas hydrophila.

As an intermediate substance of the tricarboxylic acid cycle and a precursor substance of glutamic acid synthesis, the effect of alpha-ketoglutarate on growth and protein synthesis has been extensively studied. However, its prevention and treatment of pathogenic bacteria and its mechanism have not yet been noticed. To evaluate the effects of alpha-ketoglutarate on intestinal antioxidant capacity and immune response of Songpu mirror carp, a total of 360 fish with an average initial weight of 6.54 ± 0.08 g were fed diets containing alpha-ketoglutarate with 1% for 8 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results indicated that alpha-ketoglutarate supplementation significantly increased the survival rate of carp after infection with Aeromonas hydrophila (P < 0.05), and the contents of immune digestion enzymes including lysozyme, alkaline phosphatase and the concentration of complement C4 were markedly enhanced after alpha-ketoglutarate supplementation (P < 0.05). Also, appropriate alpha-ketoglutarate increased the activities of total antioxidant capacity and catalase and prevented the up-regulation in the mRNA expression levels of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 (P < 0.05). Furthermore, the mRNA expression levels of toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB) were strikingly increased after infection with Aeromonas hydrophila (P < 0.05), while the TLR4 was strikingly decreased with alpha-ketoglutarate supplementation (P < 0.05). Moreover, the mRNA expression levels of tight junctions including claudin-1, claudin-3, claudin-7, claudin-11 and myosin light chain kinases (MLCK) were upregulated after alpha-ketoglutarate supplementation (P < 0.05). In summary, the appropriate alpha-ketoglutarate supplementation could increase survival rate, strengthen the intestinal enzyme immunosuppressive activities, antioxidant capacities and alleviate the intestinal inflammation, thereby promoting the intestinal immune responses and barrier functions of Songpu mirror carp via activating TLR4/MyD88/NF-κB and MLCK signaling pathways after infection with Aeromonas hydrophila.

Modulatory effects of laurel-leaf cistus (Cistus laurifolius) ethanolic extract on innate immune responses and disease resistance in common carp (Cyprinus carpio).

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.

ß-Glucan-Induced Immuno-Modulation: A Role for the Intestinal Microbiota and Short-Chain Fatty Acids in Common Carp.

Dietary supplementation of fish with ß-glucans has been commonly associated with immunomodulation and generally accepted as beneficial for fish health. However, to date the exact mechanisms of immunomodulation by ß-glucan supplementation in fish have remained elusive. In mammals, a clear relation between high-fibre diets, such as those including ß-glucans, and diet-induced immunomodulation via intestinal microbiota and associated metabolites has been observed. In this study, first we describe by 16S rRNA sequencing the active naive microbiota of common carp intestine. Based on the abundance of the genus Bacteroides, well known for their capacity to degrade and ferment carbohydrates, we hypothesize that common carp intestinal microbiota could ferment dietary ß-glucans. Indeed, two different ß-glucan preparations (curdlan and MacroGard®) were both fermented in vitro, albeit with distinct fermentation dynamics and distinct production of short-chain fatty acids (SCFA). Second, we describe the potential immunomodulatory effects of the three dominant SCFAs (acetate, butyrate, and propionate) on head kidney leukocytes, showing effects on both nitric oxide production and expression of several cytokines (il-1b, il-6, tnfα, and il-10) in vitro. Interestingly, we also observed a regulation of expression of several gpr40L genes, which were recently described as putative SCFA receptors. Third, we describe how a single in vivo oral gavage of carp with MacroGard® modulated simultaneously, the expression of several pro-inflammatory genes (il-1b, il-6, tnfα), type I IFN-associated genes (tlr3.1, mx3), and three specific gpr40L genes. The in vivo observations provide indirect support to our in vitro data and the possible role of SCFAs in ß-glucan-induced immunomodulation. We discuss how ß-glucan-induced immunomodulatory effects can be explained, at least in part, by fermentation of MacroGard® by specific bacteria, part of the naive microbiota of common carp intestine, and how a subsequent production of SFCAs could possibly explain immunomodulation by ß-glucan via SCFA receptors present on leukocytes.

Pro- and anti-inflammatory responses of common carp Cyprinus carpio head kidney leukocytes to E.coli LPS as modified by different dietary plant oils.

Dietary lipids could modify fatty acid (FA) composition in fish tissues. Long chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid (ARA), eicosapentaneoic acid (EPA) and docosahexaenoic acid (DHA) are able to modulate the immune status in fish through an inflammatory process but their availability may be limited when fish are exclusively fed plant oils. This study was conducted to evaluate how to maximise the utilisation of dietary plant oil for an efficient inflammatory response in common carp head kidney leukocytes (HKLs) exposed to a gram-negative bacterial endotoxin, Escherichia coli lipopolysaccharides (LPS). HKLs were isolated from fish fed cod liver oil (CLO), linseed oil (LO), sesame oil (SO) a blend of SO and LO (SLO, v:v 1:1), and these plant oil diets supplemented with DHA (SO + DHA, SOD) or ARA (LO + ARA, LOA) for 6 weeks. Cells were then exposed to LPS at a dose of 10 µg/mL for 4 and 24 h. Peroxidase activity, total Ig, and NO levels were measured in the culture medium, while cells were used for expression analyses of candidate genes in pattern recognition (tlr-4), eicosanoid metabolism (pge2, 5-lox), pro-inflammatory (il-1, il-6, il-8, tnf-α, nf-kb, inos, cxc), anti-inflammatory (il-10, nf-kbi, tgf-ß1) responses, and cytoprotective (gpx-1, prdx-3) processes. Results showed that LPS induced significantly inflammatory responses, evidenced by a high level of almost all the targeted humoral immune parameters and/or gene expression. Expression of inflammatory cytokines and other inflammatory mediators was upregulated after 4 h-LPS exposure and reverted to basal levels after 24 h. HKLs from fish fed SLO, LOA, or SOD diet exhibited a more efficient regulation of acute inflammatory processes than those fed CLO diet. The results indicate that the immune competence of fish fed plant oil mixture was comparable to the one of fish fed fish oil diet. Moreover, the supplementation of ARA or DHA induced similar immunomodulation in common carp.

Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological, antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus carpio against Aeromonas hydrophila.

The present study aimed to investigate the potential effects of dietary marjoram extract on growth performance, health, and disease resistance in common carp, Cyprinus carpio. To this purpose, the fish were assigned into four treatments and fed by diets supplemented with 0 (control), 100, 200, and 400 mg marjoram extract kg-1 over eight weeks and then challenged with Aeromonas hydrophila. According to the results, 200 mg kg-1 dietary marjoram extract inclusion showed the highest final weight, weight gain, and specific growth rate, and lowest feed conversion ratio (FCR). White blood cell number, Red blood cells, hematocrit, hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin were markedly increased particularly at 200 mg kg-1 treatment. Marjoram extract significantly increased plasma superoxide dismutase activity and decreased malondialdehyde level compared to the control treatment. Plasma complement and lysozyme activities and total immunoglobulin levels, mucosal complement, lysozyme and alkaline phosphatase activities and immunoglobulin levels were significantly increased compared to the control group. The lowest post-challenge survival rate was observed in the control treatment, whereas the highest value was related to the 200 mg kg-1 marjoram treatment. In conclusion, the present study demonstrated that, marjoram extract is a suitable feed supplements for common carp, as it stimulates the fish growth, antioxidant, and immune systems, which suppress the fish mortality during Aeromonas septicemia. According to the results, 200 mg marjoram extract kg-1 is recommended for carp feed formulation.

Protective effects of black seed (Nigella sativa) diet supplementation in common carp (Cyprinus carpio) against immune depression, oxidative stress and metabolism dysfunction induced by glyphosate.

Sustainable aquaculture arises as key to increase food production in the coming years. However, the sector still faces many challenges such as the exposure of the cultured animals to pesticide-contaminated water. Pesticides used in agriculture can reach aquaculture systems either directly (integrated-agriculture aquaculture practices) or indirectly (soil leakage) and cause a broad range of ecotoxicological effects on cultured fish and shellfish. Here, we studied how glyphosate affects several haematological, biochemical, and immune parameters in common carp (Cyprinus carpio) fingerlings, the fourth most important cultured fish species worldwide. We also evaluated the potential of dietary supplementation with black seed (Nigella sativa, 0.25, 0.5 and 1%) to lower glyphosate-associated toxicity. Our results showed that 14-day sub-lethal exposure of common carp fingerlings to glyphosate increases oxidative stress, decreases antioxidant defences, affects several metabolic pathways, and induced immune depression. Furthermore, we showed that fish fed with N. sativa-enriched diets at 0.25, 0.5 and 1% for 60 days coped better with glyphosate exposure than control fish and displayed more stable levels of biochemical serum parameters (total protein, albumin, triglycerides, low-density lipoprotein LDL), cholesterol and high-density lipoprotein HDL), higher levels of immune defences (lysozyme and immunoglobulin) and higher antioxidant enzymes (superoxide dismutase SOD, glutathione peroxidase GPx) than control fish. Fish fed with all enriched diets also displayed lower lipid peroxidation (malondialdehyde MDA), lower metabolic enzymes (alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase ALP) levels in blood serum and lower cortisol levels than control fish. Altogether, our results show that dietary inclusion of black seed can be used as a sustainable bio-remediation strategy, mitigating many of the negative effects of glyphosate exposure in fish.

Red elemental selenium (Se0 ) improves the immunoactivities of EPC cells, crucian carp and zebrafish against spring viraemia of carp virus.

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 µg ml-1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 µg ml-1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 µg g-1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 µg g-1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses.

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

The immune function of immune organs is extremely crucial for maintaining organism health status, which ultimately affects fish growth. Our previous study has found that dietary supplementation of (2-carboxyethyl)dimethylsulfonium Bromide (Br-DMPT) in non-fish meal (NFM) diet could promote the growth of grass carp (Ctenopharyngodon idella), whereas the underlying reason or mechanism for this results is largely unclear. Herein, we further explored whether dietary supplementation of Br-DMPT promoted fish growth is connected with the enhanced immune function in the immune organs (the head kidney, spleen and skin). In this study, 540 fish (216.49 ± 0.29 g) were irregularly distributed to six groups with three replicates (30 fish replicate-1) and fed corresponding diets group containing a fish meal (FM) diet group and five different NFM diets supplemented with gradational Br-DMPT (0-520.0 mg/kg level) group for 60 days. After the 60-days feeding trial, 8 fish from each replicate were selected and then conducted a challenge test with A. hydrophila for 14 days. Our results indicated that in the NFM diets, appropriate Br-DMPT: (1) significantly decreased the morbidity of skin haemorrhage and lesion after A. hydrophila infection (P < 0.05). (2) significantly improved the innate and adaptive immune components (lysozyme, complement 3, immunoglobulin M and antibacterial peptides et al.) (P < 0.05). (3) increased the gene expressions of main anti-inflammatory cytokines partially by referring to TOR signalling pathway, and decreased the gene expressions of main pro-inflammatory cytokines partially by referring to NF-kB signalling pathway (P < 0.05). Strikingly, no statistical difference could be found in the most of above immune parameters between 260.0 mg/kg Br-DMPT diet group and FM diet group (P > 0.05). Taken together, in non-fish meal diet, appropriate supplementation of Br-DMPT could improve the disease resistance capacity, non-specific immunity and ameliorate inflammation, and simultaneously could mitigate these adverse effects induced by the non-fish meal diet in fish immune organs. Moreover, this study may be helpful to decipher the underlying mechanisms of how Br-DMPT promote fish growth by immune organs and also provide scientific theoretical evidence for the future application of Br-DMPT as a new immunopotentiator in aquaculture industry.

Effects of dietary vitamin D3 on growth performance, antioxidant capacities and innate immune responses in juvenile black carp Mylopharyngodon piceus.

The aim of this experiment was used to investigate the effects of different contents of dietary vitamin D3 on the growth performance and antioxidant and innate immune responses in juvenile black carp Mylopharyngodon piceus. Black carp juveniles were fed six levels of dietary vitamin D3 (VD3) (96, 220, 412, 840, 1480, and 3008 IU/Kg) for 9 weeks. Results showed that highest weight gain (WG) and special growth ratio (SGR) were obtained at 534.2 IU/Kg dietary VD3 according to the second-order polynomial regression model. The protein efficiency ratio (PER) of black carp could be significantly increased by 412, 840, and 1480 IU/Kg dietary VD3 (p < 0.05), while the feed conversion ratio (FCR) were reduced by 412, 840, and 1480 IU/Kg dietary VD3 (p < 0.05). Adequate dietary VD3 content (412, 840, and 1480 IU/Kg) could significantly upregulate expression levels of lipoxygenase 5 (LPO 5); increase the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR); and improve GSH contents and total antioxidant capacities (T-AOC) in the liver of black carp. However, glutathione S-transferase (GST) activities and malondialdehyde (MDA) levels were significantly reduced by adequate dietary VD3 content (412, 840, and 1480 IU/Kg) in the fish liver. In addition, 412, 840, and 1480 IU/Kg dietary VD3 could significantly upregulate the mRNA expression levels of interferon-α (IFN-α), lysozyme (LYZ), hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), and complement component 3 (C3) and C9 in the hemocytes and liver of black carp juveniles compared with the VD3-deficient diet (96 IU/Kg). Meanwhile, higher contents of dietary VD3 could increase serum LYZ and ACP activities and C3 and C4 contents in black carp juveniles compared with the groups fed VD3-deficient diet. In conclusion, these results suggest that adequate dietary VD3 could increase growth performances, improve antioxidant capacities, and then enhance innate immune parameters in black carp juveniles.

Effects of dietary fern (Adiantum capillus-veneris) leaves powder on serum and mucus antioxidant defence, immunological responses, antimicrobial activity and growth performance of common carp (Cyprinus carpio) juveniles.

A 56-day research was performed to examine the influence of graded levels (0 (control), 0.5, 1 and 2%) of Fern (Adiantum capillus-veneris) leaves powder (FLP) in diet on immune competence and growth of common carp (Cyprinus carpio, initial weight = 20 g). The serum total immunoglobulins content and lysozyme activity in the 1 and 2% FLP groups remarkably increased compared to the other groups (P < 0.05). The skin mucosal lysozyme activity enhanced with increasing dietary FLP level in a dose-response manner. Fish fed on the FLP-supplemented diets had higher skin-mucosal superoxide dismutase activity than the control (P < 0.05). However, serum antioxidant enzymes were not affected by dietary fern (P > 0.05). The serum bactericidal activity against human and fish pathogens increased with enhancing the FLP level in diet against Staphylococcus aureus, Escherichia coli (EHEC ATCC 43895), Escherichia coli (CI), Pseudomonas aeruginosa, Klebsiella pneumonia and Aeromonas hydrophila. The serum antibacterial activity against Yersinia ruckeri in the 2% FLP group was higher than the other treatments. Furthermore, the serum bactericidal activity against P. aeruginosa (ATCC 27853) only observed in fish fed on the 1 and 2% FLP-supplemented diets. The skin mucosal bactericidal activity and inhibitory effects increased with enhancing the FLP level in diet against E. coli, K. pneumonia, Y. ruckeri and A. hydrophila in a dose response manner. Moreover, the skin mucosal bactericidal activity against S. aureus only observed in fish fed on 1 and 2% FLP-supplemented diets. The weight gain values in the 1 and 2% FLP groups were higher than the other treatments (P < 0.05). Feed conversion ratio (FCR) improved with increasing FLP level in diet in a dose-response manner (P < 0.05). By considering serum and mucosal bactericidal activities against different pathogenic bacteria, the supplementation of 2% FLP in diet is recommended for C. carpio during grow-out phase.

Amelioration of hexavalent chromium-induced bioaccumulation, oxidative stress, tight junction proteins and immune-related signaling factors by Allium mongolicum Regel flavonoids in Ctenopharyngodon idella.

Chromium (Cr) is the most common heavy metal and that becomes toxic when present at higher concentrations in aquatic environments. Allium mongolicum Regel flavonoids (AMRF) has been documented to possess detoxification, antioxidant and anti-inflammatory properties. The aim of this study was to explore the potential of dietary AMRF and Cr exposure on bioaccumulation, oxidative stress, and immune response in Ctenopharyngodon idella. After acclimation, 360 fish were randomly distributed into six groups. The fish were fed with diets supplemented with Cr and/or AMRF for 4 weeks (28 days), the Cr concentrations were 0, 120, and 240 mg/kg and the concentrations of AMRF were 0 or 40 mg/kg, respectively. The results shown that Cr accumulation in the kidney, liver, spleen, intestine and gill were significantly increased following Cr exposure, dietary AMRF supplementation attenuated the increased in Cr accumulation. Dietary AMRF supplementation significantly reduced the levels of malondialdehyde (MDA) and protein carbonyl (PC) in liver, spleen and gill compared with the same Cr dose groups. When fish were supplemented with AMRF significantly increased lysozyme activity (LZM), complement 3 (C3) in kidney and intestine compared with the same Cr dose groups. Serum glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were significantly increased following exposure to Cr. Dietary AMRF supplementation significantly decreased GOT and GPT activity in the serum. In addition, AMRF supplementation can decrease the expression of inflammatory (NF-κB p65, TNF-α and IL-1ß) and increased the expression of tight junction proteins (occludin and ZO-1) following Cr exposure in C. idella. These results indicate that AMRF has the potential to alleviate the effects of Cr toxicity in C. idella.

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.

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.