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Vitamin D 3 (VD 3 ), an essential nutrient for animals, has been demonstrated to stimulate the uptake of certain amino acids. However, the role of VD 3 in the intestine, the primary site for digestion and absorption of nutrients, remains poorly characterized. Here, the grass carp ( Ctenopharyngodon idella ) was studied to assess the influence of different doses of VD 3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) on growth performance, intestinal morphology, digestive absorption, amino acid transport, and potential signaling molecule levels in a feeding experiment. As a result, dietary VD 3 improved growth performance, intestinal structure, and digestive and brush border enzyme activities. Additionally, most intestinal free amino acids and their transporters were upregulated after VD 3 intake, except for Ala, Lys, Asp, Leu, solute carrier ( SLC ) 7A7 , SLC1A5 , and SLC1A3 mRNA in different segments, Leu and SLC6A1 4 mRNA in the proximal intestine, and SLC7A5 mRNA in the mid and distal intestine. In the crucial target of rapamycin (TOR) signal pathway of amino acid transport, the gene and protein expression of TOR, S6 kinase 1, and activating transcription factor 4 were elevated, whereas 4E-binding protein 1 was decreased, further suggesting an advanced amino acid absorption capacity in the fish due to VD 3 supplementation. Based on percentage weight gain, feed efficiency, and trypsin activity, the VD 3 requirements of on-growing grass carp were estimated to be 968.33, 1,005.00, and 1,166.67 IU/kg, respectively. Our findings provide novel recommendations for VD 3 supplementation to promote digestion and absorption capacities of fish, contributing to the overall productivity of aquaculture., (© 2022 Chinese Association of Animal Science and Veterinary Medicine. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.)

Mannan oligosaccharides (MOS) are a type of functional oligosaccharide which have received increased attention because of their beneficial effects on fish intestinal health. However, intestinal structural integrity is a necessary prerequisite for intestinal health. This study focused on exploring the protective effects of dietary MOS supplementation on the grass carp’s (Ctenopharyngodon idella) intestinal structural integrity (including tight junction (TJ) and adherent junction (AJ)) and its related signalling molecule mechanism. A total of 540 grass carp (215.85 ± 0.30 g) were fed six diets containing graded levels of dietary MOS supplementation (0, 200, 400, 600, 800 and 1000 mg/kg) for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila for 14 days. We used ELISA, spectrophotometry, transmission electron microscope, immunohistochemistry, qRT-PCR and Western blotting to determine the effect of dietary MOS supplementation on intestinal structural integrity and antioxidant capacity. The results revealed that dietary MOS supplementation protected the microvillus of the intestine; reduced serum diamine oxidase and d-lactate levels (p < 0.05); enhanced intestinal total antioxidant capacity (p < 0.01); up-regulated most intestinal TJ and AJ mRNA levels; and decreased GTP-RhoA protein levels (p < 0.01). In addition, we also found several interesting results suggesting that MOS supplementation has no effects on ZO-2 and Claudin-15b. Overall, these findings suggested that dietary MOS supplementation could protect intestinal ultrastructure, reduce intestinal mucosal permeability and maintain intestinal structural integrity via inhibiting MLCK and RhoA/ROCK signalling pathways.

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp ( Ctenopharyngodon idella ) infected with Flavobacterium columnare. The fish were fed different concentrations of vitamin A diets for 10 weeks and then infected with F. columnare by immersion. We observed that optimal vitamin A significantly prevented gill rot morbidity in fish infected with F. columnare . Further investigations revealed that vitamin A boosted the gill immunity by increasing the contents of complements (C3 and C4), activities of acid phosphatase (ACP) and lysozyme, mRNAs of β-defensin-1, liver-expressed antimicrobial peptide 2A and 2B (LEAP-2A and LEAP-2B) , hepcidin , and anti-inflammatory cytokines like transforming growth factor β1 (TGF-β1), TGF-β2, interleukin-10 (IL-10), and IL-11 . It also enhanced the levels of various related signaling molecules including inhibitor protein κBα (IκBα), target of rapamycin (TOR), and ribosome protein S6 kinase 1 (S6K1) but downregulated the expression of pro-inflammatory cytokines including IL-1β, IL-8, tumor necrosis factor α (TNF-α) , and interferon γ2 (IFN-γ2) and related signaling molecules including nuclear factor κB p65 (NF-κB p65) (rather than NF-κB p52), IκB kinase β (IKKβ), IKKγ (rather than IKKα), eIF4E-binding protein 1 (4E-BP1), and 4E-BP2 mRNA levels in fish gills. In addition, dietary vitamin A markedly lowered the concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC), increased both the activities and mRNAs of copper/zinc superoxide dismutase (Cu/ZnSOD), MnSOD, glutathione transferases (GSTs), glutathione peroxidase (GPx), and glutathione reductase (GR) associated with upregulation of NF-E2-related factor 2 (Nrf2) mRNAs and downregulation of Kelch-like-ECH-associated protein (Keap1a) and Keap1b mRNAs. Moreover, vitamin A decreased the mRNAs of different apoptotic mediators [ caspases 8, 9, 3 (rather than 7)] associated with downregulation of signaling molecule p38 mitogen-activated protein kinase (p38MAPK) mRNAs in fish gills. Besides, vitamin A promoted tight junction (TJ) complex mRNAs [including claudin-b, -c, -3, -7, -12, occludin , and zonula occludens-1 (ZO-1) ] that have been linked to the downregulation of myosin light chain kinase (MLCK) signaling. Taken together, the current study demonstrated for the first time that vitamin A markedly enhanced gill health associated with immune modulation and physical barrier protection. Based on protecting fish against gill rot morbidity, ACP activity, and against lipid peroxidation, optimum vitamin A concentrations in on-growing grass carp (262-997 g) were found to be 1,991, 2,188, and 2,934 IU/kg diet, respectively., Competing Interests: S-YK, S-WL, and LT are employed by Sichuan Animtech Feed Co., Ltd. H-FM and LZ are employed by Tongwei Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jiang, Zhang, Feng, Wu, Liu, Kuang, Li, Tang, Mi, Zhang and Zhou.)

This study aims to investigate the effects of threonine (Thr) on immunoregulation in vivo and in vitro of teleost grass carp ( Ctenopharyngodon idella ). Juveniles (9.53 ± 0.02 g) were reared for 8 weeks with respective Thr diet (3.99, 7.70, 10.72, 14.10, 17.96, and 21.66 g/kg) and then challenged with Aeromonas hydrophila for in vivo study. Macrophages isolated from head kidney were treated in vitro for 48 h with L-Thr (0, 0.5, 1.0, 2.0, 4.0, and 8.0 mM) after 6 h of lipopolysaccharide induction. The results showed that, compared with Thr deficiency (3.99 g/kg), the optimal dietary Thr (14.10g/kg) affected the immunocyte activation in the head kidney (HK) and spleen (SP) by downregulating the mRNA expressions of MHC-II and upregulating CD4 (not CD8), and it mediated the innate immune by enhancing the activities of lysozyme (LZ), acid phosphatase content of complement 3 (C3) and C4, increasing the mRNA abundances of hepcidin, liver expressed antimicrobial peptide-2A (LEAP-2A), LEAP-2B, β-defensin1, downregulating tumor necrosis factor α (TNF-α), IL-6, IL-1β, IL-12p35, IL-12p40, IL-17AF1, and IL-17D partly by attenuating RORγ1 transcriptional factor and nuclear factor kappa B p65 (NF-κBp65) signaling cascades [IKKβ/IκBα/NF-κBp65] and upregulating transforming growth factor β1 (TGF-β1), IL-4/13A, -4/13B, IL-10, and IL-22 partly by GATA-3. Besides these, the optimal dietary Thr regulated the adaptive immune by upregulating the mRNAs of immunoglobulin M (IgM) and IgZ (not IgD). Moreover, 2 mM Thr downregulated in vitro the mRNA abundances of colony stimulating factor-1, inducible nitric oxide synthase, mannose receptor 1, matrix metalloproteinase2 (MMP-2), and MMP-9 significantly ( P < 0.05), indicating that Thr could attenuate the M1-type macrophages' activation. Moreover, L-Thr downregulated the mRNA transcripts of TNF-α, IL-6, and IL-1β associated with impairing the SOCS1/STAT1 signaling and upregulated IL-10 and TGF-β1 partly by accentuating the SOCS3/STAT3 pathway. The above-mentioned observations suggested that Thr improved the immune status in the immune organs of fish by enhancing the immune defense and mediating the inflammation process. Finally, based on the immune indices of LZ activity in HK and C3 content in SP, the optimal Thr for immune enhancement in juvenile grass carp (9.53-53.43 g) was determined to be 15.70 g/kg diet (4.85 g/100 g protein) and 14.49 g/kg diet (4.47 g/100 g protein), respectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dong, Jiang, Wu, Liu, Kuang, Tang, Tang, Zhou and Feng.)

The occurrence of immuno-compromised status in animals with zearalenone (ZEA) exposure may be a critical contributor to associated mucosal (gastrointestinal tract) diseases. However, it is difficult to assess the associated risks with limited reference data. This study comprehensively discussed the effects of ZEA on intestinal immune components, cytokines and molecular mechanism of juvenile grass carp infected with Aeromonas hydrophila. Specifically, the fish were fed six graded levels of dietary ZEA (0-2507 μg kg -1 diet) for 70 d. The results pointed out that the average residual amount of ZEA in the intestines increased with dose level after ZEA feeding. We further performed an infection assay using A. hydrophila. After 14 d, ZEA groups increased enteritis morbidity rate compared with controls. The acid phosphatase (ACP), lysozyme (LZ) activities and immunoglobulin M (IgM) content were significantly decreased in three intestinal segments. Furthermore, ZEA could reduce the transcription of β-defensin-1, Hepcidin, liver expressed antimicrobial peptide 2A/2B (LEAP-2A/2B) and Mucin-2. We next confirmed the loss of these immune components accompanied by the invasion of the intestinal barrier by bacteria, as indicated by activation of the nuclear factor κB (NF-κB) and the expression of downstream cytokines. Notably, the phosphorylated target of rapamycin (TOR) plays an important role in regulating these genes, thus indicating a possible target caused by ZEA. In summary, the extensive inhibition of immune components by ZEA promotes the spread of pathogens, which may increase the possibility of intestinal mucosa exposure and the risk of transforming disease., (Copyright © 2021. Published by Elsevier Inc.)

The objective of this study was to evaluate the efficacy of dietary Mannan oligosaccharides (MOS) supplementation on skin barrier function and the mechanism of on-growing grass carp ( Ctenopharyngodon idella ). Five hundred forty grass carp were fed for 60 days from the growing stage with six different levels of MOS diets (0, 200, 400, 600, 800, and 1,000 mg kg -1 ). At the end of the growth trial, the 14-day Aeromonas hydrophila challenge experiment has proceeded. The obtained data indicate that MOS could (1) decline skin lesion morbidity after being challenged by the pathogenic bacteria; (2) maintain physical barrier function via improving antioxidant ability, inhibiting excessive apoptosis, and strengthening the tight junction between the epithelial cell and the related signaling pathway (Nrf2/Keap1, p38MAPK, and MLCK); and (3) regulate immune barrier function by modulating the production of antimicrobial compound and expression of involved cytokines and the related signaling pathway (TOR and NFκB). Finally, we concluded that MOS supplementation reinforced the disease resistance and protected the fish skin barrier function from Aeromonas hydrophila infection., Competing Interests: S-YK, LT, S-WL, X-AL and C-BZ were employed by Sichuan Animtech Feed Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Feng, Jiang, Wu, Liu, Jiang, Kuang, Tang, Li, Liu, Zhong and Zhou.)

The present study evaluated the effect of cinnamaldehyde (CIN) on the growth performance and digestion and absorption capacity of grass carp (Ctenopharyngodon idella). Fish were fed five diets including graded levels of CIN for 60 days. The results indicated that (1) appropriate CIN supplementation increased the growth performance and promoted the intestine growth of grass carp; (2) dietary appropriate CIN supplementation increased the digestion and absorption capacity by increasing the activities of intestinal and hepatopancreas digestive enzymes (lipase, chymotrypsin, trypsin, and amylase) and intestinal brush border enzymes (creatine kinase (CK), Na + /K + -ATPase, γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (AKP)); (3) dietary CIN increased the absorption capacity which may be associated with the upregulated messenger RNA (mRNA) abundances of their amino acid transporters (AATs) in the intestine, which might be associated with activating the target of rapamycin (TOR) signaling pathway. The best CIN supplementation in the diets of grass carp was estimated to be 76.40 mg kg -1 diet based on the best percent weight gain (PWG). In general, CIN increased the digestion and absorption capacity of grass carp and raised the mRNA abundances of AATs which may be partly related to activation of the TOR signaling pathway.

The present study aimed to investigate the dynamic process of soybean β-conglycinin in digestion, absorption, and metabolism in the intestine of grass carp (Ctenopharyngodon idella). Fish fed with 80 g β-conglycinin/kg diet for 7 weeks, the intestinal digestive enzyme was extracted to hydrolyze β-conglycinin in vitro, the free amino acid and its metabolism product contents in intestinal segments were analyzed. The present study first found that β-conglycinin cannot be thoroughly digested by fish intestine digestive enzyme and produces new products (about 60- and 55-kDa polypeptides). The indigestible β-conglycinin further caused the free amino acid imbalance, especially caused free essential amino acid deficiency in the proximal intestine but excess in the distal intestine. Moreover, these results might be partly associated with the effect of β-conglycinin in amino acid transporters and tight junction-regulated paracellular pathway. Finally, dietary β-conglycinin increased the content of amino acid catabolism by-product ammonia while decreased the amino acid anabolism product carnosine content in the proximal intestine and distal intestine. Thus, the current study first and systemically explored the dynamic process of β-conglycinin in digestion, absorption, and metabolism, which further supported our previous study that dietary β-conglycinin suppressed fish growth and caused intestine injure.

Deoxynivalenol (DON) is a common mycotoxin existed in animal feed, and lead to significant economic loss due to its negative impacts on animal growth performance and animal health. The gill is a primary mucosal immune organ in teleosts, and the structural integrity of the gill is closely relevant with fish healthy growth. Hence, this study assessed the influences of DON on the gill structural integrity of juvenile grass carp, Ctenopharyngodan idella (initial average weight 12.17 ± 0.01 g), when offered with six different diets which contained various content of DON (27, 318, 636, 922, 1243 and 1515 μg/kg diet) for 60 days. Our research firstly systematically elaborated that DON caused histopathological lesions, oxidative injury, reduction of antioxidant ability, apoptosis as well as damages of tight junctions in fish gills. Comparing these data to the control, we found that DON at dose of more than 318 μg/kg diet led to oxidative injury, apoptosis and disruption of tight junctions in fish gill, which were likely to be relevant with Nrf2, JNK and MLCK signalling pathways, respectively. It was worth noting that DON was not found to affect the gene expressions of Keap1b (rather than Keap1a), claudin-b, claudin-3c and claudin-15b (not claudin-15a) in fish gills. Furthermore, based on MDA and T-AOC activities in the gill, the maximum permissible levels of DON were evaluated to be 375.60 as well as 412.91 μg/kg diet in grass carp, respectively., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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

In aquaculture, the occurrence of enteritis has increased and dietary nutrition is considered as one of the major strategies to solve this problem. In the present study, we assume that dietary phosphorus might enhance intestinal immune barrier and physical barrier function to reduce the occurrence of enteritis in fish. To test this assumption, a total of 540 grass carp (Ctenopharyngodon idella) were investigated by feeding graded levels of available phosphorus (0.95-8.75 g/kg diet) and then infection with Aeromonas hydrophila. The results firstly showed that phosphorus deficiency decreased the ability to combat enteritis, which might be related to the impairment of intestinal immune barrier and physical barrier function. Compared with optimal phosphorus level, phosphorus deficiency decreased fish intestinal antimicrobial substances activities or contents and down-regulated antimicrobial peptides mRNA levels leading to the impairment of intestinal immune response. Phosphorus deficiency down-regulated fish intestinal anti-inflammatory cytokines mRNA levels and up-regulated the mRNA levels of pro-inflammatory cytokines [except IL-1β and IL-12p35 in distal intestine (DI) and IL-12p40] causing aggravated of intestinal inflammatory responses, which might be related to the signalling molecules target of rapamycin and nuclear factor kappa B. In addition, phosphorus deficiency disturbed fish intestinal tight junction function and induced cell apoptosis as well as oxidative damage leading to impaired of fish intestinal physical barrier function, which might be partially associated with the signalling molecules myosin light chain kinase, c-Jun N-terminal protein kinase and NF-E2-related factor 2, respectively. Finally, based on the ability to combat enteritis, dietary available phosphorus requirement for grass carp (254.56-898.23 g) was estimated to be 4.68 g/kg diet., (Copyright © 2017. Published by Elsevier Ltd.)

This study was conducted to investigate the effects of methionine hydroxy analogue (MHA) on the physical barrier and immune defence in the gill of young grass carp (Ctenopharyngodon idella). A total 630 young grass carp with an average initial weight of 259.70 ± 0.47 g were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one DL-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. After feeding trial, 15 fish from each treatment were challenged with Flavobacterium columnare. Compared to the basal diet, optimal MHA improved cellular structure integrity of gill via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < .05). Simultaneously, optimal MHA supplementation improved cellular structure integrity of gill via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < .05). Besides, optimal MHA improved intercellular structure integrity of immune organs via up-regulating the mRNA levels of intercellular tight junctions-related genes, which might be owing to the down-regulation of myosin light chain kinase (MLCK) mRNA levels (P < .05). Summarily, MHA could improve the physical barrier of fish gill. In addition, optimal MHA supplementation increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M contents and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin and β-defensin, suggesting that MHA could enhance antimicrobial ability of fish gill. Meanwhile, optimal MHA supplementation enhanced the immune defence of gill via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase 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 < .05). In conclusion, the positive effect of MHA on gill health is associated with the improvement of the defence against apoptosis, antioxidant status, tight junctions and immune defence of fish gill. Meanwhile, MHA was superior to DLM on improving the physical barrier of fish gill. For the direction to healthy breeding of young grass carp, the optimal MHA supplementation levels on the premise of 4.01 g/kg methionine basal were estimated by quadratic regression curve, such as 5.49, 6.17 and 6.02 g/kg diet bases on the defence against gill-rot, malondialdehyde content and LZ activity in the gill, respectively., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this study was to assess the effects of dietary pyridoxine (PN) deficiency on intestinal antioxidant capacity, cell apoptosis and intercellular tight junction in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (231.85 ± 0.63 g) were fed six diets containing graded levels of PN (0.12-7.48 mg/kg diet) for 10 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results showed that compared with the optimal PN level, PN deficiency (1) increased the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC), decreased the activities and mRNA levels of antioxidant enzymes such as copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) (P < .05); (2) up-regulated the mRNA levels of cysteinyl aspartic acid-protease-3 (caspase-3), caspase-7, caspase-8, caspase-9, Bcl-2 associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1) and Fas ligand (FasL), and down-regulated the mRNA levels of inhibitor of apoptosis proteins (IAP), B-cell lymphoma protein-2 (Bcl-2) and myeloid cell leukaemia-1 (Mcl-1) (P < .05); (3) down-regulated the mRNA levels of ZO-1, occludin [only in middle intestine (MI)], claudin-b, claudin-c, claudin-f, claudin-3c, claudin-7a, claudin-7b and claudin-11, and up-regulated the mRNA levels of claudin-12 and claudin-15a (P < .05), which might be partly linked to Kelch-like-ECH-associated protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2), p38 mitogen-activated protein kinase (p38MAPK) and myosin light chain kinase (MLCK) signalling in the intestines of fish. However, the activities and mRNA levels of MnSOD, the mRNA levels of Keap1b, c-Jun N-terminal protein kinase (JNK) and claudin-15b in three intestinal segments, and the mRNA levels of occludin in the proximal intestine (PI) and distal intestine (DI) were not affected by graded levels of PN. These data indicate that PN deficiency could disturb the intestinal physical barrier function of fish. Additionally, based on the quadratic regression analysis for MDA content and GST activity, the dietary PN requirements for young grass carp were estimated as 4.85 and 5.02 mg/kg diet, respectively., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the effects and potential mechanisms of dietary iron on immune function and structural integrity in gill of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (242.32 ± 0.58 g) were fed diets containing graded levels of iron at 12.15 (basal diet), 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg/kg for 60 days. Subsequently, a challenge test was conducted by infection with Flavobacterium columnare to investigate the effects of dietary iron on gill immune function and structural integrity in young grass carp. First, the results indicated that compared with the optimal iron level, iron deficiency decreased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antibacterial peptides, anti-inflammatory cytokines (except IL-4/13B), inhibitor of κBα (IκBα), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1). In contrast, iron deficiency up-regulated the mRNA levels of pro-inflammatory cytokines (except IL-6 and IFN-γ2), nuclear factor κB p65 (NF-κBp65), IκB kinases α (IKK), IKKβ, IKKγ, eIF4E-binding protein 1 (4E-BP1) and 4E-BP2 in gill of young grass carp, indicating that iron deficiency could impair immune function in fish gill. Second, iron deficiency down-regulated the mRNA levels of inhibitor of apoptosis protein (IAP) and myeloid cell leukemia 1 (Mcl-1), decreased activities and mRNA levels of antioxidant enzymes, down-regulated the mRNA levels of NF-E2-related factor 2 (Nrf2) and tight junction proteins (except claudin-12 and -15), and simultaneously increased malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) contents. Iron deficiency also up-regulated mRNA levels of cysteinyl aspartic acid-protease (caspase) -2, -7, -8, -9, Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), B-cell-lymphoma-2 associated X protein (Bax), p38 mitogen-activated protein kinase (p38MAPK), Kelch-like ECH-associating protein (Keap) 1a, Keap1b, claudin-12, -15 and MLCK, indicating that iron deficiency could disturb the structural integrity of gill in fish. Third, iron excess impaired immune function and structural integrity in gill of young grass carp. Forth, there was a better effect of ferrous fumarate than ferrous sulfate in young grass carp. Finally, the iron requirements based on ability against gill rot, ACP activity and MDA content in gill of young grass carp were estimated to be 76.52, 80.43 and 83.17 mg/kg, respectively., (Copyright © 2018. Published by Elsevier Ltd.)

The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg(-1) diet) for 56 days. The test diets (28% CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg(-1) diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95% maximum response indicated lysine requirement was 10.9 g kg(-1) diet. Activities of trypsin, chymotrypsin, lipase, Na(+), K(+)-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg(-1) diet, respectively.