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

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

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

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

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine.

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63 mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81 mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32 mg/kg diet.

Novel Perspective on Mechanism in Muscle Growth Inhibited by Ochratoxin A Associated with Ferroptosis: Model of Juvenile Grass Carp (Ctenopharyngodon idella) In Vivo and In Vitro Trials.

Ochratoxin A (OTA) is a common mycotoxin in food and feed that seriously harms human and animal health. This study investigated the effect of OTA on the muscle growth of juvenile grass carp (Ctenopharyngodon idella) and its possible mechanism in vitro. Our results have the following innovative findings: (1) Dietary OTA increased the expression of increasing phase I metabolic enzymes and absorbing transporters while reducing the expression of efflux transporters, thereby increasing their residue in muscles; (2) OTA inhibited the expressions of cell cycle and myogenic regulatory factors (MyoD, MyoG, and MyHC) and induced ferroptosis by decreasing the mRNA and protein expressions of FTH, TFR1, GPX4, and Nrf2 both in vivo and in vitro; and (3) the addition of DFO improved OTA-induced ferroptosis of grass carp primary myoblasts and promoted cell proliferation, while the addition of AKT improved OTA-inhibited myoblast differentiation and fusion, thus inhibiting muscle growth. Overall, this study provides a potential research target to further mitigate the myotoxicity of OTA.

Emerging role of vitamin D3 in alleviating intestinal structure injury caused by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella).

Bacterial pathogens destroy the structural integrity of functional organs in fish, leading to severe challenges in the aquaculture industry. Vitamin D3 (VD3) prevents bacterial infections and strengthens immune system function via vitamin D receptor (VDR). However, the correlation between VD3/VDR and the structural integrity of functional organs remains unclarified. This study aimed to investigate the influence of VD3 supplementation on histological characteristics, apoptosis, and tight junction characteristics in fish intestine during pathogen infection. A total of 540 healthy grass carp (257.24 ± 0.63 g) were fed different levels of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) for 70 d. Subsequently, fish were challenged with Aeromonas hydrophila, a pathogen that causes intestinal inflammation. Our present study demonstrated that optimal supplementation with VD3 (1) alleviated intestinal structural damage, and inhibited oxidative damage by reducing levels of oxidative stress biomarkers; (2) attenuated excessive apoptosis-related death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling (P < 0.05); (3) enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling (P < 0.05); and (4) elevated VDR isoform expression in fish intestine (P < 0.05). Overall, the results demonstrated that VD3 alleviates oxidative injury, apoptosis, and the destruction of tight junction protein under pathogenic infection, thereby strengthening pathogen defenses in the intestine. This finding supports the rationale for VD3 intervention as an essential practice in sustainable aquaculture.

The role of vitamin E in polyunsaturated fatty acid synthesis and alleviating endoplasmic reticulum stress in sub-adult grass carp (Ctenopharyngodon idella).

Vitamin E (VE) is an essential lipid-soluble vitamin that improves the fish flesh quality. However, the underlying molecular mechanisms remain unclear. This study aimed to investigate the effects of VE on growth performance and flesh quality in sub-adult grass carp (Ctenopharyngodon idella). A total of 450 fish (713.53 ± 1.50 g) were randomly divided into six treatment groups (three replicates per treatment) and fed for nine weeks with different experimental diets (dietary lipid 47.8 g/kg) that contained different levels of VE (5.44, 52.07, 96.85, 141.71, 185.66, and 230.12 mg/kg diet, supplemented as dl-α-tocopherol acetate). Notably, the treatment groups that were fed with dietary VE ranging from 52.07 to 230.12 mg/kg diet showed improvement in the percent weight gain, special growth rate, and feed efficiency of grass carp. Moreover, the treatment groups supplemented with dietary VE level of 141.71, 185.66, and 230.12 mg/kg diet showed enhancement in crude protein, lipid, and α-tocopherol contents in the muscle, and the dietary levels of VE ranging from 52.07 to 141.71 mg/kg diet improved muscle pH24h and shear force but reduced muscle cooking loss in grass carp. Furthermore, appropriate levels of VE (52.07 to 96.85 mg/kg diet) increased the muscle polyunsaturated fatty acid content in grass carp. Dietary VE also increased the mRNA levels of fatty acid synthesis-related genes, including fas, scd-1, fad, elovl, srebp1, pparγ, and lxrα, and up-regulated the expression of SREBP-1 protein. However, dietary VE decreased the expression of fatty acid decomposition-related genes, including hsl, cpt1, acox1, and pparα, and endoplasmic reticulum stress-related genes, including perk, ire1, atf6, eif2α, atf4, xbp1, chop, and grp78, and down-regulated the expression of p-PERK, p-IRE1, ATF6, and GRP78 proteins. In conclusion, dietary VE increased muscle fatty acid synthesis, which may be partly associated with the alleviation of endoplasmic reticulum stress, and ultimately improves fish flesh quality. Moreover, the VE requirements for sub-adult grass carp (713.53 to 1590.40 g) were estimated to be 124.9 and 122.73 mg/kg diet based on percentage weight gain and muscle shear force, respectively.

Tea polyphenols protect against Flavobacterium columnare-induced gill injury via suppression of oxidative stress, inflammation, and apoptosis in grass carp.

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.

A new insight on copper: Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella).

Copper (Cu) is a trace element, essential for fish growth. In the current study, in addition to growth performance, we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella). A total of 1080 fish (11.16 ± 0.01 g) were randomly divided into 6 treatments (3 replicates per treatment) to receive five doses of organic Cu, which were Cu citrate (CuCit) at 0.99 (basal diet), 2.19, 4.06, 6.15, and 8.07 mg/kg, and one dose of inorganic Cu (CuSO4·5H2O at 3.15 mg/kg), for 9 weeks. The results showed appropriate Cu level (4.06 mg/kg) enhanced growth performance, improved nutritional Cu status, and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas, intestine, and muscle of juvenile grass carp. Meanwhile, collagen content in fish muscle was increased after Cu intake, which was probably due to the following pathways: (1) activating CTGF/TGF-ß1/Smads signaling pathway to regulate collagen transcription; (2) upregulating of La ribonucleoprotein domain family 6 (LARP6) mRNA levels to regulate translation initiation; (3) increasing proline hydroxylase, lysine hydroxylase, and lysine oxidase activities to regulate posttranslational modifications. In addition, optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter >50 µm, which might be associated with upregulation of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen, myogenic determining factor (MyoD), myogenic factor 5, myogenin (MyoG), myogenic regulatory factor 4 and myosin heavy chain (MyHC) and downregulation of myostatin mRNA levels, increasing protein levels of MyoD, MyoG and MyHC in fish muscle. Finally, based on percentage weight gain (PWG), serum ceruloplasmin (Cp) activity and collagen content in fish muscle, Cu requirements were determined as 4.74, 4.37 and 4.62 mg/kg diet (CuCit as Cu source) of juvenile grass carp, respectively. Based on PWG and Cp activity, compared to CuSO4·5H2O, the efficacy of CuCit were 131.80% and 115.38%, respectively. Our findings provide new insights into Cu supplementation to promote muscle growth in fish, and help improve the overall productivity of aquaculture.

New perspective on mechanism in muscle toxicity of ochratoxin A: Model of juvenile grass carp (Ctenopharyngodon idella).

Ochratoxin A (OTA) is a common fungal toxin that pollutes raw materials of aquatic feeds (such as corn, soybean meal, and wheat). This study explored the effects of OTA through diet on muscle toxicity in juvenile grass carp (Ctenopharyngodon idella). The following results were obtained for the muscle. (1) With an increase in dietary OTA, the residue of OTA in muscle increased, muscle fiber diameter and density decreased, and even muscle fiber breakage. (2) OTA caused oxidative stress by downregulating GPx1 (a, b) and Trx via inhibited the PGC1-α/Nrf2 signaling pathway. (3) OTA exacerbated endoplasmic reticulum stress in the muscle by causing endoplasmic reticulum expansion (results of transmission electron microscopy) and upregulating the expression of GRP78, eIF2α, ATF6, PERK, and CHOP. (4) OTA reduced muscle fiber diameter by inhibiting protein synthesis (AKT, TOR, and S6K1) and promoting the mRNA expression of protein degradation-related genes (MURF1, MAFBX, and FoxO3a), as well as by reducing AKT and promoting the immunofluorescence expression of FoxO3. (5) OTA inhibits collagen deposition by downregulating TGF-ß1, TGF-ßR1, Smad2, Smad3, Smad4, CTGF, TIMP, PHD, and LOX mRNA expressions as well as the CTGF immunofluorescence expression. Moreover, based on the GSH and collagen content contents, the upper safe dose for OTA-induced toxicity was 963.6 and 1129.6 µg/kg diet, respectively. Using the example of OTA, our research has provided new insights that raise concerns about the quality of aquatic products by exploring muscle toxicity caused by mycotoxins.

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

The vertebrate mucosal barrier comprises physical and immune elements, as well as bioactive molecules, that protect organisms from pathogens. Vitamin D is a vital nutrient for animals and is involved in immune responses against invading pathogens. However, the effect of vitamin D on the mucosal barrier system of fish, particularly in the skin, remains unclear. Here, we elucidated the effect of vitamin D supplementation (15.2, 364.3, 782.5, 1167.9, 1573.8, and 1980.1 IU/kg) on the mucosal barrier system in the skin of grass carp (Ctenopharyngodon idella) challenged with Aeromonas hydrophila. Dietary vitamin D supplementation (1) alleviated A. hydrophila-induced skin lesions and inhibited oxidative damage by reducing levels of reactive oxygen species, malondialdehyde, and protein carbonyl; (2) improved the activities and transcription levels of antioxidant-related parameters and nuclear factor erythroid 2-related factor 2 signaling; (3) attenuated cell apoptosis by decreasing the mRNA and protein levels of apoptosis factors involved death receptor and mitochondrial pathway processes related to p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signaling; (4) improved tight junction protein expression by inhibiting myosin light-chain kinase signaling; and (5) enhanced immune barrier function by promoting antibacterial compound and immunoglobulin production, downregulating pro-inflammatory cytokine expression, and upregulating anti-inflammatory cytokines expression, which was correlated with nuclear factor kappa B and the target of rapamycin signaling pathways. Vitamin D intervention for mucosal barrier via multiple signaling correlated with vitamin D receptor a. Overall, these results indicate that vitamin D supplementation enhanced the skin mucosal barrier system against pathogen infection, improving the physical and immune barriers in fish. This finding highlights the viability of vitamin D in supporting sustainable aquaculture.

Promotion of fatty acid metabolism and glucose metabolism in the muscle of sub-adult grass carp (Ctenopharyngodon idella): The role of alpha-linoleic acid/linoleic acid (ALA/LNA) ratios.

The n6/n3 ratios improved meat quality of terrestrial animals, but alpha-linolenic acid/linoleic acid (ALA/LNA) ratios were rarely studied in aquatic animals. In this study, sub-adult grass carp (Ctenopharyngodon idella) were fed diets fed diets containing six varying ALA/LNA ratios (0.03, 0.47, 0.92, 1.33, 1.69, and 2.15) for 9 weeks and the total value of n3 + n6 (1.98) was kept constant for all six treatments. The results indicated optimal ALA/LNA ratio improved growth performance, changed fatty acid composition in grass carp muscle, and promoted glucose metabolism. Additionally, optimal ALA/LNA ratio improved chemical attributes by increasing crude protein and lipid contents, and technological attributes by increasing pH24h value and shear force in grass carp muscle. The signaling pathways related to fatty acid metabolism and glucose metabolism (LXRα/SREBP-1, PPARα, PPARγ, AMPK) might be responsible for these changes. Dietary optimal ALA/LNA ratio based on PWG, UFA and glucose contents was 1.03, 0.88 and 0.92, respectively.

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

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

Dietary threonine improves muscle nutritional value and muscle hardness associated with collagen synthesis in grass carp (Ctenopharyngodon idella).

To further explain the improvement effect of threonine (Thr) on the fillet quality of fish, a 9-week feeding experiment was conducted. After feeding graded levels of Thr (2.38, 5.38, 8.38, 11.38, 14.38 and 17.38 g/kg), the compositions of fillet hydrolyzed amino acid and fatty acid, and the muscle hardness associated with collagen biosynthesis were mainly analyzed in grass carp (Ctenopharyngodon idella). The results showed that Thr increased the pH value, changed the amino acids and fatty acid composition of fillets, especially essential amino acid (EAA), C22:6n3 (DHA) and C20:5n3 (EPA). Furthermore, this study revealed for the first time that the improvement of muscle hardness by Thr was associated with collagen biosynthesis, and the TGF-ß1/Smads, LARP6a and Hsp47 regulate transcriptional processes, translation initiation and post-translational modifications in collagen biosynthesis, respectively. This study offered a basis for exploring the contribution of Thr in improving muscle quality in sub-adult grass carp.

Aflatoxin B1 exposure induced developmental toxicity and inhibited muscle development in zebrafish embryos and larvae.

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.

Aflatoxin B1 damaged structural barrier through Keap1a/Nrf2/ MLCK signaling pathways and immune barrier through NF-κB/ TOR signaling pathways in gill of grass carp (Ctenopharyngodon idella).

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 µg/kg diet.

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

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

Dietary cinnamaldehyde improves muscle protein content by promoting muscle fiber growth via PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling pathway in grass carp (Ctenopharyngodon idella).

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.

Dietary isoleucine affects muscle fatty acid and amino acid profiles through regulating lipid metabolism and autophagy in hybrid catfish (Pelteobagrus vachelli ♀ × Leiocassis longirostris ♂).

The present study explored the impacts of Ile on muscle fatty acid and amino acid profiles, lipid metabolism, and autophagy in hybrid catfish. Seven isonitrogenous (387.8 g/kg protein) semi-purified diets were formulated to contain 5.0 (control), 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 g Ile/kg diet respectively. The fish (initial weight of 33.11 ± 0.09 g) were randomly assigned to 7 groups for a 56-day trial. Each group has 3 replicates with 30 fish per replicate, fed at 08:00 and 18:00 each day. Results showed that muscle protein and lipid, C14:0, C18:0, C22:0, C14:1, C18:1n-9, polyunsaturated fatty acid (PUFA), Arg, Ile, Ala, Cys, Gly, Tyr, essential amino acid (EAA), and total amino acid (TAA) contents and flavor amino acid (FAA)/TAA in muscle had positive linear and/or quadratic responses to dietary Ile levels (P < 0.05). Fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), acetyl-CoA carboxylase (ACC), and lipoprotein lipase (LPL) activities had positive linear and/or quadratic responses, but carnitine palmitoyl transferase 1 (CPT1) activity had a negative response with increasing dietary Ile levels (P < 0.05). The mRNA expressions of FAS, SCD, ACC, LPL, fatty acid binding protein 4 (FABP4), FATP1, sterol response element-binding protein 1c (SREBP-1c), sequestosome 1 (SQSTM1), and adenosine 5'-monophosphate-activated protein kinase (AMPK) had positive linear and/or quadratic responses to dietary Ile levels (P < 0.05). The mRNA expressions of hormone-sensitive lipase (HSL), CPT1, peroxisome proliferator-activated receptor α (PPARα), PPARγ, uncoordinated 51-like kinase 1 (ULK1), beclin1 (Becn1), autophagy-related protein 9α (Atg9α), Atg4b, Atg7, autophagy marker light chain 3 B (LC3B), and SQSTM1 in muscle had negative linear and/or quadratic responses to dietary Ile levels (P < 0.05). The p-AMPK and ULK1 protein levels, and p-AMPK/AMPK were decreased by 12.5 g Ile/kg in the diet (P < 0.05). Finally, SQSTM1 protein level had the opposite effect (P < 0.05). The above results indicate that dietary Ile improves fish muscle fatty acid and amino acid profiles potentially via respectively regulating lipid metabolism and autophagy. The Ile requirement of hybrid catfish (33 to 72 g) were estimated to be 12.63, 13.77, 13.75, 11.45, 10.50, 12.53 and 12.21 g/kg diet based on the regression analysis of protein, lipid, SFA, PUFA, FAA, EAA, and TAA muscle contents, respectively.

Dietary Aflatoxin B1 attenuates immune function of immune organs in grass carp (Ctenopharyngodon idella) by modulating NF-κB and the TOR signaling pathway.

Aflatoxin B1 (AFB1) is kind of a common mycotoxin in food and feedstuff. Aquafeeds are susceptible to contamination of AFB1. In teleost fish, the spleen and head kidney are key immune organ. Moreover, the fish skin is a critical mucosal barrier system. However, there was little study on the effects of dietary AFB1 on the immune response of these immune organs in fish. This study aimed to explore the impacts of oral AFB1 on the immune competence and its mechanisms in the skin, spleen, and head kidney of grass carp. Our work indicated that dietary AFB1 reduced antibacterial compounds and immunoglobulins contents, and decreased the transcription levels of antimicrobial peptides in grass carp immune organs. In addition, dietary AFB1 increased the transcription levels of pro-inflammatory cytokines and reduced the transcription levels of anti-inflammatory cytokines in the grass carp immune organs, which might be regulated by NF-κB and TOR signaling, respectively. Meanwhile, we evaluated the content of AFB1 in the grass carp diet should not exceed 29.48 µg/kg diet according to the levels of acid phosphatase and lysozyme. In summary, dietary AFB1 impaired immune response in grass carp skin, spleen, and head kidney.

Physicochemical property optimization and nutrient redistribution in the muscle of sub-adult grass carp (Ctenopharyngodon idella) by conjugated linoleic acid.

We studied the effects of conjugated linoleic acid (CLA) on the amount of nutrients, flavour substances, and healthcare fatty acids, the physicochemical properties, and the potential molecular mechanisms in the muscles of sub-adult grass carp (Ctenopharyngodon idella). Fish were fed graded levels of CLA (0.0, 3.1, 6.4, 9.6, 12.7, and 15.9 g/kg diets) for 60 days. Protein, glutamic acid, alanine, inosine monophosphate (IMP), eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3), and total CLA contents (p < 0.05) increased in CLA 3.1 âˆ¼ 12.7, 6.4 âˆ¼ 9.6, 6.4 âˆ¼ 9.6, 6.4 âˆ¼ 15.9, 3.1 âˆ¼ 9.6, 3.1 âˆ¼ 9.6, and 3.1 âˆ¼ 15.9 g/kg diet, respectively (p < 0.05). In addition, optimal CLA significantly increased pH24, shear force, collagen content, and myofibre density in the muscle (P < 0.05); however, it decreased myofibre diameter (p < 0.05). We concluded that 6-9 g/kg CLA in the diet could improve the flesh quality of sub-adult grass carp.