Lin Feng

Abstract We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia- positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.

The present research evaluated the effects of dietary tryptophan (Trp) on growth performance, intestinal mucosal immune, barrier function and antioxidant capacity and gene expression of young grass carp (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of Trp at 0.7(control), 1.7, 3.1, 4.0, 5.2 and 6.1 g kg−1 diet for 8 weeks. The results showed that Trp supplementation significantly enhanced the percent weight gain (PWG), feed intake and feed efficiency (P < 0.05), and decreased the plasma ammonia content (PAC) (P < 0.05). After the 8-week feeding trail, an environmental copper exposure trail was conducted for 4 days. Results from the copper exposure trail showed that dietary Trp enhanced the lysozyme, acid phosphatase activities and complement 3 contents in the intestine of young grass carp (P < 0.05). In addition, Trp supplementation increased the copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx) activities and glutathione contents (P < 0.05), and decreased the protein carbonyl and malondialdehyde contents (P < 0.05). Furthermore, the relative gene expression levels of interleukin 10, transforming growth factor-β1, occludin, zonula occludens 1, claudin-b, -c, and −3, SOD1, GPx and NF-E2-related factor 2 in the intestine were significantly up-regulated with increasing of dietary Trp up to a certain level (P < 0.05). Conversely, the mRNA levels of tumor necrosis factor α, interleukin 8, target of rapamycin, Kelch-like-ECH-associated protein 1, claudin-12 and -15a in the intestine were significantly down-regulated by Trp (P < 0.05). Collectively, appropriate dietary Trp level improves fish growth, intestinal immune response, barrier function and antioxidant status, and regulated the mRNA levels of related signal molecules of young grass carp. Based on the quadratic regression analysis of the PWG and PAC, the dietary Trp requirement of young grass carp (287–699 g) was estimated to be 3.81 g kg−1 diet (12.7 g kg−1 protein) and 3.89 g kg−1 diet (13.0 g kg−1 protein), respectively.

Growth performance, flesh quality, antioxidant status and antioxidant-related signalling molecule expression in the muscle of young grass carp, which were fed graded levels of arginine (6.9-24.5 g/kg diet) for eight weeks, were investigated. Muscle protein, lipid and nitric oxide contents, shear force, hydroxyproline concentration, and pH were significantly improved by appropriate arginine. Cooking loss, lactate content, cathepsins activities, malondialdehyde and protein carbonyl contents exhibited an opposite tendency. Additionally, optimum arginine significantly enhanced glutathione content and the activities and gene expression of copper/zinc superoxide dismutase, catalase and glutathione peroxidase in muscle. Moreover, the expression levels of glutamate-cysteine ligase, target of rapamycin, ribosome protein S6 kinase 1, casein kinase 2 and NF-E2-related factor 2 in muscle were significantly elevated by appropriate arginine. However, optimum arginine significantly decreased Kelch-like ECH-associated protein 1 mRNA levels in muscle. In conclusion, arginine improved the flesh quality and muscle antioxidant capacity and regulated antioxidant-related signalling molecule expression.

The present study evaluated the effect of dietary sodium butyrate (SB) supplementation on the growth and immune function in the proximal intestine (PI), middle intestine (MI) and distal intestine (DI) 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. The results indicated that optimal SB supplementation improved the fish growth performance (percent weight gain, specific growth rate, feed intake and feed efficiency) and intestinal growth and function (intestine weight, intestine length, intestinal somatic index, folds height, trypsin, chymotrypsin, lipase and amylase activities), increased beneficial bacteria lactobacillus amount and butyrate concentration, decreased baneful bacteria Aeromonas and Escherichia coli amounts, reduced acetate and propionate concentrations, elevated lysozyme and acid phosphatase activities, increased complement (C3 and C4) and immunoglobulin M contents, and up-regulated β-defensin-1 (rather than DI), hepcidin, liver expressed antimicrobial peptide 2B (LEAP-2B) (except LEAP-2A), Mucin2, interleukin 10 (IL-10), IL-11 (rather than PI), transforming growth factor β1 (rather than PI), transforming growth factor β2 (rather than PI), IL-4/13A, IL-4/13B and inhibitor of κBα (IκBα) mRNA levels, whereas it down-regulated tumor necrosis factor α, interferon γ2, IL-1β (rather than PI), IL-6, IL-8, IL-15 (rather than PI), IL-17D (rather than PI), IL-12p35, IL-12p40 (rather than PI or MI), nuclear factor kappa B p65 (NF-κB p65) (except NF-κB p52), c-Rel (rather than PI or MI), IκB kinase β (IKKβ) (rather than PI), IKKγ (except IKKα), p38 mitogen-activated protein kinase (p38MAPK) and MAPK kinase 6 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that SB supplementation improves growth and intestinal immune function of fish. Furthermore, according to the positive effect, MSB was superior to PSB on improving growth and enhancing intestinal immune function of fish, and based on feed efficiency of young grass carp, the efficacy of MSB was 3.5-fold higher than that of PSB. Finally, based on percent weight gain, protecting fish against enteritis morbidity and lysozyme activity, the optimal SB supplementation (MSB as SB source) of young grass carp were estimated to be 160.8, 339.9 and 316.2 mg kg−1 diet, respectively.

Abstract T cells expressing gamma delta TCR may have evolved to recognize Ag in a different manner as well as perform a broader set of functions than T cells with alpha beta TCR. In this study, we tested the hypothesis that dendritic epidermal T cells (DETC) bearing the invariant V gamma 3V delta 1 TCR may be able to signal the migration of peripheral alpha beta T cells to the epidermis by secreting specific chemokines. Expression of macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, RANTES, and lymphotactin was inducible in DETC 7-17 cells, whereas mRNA for monocyte chemoattractant protein (MCP)-1 could not be detected. Strikingly, lymphotactin was the most abundant chemokine produced by activated DETC 7-17 cells. Activated primary DETC cultures also produced copious amounts of lymphotactin mRNA. Similarly, freshly isolated and activated intestinal intraepithelial T cells (i-IEL) with gamma delta TCR expressed high levels of lymphotactin mRNA. In contrast, lymphotactin mRNA was present in activated spleen gamma delta T cells at low basal levels. Migration of CD8+ T cells induced by culture supernatants from stimulated DETC 7-17 cells was strongly reduced in the presence of a neutralizing anti-lymphotactin antiserum and to a lesser extent by neutralizing anti-MIP-1 alpha, anti-MIP-1 beta, or anti-RANTES antiserum. The presence of lymphotactin in supernatants from activated DETC 7-17 cultures was directly demonstrated by Western blot analysis. These observations are consistent with a model in which gamma delta IEL play an active multi-faceted role in the maintenance of epithelia homeostasis.

We prospectively studied 98 chronic myeloid leukemia (CML) patients after bone marrow transplantation by competitive polymerase chain reaction to detect and quantify leukemia-specific BCR-ABL mRNA. Of 69 patients who had persistently undetectable, decreasing, or low BCR-ABL levels ( &lt; 50 transcripts/microgram RNA) on sequential analysis, one (1%) subsequently relapsed. Of 29 patients who had increasing or persistently high BCR-ABL (&gt; 50 transcripts/microgram RNA) on sequential analysis, 21 (72%) have relapsed (P &lt; .00001). In 19 patients studied sequentially, a constant logarithmic increase in the number of BCR-ABL transcripts was found before relapse, indicating a constant BCR-ABL transcript doubling time. The doubling time for patients relapsing cytogenetically or into chronic phase (median, 24.7 days) was significantly longer than that of patients relapsing into advanced phases (median, 14.7 days; P = .005). Eight patients were treated for relapse by donor leukocyte transfusions. The doubling time of responders was significantly longer than that of nonresponders (P = .017). We conclude that quantification of BCR-ABL transcripts after bone marrow transplantation (BMT) is valuable in predicting relapse: a more rapid BCR-ABL transcript doubling time before relapse might indicate a more aggressive disease.

Gene expression microarrays are being used to develop new prognostic and predictive tests for breast cancer, and might be used at the same time to confirm oestrogen-receptor status and ERBB2 status. Our goal was to establish a new method to assign oestrogen receptor and ERBB2-receptor status to breast carcinoma based on mRNA expression measured using Affymetrix U133A gene-expression profiling. We used gene expression data of 495 breast cancer samples to assess the correlation between oestrogen receptor (ESR1) and ERBB2 mRNA and clinical status of these genes (as established by immunohistochemical [IHC] or fluorescence in-situ hybridisation [FISH], or both). Data from 195 fine-needle aspiration (FNA) samples were used to define mRNA cutoff values that assign receptor status. We assessed the accuracy of these cutoffs in two independent datasets: 123 FNA samples and 177 tissue samples (ie, resected or core-needle biopsied tissues). Profiling was done at two institutions by use of the same platform (Affymetrix U133A GeneChip). All data were uniformly normalised with dCHIP software. ESR1 and ERBB2 mRNA levels correlated closely with routine measurements for receptor status in all three datasets. Spearman's correlation coefficients ranged from 0·62 to 0·77. An ESR1 mRNA cutoff value of 500 identified oestrogen-receptor-positive status with an overall accuracy of 90% (training set), 88% (first validation set), and 96% (second validation set). An ERBB2 mRNA threshold of 1150 identified ERBB2-positive status with the overall accuracy of 93% (training set), 89% (first validation set), and 90% (second validation set). Reproducibility of mRNA measurements in 34 replicate experiments was high (correlation coefficient 0·975 for ESR1, 0·984 for ERBB2). Amounts of ESR1 and ERBB2 mRNA as measured by the Affymetrix GeneChip reliably and reproducibly establish oestrogen-receptor status and ERBB2 status, respectively.

The brain is the center of the nervous system in all vertebrates, and homeostasis of the brain is crucial for fish survival. Copper (Cu) is essential for normal cellular processes in most eukaryotic organisms but is toxic in excess. Although Cu is indicated as a potent neurotoxicant, information regarding its threat to fish brain and underlying mechanisms is still scarce. In accordance, the objective of this study was to assess the effects and the potential mechanism of Cu toxicity by evaluating brain oxidative status, the enzymatic and mRNA levels of antioxidant genes, as well as the Nrf2/ARE signaling in the brain of fish after Cu exposure. The protective effects of myo-inositol (MI) against subsequent Cu exposure were also investigated. The results indicate that induction of oxidative stress by Cu is shown by increases in brain ROS production, lipid peroxidation and protein oxidation, which are accompanied by depletions of antioxidants, including total superoxide dismutase (T-SOD), CuZnSOD, glutathione-S-transferase (GST) and glutathione reductase (GR) activities and glutathione (GSH) content. Cu exposure increased the catalase (CAT) and glutathione peroxidase (GPx) activities. Further molecular results showed that Cu exposure up-regulated CuZnSOD, GPx1a and GR mRNA levels, suggesting an adaptive mechanism against stress. Moreover, Cu exposure increased fish brain Nrf2 nuclear accumulation and increased its ability of binding to ARE (CuZnSOD), which supported the increased CuZnSOD mRNA levels. In addition, Cu exposure caused increases of the expression of the Nrf2, Maf G1 (rather than Maf G2 gene) and PKCd genes, suggesting that de novo synthesis of those factors is required for the protracted induction of such antioxidant genes. However, the modulation of Keap1a (rather than Keap1b) of fish brain under Cu exposure might be used to turn off of the signaling cascade and avoid harmful effects. Interestingly, pre-treatment of fish with MI prevented the fish brain from Cu-induced oxidative damages mainly by increasing the GSH content and CuZnSOD and GST activities. Summarily, this study indicates that although Cu stimulates adaptive increases in the expression of some antioxidant enzyme genes through Nrf2/ARE signaling, it also induces oxidation and the depletion of most of antioxidant enzyme activities and GSH content due to the increase of ROS production, and MI protects the fish brain against Cu toxicity.

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

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2(V617F)-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10(-10)) and rs2201862 (MECOM; meta-analysis P=1.96 × 10(-9)). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2(V617F)-positive cases. rs9376092 has a stronger effect in JAK2(V617F)-negative cases with CALR and/or MPL mutations (Breslow-Day P=4.5 × 10(-7)), whereas in JAK2(V617F)-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ(2) P=7.3 × 10(-7)). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype.

The muscle is the main portion of fish that is consumed by humans. Copper (Cu) can induce oxidative damage in fish muscle. However, the effects of Cu exposure on the muscle antioxidant system and molecular patterns and preventive measures against these effects remain unclear. In this study, ROS production, enzymatic and mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2) signaling-related molecules, antioxidant response element (ARE) binding ability, DNA fragmentation and caspase-3 activities were analyzed in fish muscle following Cu exposure or myo-inositol (MI) pre-administration. The results indicated that contamination due to copper exposure caused an approximately three-fold increase in ROS production, induced lipid peroxidation and protein oxidation, and resulted in depletion of the glutathione (GSH) content of fish muscle. Moreover, Cu exposure caused decreases in the activities of total superoxide dismutase (T-SOD), CuZnSOD, and glutathione peroxidase (GPx) that were accompanied by decreases in CuZnSOD, GPx1a, GPx1b and signaling factor protein kinase C delta mRNA levels. The decreases in the antioxidant enzyme gene mRNA levels were confirmed to be partly due to the reduced nuclear Nrf2 protein levels, poor ARE binding ability and increased caspase-3 signaling-modulated DNA fragmentation in the fish muscle. Interestingly, MI pre-treatment prevented fish muscle from Cu-induced oxidative damages mainly through increasing the GSH content, and increasing the CuZnSOD and GPx activities and corresponding mRNA levels and ARE binding ability. Taken together, our results show for the first time that Cu exposure caused oxidative damage to the muscle by decreasing the antioxidant enzyme activities via the down-regulation of the expression of genes related to the disruption of the Nrf2/ARE signaling, and this down-regulation was partially caused by caspase-3-regulated DNA fragmentation. Finally, MI protects fish against Cu toxicity.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.

The pro-inflammatory functions of NF-kappaB must be tightly regulated to prevent inappropriate tissue damage and remodelling caused by activated inflammatory and wound-healing cells. The p50 subunit of NF-kappaB is emerging as an important repressor of immune and inflammatory responses, but by mechanisms that are poorly defined. This study aims to delineate p50 target genes in activated hepatic stellate cells and to outline mechanisms utilised in their repression.Hepatic stellate cells were isolated from nfkb1(p50)-deficient or Wt mice and gene expression compared using microarray. Target genes were verified by qRT-PCR and p50-mediated HDAC-1 recruitment to the target genes demonstrated using chromatin immunoprecipitation.We identify p50 as transcriptional repressor of multiple pro-inflammatory genes including Ccl2, Cxcl10, Gm-csf, and Mmp-13. These genes are over-expressed in nfkb1(p50)-deficient mice suffering from chronic hepatitis and in fibrogenic/inflammatory hepatic stellate cells isolated from nfkb1(-/-) liver. We identify Mmp-13 as a bona-fide target gene for p50 and demonstrate that p50 is required for recruitment of the transcriptional repressor histone deacetylase (HDAC)-1 to kappaB sites in the Mmp-13 promoter. Chromatin immunoprecipitations identified binding of HDAC-1 to specific regulatory regions of the Ccl2, Cxcl10, Gm-csf genes that contain predicted kappaB binding motifs. Recruitment of HDAC-1 to these genes was not observed in nfkb1(-/-) cells suggesting a requirement for p50 in a manner similar to that described for Mmp-13.Recruitment of HDAC-1 to inflammatory genes provides a widespread mechanism to explain the immunosuppressive properties of p50.

The present study explored the effects of glutamine on hydrogen peroxide (H2O2)-induced oxidative stress in isolated carp enterocytes. In order to select an optimal H2O2 concentration to induce oxidative stress in enterocytes, cultures were treated with different concentrations of H2O2 (0–100 μM) for 16 h. The results showed that exposure to H2O2 increased lactate dehydrogenase activity and malondialdehyde levels in a dose-dependent manner in the culture medium, suggesting increase toxicity of H2O2. Thus, 100 μM was an appropriate concentration for inducing oxidative stress. We then examined the cytoprotective effects of glutamine under conditions of oxidative stress. Cells were treated with glutamine (0–20 mM) in the presence of H2O2 (100 μM) for 16 h. The control cells were kept in the glutamine-free MEM only. Results showed that glutamine completely blocked H2O2-stimulated release of lactate dehydrogenase. Furthermore, glutamine reduced H2O2-induced increase in malondialdehyde level and protein carbonyls content to the level seen in the control culture. In addition, glutamine treatment completely prevented the decrease in Na+-K+-ATPase, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase activities as well as reduced glutathione concentration and the ratio between reduced and oxidized glutathione induced by H2O2. No significant difference was observed between cells incubated with glutamine in the presence of H2O2 and control cells in values for most of the markers mentioned above. Complete protection was obtained at a concentration of glutamine as small as 4 mM. The present results indicate that glutamine is effective in protecting against H2O2-induced oxidative stress in carp intestinal epithelial cells.

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

This study was conducted to investigate the effects of dietary isoleucine (Ile) on the immune response, antioxidant status, tight junctions, and microbial population in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with average initial weight 6.9 ± 0.03 g were fed semi-purified isonitrogenous diets containing 4.2 (unsupplemented control group), 7.0, 9.5, 11.9, 13.9 and 16.9 g Ile kg(-1) diet for 60 days. Results indicated that Ile supplementation decreased malondialdehyde (MDA) and protein carbonyl content, and the amounts of Escherichia coli and Aeromonas in the intestine (P < 0.05), and increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), glutathione content and the amounts of Lactobacillus and Bacillus in the intestine (P < 0.05). Furthermore, real time polymerase chain reaction revealed that relative mRNA expression of copper/zinc superoxide dismutase (Cu-ZnSOD), manganese superoxide dismutase (MnSOD), CAT, NF-E2-related factor 2 (Nrf2), p38 mitogen-activated protein kinases (p38MAPK) in the intestine were increased with increasing of dietary Ile up to a certain point (P < 0.05). Conversely, the relative mRNA expression of occludin, claudin-3, claudin-7, TNF-α, IL-10, Kelch-like-ECH- associated protein 1 (Keap1), extracellular signal-regulated kinase 1 (ERK1) in the intestine showed a downward trend (P < 0.05). In conclusion, dietary Ile improves intestinal immune function, antioxidant capacity and microbial population, and regulates gene expression of antioxidant enzyme, tight junctions, Nrf2, Keap1, p38 and ERK1 in the intestine of Jian carp.

A 105-day feeding trial was carried out to investigate the effects of dietary curcumin (CM) supplementation on growth performance, digestive enzyme activities, and intestinal antioxidant capacity of crucian carp (Carassius auratus). A total of 585 crucian carp with average initial weight of 76.3 ± 0.10 g were fed three diets supplemented with 0 (basal group), 1, or 5 g kg− 1 CM. Fish fed the diet supplemented with 5 g kg− 1 CM showed significantly higher final body weight (FW), percent weight gain (PWG), and feed efficiency (FE) compares to those fed the basal or 1 g kg− 1 CM diet (P < 0.05). Feed intake and body composition were not significantly different among dietary groups (P > 0.05). The hepatopancreas weight, hepatopancreas protein content, intestinal weight, intestinal somatic index, intestine protein content, trypsin and lipase activities in hepatopancreas and intestine, amylase activity in hepatopancreas, Na+, K+-ATPase (NKA), alkaline phosphatase (AKP), gamma-glutamyl transpeptidase (γ-GT), and creatine kinase (CK) activities in intestine were significantly increased with dietary CM supplementation at 5 g kg− 1 (P < 0.05). Fish fed the 5 g kg− 1 CM diet showed significantly lower malondialdehyde and protein carbonyl contents in the intestine (P < 0.05). Superoxide anion and hydroxy radical scavenging ability, glutathione reducase (GR), catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) activities, and glutathione (GSH) content in the intestine were significantly increased with increased CM. The relative mRNA expressions of trypsin, lipase, NKA, AKP, γ-GT, CK, SOD1, CAT, GPx, GST, and GR in intestine were significantly up-regulated by dietary CM supplementation at 5 g kg− 1. These results suggested that dietary CM supplementation increased intestinal antioxidant capacity, digestive and absorptive ability, and promoted fish growth.

The effects of low salt and sugar dry-curing on the quality changes of black carp (Mylopharyngodon piceus) fillets stored at 4 °C were evaluated by sensory, physical, chemical, and microbiological methods. Fish samples were left untreated (control), or were dry-cured with 1.5% salt (T1) or 1.5% salt + 1.2% sugar (T2). Curing treatments reduced chemical changes reflected in HxR, Hx, pH, and total volatile base nitrogen (TVB-N); decreased cooking loss; and increased overall sensory quality of fish (p < 0.05) compared to untreated samples. Significantly lower values of cadaverine and putrescine were observed in T1 and T2 compared to the control after the 2nd and 4th day, respectively (p < 0.05). There were significant differences (p < 0.05) between T1 and T2 for pH, TVB-N, total aerobic counts (TAC), and sensory characteristics. Sensory characteristics were significantly correlated with TAC, TVB-N, putrescine, and cadaverine in all samples (p < 0.01).

β-conglycinin has been identified as one of the major feed allergens. However, studies of β-conglycinin on fish are scarce. This study investigated the effects of β-conglycinin on the growth, digestive and absorptive ability, inflammatory response, oxidative status and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and their enterocytes in vitro. The results indicated that the specific growth rate (SGR), feed intake, and feed efficiency were reduced by β-conglycinin. In addition, activities of trypsin, chymotrypsin, lipase, creatine kinase, Na+,K+-ATPase and alkaline phosphatase in the intestine showed similar tendencies. The protein content of the hepatopancreas and intestines, and the weight and length of the intestines were all reduced by β-conglycinin. β-conglycinin increased lipid and protein oxidation in the detected tissues and cells. However, β-conglycinin decreased superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the intestine and enterocytes. Similar antioxidant activity in the hepatopancreas was observed, except for GST. The expression of target of rapamycin (TOR) gene was reduced by β-conglycinin. Furthermore, mRNA levels of interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) genes were increased by β-conglycinin. However, β-conglycinin increased CuZnSOD, MnSOD, CAT, and GPx1b gene expression. In conclusion, this study indicates that β-conglycinin induces inflammation and oxidation, and causes dysfunction of intestinal digestion and absorption in fish, and finally reduces fish growth. The results of this study provide some information to the mechanism of β-conglycinin-induced negative effects.

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

Flesh quality, muscle antioxidant status and related signalling molecule expressions were investigated in young grass carp fed six levels of tryptophan (Trp) for 8 weeks. The results indicated that fish fed 0.7 (deficiency) and 6.1 g Trp g/kg (excess) diets exhibited lower muscle water-holding capacity, tenderness, cathepsin activity, protein levels, lipids and collagen contents. Optimal Trp reversed these negative effects, which were related to enhanced glutathione (GSH) content and glutathione peroxidase (GPx) activities regulated at gene transcription levels, rather than to superoxide dismutase (SOD) or catalase (CAT). The expression of signalling molecules [Kelch-like ECH-associated protein 1, target of rapamycin (TOR) and ribosomal S6 protein kinase 1] involved in the NF-E2-related factor 2 (Nrf2) pathway revealed a potential method of Trp-enhanced antioxidant defence. Collectively, the present study indicated that appropriate Trp levels improved flesh quality partly related to the enhancement of antioxidant ability through Nrf2 and TOR signalling.

This study was conducted to evaluate the effects of dietary isoleucine (Ile) on the immune response, antioxidant status and gene expression in the head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Six semi-purified isonitrogenous diets (4.2, 7.0, 9.5, 11.9, 13.9 and 16.9 g Ile kg−1 diet) were fed to Jian carp (6.9 ± 0.03 g) for 60 days. The results showed that Ile supplementation improved the head kidney index, red and white blood cell counts, anti-hydroxyl radical capacity and the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase (P < 0.05), and decreased the malondialdehyde, protein carbonyl and glutathione contents in the head kidney (P < 0.05). After a 60 day feeding trial, an Aeromonas hydrophila challenge study was conducted for 17 days. Differences in survival rate, leucocyte phagocytic activity, serum lysozyme activity, acid phosphatase activity, haemagglutination titre, complement components 3 and 4, immunoglobulin M level and A. hydrophila agglutination antibody titre followed the same trend as that of the head kidney index (P < 0.05). Furthermore, real time polymerase chain reaction revealed that relative mRNA expression of transforming growth factor β2 and target of rapamycin (TOR) in the head kidney significantly increased with increasing Ile levels (P < 0.05). Conversely, the relative mRNA expression of tumour necrosis factor α, interleukin 10 and eIF4E-binding protein (4E-BP) in the head kidney showed a downward trend (P < 0.05). Collectively, this study indicates that dietary Ile improves the fish immune response, regulates the antioxidant status and cytokine, TOR and 4E-BP gene expression in the head kidney.

Small ubiquitin-like modifier (SUMO) modification has emerged as an important regulatory mechanism during embryonic development. However, it is not known whether SUMOylation plays a role in the development of the immune system. Here, we show that SUMO-specific protease 1 (SENP1) is essential for the development of early T and B cells. STAT5, a key regulator of lymphoid development, is modified by SUMO-2 and is specifically regulated by SENP1. In the absence of SENP1, SUMO-2 modified STAT5 accumulates in early lymphoid precursors, resulting in a block in its acetylation and subsequent signaling. These results demonstrate a crucial role of SENP1 in the regulation of STAT5 activation during early lymphoid development.

Immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of methionine hydroxy analogue (MHA) (0, 5.1, 7.6, 10.2, 12.7, 15.3 g kg−1 diet) for 60 days were investigated. Results indicated that head kidney index, spleen index, red and white blood cell counts significantly increased with increasing MHA levels up to a point (P < 0.05), whereupon decreased (P < 0.05). Glutathione reductase activity in head kidney and spleen, anti-hydroxy radical and glutathione-S-transferase activities in spleen, catalase activity and GSH content in head kidney significantly increased by MHA supplement, while malondialdehyde content, anti-superoxide anion, superoxide dismutase, glutathione peroxidase activities in head kidney and spleen, protein carbonyl content and catalase activity in spleen, anti-hydroxy radical activity in head kidney significantly decreased by MHA supplement. However, protein carbonyl content and glutathione-S-transferase activity in head kidney, GSH content in spleen remained unaffected. After 60-day feeding trial, a challenge study was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that survival rate, leukocytes phagocytic activity, lysozyme activity, acid phosphatase activity, total iron-binding capacity, haemagglutination titre, complement 3, 4 and immunoglobulin M contents significantly increased by optimal dietary MHA supplement (P < 0.05). These data suggested that MHA affected antioxidant status of immune organs and promoted immune response in juvenile Jian carp.

The aim of the study was to evaluate the effects of dietary leucine on growth performance, intestinal antioxidant status and relative expression of Nrf2 and its target genes in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (295.85 ± 2.07 g) were fed six isonitrogenous diets with graded levels of leucine (7.1, 8.9, 11.0, 13.3, 15.2 and 17.1 g kg− 1 diet) for 8 weeks. Results indicated that percent weight gain (PWG), feed intake, feed efficiency, intestine length, intestine weight and intestine protein content were significantly improved by dietary leucine. Similarly, optimum leucine supplementation significantly increased intestinal glutathione content, copper/zinc superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPx) activities as compared with the control group. The changes in intestinal CuZnSOD, GPx and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels induced by leucine were similar to those noted in the enzyme activities. Plasma ammonia concentration (PAC), intestinal malondialdehyde (MDA) and protein carbonyl contents, as well as the mRNA level of Kelch-like ECH-associated protein 1 (Keap1) exhibited an opposite tendency. Interestingly, dietary leucine level higher than 13.3 or 15.2 g kg− 1 diet has adverse effect on intestinal antioxidant status. Collectively, our results indicated that optimum leucine supplementation improved fish growth and antioxidant capacity, which may be partly related to the Nrf2 signalling pathway. Based on PWG, PAC and intestinal MDA, the optimum dietary leucine requirements of young grass carp (296–690 g) were estimated to be 13.0, 12.9 and 13.2 g kg− 1 diet, corresponding to 42.4, 42.0 and 43.0 g kg− 1 of dietary protein, respectively.

The present work evaluates the effects of various levels of dietary choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed with six different experimental diets containing graded levels of choline at 165 (choline-deficient control), 310, 607, 896, 1167 and 1820 mg kg(-1) diet for 65 days. At the end of the feeding trail, fish were challenged with Aeromonas hydrophila and mortalities were recorded over 17 days. Dietary choline significantly decreased malondialdehyde and protein carbonyl contents in spleen and head kidney. However, anti-superoxide anion and anti-hydroxyl radical activities in spleen and head kidney also decreased. Interestingly, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) in spleen, GPx activity in head kidney, and glutathione contents in spleen and head kidney were decreased with increase of dietary choline levels up to a certain point, whereas, activities of SOD, GST and GR in head kidney showed no significantly differences among groups. Similarly, expression levels of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR gene in spleen and head kidney were significantly lower in group with choline level of 607 mg kg(-1) diet than those in the choline-deficient group. The relative gene expressions of Nrf2 in head kidney and Keap1a in spleen and head kidney were decreased with increasing of dietary choline up to a certain point. However, the relative gene expression of Nrf2 in spleen were not significantly affected by dietary choline. In conclusion, dietary choline decreased the oxidant damage and regulated the antioxidant system in immune organs of juvenile Jian carp.

The present research studied the effects of dietary isoleucine (Ile) on growth performance, the digestion and absorption capacity, as well as gene expression in hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp (Cyprinus carpio var. Jian) (6.9 ± 0.03 g) were randomly distributed into six groups with four replicates each, fed semi-purified isonitrogenous diets (335.8 g crude protein/kg diet) containing graded levels of Ile (4.2, 7.0, 9.5, 11.9, 13.9 and 16.9 g/kg diets) for 60 days. The relative expression of gene was performed by real-time quantitative PCR. Compared with the control group, Ile supplementation increased (P < 0.05): (1) specific growth rate (SGR) and feed intake, (2) body protein content and protein retention value, (3) intestine fold height, (4) activities of trypsin, chymotrypsin, lipase and amylase in hepatopancreas and intestine, (5) activities of alkaline phosphatase in distal intestine, Na+/K+-ATPase and γ-glutamyl transpeptidase in intestine, (6) glutamate–oxaloacetate transaminase activity in hepatopancreas, and (7) relative mRNA expression of chymotrypsin, lipase and amylase in hepatopancreas, γ-glutamyl transpeptidase in mid intestine, Na+/K+-ATPase in intestine and target of rapamycin (TOR) in hepatopancreas and mid intestine. However, Ile supplementation decreased (P < 0.05): (1) feed conversion ratio, (2) glutamate–pyruvate transaminase activity in hepatopancreas, and (3) relative mRNA expression of trypsin in hepatopancreas, alkaline phosphatase in intestine, γ-glutamyl transpeptidase in distal intestine and eIF4E-binding protein (4E-BP) in hepatopancreas, proximal and mid intestine. Collectively, this study indicated that dietary Ile improves fish growth, promotes the digestive and absorptive ability and regulates gene expression of the digestive and brush border enzymes, TOR and 4E-BP. Based on the quadratic regression analysis of SGR, the Ile requirement of juvenile Jian carp (6.90–63.39 g) was estimated to be a 12.9 g/kg diet, corresponding to 38.4 g/kg of dietary protein.

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

Abstract Background Glioma-associated microglia/macrophages (GAMs) comprise macrophages of peripheral origin and brain-intrinsic microglia, which support tumor progression. Chemokine C-C ligand 5 (CCL5) is an inflammatory mediator produced by immune cells and is involved in tumor growth and migration in several cancers, including glioma. However, the mechanisms detailing how CCL5 facilitates glioma invasion remain largely unresolved. Methods Glioma migration and invasion were determined by wound healing, transwell assay, and 3D µ-slide chemotaxis assay. The expression levels of CCL5, CD68, matrix metalloproteinase 2 (MMP2), phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII), p-Akt, and phosphorylated proline-rich tyrosine kinase 2 were determined by cytokine array, quantitative PCR, western blot, or immunohistochemistry. Zymography and intracellular calcium assays were used to analyze MMP2 activity and intracellular calcium levels, respectively. Results CCL5 modulated the migratory and invasive activities of human glioma cells in association with MMP2 expression. In response to CCL5, glioma cells underwent a synchronized increase in intracellular calcium levels and p-CaMKII and p-Akt expression levels. CCL5-directed glioma invasion and increases in MMP2 were suppressed after inhibition of p-CaMKII. Glioma cells tended to migrate toward GAM-conditioned media activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in which CCL5 was abundant. This homing effect was associated with MMP2 upregulation, and could be ameliorated either by controlling intracellular and extracellular calcium levels or by CCL5 antagonism. Clinical results also revealed the associations between CCL5 and GAM activation. Conclusion Our results suggest that modulation of glioma CaMKII may restrict the effect of CCL5 on glioma invasion and could be a potential therapeutic target for alleviating glioma growth.

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

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

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

Growth, muscle composition, meat quality characteristics and antioxidant capacity in muscle of young grass carp (Ctenopharyngodon idella) (initial weight 282.9 ± 3.3 g) fed graded levels of phosphorus (1.0, 2.5, 3.8, 5.6, 7.8 and 9.5 g/kg diet) for 8 wk were investigated. Results indicated that percentage weight gain, feed intake, feed efficiency, serum phosphorus and alkaline phosphatase were improved with optimal phosphorus supplementations (P < 0.05). Muscle protein content and water holding capacity were significantly elevated, while moisture, lipid and ash contents were significantly decreased with dietary phosphorus to a certain level (P < 0.05). The meat shear force value and hydroxyproline content were not influenced by different levels of phosphorus. Muscle anti-hydroxyl radical, superoxide dismutase, catalase, glutathione S-transferase activities and glutathione content were significantly improved (P < 0.05). Conversely, anti-superoxide anion, glutathione reducase and glutathione peroxidase activities were decreased (P < 0.05) with dietary phosphorus to a certain level. These results indicated that suitable dietary phosphorus improved growth performance, meat quality and muscle antioxidant capacity. Dietary available phosphorus requirement of young grass carp for percentage weight gain was 4.0 g/kg diet.

Growth performance, digestive and absorptive capacities and target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and eIF4E-binding protein (4E-BP) gene expression in the hepatopancreas and intestine of juvenile grass carp (Ctenopharyngodon idellus) fed graded ratios of dietary alpha-linolenic acid/linoleic acid (ALA/LNA) (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days were investigated. The results showed that ALA/LNA ratio of 1.03 significantly improved (i) per cent weight gain (PWG) and feed efficiency, (ii) hepatopancreatic trypsin, chymotrypsin, lipase, amylase and intestinal creatine kinase (CK) activities, (iii) hepatopancreatic trypsinogen-2 and chymotrypsinogen mRNA levels. Meanwhile, fish fed with ALA/LNA ratio of 0.68 significantly enhanced, (iv) Na+/K+-ATPase and γ-glutamyl transpeptidase activities in whole intestine, and alkaline phosphatase activities in the proximal intestine (PI) and distal intestine, (v) amylase, intestinal Na+/K+-ATPase alpha-subunit isoform 1, Na+/K+-ATPase alpha-subunit isoform 8 and CK mRNA abundances, (vi) TOR and S6K1 gene expression in the hepatopancreas and intestine of juvenile grass carp. Based on the quadratic regression analysis of PWG, cholecystokinin and leptin contents in the PI, optimal dietary ALA/LNA ratio of juvenile grass carp (8.78–72.00 g) was estimated to be 1.08, 1.19 and 1.05, respectively.

Abstract 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 β-defensin1 (except in the PI) ( P &lt;0·05); (2) could up-regulate intestinal pro-inflammatory cytokines TNF-α , IL-1β , 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 ) -β 1, TGF-β2 , 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.

This study was conducted to investigate the effects of dietary phospholipids (PL) on the growth performance, intestinal enzyme activity and immune response and intestinal physical barrier of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juvenile grass carp with an average initial weight of 9.34 ± 0.03 g were fed six semi-purified diets containing 0.40% (unsupplemented control group), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% PL for 2 months. Results indicated that 3.29% PL increased lysozyme (LZ) and acid phosphatase (ACP) activities and complement component 3 (C3) content (P < 0.05), up-regulated the mRNA relative expression levels of interleukin 10, transforming growth factor β1 (TGF-β1), inhibitor protein κBα (IκBα), target of rapamycin (TOR) and casein kinase 2 (CK2) (P < 0.05), and down-regulated tumor necrosis factor α (TNF-α), interleukin 1β, nuclear factor κB p65 (NF-κB p65), IκB kinase β (IKKβ) and IκB kinase γ (IKKγ) mRNA relative expression levels (P < 0.05) in the intestine, suggesting that optimum PL could improve fish intestinal immunity. In addition, 3.29% PL increased the activities of anti-superoxide anion (ASA), anti-hydroxyl radical, copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx) and glutathione reductase (GR), the content of glutathione (P < 0.05), and the mRNA relative expression levels of occludin, zonula occludens 1 (ZO-1), claudin 3, claudin 12, claudin b, claudin c, SOD1, GPx, GR and NF-E2-related factor 2 (Nrf2) and decreased malondialdehyde (MDA), protein carbonyl (PC) and ROS content (P < 0.05), the mRNA relative expression levels of Kelch-like-ECH-associated protein 1a (Keap1a), myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) in the intestine, indicating that the optimum PL could improve fish intestinal physical barrier. Finally, based on the PWG, C3 content in the DI, ACP activity in the DI, intestinal PC content and intestinal ASA activity, the optimal dietary PL levels for juvenile grass carp (9.34-87.50 g) were estimated to be 3.46%, 3.79%, 3.93%, 3.72%, and 4.12%, respectively.

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

Our study investigated the effects of dietary zinc (Zn) deficiency on growth performance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (244.14 ± 0.40 g) were fed graded levels of zinc lactate (10.71, 30.21, 49.84, 72.31, 92.56, 110.78 mg Zn/kg diet) and one zinc sulfate group (56.9 mg Zn/kg diet) for 60 days. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. These results indicated that compared with optimal dietary Zn level, dietary Zn deficiency (10.71 mg/kg diet) decreased the production of antibacterial compounds, up-regulated pro-inflammatory cytokines related to nuclear factor kappa B (NF-κB) and down-regulated anti-inflammatory cytokines related to target of rapamycin (TOR) in three intestinal segments of young grass carp (P < 0.05), suggesting that dietary Zn deficiency could impair intestinal immune barrier of fish; decreased the activities and mRNA levels of antioxidant enzymes related to NF-E2-related factor 2 (Nrf2), up-regulated the mRNA levels of caspase-3, -7, -8, -9 related to p38 mitogen activated protein (p38 MAPK) and c-Jun N-terminal protein kinase (JNK), down-regulated the mRNA levels of tight junction complexes (TJs) related to myosin light chain kinase (MLCK) in three intestinal segments of young grass carp (P < 0.05), demonstrating that dietary Zn deficiency could injury intestinal physical barrier of fish. Besides, the Zn requirements (zinc lactate as Zn source) based on percent weight gain (PWG), against enteritis morbidity, acid phosphatase (ACP) activity in the proximal intestine (PI) and malondialdehyde (MDA) content in the PI of young grass carp was estimated to be 61.2, 61.4, 69.2 and 69.5 mg/kg diet, respectively. Finally, based on specific growth rate (SGR), feed efficiency (FE) and against enteritis morbidity of young grass carp, the efficacy of zinc lactate relative to zinc sulfate were 132.59%, 135.27% and 154.04%, respectively.

Flesh quality, amino acid and fatty acid composition, antioxidant status and related molecule expression in fish muscle were estimated by feeding grass carp with diets containing 3.65-27.86mg/kg diet of manganese (Mn) for 8weeks. Results demonstrated that optimal Mn increased toughness, collagen content, and pH, and decreased the cooking loss, and cathepsin B and L activities to enhance the flesh quality of fish. Meanwhile, optimal Mn increased the protein, lipid, the total essential amino acid (AA) (especially umami AA), and healthcare fatty acids, C18: 1c+t, C20: 3n-3, C20: 4 and DHA contents. These might be partially related to the decreased lipid peroxidation and protein oxidation, and the enhanced activities of Mn superoxide dismutase (MnSOD), catalase (CAT) and glutathione peroxidase (GPx) modulated by their gene expression, Nrf2 and TOR signaling. We firstly demonstrated that Mn improved flesh quality, flavor and healthcare function in fish muscle.

Our study explored the effect of dietary lipids on growth and immunity and structure (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ± 0.53 g were fed diets containing six graded levels of lipids at 5.9–80.1 g/kg diet for 8 weeks. After that, a challenge trial was conducted by injection of Aeromonas hydrophila over 2 weeks. The results indicated that compared with optimal lipids supplementation, low and excess levels of lipids down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα) and ribosomal p70S6 kinase (S6K1), and up-regulated pro-inflammatory cytokines, nuclear factor κB p65 (NF-κB p65), NF-κB c-Rel (not p52), IκB kinase α (IKKα), IKKβ, IKKγ, and eIF4E-binding protein (4EBP) mRNA levels in the head kidney and spleen of young grass carp (P < 0.05). Low or excess levels of lipids also increased reactive oxygen species (ROS) production and malondialdehyde (MDA) and protein carbonyl (PC) contents, reduced the activities of antioxidant enzymes (P < 0.05), down-regulate the relative mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2), and up-regulated the expression levels of Kelch-like ECH-associating protein 1a (Keap1a) and Keap1b in the head kidney and spleen. In addition, low or excess levels of lipids down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) in the head kidney and spleen, whereas up-regulated the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), caspase 3, 7, 8 and 9 mRNA levels in the head kidney and spleen and Fas ligand (FasL) mRNA levels in the spleen of young grass carp, suggesting that low or excess levels of lipids could decrease the head kidney and spleen immune function, induce oxidative damage and apoptosis and impair antioxidant system of young grass carp. At last, low or excess levels of lipids also impaired the immune function and structure in the skin of young grass carp. Based on the quadratic regression analysis for PWG, skin haemorrhage and lesions morbidity and IgM content, the dietary lipids requirements for young grass carp were estimated to be 43.7, 60.2, 55.0 and 52.1 g/kg diet, respectively.

This study investigated the effects of dietary vitamin E on growth, disease resistance and the immunity and structural integrity of head kidney, spleen and skin in grass carp (Ctenopharyngodon idella). The fish were fed six diets containing graded levels of vitamin E (0, 45, 90, 135, 180 and 225 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. The results showed that compared with optimal vitamin E supplementation, vitamin E deficiency caused depressed growth, poor survival rates and increased skin lesion morbidity in grass carp. Meanwhile, vitamin E deficiency decreased lysozyme and acid phosphatase activities, complement component 3 and complement component 4 contents in the head kidney, spleen and skin of grass carp (P < 0.05). Moreover, vitamin E deficiency down-regulated antimicrobial peptides (Hepcidin, liver-expressed antimicrobial peptide-2A, -2B, β-defensin), IL-10, TGFβ1, IκBα, TOR and S6K1 mRNA levels (P < 0.05) and up-regulated IL-1β, IL-6, IL-8, IFN-γ2 and TNFα, NF-κB p65, IKKα, IKKβ and 4EBP1 (not in the head kidney) mRNA levels (P < 0.05). In addition, vitamin E deficiency caused oxidative damage, decreased superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) activities, and down-regulated the mRNA levels of antioxidant enzymes and signaling molecules Nrf2 (P < 0.05). Vitamin E deficiency also induced apoptosis by up-regulating capase-2, -3, -7, and -8 mRNA levels in the head kidney, spleen and skin of grass carp. In conclusion, this study indicated that dietary vitamin E deficiency depressed fish growth, impaired the immune function and disturbed the structural integrity of the head kidney, spleen and skin in grass carp, but optimal vitamin E supplementation can reverse those negative effects in fish. The optimal vitamin E requirements for young grass carp (266.39-1026.63 g) to achieve optimal growth performance and disease resistance based on the percent weight gain (PWG) and skin lesion morbidity were estimated to be 116.2 and 130.9 mg/kg diet, respectively. Meanwhile, based on immune indicator (LA activity in the head kidney) and antioxidant indicator (protection of spleen against MDA), the optimal vitamin E requirements for young grass carp were estimated to be 123.8 and 136.4 mg/kg diet, respectively.

Heptamethine cyanine dyes (Cy7) have attracted much attention in the field of biological application due to their unique structure and attractive near infrared (NIR) photophysical properties. In this review, the influences of different modification sites on the absorption characteristics, photostability, Stokes shift, fluorescence characteristics, water solubility, and singlet oxygen generation efficiency of this class of dyes are summarized, and the application development of the corresponding dyes in the field of biological application is introduced, which will provide a reference for the optimization and improvement of heptamethine cyanine dyes in the future.

This study evaluates the effects of dietary phosphorus on the growth, immune function and structural integrity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) that were fed graded levels of available phosphorus (0.95–8.75 g/kg diet). Results indicated that phosphorus deficiency decreased the growth performance of young grass carp. In addition, the results first demonstrated that compared with the optimal phosphorus level, phosphorus deficiency depressed the lysozyme (LZ) and acid phosphatase (ACP) activities and the complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα) and target of rapamycin (TOR), whereas it up-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65 and NF-κB p52 mRNA levels to decrease fish head kidney and spleen immune functions. Moreover, phosphorus deficiency up-regulated the mRNA levels of Kelch-like-ECH-associated protein 1a (Keap1a), Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), caspase −2, −3, −7, −8 and −9, p38 mitogen-activated protein kinase (MAPK) and myosin light chain kinase (MLCK), whereas it depressed the glutathione (GSH) contents and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes, NF-E2-related factor 2 (Nrf2), B-cell lymphoma protein-2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and tight junction complexes to attenuate fish head kidney and spleen structural integrity. In addition, phosphorus deficiency increased skin hemorrhage and lesions morbidity. Finally, based on the percent weight gain (PWG) and the ability to combat skin hemorrhage and lesions, the dietary available phosphorus requirements for young grass carp (254.56–898.23 g) were estimated to be 4.10 and 4.13 g/kg diet, respectively. In summary, phosphorus deficiency decreases the growth performance, and impairs immune function and structural integrity in the head kidney, spleen and skin of young grass carp.

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.

Fish scales are usually discarded or used to produce fish meal, etc. In order to enhance their utility, we produced the gelatin hydrolysates from fish scales (FSGH) and they were heated with glucose, xylose, and ribose to prepare sugar-FSGH Maillard reaction products (MRPs). The antioxidant capacity and sensory property of MRPs were evaluated. The results showed that ribose-FSGH MRPs exhibited higher antioxidant capacity than glucose- and xylose-FSGH MRPs. After simulated gastrointestinal digestion, the DPPH and ABTS scavenging activity of ribose-FSGH MRPs were 25.32 μM and 193.37 μM Trolox equivalent/g sample, respectively, and the reducing power was 0.509. Flavor compounds (such as butanal, benzaldehyde, 2-methyl-3-furanthiol, and maltol) of ribose-FSGH MRPs were produced in abundance after 5 h of heating and ribose-FSGH MRPs exhibited flavor enhanced effect on caramel-like and mouthfulness sensory attributes. These results suggest that ribose-FSGH MRPs can be potentially used as food antioxidants.

A 60 days feeding trial was conducted to investigate the effects of dietary nucleotides levels on growth performance, proximate composition, physicochemical properties, amino acids constituents, fatty acids composition, apoptosis and antioxidant status related mechanism of grass carp (Ctenopharyngodon idella) (initial weight: 200.00 ± 1.00 g). Six graded levels of dietary nucleotides were designed as 0, 200.0, 400.0, 600.0, 800.0 and 1000.0 mg/kg, respectively. The results showed that, compared with control group, dietary nucleotides supplementation increased the growth of grass carp with higher final body weight, percentage weight gain and specific growth rate, decreased moisture but increased protein and lipid in muscle. However, dietary nucleotides had no effect on ash, calcium and phosphorus in fish fillets (P > .05). Dietary nucleotides supplementation increased the content of umami and sweetness-associated amino acids, flavor nucleotide (inosine mono-phosphate), healthcare n-3 PUFAs (EPA and DHA) and the ratio of n-3/n6 in fish muscle. Meanwhile, dietary nucleotides increased water holding capacity, tenderness and pH to enhance flesh quality, which were partly related to inhibited apoptosis with regulation of apoptosis-related gene expression, decreased hydroxyproline with increase of cathepsins activities and decreased lactate in muscle, respectively. In addition, dietary nucleotides enhanced flesh quality of grass carp fillets concerning increasing antioxidant enzymes activities and its gene expression, which were modulated by signaling molecules (TOR and Nrf2) gene and protein expression in grass carp muscle. In summary, dietary nucleotides supplementation increased growth performance, flavor characteristics, healthy fatty acid contents, physicochemical properties (water holding capacity, tenderness and pH), anti-apoptotic ability and antioxidant capacity in fish. This study demonstrated that nucleotides was a promising feed additives to enhance flesh quality in fish.

Flesh quality is evaluated according to nutritional value and sensory quality. Cinnamaldehyde (CIN) improves mammalian meat quality, but research relating this to aquaculture is scarce. In this study, five doses of CIN (0, 36, 72, 108, 144 mg/kg diet) were fed to grass carp (Ctenopharyngodon idella) for 60 days. The results show that CIN supplementation increased nutritional value by increasing crude protein content. CIN also improved the sensory quality by increasing the pH and collagen content, decreasing shear force, lactate, and cooking loss. These changes may be related to changes in muscle fiber growth by increasing myofiber diameter. The increased myofiber diameter induced by CIN is associated with TOR mRNA and protein levels, and down-regulated FOXO3a mRNA levels, which might be associated with PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling. Based on muscle crude protein content, optimal CIN supplementation dosage was 88.01 mg/kg.

The prevalence of aflatoxin B1 (AFB1), one of the most toxic mycotoxins that contaminates feedstock and food is increasing worldwide. AFB1 can cause various health problems in humans and animals, as well as direct embryotoxicity. However, the direct toxicity of AFB1 on embryonic development, especially foetal foetus muscle development, has not been studied in depth. In the present study, we used zebrafish embryos as a model to study the mechanism of the direct toxicity of AFB1 to the foetus, including muscle development and developmental toxicity. Our results showed that AFB1 caused motor dysfunction in zebrafish embryos. In addition, AFB1 induces abnormalities in muscle tissue architecture, which in turn causes abnormal muscle development in larvae. Further studies found that AFB1 destroyed the antioxidant capacity and tight junction complexes (TJs), causing apoptosis in zebrafish larvae. In summary, AFB1 may induce developmental toxicity and inhibit muscle development through oxidative damage, apoptosis and disruption of TJs in zebrafish larvae. Our results revealed the direct toxicity effects of AFB1 on the development of embryos and larvae, including inhibition of muscle development and triggering neurotoxicity, induction of oxidative damage, apoptosis and disruption of TJs, and fills the gap in the toxicity mechanism of AFB1 on foetal development.

Flavobacterium columnare (F. columnare) is one of the deadliest fish pathogens causing bacterial gill rot disease in various freshwater fish species globally. Tea polyphenols (TPs) are an inexpensive product extracted from tea that have received much attention as a feed additive in aquaculture. The current study was designed to investigate the underlying mechanisms and protective effects of dietary TPs against F. columnare-induced gill injury via suppression of oxidative stress, apoptosis, and inflammation in grass carp. TPs were not supplemented to the diet (control) and were supplemented at 40, 80, 120, 160 or 200 mg/kg diet. The feeding experiment was carried out for 60 days, followed by a 3-Day F. columnare challenge test. The results showed that 120 mg/kg TPs in the diet exerted the following five protective effects in fish gill: (1) control gill-rot disease and improved histopathology, (2) inhibit excessive apoptosis, (3) enhance the activity of antioxidant enzymes and upregulate related gene expression via the Nrf2/Keap1 pathway, (4) increase the activity of immune enzymes, And (5) mediate inflammatory cytokine gene expression via the JAK/STAT3 pathway. Taken together, dietary supplementation with TPs is a compelling approach to protect the gill function of fish against F. columnare.

One hundred and forty-three patients with p210 BCR-ABL-positive leukemia were studied for coexpression of p190 BCR-ABL mRNA. p190 mRNA was detected in 14 of 16 (88%) patients with chronic-phase chronic myeloid leukemia (CML) at diagnosis, in 10 of 10 (100%) CML patients in blast crisis, in 75 of 107 (70%) CML patients receiving interferon- alpha (IFN-alpha), and 10 of 10 (100%) patients with p210 BCR-ABL- positive acute lymphoblastic leukemia (ALL). Neither p210 nor p190 BCR- ABL transcripts were detected in normal healthy adults (n = 20). The numbers of p190 transcripts determined by competitive PCR in patients with CML were low compared with the numbers of p210 transcripts. The median numbers of p210 and p190 transcripts per unit volume of cDNA in positive samples were 1.0 x 10(5) (range, 15 to 1.4 x 10(6)) and 10 (range, 10 to 2.9 x 10(3)), respectively. The numbers of p190 and p210 transcripts were significantly correlated in individual samples (r = .65, P &lt; .001). The median number of p210 BCR-ABL transcripts was significantly lower in samples negative for p190 BCR-ABL transcripts than in samples in which p190 BCR-ABL transcripts were identified (3.1 x 10(3)[n = 73] v 1.0 x 10(5)[n = 115]; P &lt; .0001). The median ratio of p190 to p210 BCR-ABL mRNA was not significantly different between chronic phase CML (1.9 x 10(-4)) and CML in blast crisis (1.7 x 10(- 4)). The median ratio in p210 ALL was also low (1.9 x 10(-3)) but significantly higher than that of CML. We conclude that pl90 BCR-ABL transcripts are frequently present at a low level in p210 BCR-ABL- positive leukemias. p190 mRNA may arise through alternative or missplicing and its presence is probably of no pathogenetic significance.

Lipid peroxidation, protein oxidant and antioxidant status of muscle, intestine and hepatopancreas in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of myo-inositol (MI) (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg/kg diet) for 60 days were investigated. Total tissue malondialdehyde (MDA) and protein carbonyl (PC) content showed a downward trend to a point (P < 0.05). Conversely, total tissue anti-hydroxyl radical (AHR), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reducase (GR) activities and glutathione (GSH) content were generally higher in MI-supplemented diets than MI-unsupplemented diet (P < 0.05). Muscle and intestinal superoxide dismutase (SOD), and intestinal anti-superoxide anion (ASA) were increased by MI supplementation (P < 0.05), whereas these parameters in the other tissue showed no alterations (P > 0.05). These results indicated that antioxidant status was improved, and lipid peroxidation and protein oxidant were depressed in muscle, intestine and hepatopancreas by MI.

An 8-week feeding experiment was conducted to determine the effect of dietary methionine supplementation on intestinal microflora and humoral immune of juvenile Jian carp (initial weight of 9.9 ± 0.0 g) reared in indoor flow-through and aerated aquaria. Eight amino acid test diets (350 g kg−1 crude protein, CP), using fish meal, soybean-condensed protein and gelatin as intact protein sources supplemented with crystalline amino acids, were formulated to contain graded levels of methionine (0.6–22.0%) at a constant dietary cystine level of 3 g kg−1. Each diet was randomly assigned to three aquaria. Growth performance and feed utilization were significantly influenced by the dietary methionine levels (P < 0.05). Maximum weight gain, feed intake occurred at 12 g kg−1 dietary methionine (P < 0.05). Methionine supplementation improved hepatopancreas and intestine weight, hepatosomatic and intestine index, intestinal γ-glutamyltransferase and creatine kinase activity, Lactobacillus count, Bacillus count, lysozyme activities, lectin potency, sim-immunoglobulin M content, addiment C3,C4 contents and serum total iron-binding capacity and declined Escherichia coli and Aeromonas counts. Quadratic regression analysis of weight gain against dietary methionine levels indicated that the optimal dietary methionine requirement for maximum growth of juvenile Jian carp is 12 g kg−1 of the dry diet in the presence of 3 g kg−1 cystine.

Although oxidative stress has been demonstrated to be involved in copper (Cu)-induced toxicity, information regarding the effect of antioxidants on Cu toxicity is still scarce. This study assessed the possible protective effects of myo-inositol (MI) against subsequent Cu exposure in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in their enterocytes in vitro. First, oxidative stress was established by exposing fish to different concentrations of Cu (0–7.2 mg Cu/L water) for 4 days. Next, the protective effects of MI (administered as a dietary supplement for 60 days) against subsequent Cu exposure (0.6 mg Cu/L water for 4 days) were studied in fish. The third trial determined the effects of Cu exposure (0–6.0 mg Cu/L of medium for 24 h) on enterocytes in vitro. Finally, enterocytes were pre-incubated with graded levels of MI (0–75 mg MI/L of medium) for 72 h and exposed to 6.0 mg Cu/L of medium for 24 h. The results indicated that ≥0.6 mg Cu/L water could induce oxidative stress in fish (P < 0.05). Cu exposure significantly induced increases in lipid peroxidation and protein oxidation in the gill, hepatopancreas and intestine in fish. However, these oxidative effects were prevented by MI pre-supplementation. MI also prevented the toxic effects of Cu on anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in these organs. In vitro, enterocytes exposed to Cu displayed a dose-dependent injury. Moreover, cell viability, protein retention (PR), alkaline phosphatase, total-SOD (T-SOD) and Cu/ZnSOD activities were all depressed by Cu (P < 0.05). Interestingly, the final experiment showed that MI pre-supplementation could block the toxic effects of Cu on the antioxidant system, and thus protect enterocytes from Cu-induced oxidative damage. All of these results indicated that the induction of key antioxidant defenses by MI pre-supplementation, including SOD, CAT, GPx, GST and GSH, may play an important role in the protection of fish against oxidative stress.

Aquaculture NutritionVolume 15, Issue 4 p. 409-414 Influence of glutamine and vitamin E on growth and antioxidant capacity of fish enterocytes J. JIANG, J. JIANG Animal Nutrition Institute, Sichuan Agricultural University, Yaan, China Key Laboratory for Animal Disease-Resistance Nutrition of Ministry of Education, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorT. ZHENG, T. ZHENG Animal Nutrition Institute, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorX.-Q. ZHOU, X.-Q. ZHOU Animal Nutrition Institute, Sichuan Agricultural University, Yaan, China Key Laboratory for Animal Disease-Resistance Nutrition of Ministry of Education, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorY. LIU, Y. LIU Animal Nutrition Institute, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorL. FENG, L. FENG Animal Nutrition Institute, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this author J. JIANG, J. JIANG Animal Nutrition Institute, Sichuan Agricultural University, Yaan, China Key Laboratory for Animal Disease-Resistance Nutrition of Ministry of Education, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorT. ZHENG, T. ZHENG Animal Nutrition Institute, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorX.-Q. ZHOU, X.-Q. ZHOU Animal Nutrition Institute, Sichuan Agricultural University, Yaan, China Key Laboratory for Animal Disease-Resistance Nutrition of Ministry of Education, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorY. LIU, Y. LIU Animal Nutrition Institute, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this authorL. FENG, L. FENG Animal Nutrition Institute, Sichuan Agricultural University, Yaan, ChinaSearch for more papers by this author First published: 06 July 2009 https://doi.org/10.1111/j.1365-2095.2008.00605.xCitations: 75 Zhou Xiao-qiu, Animal Nutrition Institute, Sichuan Agricultural University, Yaan 625014, Sichuan, China. E-mail: zhouxq@sicau.edu.cn Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract The present study explored the effect of glutamine and vitamin E on growth and anti-oxidation capacity of isolated fish enterocytes. Fish enterocytes were cultured with six medium, respectively, containing 0, 2.0, 4.7, 6.8, 8.1, 9.2 mmol L−1 glutamine for 64 h. The results showed that glutamine could promote fish enterocytes proliferation and differentiation. Fish enterocytes were cultured with different medium containing 0, 2.5, 3.5, 4.5, 6.0, 7.0 μg mL−1 vitamin E for 96 h. The results showed that cells proliferation and differentiation were not significantly enhanced, but anti-superoxide anion activity, anti-hydroxy radical activity, reduced glutathione concentration, the ratio between reduced and total glutathione in the cells were significantly enhanced, and the malondialdehyde concentration in the culture medium was significantly depressed with the vitamin E treatment. In the whole, the present results firstly indicated that glutamine could promote fish enterocytes growth, but vitamin E could not. Vitamin E could promote fish enterocytes antioxidant capacity and cellular structural integrity. These data would be instructive for glutamine and vitamin E supplement in aquaculture diets. Citing Literature Volume15, Issue4August 2009Pages 409-414 RelatedInformation

A 60-day feeding trial was carried out with juvenile Jian carp (Cyprinus carpio var. Jian) to study the effects of myo-inositol (MI) on the growth, digestive enzyme and intestinal microbial population. Diets with seven levels of inositol (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg MI kg−1 diet) were fed to Jian carp (initial weight 22.28±0.07 g). Per cent weight gain (PWG) was improved with increasing inositol levels up to 535.8 mg MI kg−1 diet (P<0.05), and plateaued (P>0.05). The protein production value, lipid production value and ash production value were increased with increasing dietary inositol levels up to 384.2, 838.8 and 838.8 mg MI kg−1 diet respectively (P<0.05). Although intestinal protein content and trypsin activity were not affected by inositol levels (P>0.05), chymotrypsin, lipase and amylase activities in intestine were the lowest for fish fed the MI-unsupplemented diet (P<0.05). Alkaline phosphatase, Na+, K+-ATPase, γ-glutamyl transpeptidase and creatinkinase activities in the intestine were increased with an increase in the inositol levels up to 384.2–687.3 mg MI kg−1 diet (P<0.05). Intestinal Aeromonas hydrophila and Escherichia coli decreased with an increase in the levels of dietary inositol up to 232.7 and 687.3 mg MI kg−1 diet respectively (P<0.05), while Lactobacillus in the intestine increased with an increase in inositol levels up to 990.3 mg MI kg−1 diet (P<0.05). In conclusion, inositol improved growth, digestive capacity and intestinal microbial population of juvenile Jian carp, and the dietary inositol requirement for PWG of juvenile Jian carp is 518.0 mg MI kg−1 diet.

The effects of dietary choline on growth, digestive and absorptive capacities, and target of rapamycin (TOR) and eIF4E-binding protein2 (4E-BP2) gene expression in muscle, hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian) were assessed. A total of 1200 juvenile Jian carp with an average initial weight of 7.94 ± 0.03 g were fed six semi-purified diets containing 165 (unsupplemented control group), 310, 607, 896, 1167 and 1820 mg choline kg− 1 diet for 65 days. Specific growth rate (SGR), feed intake, feed efficiency, protein retention value, protein content and lipid content of fish carcasses were significantly improved by dietary choline supplementation; whereas, protein efficiency ratio and body ash content were not significantly different among dietary groups. Patterns of differences in intestine length, relative gut length, intestine weight, hepatopancreas protein content, intestinal folds height in three intestinal segments, trypsin and chymotrypsin activities in intestine and hepatopancreas, α-amylase and creatine kinase activities in intestine, Na+/K+-ATPase activity in distal intestine and relative expression of TOR gene in distal intestine and 4E-BP2 gene in muscle responded similar to SGR; whereas, the trends of hepatosomatic index, lipase activity in intestine and hepatopancreas, Na+/K+-ATPase activity in proximal intestine and mid intestine, TOR gene expression in muscle and hepatopancreas, and 4E-BP2 gene expression in proximal intestine, mid intestine and distal intestine were opposite. Alkaline phosphatase and γ-glutamyl transpeptidase activities in intestinal segments were not significantly different among dietary groups. These results indicated that dietary choline could improve fish growth, enhance digestive and absorptive ability and regulate TOR and 4E-BP2 gene expression in tissues. The dietary choline requirement of Jian carp estimated by the broken-line model based on SGR was 566 mg choline kg− 1 diet in the form of choline chloride.

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

A 9-week feeding trial was carried out with juvenile Jian carp to study the effect of dietary pantothenic acid (PA) on growth, body composition and intestinal enzyme activities. Semi-purified diets with seven levels (4.0, 15.5, 25.6, 36.1, 45.9, 56.1 and 65.9 mg PA kg−1) of supplemental calcium d-pantothenate were fed to Jian carp (13.0 ± 0.0 g). PA improved specific growth rate (SGR), protein productive value (PPV), protein efficiency ratio (PER) and lipid production value (LPV) (P < 0.05). Fish fed the control diet had significantly lower feed efficiency (FE) than that in any other group (P < 0.05). Body protein content increased with increasing PA levels (P < 0.05), but moisture, lipid and ash of fish carcasses were negatively related to the graded PA levels (P < 0.05). Intestine protein content (IPC), hepatopancreas protein content (HPC) and activity of α-amylase, lipase, trypsin, Na+,K+-ATPase, alkaline phosphatase (AKP) and gamma-glutamyl transpeptidase (γ-GT) were all positively affected by the dietary PA levels (P < 0.05), while intestine index (ISI) and hepatopancreas index (HSI) decreased with the increment of supplemental levels of PA (P < 0.05). These results suggested that PA could enhance fish growth and intestinal enzyme activities. The dietary PA requirement of juvenile Jian carp, Cyprinus carpio var. Jian (13.0–73.0 g), for optimal growth estimated by the broken-line analysis was 23.0 mg PA kg−1 diet.

This study was conducted both in vivo and in vitro to investigate the effects of tryptophan on growth performance, digestive and absorptive function and protein synthesis of juvenile Jian carp (Cyprinus carpio var. Jian). 1050 juvenile Jian carp (initial weight 7.73 ± 0.03 g) were fed seven isonitrogenous diets with graded concentrations of tryptophan (1.1, 1.7, 2.5, 3.8, 4.9, 6.0, 6.9 g/kg diet) for 8 weeks. Percent weight gain, feed intake and protein retention value were markedly improved, with increases in dietary tryptophan up to 3.8 g/kg diet. Similar trend was found in glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT) activities, trypsin, lipase and α-amylase activities, Na+/K+-ATPase, alkaline phosphatase (AKP), γ-glutamyl transpeptidase and creatine kinase activities, and relative expression of eIF4E-binding protein (4E-BP). On the other hand, feed conversion ratio, plasma ammonia concentration and the relative expression of target of rapamycin (TOR) in different tissue showed an opposite pattern. A series of experiments in vitro were then carried out. Compared with the control group, tryptophan supplementation increased 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) OD value, protein content and activity of AKP, GOT, GPT and Na+/K+-ATPase in enterocytes and decreased lactate dehydrogenase activity and ammonia concentration in the culture medium. Protein synthesis rate was 17% higher and relative expression of TOR was 28% lower in tryptophan-supplemented than in control carp enterocytes. In conclusion, our results indicate that tryptophan improved fish growth, digestive and absorptive function as well as protein synthesis, which may be partly related to the TOR signaling pathway.

A 6-week trial was carried out with 900 juvenile Jian carp (Cyprinus carpio var. Jian) to investigate the effects of dietary zinc on growth, body composition and intestinal enzyme activities. Diets supplemented with increasing levels (15.3, 26.9, 40.8, 58.2, 68.9 and 92.5 mg Zn kg−1) of zinc lactate were fed to Jian carp (mean initial weight 15.7 ± 0.01 g). Results indicated that per cent weight gain (PWG), feed efficiency (FE), protein efficiency ratio (PER) and lipid productive value (LPV) enhanced with dietary zinc levels up to 40.8 mg kg−1 diet (P < 0.05), and plateaued thereafter (P > 0.05). Feed intake (FI) was similar to that observed for PWG. Intestosomatic index (ISI), relative gut length (RGL), hepatopancreas protein content (HPC), intestine protein content (IPC), trypsin, chymotrypsin, lipase, amylase, alkaline phosphatase (AKP), Na+, K+-ATPase and γ-glutamyl transpeptidase (γ-GT) activities were all higher by dietary zinc supplementation than zinc un-supplementation (P < 0.05). These results suggested that zinc could promote growth and increase nutrient deposition and intestinal enzyme activities. The dietary zinc requirements (use zinc lactate as zinc source) of juvenile Jian carp (15.7–42.2 g) based on PWG and serum zinc were 48.7 and 43.2 mg Zn kg−1 diet, respectively.

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

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

A total of 900 juvenile Jian carp (Cyprinus carpio var. Jian) (7.99 ± 0.02 g) were fed diets containing graded levels of xylanase at 220 (unsupplemented control), 650, 1070, 1480, 1810 and 2470 U kg−1 diet for 10 weeks to investigate the effects of xylanase levels on growth performance, intestinal enzyme activities and microflora. The per cent weight gain, feed efficiency, protein efficiency ratio, protein production value, lipid production value, ash production value, calcium production value and phosphorus retention ratio were significantly improved with increasing levels of xylanase up to a point, and thereafter declined (P < 0.05). The activities of trypsin, chymotrypsin, lipase and amylase in the hepatopancreas and intestine, activities of alkaline phosphatase, Na+, K+-ATPase, creatine kinase and γ-glutamyl transpeptidase in three intestinal segments were improved by dietary xylanase (P < 0.05). The amounts of Lactobacillus, Escherichia coli and Aeromonas were significantly affected by dietary xylanase levels (P < 0.05). In conclusion, xylanase supplementation improved growth performance, enhanced intestinal enzyme activities and influenced the balance of intestinal microflora of juvenile Jian carp. The optimal level of xylanase in juvenile Jian carp (7.99–99.16 g) based on PWG was 1259 U kg−1 diet by the quadratic regression analysis.

This study aimed to investigate the effects of threonine (Thr) on the digestive and absorptive ability, proliferation and differentiation of enterocytes, and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian). First, seven isonitrogenous diets containing graded levels of Thr (7.4-25.2 g/kg diet) were fed to the fishes for 60 days. Second, enterocyte proliferation and differentiation were assayed by culturing enterocytes with graded levels of Thr (0-275 mg/l) in vitro. Finally, enterocytes were cultured with 0 and 205 mg/l Thr to determine protein synthesis. The percent weight gain (PWG), specific growth rate, feed intake, feed efficiency, protein retention value, activities of trypsin, lipase and amylase, weights and protein contents of hepatopancreas and intestine, folds heights, activities of alkaline phosphatase (AKP), γ- glutamyl transpeptidase and Na(+)/K(+)-ATPase in all intestinal segments, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities in hepatopancreas, and 4E-BP2 gene expression in muscle, hepatopancreas and intestinal segments were significantly enhanced by Thr (p<0.05). However, the plasma ammonia concentration and TOR gene expression decreased (p<0.05). In vitro, Thr supplement significantly increased cell numbers, protein content, the activities of GOT, GPT, AKP and Na(+)/K(+)-ATPase, and protein synthesis rate of enterocytes, and decreased LDH activity and ammonia content in cell medium (p<0.05). In conclusion, Thr improved growth, digestive and absorptive capacity, enterocyte proliferation and differentiation, and protein synthesis and regulated TOR and 4E-BP2 gene expression in juvenile Jian carp. The dietary Thr requirement of juvenile Jian carp was 16.25 g/kg diet (51.3 g/kg protein) based on quadratic regression analysis of PWG.

Two Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) variants are naturally expressed in breast epithelia. Breast cancer (BCa) onset down-regulates the short SENP7 splice variant (SENP7S) and enhances the long transcript (SENP7L). Here, we show that SENP7L induction promotes gene expression profiles that favor aberrant proliferation and initiate epithelial-mesenchymal transition (EMT). SENP7L exhibits an interaction domain for the epigenetic remodeler heterochromatin protein 1 α (HP1α) and isopeptidase activity against SUMO-modified HP1α. Loss of this interaction domain, as observed with SENP7S, favors HP1α SUMOylation. SUMOylated HP1α is enriched at E2F-responsive and mesenchymal gene promoters, silences transcription of these genes, and promotes cellular senescence. Elevated SENP7L renders HP1α hypo-SUMOylated, which relieves transcriptional repression of the same genes and concurrently decreases transcription of epithelial-promoting genes via an HP1α-independent mechanism. Consequently, SENP7L levels correlate with EMT, motility, and invasiveness of BCa cells. Stable knockdown of elevated SENP7L levels lessens the dissemination of highly metastatic BCa cells to the lungs from primary implantation sites in in vivo studies. Thus, differential splicing of the SENP7 regulates either tumor suppression or progression.

This study investigated the effects of dietary pantothenic acid (PA) on the growth, intestinal mucosal immune and physical barrier, and relative mRNA levels of signaling molecules in the intestine of grass carp (Ctenopharyngodon idella). A total of 540 grass carp (253.44 ± 0.69 g) were fed six diets with graded levels of PA (PA1, PA15, PA30, PA45, PA60 and PA75 diets) for 8 weeks. The results indicated that compared with PA deficiency (PA1 diet) and excess (PA75 diet) groups, optimal PA supplementation increased (P < 0.05): (1) percent weight gain (PWG), feed intake and feed efficiency; (2) lysozyme activity, complement 3 content, liver-expressed antimicrobial peptide 2 and hepcidin, interleukin 10, transforming growth factor β1 and inhibitor of κBα mRNA levels in some intestinal segments; (3) activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase, and NF-E2-related factor 2 (Nrf2) mRNA level in the whole intestine; (4) Claudin b, Claudin 3, Claudin c, Occludin and ZO-1 mRNA levels in some intestinal segments of grass carp. Conversely, optimal PA supplementation decreased (P < 0.05): (1) tumor necrosis factor α, interleukin 1β, interferon γ2, interleukin 8, nuclear factor κB P65 (NF-κB P65), IκB kinase α, IκB kinase β, IκB kinase γ and target of rapamycin (TOR) mRNA expression levels in some intestinal segments; (2) reactive oxygen species, malondialdehyde and protein carbonyl contents, and Kelch-like ECH-associating protein 1a, Kelch-like ECH-associating protein 1b in the intestine; (3) Claudin 12, Claudin 15a and myosin light-chain kinase (MLCK) mRNA levels in some intestinal segments of grass carp. In conclusion, optimum PA promoted growth, intestinal mucosal immune and physical function, as well as regulated mRNA levels of signaling molecules NF-κB P65, TOR, Nrf2 and MLCK in grass carp intestine. Based on the quadratic regression analysis of PWG and intestinal lysozyme activity, the optimal PA levels in grass carp (253.44-745.25 g) were estimated to be 37.73 mg/kg and 41.38 mg/kg diet, respectively.

Our study investigated the effect of dietary methionine hydroxy analogue (MHA) on growth and immunity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (259.70 ± 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. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. The results indicated that optimal MHA increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and up-regulated mRNA levels of liver expressed antimicrobial peptide 2, hepcidin (head kidney), β-defensin-1 in the immune organs (P < 0.05), suggesting that MHA could enhance antimicrobial ability of fish. Meanwhile, optimal MHA enhanced the immune function of immune organs 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 β, 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 < 0.05). In addition, optimal MHA improved cellular structure integrity of immune organs 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 < 0.05). Simultaneously, optimal MHA improved cellular structure integrity of immune organs 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 < 0.05). 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 mRNA levels (P < 0.05). In conclusion, MHA exerted a positive effect on the immune function and structural integrity of immune organs in fish. Furthermore, according to the positive effect, MHA was superior to DLM in grass carp. However, based on the growth performance, the efficacy of MHA relative to DLM was 97%. Finally, on the premise of the basal diet containing 4.01 g/kg methionine, the optimal MHA supplementation levels based on feed intake, PWG, defense against skin hemorrhage and lesion, LZ and ACP activities, IgM content, against malondialdehyde, protein carbonyl and ROS in the head kidney of young grass carp were 5.07, 5.21, 5.76, 5.90, 5.88, 5.80, 6.22, 5.68 and 6.85 g/kg diet, respectively.

This study aimed to investigate the effects of dietary selenium on resistance to skin haemorrhages and lesions and on immune function as well as the underlying mechanisms of those effects in the head kidney, spleen and skin of young grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp with initial body weight (226.48 ± 0.68 g) were randomly divided into six groups and fed six separate diets with graded dietary levels of selenium (0.025, 0.216, 0.387, 0.579, 0.795 and 1.049 mg/kg diet) for 80 days. After the feeding period, an immunization trial was performed by infection with Aeromonas hydrophila for 14 days. The results showed that, compared with the optimal selenium level, (1) selenium deficiency impaired the production of antibacterial compounds and immunoglobulins and down-regulated the transcript abundances of antimicrobial peptides and selenoproteins; (2) selenium deficiency aggravated inflammatory responses in part by up-regulating pro-inflammatory cytokines and down-regulating anti-inflammatory cytokines mRNA levels, which were partially related to [IKKα, β, γ/IκBα/NF-κB] signalling and [TOR/(S6K1, 4E-BP1)] signalling, respectively. Interestingly, selenium deficiency had no effect on the expression of TGF-β2, IL-4/13B, IL-10, IL-12p35, IL-15 (skin only) or 4E-BP2 in the head kidney, spleen and skin of young grass carp. Finally, based on the percent weight gain (PWG), the morbidity of skin haemorrhages and lesions, the ACP activity in the head kidney and the lysozyme activity in spleen, the optimal dietary selenium requirements for young grass carp were estimated to be 0.546–0.604 mg/kg diet. In summary, selenium deficiency decreased the growth performance and impaired the immune function in the head kidney, spleen and skin of young grass carp.

The present study was designed to assess the possible protective effects of arginine (Arg) against lipopolysaccharide (LPS) induced inflammatory response in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in enterocytes in vitro. Firstly, inflammatory response was established by exposing enterocytes to different concentrations of LPS for 24 h. Secondly, the protective effects of Arg against subsequent LPS exposure were studied in enterocytes. Finally, we investigated whether dietary Arg supplementation could attenuate immune challenge induced by LPS in vivo. The result indicated that 10 mg/L LPS could induced inflammatory response in enterocytes. Cells exposed to LPS (10–30 mg/L) alone for 24 h resulted in a significant increase in lactate dehydrogenase release (LDH) (P < 0.05). The cell viability, protein content, alkaline phosphatase activity were decreased by LPS (P < 0.05). Moreover, LPS exposure significantly increased TNF-α, IL-1β, and IL-6 mRNA expression in vitro (P < 0.05). However, pre-treatment with Arg remarkably prevented the increase of TNF-α, IL-1β, and IL-6 by inhibiting the excessive activation of TLR4-Myd88 signaling pathway through down-regulating TLR4, Myd88, NFκB p65, and MAPK p38 mRNA expression (P < 0.05). Interestingly, the experiment in vivo showed that Arg pre-supplementation could attenuate immune challenge induced by LPS via TLR4-Myd88 signaling pathway, and thus protect fish against LPS-induced inflammatory response. In conclusion, all of these results indicated pre-supplementation with Arg decreased LPS induced immune damage and regulated TLR4-Myd88 signaling pathway in juvenile Jian carp in vivo and in enterocytes in vitro.

Abstract The present study investigated the effects of dietary vitamin A on immune function in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of young grass carp ( Ctenopharyngodon idella ). Fish were fed graded levels of dietary vitamin A for 10 weeks, and then a challenge test using an injection of Aeromonas hydrophila was conducted for 14 d. The results showed that, compared with the optimum vitamin A level, vitamin A deficiency significantly decreased fish growth performance, increased enteritis morbidity, decreased intestinal innate humoral immune response and aggravated intestinal inflammation. However, liver-expressed antimicrobial peptide 2A/B mRNA in the DI and IL-6 , IL-17D , IL-10 , transforming growth factor ( TGF ) -β1 and TGF-β2 mRNA in the PI were not affected by vitamin A levels. Meanwhile, vitamin A deficiency disturbed inflammatory cytokines in the PI, MI and DI, which might be partly linked to p38 mitogen-activated protein kinase ( p38MAPK ) signalling and NF-κB canonical signalling pathway (I κ B kinase β ( IKKβ ), IKKγ , inhibitor of κ B α , NF-κB p65 and c-Rel ) rather than NF-κB non-canonical signalling pathway ( NF-κB p52 and IKKα ). However, the signalling molecules NF-κB p65 and p38MAPK did not participate in regulating cytokines in the PI. These results suggested that vitamin A deficiency decreased fish growth and impaired intestinal immune function, and that different immune responses in the PI, MI and DI were mediated partly by NF-κB canonical signalling and p38MAPK signalling pathways. On the basis of percentage of weight gain, to protect fish against enteritis morbidity and acid phosphatase activity, the optimum dietary vitamin A levels were estimated to be 0·664, 0·707 and 0·722 mg /kg, respectively.

The present study investigated the effects of dietary DL-methionyl-DL-methionine (Met-Met) on growth performance, intestinal immune function and the underlying signalling molecules in juvenile grass carp (Ctenopharyngodon idella). Fish were fed one DL-methionine (DL-Met) group (2.50 g/kg diet) and six graded levels of Met-Met groups (0, 0.79, 1.44, 1.84, 2.22 and 2.85 g/kg diet) for 10 weeks, and then challenged with Aeromonas hydrophila for 14 days. Results indicated that the optimal Met-Met supplementation: (1) increased fish growth performance, intestinal lysozyme (LZ) and acid phosphatase (ACP) activities, complement (C3 and C4) and immunoglobulin M (IgM) contents, up-regulated hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, β-defensin-1 and Mucin2 mRNA levels; (2) down-regulated tumour necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), interleukin 1β (IL-1β), IL-8 [only in the distal intestine (DI)], IL-12p35, IL-12p40 and IL-15 (not IL-17D) mRNA levels partially related to the down-regulation of IκB kinase β (IKKβ) and IKKγ (rather than IKKα), nuclear factor kappa B (NF-κB) p65 and c-Rel (rather than NF-κB p52) mRNA levels and the up-regulation of inhibitor of κBα (IκBα) mRNA levels; (3) up-regulated IL-4/13A, IL-4/13B, IL-6, IL-10, IL-11 and transforming growth factor (TGF)-β1 (not TGF-β2) mRNA levels partially associated with the target of rapamycin (TOR) signalling pathway [TOR/ribosomal protein S6 kinases 1 (S6K1), eIF4E-binding proteins (4E-BP)] in three intestinal segments of juvenile grass carp. These results suggest that Met-Met supplementation improves growth and intestinal immune function in fish. Furthermore, according to a positive effect, the optimal Met-Met supplementation was superior to the optimal DL-Met supplementation at improving the growth performance and enhancing the intestinal immune function in fish. Finally, based on percent weight gain (PWG), protection against enteritis morbidity and immune index (LZ activity), the optimal Met-Met supplementation for juvenile grass carp was estimated as 1.61, 1.64 and 1.68 g/kg diet, respectively, as the basal diet contains 8.03 g/kg total sulfur amino acids (TSAA) (4.26 g methionine/kg and 3.77 g cysteine/kg).

This study was conducted to investigate the effects of dietary iron on the growth, and immune function and structural integrity in head kidney, spleen and skin as well as the underlying signaling of young grass carp (Ctenopharyngodon idella). Total 630 grass carp (242.32 ± 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 mg/kg (diets 2–6 were added with ferrous fumarate) and 73.50 mg/kg (diet 7 was added with ferrous sulfate) diet for 60 days. Then, a challenge test was conducted by infection of Aeromonas hydrophila for 14 days. The results firstly showed that compared with 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, inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), whereas up-regulated the mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65, IκB kinases β (IKKβ) and eIF4E-binding protein (4E-BP) in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired immune function in head kidney and spleen of fish. Secondly, iron deficiency down-regulated the mRNA levels of B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), and inhibitor of apoptosis protein (IAP), and decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction complexes, and up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -3, -7, -8, -9, apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), Fas ligand (FasL), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12 and myosin light chain kinase (MLCK), and increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents in head kidney and spleen of young grass carp (P < 0.05), indicating that iron deficiency impaired structural integrity in head kidney and spleen of fish. Thirdly, iron deficiency increased skin hemorrhage and lesion morbidity, and impaired immune function and structural integrity in skin of fish. Fourthly, iron excess decreased growth and impaired the immune function and structural integrity in head kidney, spleen and skin of fish. Besides, in young grass carp, based on PWG and ability against skin hemorrhage and lesion, the efficacy of ferrous fumarate relative to ferrous sulfate was 140.32% and 126.48%, respectively, and the iron requirements based on PWG, ability against skin hemorrhage and lesion, ACP activities and MDA contents in head kidney and spleen were estimated to be 75.65, 87.03, 79.74, 78.93, 83.17 and 82.14 mg/kg diet (based on ferrous fumarate), respectively.

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

Atherosclerosis is the major cause of stroke and heart disease. However, the course and pathogenesis of atherosclerosis remains unknown. The proliferation and migration of endothelial cell play important roles in the inition and pathological progression of atherosclerosis. In this study, we demonstrated that long noncoding RNA (lncRNA) HOXA transcript at the distal tip (HOTTIP) expression level was higher in coronary artery disease (CAD) tissues than in normal arterial tissues. The expression level of HOTTIP was upregulated in the proliferating endothelial cells induced by TNF-α or PDGF-BB. Ectopic expression of HOTTIP promoted endothelial cell proliferation and also increased the expression of proliferating makers cyclin D1 and PCNA. Moreover, elevated expression of HOTTIP promoted endothelial cell migration. Downregulation expression of HOTTIP suppressed endothelial cell proliferation and migration. Furthermore, we determined that overexpression of HOTTIP induced β-catenin expression and enhanced the downstream protein c-Myc expression in the endothelial cell. Ectopic expression of HOTTIP increased endothelial cell proliferation and migration via activation of the Wnt/β-catenin pathway. These results suggested that HOTTIP might manipulate the endothelial cell proliferation and migration via activation of the Wnt/β-catenin pathway.

This study was conducted to investigate the effects of the dietary vitamin myo-inositol (MI), on the immunity and structural integrity of the head kidney and spleen following infection of fish with the major freshwater pathogen bacterial Aeromonas hydrophila. The results demonstrated for the first time that MI deficiency depressed the lysozyme and acid phosphatase (ACP) activities and the complement 3 (C3) and C4 contents in the head kidney and spleen compared with the optimal MI levels, indicating that MI deficiency decreased the immunity of these important fish immune organs. The depression in immunity due to MI deficiency was partially related to oxidative damage [indicated by increases in the malondialdehyde (MDA) and protein carbonyl (PC) contents] that was in turn partially due to the decreased glutathione (GSH) content and the disturbances in antioxidant enzyme activities [total superoxide dismutase (T-SOD), CuZnSOD, MnSOD, catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR)]. MI deficiency inhibited the antioxidant-related gene transcription [CuZnSOD, MnSOD, CAT, GPx1a, GR and NF-E2-related factor 2 (Nrf2)] in the head kidney and spleen following infection of the fish with A. hydrophila. The oxidative damage due to MI deficiency also resulted in the inhibition of proliferation-associated signalling (cyclin D1, cyclin A, cyclin E and E2F4). Thus, MI deficiency partially inhibited damage repair. Excessive MI exhibited negative effects that were similar to MI deficiency, whereas the optimal MI content reversed those indicators. These observations indicated that an MI deficiency or excess could cause depression of the immune system that might be partially related to oxidative damage, antioxidant disturbances, and the inhibition of the proliferation-associated signalling in the head kidney and spleen following infection of fish with A. hydrophila. Finally, the optimal MI levels were 660.7 (based on ACP) and 736.8 mg kg−1 diet (based on MDA) in the head kidney and 770.5 (based on ACP) and 766.9 mg kg−1 diet (based on MDA) in the spleen of juvenile Jian carp.

This study was conducted to investigate the effects of dietary phospholipids (PL) on the gill immune response and physical barrier of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juvenile grass carp with an average initial weight of 9.34 ± 0.03 g were fed six semi-purified diets containing 0.40% (unsupplemented control group), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% PL for 2 months. Compared with the control group, optimal PL supplementation increased (P < 0.05): (1) the lysozyme activity, acid phosphatase activity, complement component 3 (C3) content, liver expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 mRNA expression; (2) the relative mRNA expression of interleukin 10, transforming growth factor β1, inhibitor factor κBα (IκBα) and target of rapamycin (TOR); (3) the activities of anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), copper/zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), glutathione content and mRNA levels of SOD1, CAT, GPx, GR and NF-E2-related factor 2 (Nrf2) genes; (4) the transcription abundance of occludin, claudin b, claudin c, claudin 12 and zonula occludens 1 genes. At the same time, appropriate PL supplementation decreased (P < 0.05): (1) tumor necrosis factor α, interleukin 1β, nuclear factor κB p65 (NF-κB p65), IκB kinase β (IKKβ) and IκB kinase γ (IKKγ) mRNA expression; (2) malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) content and the relative mRNA expression of Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b; (3) the transcription abundance of myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) genes. In conclusion, the positive effect of PL on gill health is associated with the improvement of the immunity, antioxidant status and tight junction barrier of fish gills. Finally, based on ACP activity, C3 content, PC content and ASA activity in the gills, the optimal dietary PL level for juvenile grass carp (9.34-87.50 g) was estimated to be 3.62%, 4.30%, 3.91% and 3.86%, respectively.

This study investigated the effects of dietary protein levels on the flesh quality, fatty acids, antioxidant capacity and antioxidant-related signalling molecule gene expression in the muscle 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. The results indicated that optimal dietary protein levels: (1) improved physical (or chemical) and flavor quality of grass carp fillets by increasing shear force, pH, hydroxyproline, protein, lipid, free amino acids (aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, lysine, histidine, arginine and proline) and nucleotide (5′-inosinic acid) contents, and decreasing cooking loss, cathepsin B and L activities and lactate content. (2) enhanced the potential health benefits to humans of grass carp fillets by decreasing the saturated fatty acid (C14:0, C15:0, C16:0, C18:0 and C20:0) concentrations, and increasing unsaturated fatty acids [monounsaturated fatty acids (C14:1 and C16:1) and polyunsaturated fatty acids (C18:2c+t, C18:3n-3, C20:4 and C22:6)] concentrations. (3) enhanced antioxidant capacity of grass carp fillets by decreasing reactive oxygen species, malondialdehyde and protein carbonyl contents, and increasing the activities and gene expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase and the content of glutathione. (4) regulated antioxidant-related signalling molecules gene expression by up-regulating the mRNA levels of NF-E2-related factor 2, casein kinase 2α and 2β, and decreasing Kelch-like-ECH-associated protein 1a and 1b in the muscle of grass carp. In conclusion, optimal dietary protein level improved the physical and flavor characteristics, fatty acid profile, antioxidant capacity and regulated the gene expression of antioxidant-related signalling molecules in fish fillets.

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

For many years, feed-borne mycotoxin contamination has been a serious and unavoidable problem in the aquaculture industry. This problem is extensively aggravated due to the increasing replacement of fish meal by plant protein. Aflatoxin B1 (AFB1) is a mycotoxin that has received a great deal of attention, owing to its prevalence in plant feedstuffs and detrimental effects on animals. The objective of this study was to investigate the effect of AFB1 on growth and health. The growth performance and structural integrity of immune organs were evaluated after feeding grass carp diets with 0, 29, 59, 86, 110 and 147 μg AFB1/kg for 60 days. The results indicated that dietary AFB1 caused poor growth performance and deformities in juvenile grass carp. In addition, we are the first to observe that AFB1 was detectable in the head kidney and spleen of juvenile grass carp and damages the structural integrity of those organs. And we, for the first time, explore potential mechanisms of this destruction effect to immune organs of fish, which might be partly involved in (1) attenuating antioxidant ability through up-regulation of Keap1a (not Keap1b) to suppress Nrf2 signalling leading to decrease of mRNA levels and activities of antioxidant enzymes (except GSTP1 mRNA level); (2) aggravating apoptosis partly by activating p38 MAPK signalling to activate mitochondria pathway and death receptor pathway; and (3) weakening TJs structure by promoting MLCK signalling to down-regulate the mRNA of TJs protein (claudin-12 was up-regulated) in the head kidney and spleen. Finally, based on the oxidative injury-related indexes (contents of MDA in the head kidney and PC in the spleen), the safe upper doses of AFB1 for grass carp were first estimated to be 30 and 29 μg/kg diet, respectively.

To improve the utilization of cod skin collagen peptides (CSCP), we heated them with xylose at 80 °C, 100 °C, and 120 °C for up to 150 min to prepare xylose-CSCP Maillard reaction products (MRPs), and then investigated their physicochemical and functional properties. The results showed that Arg, Lys, Phe, and Asp were the major amino acids involved in the Maillard reaction. After being heated at 120 °C for 150 min, the ABTS scavenging activity and reducing power of xylose-CSCP MRPs were 99.59% and 0.887 absorbance units, respectively. Xylose-CSCP MRPs had better emulsifying properties and foaming properties than CSCP. Furthermore, 26 volatile compounds, including 2,5-dimethyl-pyrazine and 2-ethyl-3,5-dimethylpyrazine, were identified from xylose-CSCP MRPs by gas chromatography-ion mobility spectrometry. Newly formed heterocyclic compounds might be responsible for the flavor and antioxidant capacity of xylose-CSCP MRPs. These results suggest the potential for xylose-CSCP MRPs to serve as functional food ingredients.

This study investigated the effects of bile acid (BA) supplementation on growth performance, intestinal immune function and the mRNA expression of the related signalling molecules in on-growing grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp (mean weight 179.85 ± 1.34 g) were fed a normal protein and lipid (NPNL) diet containing 29% crude protein (CP) and 5% ether extract (EE), and five low-protein and high-lipid (LPHL) diets (26% CP, 6% EE) with graded levels of BA (0–320 mg/kg diet) for 50 days. The fish were then challenged with Aeromonas hydrophila for 14 days. The results indicated that compared with the NPNL diet, the LPHL diet (unsupplemented BA) suppressed the growth performance, intestinal development and enteritis resistance capability and impaired the partial intestinal immune function of on-growing grass carp. Whereas in the LPHL diet, optimal BA supplementation significantly improved fish growth performance (percent weight gain, specific growth rate, feed intake and feed efficiency) and intestinal growth and function (intestine weight, intestine length and intestosomatic index), increased beneficial bacteria Lactobacillus and Bifidobacterium amounts, decreased harmful bacteria Aeromonas and Escherichia coli amounts, elevated lysozyme and acid phosphatase activities, increased complement (C3 and C4) and immunoglobulin M contents, and upregulated β-defensin-1, hepcidin, liver expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, Mucin2, interleukin 10 (IL-10), IL-11, transforming growth factor (TGF)-β1, TGF-β2, IL-4/13A (not IL-4/13B), TOR, S6K1 and inhibitor of κBα (IκBα) mRNA levels. In addition, optimal BA supplementation in the LPHL diet downregulated tumour necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), IL-1β, IL-6, IL-8, IL-15, IL-17D, IL-12p35, IL-12p40 (rather than proximal intestine (PI) or mid intestine (MI), nuclear factor kappa B p65 (NF-κB p65) (except NF-κB p52), c-Rel, IκB kinase β (IKKβ), IKKγ (except IKKα), eIF4E-binding proteins (4E-BP)1 and 4E-BP2 mRNA levels in all three intestinal segments of on-growing grass carp (P < 0.05). These findings suggest that BA supplementation in the LPHL diet improves growth and intestinal immune function of fish. Furthermore, 240 mg/kg BA supplementation in the LPHL diet was superior to the NPNL diet in improving growth and enhancing intestinal immune function of fish. Finally, based on percent weight gain, feed intake, protecting fish against enteritis, lysozyme activity in MI and acid phosphatase activity in distal intestine (DI), the optimal BA supplementation for on-growing grass carp were estimated to be 168.98, 170.23, 166.67, 176.50 and 191.97 mg/kg diet, respectively.

The present study explored the effects of dietary gossypol on the gut health of on-growing grass carp. The fish were fed six diets containing different levels of free gossypol (0, 121.38, 243.94, 363.89, 759.93 and 1162.06 mg/kg diet) from gossypol-acetic acid for 60 days and then challenged with Aeromonas hydrophila for 14 days. The results showed that dietary gossypol (1) could aggravate enteritis and damage the structure of intestinal epithelial cells, (2) decreased the lysozyme (LZ) and Acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and it down-regulated the Hepcidin (rather than distal intestine (DI)), immunoglobulin Z (IgZ), liver-expressed antimicrobial peptide (LEAP)-2B, Mucin2 and β-defensin-1 mRNA levels in the proximal intestine (PI), mid intestine (MI) and DI, (3) up-regulated intestinal pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), interleukin 1β (IL-1β), IL-6 (only in PI), IL-8 and IL-12p35 mRNA levels partly related to nuclear factor kappa B (NF-κB) signalling, and (4) down-regulated the mRNA levels of anti-inflammatory cytokines such as transforming growth factor (TGF)-β1, TGF-β2, interleukin 4/13A (IL-4/13A) (except IL-4/13B), IL-10 and IL-11 partly relating to target of rapamycin (TOR) signalling in the intestines of on-growing grass carp. Moreover, the dietary gossypol had no impact on the LEAP-2A, IL-12P40, IL-17D, IL-10, NF-κBp52, IKKα and eIF4E-binding proteins 2 (4E-BP2) mRNA levels in the intestines. Finally, based on the intestinal histopathological results, enteritis morbidity, LZ activity and IgM content, the safe dose of gossypol in the diets for on-growing grass carp should be less than 103.42 mg/kg diet.

(1) Background: l-leucine (Leu) plays a positive role in regulating protein turnover in skeletal muscle in mammal. However, the molecular mechanism for the effects of Leu on muscle growth and protein deposition is not clearly demonstrated in fish. This study investigated the effects of dietary Leu on growth performance and muscle growth, protein synthesis, and degradation-related signaling pathways of hybrid catfish (Pelteobagrus vachelli♀ × Leiocassis longirostris♂). (2) Methods: A total of 630 hybrid catfish (23.19 ± 0.20 g) were fed 6 different experimental diets containing graded levels of Leu at 10.0 (control), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g Leu kg-1 for 8 weeks. (3) Results: Results showed that dietary Leu increased percent weight gain (PWG), specific growth rate (SGR), FI (feed intake), feed efficiency (FE), protein efficiency ratio (PER), muscle fibers diameter, and muscle fibers density; up-regulated insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), proliferating cell nuclear antigen (PCNA), myogenic regulation factors (MyoD, Myf5, MyoG, and Mrf4), and MyHC mRNA levels; increased muscle protein synthesis via regulating the AKT/TOR signaling pathway; and attenuated protein degradation via regulating the AKT/FOXO3a signaling pathway. (4) Conclusions: These results suggest that Leu has potential role to improve muscle growth and protein deposition in fish, which might be due to the regulation of IGF mRNA expression, muscle growth related gene, and protein synthesis and degradation-related signaling pathways. Based on the broken-line model, the Leu requirement of hybrid catfish (23.19-54.55 g) for PWG was estimated to be 28.10 g kg-1 of the diet (73.04 g kg-1 of dietary protein). These results will improve our understanding of the mechanisms responsible for muscle growth and protein deposition effects of Leu in fish.

Patients who have diabetes must receive insulin administration two to three times every day via subcutaneous (SC) injection after measurement of blood glucose levels (BGLs) using lancet/blood-glucose meter with the side effects of pain and lipodystrophy. Achieving instrument-free glucose-biosensing and persistent glycaemic control in a painless and safe way is the ultimate goal of diabetes management. Here, we report the design of a microneedles (MNs) set comprising a glucose-biosensing microneedle patch (GBMP) and an insulin-delivery MP (IDMP). Once in the skin, the skin interstitial fluid (ISF) would diffuse into GBMP from epidermis to develop colour in abnormal glucose concentration which then quantified by smartphone. Subsequently, the IDMP composing of free insulin and biodegradable insulin-loaded glucose-responsive nanovesicles (IG-NVs) as an artificial on-skin pancreas, could be applied for hyperglycaemia-triggered insulin release to self-regulate BGLs and improve the health and quality of life for patients with type 1 and advanced type 2 diabetes. In chemically induced type 1 diabetic rats, the BGLs can be rapidly measured by GBMP and the BGLs can also be immediately reduced to and kept at normoglycaemic levels for up to 13 h by IDMP. The whole system permits the syringe-free diabetes management, avoiding both hyperglycaemia and hypoglycaemia.

Fish is an important animal-source food for humans. However, the oxidative stress-induced by intensive aquaculture usually causes deterioration of fish meat quality. The nutritional way has been considered to be a useful method for improving fish flesh quality. This study using the same growth experiment as our previous study was conducted to investigate whether vitamin A could improve flesh quality by enhancing antioxidative ability via Nrf2/Keap1 signaling in fish muscle. Six diets with different levels of vitamin A were fed to grass carp (Ctenopharyngodon idella) (262.02 ± 0.45 g) for 10 weeks. Dietary vitamin A significantly improved flesh sensory appeal and nutritional value, as evident by higher pH24h value, water-holding capacity, shear force, contents of protein, lipid, four indispensable amino acids (lysine, methionine, threonine, and arginine) and total polyunsaturated fatty acid in the muscle. Furthermore, dietary vitamin A reduced oxidative damage, as evident by decreased levels of muscle reactive oxygen species, malondialdehyde, and protein carbonyl, enhanced activities of antioxidative enzyme (catalase, copper/zinc superoxide dismutase (CuZnSOD), MnSOD, glutathione peroxidase, and glutathione reductase), as well as increased content of glutathione, which was probably in relation to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. These findings demonstrated that dietary vitamin A improved flesh quality probably by enhancing antioxidant ability through Nrf2/Keap 1a signaling in fish.

Vitamin D regulates biological processes via vitamin D receptors (VDRs) and exerts a pleiotropic effect in the prevention of pathogen infections. In various fish, the existence of two unique VDR isoforms (VDRa and VDRb) has been reported; however, the effects of vitamin D and VDR isoforms on the function of immune-related organs (e.g., the head kidney and spleen) remain poorly understood. In the present study, diets containing different vitamin D concentrations (0, 364.3, 782.5, 1167.9, 1573.8, and 1980.1 IU/kg) were provided to on-growing grass carp for 70 d. Subsequently, each group was subjected to a challenge trial by infection with Aeromonas hydrophila for 14 d. Dietary vitamin D decreased the oxidative biomarker content and upregulated the transcriptional responses of antioxidant enzymes potentially involved in Nrf2 signaling; alleviated cell apoptosis by down-regulating caspase expression, potentially in relation to the inhibition of p38MAPK molecules; enhanced immune component activity; downregulated the transcriptional levels of pro-inflammatory cytokines; and upregulated those of anti-inflammatory cytokines. Furthermore, the optimal vitamin D dose decreased the expression of nuclear factor kappa B p65, c-Rel, IKKα, IKKβ, and IKKγ and increased the transcriptional levels of iκBα, VDRb (but not VDRa), macrophage stimulating 1 (MST1), and signal transducers and activators of transcription (STAT) 3b1 and 3b2. Moreover, the protein levels of VDR, p-STAT3Tyr705, and p-MST1Thr108 were elevated both in the head kidney and spleen after treatment. In summary, vitamin D protected the immune organs from oxidative stress, apoptosis, and immunological injury. Based on lysozyme activity in the head kidney and spleen, the vitamin D requirements of on-growing grass carp (256.89–1129.67 g) were estimated to be 1150.71 and 1355.37 IU/kg, respectively. In conclusion, our findings confirmed that vitamin D can regulate molecular pathways related to immune organ function and pathogen defense in an aquaculture setting, supporting the feasibility of vitamin D supplementation to support immunological functions under infection conditions.

Because fish is a vital protein source, fish products are strongly linked to the health of humans. However, the survival of fish is seriously threatened by environmental hypoxia. The present study reported that an additive named tea tree oil (TTO) can prevent damage to the grass carp gill from hypoxia stress. The fish was fed with six graded levels of TTO (0, 40, 80, 120, 160, and 200 mg/kg) diets for 60 days and afterward subjected to a 96-h hypoxia stress trial. Our research elaborated that TTO alleviated the deterioration of serum parameters (cortisol, glucose, lactic dehydrogenase, and lactic acid) and reduced oxidative damage to the gills and apoptosis caused by hypoxia stress, thereby improving the structural integrity of the gills. In addition, TTO relieved gill endoplasmic reticulum stress (ERS), which could be associated with reduced levels of glucose-regulated protein 78 (GRP78) protein and activating its downstream pathways. Furthermore, TTO decreased the levels of LC3-II protein and the related ATG mRNA, suggesting that gill autophagy was suppressed and was probably related to the HIF–BNIP3 pathway. In summary, TTO reduced gill damage caused by hypoxia stress via reducing oxidative damage, endoplasmic reticulum stress (ERS), and autophagy in fish, implying TTO mitigated the negative effects of hypoxia. Based on oxidative damage biomarkers reactive oxygen species (ROS) and malonaldehyde (MDA) contents in the gills, the optimal addictive TTO concentration of grass carp was determined as 89.63 and 90.25 mg/kg, respectively.

Knowledge of RNA molecules regulating testicular development and spermatogenesis in bulls is essential for elite bull selection and an ideal breeding program. Herein, we performed direct RNA sequencing (DRS) to explore the functional characterization of RNA molecules produced in the testicles of 9 healthy Simmental bulls at three testicular development stages (prepuberty, puberty, and postpuberty). We identified 5,043 differentially expressed genes associated with testicular weight. These genes exhibited more alternative splicing at sexual maturity, particularly alternative 3' (A3) and 5' (A5) splice sites usage and exon skipping (SE). The expression of hub genes in testicular developmental stages was also affected by both m6A and m5C RNA modifications. We found m5C-mediated splicing events significantly (p < 0.05) increased MAEL gene expression at the isoform level, likely promoting spermatogenesis. Our findings highlight the complexity of RNA processing and expression as well as the regulation of transcripts involved in testicular development and spermatogenesis.

Selenium is an indispensable trace element in animals. Previous findings have shown that selenium deficiency can lead to various muscle diseases. Muscle is the major edible portion of animals and the largest tissue in fish. Therefore, the aim of this experiment was to investigate how dietary selenium affects muscle growth in juvenile grass carp (Ctenopharyngodon idella). The 2520 healthy juvenile grass carp (9.76 ± 0.005 g) were randomly assigned to 7 test groups of 6 replicates each and fed graded selenomethionine (SeMet) (0.05, 0.20, 0.40, 0.61, 0.77, and 0.98 mg selenium/kg diet) and sodium selenite (SS) (0.79 mg selenium/kg diet) for 10 weeks. Results showed that optimal dietary selenium levels (0.40 mg/kg) increased growth performance, whole-body and muscle protein content, serum and muscle hormone levels, and selenium status in juvenile grass carp; optimal dietary selenium levels (0.40–0.61 mg/kg) increased the mean diameter of myofibers and the frequency of myofibers with diameter > 50 μm but decreased the frequency of myofibers with diameter < 20 μm, and promoted the expression of genes related to myoblast proliferation and differentiation; optimal dietary selenium levels (0.40–0.61 mg/kg) inhibited mRNA and protein levels of endoplasmic reticulum stress-related genes in juvenile grass carp muscle; optimal dietary selenium levels (0.40–0.61 mg/kg) increased muscle protein deposition in juvenile grass carp, which was associated with IGF-1/PI3K/AKT/TOR and IGF-1/PI3K/AKT/FoxO3a signaling pathways. In conclusion, selenium promoted muscle growth associated with myofiber hypertrophy in juvenile grass carp. And selenium-promoted myofiber hypertrophy was associated with inhibition of endoplasmic reticulum stress, promotion of myoblast proliferation and differentiation, and protein deposition. In addition, the dietary selenium requirement (SeMet as the selenium source) for juvenile grass carp (10–182 g) was 0.51 and 0.56 mg/kg, based on quadratic regression of percent weight gain (PWG) and muscle protein content, respectively.

This study was conducted to evaluate the effects of dietary myo-inositol (MI) on the antioxidant status of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1050 Jian carp (22.28±0.07 g) were randomly distributed into seven groups of three replicates each, feeding diets containing graded levels of MI (163.5, 232.7, 384.2, 535.8, 687.3, 838.8 and 990.3 mg kg−1 diet) for 60 days. Results indicated that the malondialdehyde content was the lowest for fish fed diets containing ≥384.2 mg MI kg−1, and the highest for fish fed the MI-unsupplemented basal diet (P<0.05). The protein carbonyl content was decreased with increasing dietary MI levels up to 535.8 mg kg−1 diet, and no differences were found with a further increase in the MI concentration. The anti-superoxide anion capacity (ASA) and anti-hydroxyl radical capacity (AHR) were increased with increasing MI levels up to 535.8 mg kg−1 diet, and plateaued thereafter. The superoxide dismutase and glutathione-S-transferase activities showed the same tendency with the ASA capacity. Catalase, glutathione peroxidase and glutathione reducase activities were improved with increasing MI levels up to 838.8, 384.2 and 687.3 mg kg−1 diet, respectively, and remained nearly constant thereafter. These results suggested that MI could inhibit oxygen radical generation, increase enzymatic antioxidant capacity and prevent oxidative damage of carp. Dietary MI requirements for ASA and AHR activities of juvenile Jian carp were 567.94 and 517.22 mg MI kg−1 diet respectively.

This study was conducted to study the effects of dietary zinc on lipid peroxidation, protein oxidation and antioxidant defence of juvenile Jian carp (Cyprinus carpio var. Jian) by feeding fish with increasing levels of zinc (15.3, 26.9, 40.8, 58.2, 68.9 and 92.5 mg Zn kg⁻¹) for 6 weeks. Results indicated that malondialdehyde (MDA) content and protein carbonyls (PC) in serum were the highest in fish fed diet containing 15.3 mg zinc kg⁻¹ diet (P 0.05). Serum antihydroxy radical (AHR), catalase (CAT) and glutathione-S-transferase (GST) activities followed the similar pattern to that observed in ASA. The MDA and PC levels, ASA, AHR, SOD, CAT, GPx, GR, GST activities and GSH content in intestine, hepatosomatic and muscle tissue followed the similar pattern to that observed in serum. The present results indicated that zinc decreased lipid peroxidation and protein oxidation and improved antioxidant defence in fish.

A 60-day feeding trial was carried out to investigate the effect of iron on growth, body composition and digestive enzyme activities. Diets with seven levels of iron (53.9, 90.0, 115.6, 146.1, 176.0, 215.8 and 266.0 mg iron kg−1 diet) were fed to Jian carp (initial weight 11.4 ± 0.0 g). Per cent weight gain (PWG), feed efficiency (FE) and protein efficiency ratio were the lowest in fish fed the basal diet (P < 0.05). Body protein content was increased with the increasing iron levels (P < 0.05), but moisture, lipid and ash of fish were not significantly affected by dietary iron levels (P > 0.05). Activities of trypsin, lipase, α-amylase, Na+, K+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase were improved with increasing dietary iron levels. Serum iron were significantly enhanced with dietary iron levels up to 146.1 mg iron kg−1 diet, and plateaued. In conclusion, iron improved digestive enzyme activities of juvenile Jian carp and the dietary iron requirement for serum iron of juvenile Jian carp (11.4–64.0 g) was 147.4 mg iron kg−1 diet with ferrous fumarate as the iron source.

Aquaculture NutritionVolume 17, Issue 2 p. e233-e240 Effects of dietary thiamin supplement on growth, body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var. Jian) H.-H. HUANG, H.-H. HUANG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorL. FENG, L. FENG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorY. LIU, Y. LIU Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorJ. JIANG, J. JIANG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorW.-D. JIANG, W.-D. JIANG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorK. HU, K. HU Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorS.-H. LI, S.-H. LI Animal Nutrition Institute, Sichuan Agricultural UniversitySearch for more papers by this authorX.-Q. ZHOU, X.-Q. ZHOU Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this author H.-H. HUANG, H.-H. HUANG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorL. FENG, L. FENG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorY. LIU, Y. LIU Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorJ. JIANG, J. JIANG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorW.-D. JIANG, W.-D. JIANG Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorK. HU, K. HU Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this authorS.-H. LI, S.-H. LI Animal Nutrition Institute, Sichuan Agricultural UniversitySearch for more papers by this authorX.-Q. ZHOU, X.-Q. ZHOU Animal Nutrition Institute, Sichuan Agricultural University Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, Ya'an, ChinaSearch for more papers by this author First published: 10 March 2011 https://doi.org/10.1111/j.1365-2095.2010.00756.xCitations: 42 Correspondence: Xiao-Qiu Zhou, Animal Nutrition Institute, Sichuan Agricultural University, Sichuan, Ya'an 625014, China. E-mail: [email protected]; [email protected] Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract A total of 1050 juvenile Jian carp (Cyprinus carpio var. Jian) (8.20 ± 0.02 g) were fed diets containing seven graded levels of thiamin (0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg kg−1) for 60 days to investigate the effects of thiamin on growth, body composition and digestive enzyme activities. Percent weight gain (PWG), feed intake and feed efficiency (FE) were the lowest in fish fed the basal diet (P < 0.05). Protein productive value and lipid productive value increased with increasing dietary thiamin levels up to 1.06 and 0.79 mg kg−1 diet, respectively (P < 0.05). Body protein and lipid increased with increasing dietary thiamin levels (P < 0.05), while moisture and ash of fish carcasses decreased with the increase in dietary thiamin supplementation (P < 0.05). Intestinal folds height had a similar trend to PWG (P < 0.05). Activities of α-amylase, lipase, trypsin, Na+, K+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase in intestine were all affected by the dietary thiamin (P < 0.05). In conclusion, thiamin could improve growth and intestinal enzyme activities of juvenile Jian carp. The dietary thiamin requirement of juvenile Jian carp (8.0–60.2 g) based on PWG was 1.02 mg kg−1 diet. Citing Literature Volume17, Issue2April 2011Pages e233-e240 RelatedInformation

A total of 1200 juvenile Jian carp (Cyprinus carpio var. Jian) (8.76 ± 0.02 g) were fed diets containing graded levels of histidine at 2.3 (unsupplemented control), 4.4, 6.3, 8.6, 10.8 and 12.7 g kg−1 diet for 60 days to investigate the effects of histidine levels on growth performance, body composition, intestinal enzymes activities and microflora. Specific growth rate (SGR), feed efficiency, protein efficiency ratio, protein productive value, body protein content and lipid content of fish were lowest in fish fed the basal diet (P < 0.05). Activities of glutamate-pyruvate transaminase in muscle and hepatopancreas, trypsin, chymotrypsin, amylase, lipase activities in intestine and hepatopancreas, and Na+, K+-ATPase, creatine kinase, alkaline phosphatase, γ-glutamyl transpeptidase activities in three intestinal segments were improved by dietary histidine (P < 0.05), whereas glutamate-oxaloacetate transaminase activities and plasma ammonia content followed an opposite trend. The amounts of Lactobacillus, Escherichia coli and Aeromonas were significantly affected by dietary histidine levels (P < 0.05). These results suggested that histidine could improve growth and enhance intestinal enzymes activities of juvenile Jian carp. The dietary histidine requirement of juvenile Jian carp (8.76–68.02 g) based on SGR was 7.8 g kg−1 diet or 2.38 g 100 g−1 protein by quadratic regression analysis.