High-glucose exposure impairs intestinal metabolic homeostasis and barrier integrity in fish, but the transcriptional responses associated with high-glucose adaptation in fish intestinal epithelial cells remain incompletely understood. This study investigated whether exogenous 5-methylcytosine (5MC) alleviates high-glucose-induced metabolic and epithelial stress in grass carp (Ctenopharyngodon Idella) intestinal epithelial cells and whether these responses are associated with changes in DNA methyltransferase 3 beta (dnmt3b) expression and Caudal type homeobox 1b (cdx1b)/Sodium-glucose cotransporter 1 (sglt1)-related transcriptional responses. As exploratory in silico information, molecular docking predicted candidate complex conformations of DNMT3B with CDX1B and SGLT1, with binding energies of -37.2 and -25.9 kcal/mol, respectively. Functionally, dnmt3b knockdown significantly reduced dnmt3b, Interleukin 6 (il6), and Nuclear factor kappa B (nfκb) expression, while increasing cdx1b, sglt1, Solute carrier family 2 member 3a (slc2a3a), 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4a (pfkfb4a), and Amine oxidase copper containing 1 (aoc1) expression (p < 0.05). CDX2/CDX1B-like immunoreactive protein and SGLT1 protein levels were also increased after dnmt3b knockdown (p < 0.05). Under high-glucose stress, exogenous 5MC exerted concentration-dependent effects. Specifically, 6 mM 5MC significantly reduced residual extracellular glucose, lactate dehydrogenase and diamine oxidase activities, and malondialdehyde content, while increasing glutathione content, cell viability, and cell migration (p < 0.05). These effects remained detectable after replacement with high-glucose medium for an additional 12 h. By contrast, 24 mM 5MC markedly increased lactate dehydrogenase activity and reduced cell viability, suggesting potential cytotoxicity (p < 0.05). S-adenosylmethionine (SAM) levels were significantly lower in the NC and 6 mM groups than in the HG, 12 mM, and 24 mM groups, suggesting changes in SAM-related one-carbon metabolic status rather than direct evidence of altered DNA methylation (p < 0.05). Exogenous 5MC, particularly at 6 mM, alleviated high-glucose-induced metabolic and epithelial stress in grass carp intestinal epithelial cells. These effects were accompanied by changes in several glucose metabolism- and inflammation-related genes. However, the cellular uptake, metabolic fate, DNA incorporation, methylation consequences, and causal roles of these gene-expression changes remain to be further verified.

To investigate the effects of mulberry leaf extract (MLE) on the growth performance, organ indices, and flesh quality of grass carp (Ctenopharyngodon idella), a basal diet (Control, Crude protein 30.25%, Crude lipid 7.23%) and three MLE-supplemented diets containing 1%, 2%, and 4% MLE (MLE1%, MLE2%, MLE4%) were formulated. A total of 160 grass carp (259.7 ± 1.1 g) were randomly allocated into 16 cages (1 m × 1 m × 1.2 m), with 10 fish per cage and 4 replicate cages per treatment group. Throughout the 8-week feeding trial, juvenile grass carp were fed three times daily (08:00, 12:00, and 17:00) at a daily feeding rate of 1.5-2.0% body weight. At the end of the trial, growth performance was assessed. Additionally, four fish were randomly sampled from each cage to determine the organ indices, flesh quality parameters, and gene expression levels. In the results, the final average body weight of all groups ranged from 909.00 to 942.97 g, and all the groups exhibited no significant difference in growth performance (p > 0.05). MLE-supplemented groups had significantly decreased visceral somatic index and hepatic somatic index (p < 0.05). In terms of flesh quality, all MLE-supplemented groups increased the flesh shear force, springiness, whiteness, and lightness (p < 0.05). Compared with the Control group, the MLE4% group had increased flesh hardness, gumminess, and chewiness, from 2899.52 g, 1310.62, and 516.59 to 4122.99 g, 1958.48, and 887.76, respectively (p < 0.05). MLE significantly decreased the 48 h thawing loss (MLE2%), 96 h thawing loss (MLE1%, MLE2%, MLE4%), and flesh crude lipid content (MLE2%, MLE4%) (p < 0.05). In terms of collagen metabolism, compared with the Control group, MLE significantly increased the flesh collagen content (MLE2% and MLE4%) (p < 0.05), upregulated the expression level of La ribonucleoprotein 6 (MLE4%) and tissue inhibitor of metalloproteinase 1 (MLE4%). Matrix metallopeptidase 2 was significantly downregulated in all MLE-supplemented groups (p < 0.05). In terms of myofiber development, compared with the Control group, the myofiber density was higher, and the myofiber diameter was lower in all MLE-supplemented groups (p < 0.05). MLE significantly upregulated the expression level of mammalian target of rapamycin (MLE4%), musculoskeletal embryonic nuclear protein 1 (MLE4%), myogenic differentiation 1 (MLE2%, MLE4%), and myogenic factor 5 (MLE1%, MLE2%, MLE4%) (p < 0.05). Myotubularin-related protein 8 was significantly downregulated in all MLE-supplemented groups (p < 0.05). In conclusion, dietary supplementation with 2-4% MLE improves the flesh textural properties, chroma and water-holding capacity of grass carp. MLE improves the flesh quality of grass carp by enhancing collagen deposition and myofiber development.

Ferulic acid (FA) is a green feed additive. To investigate the molecular mechanisms by which FA attenuates heat stress-induced hepatic and intestinal oxidative stress, as well as cholesterol metabolism disorders in Megalobrama amblycephala (9.75 ± 0.04 g), individuals were fed diets supplemented with 0, 100, or 200 mg/kg FA for eight weeks, followed by exposure to heat stress at 34 °C for 48 h. The results indicated that FA supplementation reduced malondialdehyde levels and downregulation genes involved in inflammatory responses (e.g., interleukin-6), apoptosis (e.g., caspase 8), and endoplasmic reticulum stress (e.g., immunoglobulin binding protein) (p < 0.05), which collectively alleviated heat stress-induced hepatic and intestinal oxidative stress. FA supplementation increased the expression of ATP-binding cassette transporter A1, apolipoprotein A1, and liver X receptor α (p < 0.05), and restored liver and plasma TC levels to pre-stress levels (p < 0.05). Additionally, FA ameliorated the heat stress-induced dysbiosis of the intestinal microbiota and modulated the composition and abundance of metabolites in intestinal contents and plasma, some of which are associated with cholesterol metabolism. In conclusion, dietary FA can alleviate heat stress-induced hepatic and intestinal oxidative stress, maintain the stability of the intestinal microbiota and regulate metabolic profiles, and improve the cholesterol metabolism disorders caused by heat stress.

The aim of this study was to explore the effects of adding Lactobacillus plantarum (LAB) to a high-starch diet on glucose and lipid metabolism, gut microbiota, and the composition of metabolites in Megalobrama amblycephala. This experiment was equipped with three isonitrogenous and isoenergetic feeds as control group (LW), high starch group (HW), and high starch with LAB group (HP). A total of 180 experimental fish (13.5 ± 0.5 g) were randomly divided into three treatments, and three floating cages (1 m × 1 m × 1 m) were set up for each treatment. A total of 20 fish per net were kept in an outdoor pond for 8 weeks. The results showed that both the HW and HP groups had an altered structure and a reduced diversity of gut microbiota. LAB increased the abundance of Cetobacterium and the ratio of Firmicutes/Bacteroidota and decreased PC (16:1/20:5) and taurochenodeoxycholic acid levels. LAB promoted the expression of genes related to the intestinal bile acid cycle (fxr, hmgcr, rxr, shp and hnf4α) and inhibited the expression of pparβ and g6pase (p < 0.05). LAB reduced the expression of genes related to transported cholesterol (lxr and ldlr) (p < 0.05) in the liver. In conclusion, LAB addition could regulate the gut microbiota disorders caused by high starch levels, promote cholesterol metabolism, produce bile acids, and reduce lipid deposition.

To examine the interactive effects of temperature and density on short-term growth and stress responses, juvenile Megalobrama amblycephala (30.18 ± 0.60 g) were reared for four weeks under two temperatures (22 °C, 32 °C) and three stocking densities (1.5, 3, and 6 kg/m³). Two-way ANOVA revealed that FBW, WGR, and SGR were significantly influenced by temperature, density and their interaction, with density being the stronger effect. The reduced growth observed at lower temperature and higher density. Plasma GLU, TC, and HDL were significantly influenced by the interaction between the two factors, with temperature being the stronger effect. The elevated temperature increased plasma lipids levels (TC and LDL), while higher density elevated plasma GLU and reduced plasma lipids levels (TC, HDL and LDL). Hepatic GSH content declined with increasing density. Temperature markedly affected hepatic genes related to ER stress and apoptosis (ire1, vdac1, nf-κb, il-6), which were upregulated at low temperature, whereas hsp90 expression was higher at high temperature. The expression of perk, grp75, and hsp70 increased with density. Temperature also significantly influenced intestinal metabolites, with 13 common differential metabolites identified among the 22 °C-1.5 kg/m³ group, 32 °C-1.5 kg/m³ group, 32 °C-6 kg/m³ group. In conclusion, both rearing temperature and stocking density exert significant and strong effects, with density exerting the dominant effect on growth performance of juvenile M. amblycephala, while temperature primarily affected plasma parameters. Furthermore, rearing temperature playing a dominant role in regulating physiological status, ER stress, and intestinal metabolites.

This study investigated the effects of plasma iron overload on hepatic ferroportin 1 (fpn1) expression, iron metabolism and oxidative stress. The experiment presents the first cloning and characterization of Megalobrama amblycephala fpn1. The open reading frame (ORF) is 1689 bp, encoding a 562-amino-acid protein predicted to adopt a canonical major facilitator superfamily (MFS) fold with 12 transmembrane domains. Tissue distribution revealed the highest fpn1 expression in the liver, followed by the trunk kidney, spleen, and posterior intestine. To investigate fpn1's role in iron metabolism, a plasma iron overload model was established by tail vein injection of sterile FeSO₄ solution (1 g/L, 0.5 mL/kg body weight). Plasma iron peaked at 6 h post-injection (hpi), while hepatic iron peaked at 12 hpi (P < 0.05). Signs of hepatic oxidative stress and injury were observed, including increased plasma lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) activities (6-48 hpi), elevated hepatic malondialdehyde (MDA) content (24-72 hpi), and decreased catalase (CAT) and superoxide dismutase (SOD) activities (6-24 hpi) (P < 0.05). Iron overload significantly upregulated hepatic hepcidin (hepc), ferritin (fer) and transferrin (tf) expression (6-24 hpi), while downregulating fpn1, transferrin receptor 1 (tfr1) and divalent metal transporter 1 (dmt1) expression (6-72 hpi) (P < 0.05). This pattern suggests disrupted iron metabolism with impaired cellular iron export, enhanced storage, and suppressed uptake. Enhancing fpn1 expression or inhibiting its degradation may promote iron efflux, activate antioxidant systems, reduce tissue iron burden, and ultimately restore iron homeostasis.

Resveratrol (RES), a natural polyphenol with lipid metabolism-regulating properties, also demonstrates remarkable efficacy in strengthening intestinal barrier integrity. In order to elucidate the mechanism by which RES ameliorates intestinal damage and lipid metabolism disturbances in Megalobrama amblycephala under a high-fat (HF) diet, a conventional diet (CON), an HF diet (HF), or an HF diet supplemented with 0.6, 3, or 6 g/kg RES (HF + 0.06%, 0.3%, or 0.6% RES) was fed to fish. After 8 weeks, RES supplementation in the HF diet significantly improved the growth performance and alleviated hepatic lipid deposition. Microbiota profiling revealed RES improved intestinal barrier function by reducing α-diversity, Actinobacteria and Bosea abundances, and enriching Firmicutes abundance. RES also maintained the integrity of the intestinal physical barrier and inhibited the inflammatory response. MeRIP-seq analysis indicated that RES modulated intestinal mRNA m6A methylation by upregulating methyltransferase-like 3 (mettl3) and downregulating fat mass and obesity-associated gene (fto) and Alk B homolog 5 (alkbh5). Combined RNA-seq and MeRIP-seq data revealed that RES alleviated endoplasmic reticulum stress (ERS) by upregulating the m6A methylation and gene level of heat shock protein 70 (hsp70). Correlation analyses identified significant associations between intestinal microbiota composition and ERS, tight junction, and inflammation. In summary, RES ameliorates lipid dysregulation via a synergistic mechanism involving intestinal microbiota, m6A modification, ERS, barrier function, and inflammatory response.

m6A methylation modification is an important genetic modification involved in biological processes such as sexual maturation, antibacterial, and antiviral in aquatic animals. However, few studies have been conducted in aquatic animals on the relationship between m6A methylation modification and autophagy-inflammation induced by lipid metabolism disorders. In the present study, a high-fat (HF) group and HF-MLP group (1 g mulberry leaf polysaccharides (MLPs)/1 kg HF diet) were set up. The mid-hind intestines of Megalobrama amblycephala juveniles from the two groups were collected for MeRIP-seq and RNA-seq after an 8-week feeding trial. The m6A peaks in the HF and HF-MLP groups were mainly enriched in the 3′ Untranslated Region (3′UTR), Stop codon, and coding sequence (CDS) region. Compared with the HF group, the m6A peaks in the HF-MLP group were shifted toward the 5′UTR region. ‘RRACH’ was the common m6A methylation motif in the HF and HF-MLP groups. Methyltransferase mettl14 and wtap expression in the intestines of the HF-MLP group were significantly higher compared with the HF group (p < 0.05). A total of 21 differentially expressed genes(DEGs) with different peaks were screened by the combined MeRIP-seq and RNA-seq analysis. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis enriched BCL2 interacting protein 3 (bnip3) to autophagy–animal and mitophagy–animal signaling pathways, etc., and nucleotide-binding domain leucine-rich repeat protein 1 (nlrp1) was enriched to the Nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. Combined MeRIP-seq and RNA-seq analysis indicated that the expression pattern of bnip3 was hyper-up and that of nlrp1 was hyper-down. Gene Set Enrichment Analysis (GSEA) analysis confirmed that the intestinal genes of HF-MLP group positively regulate lysosomal and autophagy–animal signaling pathways. In the present study, we demonstrated that m6A methylation modification plays a role in regulating autophagy-inflammatory responses induced by HF diets by MLPs, and further explored the molecular mechanisms by which MLPs work from the epigenetic perspective.

This study explored the potential protective role of mulberry leaf polysaccharides (MLPs) in mitigating intestinal damage induced by a high-fat (HF) diet in Megalobrama amblycephala juveniles. This was achieved through comprehensive multi-omics analysis on transcriptome, metabolome, and intestinal microbiota. Results showed that significant intestinal lipid deposition and endoplasmic reticulum (ER) stress were observed in juveniles of the HF group (P < 0.05). A multi-omics further analysis between the HF group and HF-MLP group (supplementing 1.00 g MLPs/kg in HF diet) was conducted. Transcriptomic analysis identified significant differential expression genes (DEGs) enrichment in the ER function and lipid metabolism pathways (P < 0.05). MLPs supplementation significantly decreased expression levels of ER stress markers (bip, chop) induced by triglyceride accumulation in HF diet-fed juverniles, likely through the suppression of srebf1 (P < 0.05). It further reduced lipid peroxidation and improved intestinal antioxidant capacity (P < 0.05). MLPs supplementation restored intestinal barrier integrity and downregulated tlr4/nf-κb pathway-related genes expressions (P < 0.05). Metabolomic analysis revealed that MLPs restored ER membrane function and preserved cell membrane integrity by reducing phosphatidylcholine, acylcarnitine, and lysophosphatidylethanolamine levels (P < 0.05). Furthermore, MLPs counteracted HF diet-induced intestinal dysbiosis by increasing the abundances of Muribaculaceae and Bacteroidales. In conclusion, MLPs supplementation regulated intestinal microbiota composition, alleviated ER stress caused by lipid accumulation, and reduced lipid peroxidation, decreased intestinal inflammation in high-fat diet- fed M. amblycephala juverniles.

This study investigated the epigenetic mechanisms through which graded levels of dietary methionine (Met) regulates growth, muscle quality, and health in juvenile Megalobrama amblycephala fed an all-plant-protein diet. Dietary Met supplementation improved growth performance in a dose-dependent manner and enhanced muscle nutritional quality, particularly protein content and amino acid composition. Optimal Met intake also strengthened hepatic antioxidant defenses, stabilized mitochondrial function, and modulated plasma metabolite profiles, including metabolites associated with antioxidant, anti-inflammatory, and antimicrobial activity. Epigenetic analysis revealed that dietary Met influenced hepatic N6-methyladenosine (m6A) RNA methylation and the expression of genes involved in autophagy and apoptosis, suggesting that these molecular pathways contribute to the observed physiological benefits. Collectively, these findings indicate that appropriate dietary Met of 10.1 g/kg not only supports growth and nutrient utilization but also promotes metabolic and cellular homeostasis through epigenetic regulation. This work provides novel insights into the nutritional and molecular strategies for improving the health and performance of juvenile M. amblycephala under plant-based feeding regimes, with potential implications for sustainable aquaculture practices.

Fish is an important source of protein for human, and lipid homeostasis is essential for fish health. As a green plant extract, ferulic acid (FA) plays a crucial role in modulating lipid metabolism. This study investigate the effects of dietary FA supplementation on lipid metabolic homeostasis, intestinal microbiota, and metabolites in plasma and intestinal contents of Megalobrama amblycephala juveniles. M. amblycephala (average weight: 9.75 +/- 0.04 g) juveniles were fed with three feeds containing 0 (Control), 100 (FA100), and 200 (FA200) mg/kg FA with equivalent nitrogen and energy levels for 8 weeks. The results indicate that FA supplementation significantly reduce the content of plasma lipids and liver lipids, and markedly increase total bile acid content in liver. It is possible that FA regulates liver fatty acid metabolism and cholesterol-bile acid metabolism by upregulating genes such as carnitine palmitoyl transferase 1 A, cholesterol 7 alpha-hydroxylase, and down-regulating genes such as sterol regulatory element-binding protein. The diversity of the intestinal microbiota is significantly enhanced, while the abundance of harmful bacteria exhibits a marked reduction in FA100 group. Further, metabolites are altered in FA100 group. Epoxy eicosatrienoic acid is a co-upregulated metabolite in both intestinal contents and plasma, and was significantly correlated with multiple indicators related to lipid metabolism. In summary, supplementing FA to feed decrease liver and plasma lipid accumulation and maintained lipid metabolic homeostasis. This may be due to the changed intestinal microbiota of M. amblycephala, as well as enrichment of intestinal and plasma metabolites.

This study evaluated the effects of short-term depuration under different salinity levels on the muscle nutritional composition and quality of Carassius auratus gibelio, aiming to provide guidance for enhancing the value of farmed crucian carp. A total of 240 fish (450 ± 50 g) were reared in recirculating aquaculture systems at salinities of 0‰ (S0), 3‰ (S3), 6‰ (S6), and 9‰ (S9) for 0 (D0), 5 (D5), and 10 days (D10). Dorsal muscle samples were analyzed for proximate composition, amino acids, fatty acids, flavor-related nucleotides, geosmin, and volatile compounds. Short-term depuration significantly improved muscle protein content, total amino acids, and umami amino acids. Culturing at 6‰ salinity for 10 days enhanced crude protein, total amino acids, umami amino acids, and lipid indices associated with cardiovascular benefits, while 9‰ salinity for 5 days increased crude lipid, total umami amino acids content, and the essential amino acid index (EAAI). Geosmin content decreased under moderate salinity but tended to accumulate at higher salinities. Amino acid scoring identified lysine, methionine, and cysteine as limiting under certain conditions, indicating a need for supplementation. Overall, short-term salinity depuration effectively improves muscle nutritional composition, fatty acid profiles, and flavor attributes, offering a practical approach to producing higher-value C. auratus gibelio with enhanced health benefits and consumer appeal.

Both silent information regulator 2 homolog 1 (sirt1) and forkhead box transcription factor 1 (foxO1) are crucial transcription factors involved in glucolipid metabolism and energy regulation. The presnt study aimed to understand their regulatory roles in glucose metabolism. Molecular cloning and sequencing of sirt1 gene of Megalobrama amblycephala (masirt1) was conducted and cellular localization of both the factors were analysed. Their effects and action patterns in the glucose metabolism of Megalobrama amblycephala (M. amblycephala) were investigated through acute and long-term glucose tolerance assays. The results revealed that the full-length masirt1 cDNA sequence was 2350 bp and closely related to Sinocyclocheilus rhinocerous. Sirt1 and foxO1 were found to be mutually dependent and localized in the nucleus. Acute glucose tolerance tests revealed that the expression levels of both factors in the liver of M. amblycephala showed an initial increase followed by a decrease. Plasma glucose levels in M. amblycephala significantly increased at 2 and 12 h (P < 0.05). In a long-term breeding experiment with high-sugar feeding, the expressions of the sirt1 and foxO1 genes in the kidney and intestine of M. amblycephala exhibited synergistic changes. The 51WS groups had significantly higher levels of sirt1 and foxO1 gene expression in the kidney and intestine compared to the 0WS and 17WS groups (P < 0.05). Overall, masirt1 is evolutionarily highly conserved, and the interaction site of sirt1 and foxO1 is located in the nucleus. In long-term hyperglycemic regulation, sirt1 and foxO1 exhibit synergistic regulatory effects in the kidney and intestine of M. amblycephala. This study provides insights into how sirt1 and foxO1 regulate glucose metabolism in M. amblycephala.

Aims to assess the effects of dietary inclusion of Antarctic krill meal (KM), five experimental diets supplemented with graded levels of KM (0 %, FM; 5 %, KM5; 10 %, KM10; 15 %, KM15; 20 %, KM20; 0 %, 13.3 %, 26.7 %, 40.0 %, and 56.7 % replacement fishmeal levels, respectively), were prepared to feed oriental river prawn ( Macrobrachium nipponense ) (0.20 +/- 0.01 g) for 8 weeks. The results revealed significant improvements in weight gain rate (WGR), specific growth rate (SGR), feed conversion rate (FCR), and protein efficiency ratio (PER) at the inclusion of 5 %-20 % KM ( P < 0.05). In the KM10 and KM15 groups, the concentrations of hemolymph total protein (TP), albumin (ALB), triglycerides (TG), high -density lipoprotein (HDL), and hepatopancreatic glutathione (GSH) were observed to be significantly higher compared to the FM group ( P < 0.05). Conversely, the contents of hepatopancreatic malondialdehyde (MDA), total nitric oxide synthase (TNOS), and inducible nitric oxide synthase (iNOS) were significantly lower in the KM10 and KM15 groups compared to the FM group ( P < 0.05). The mRNA expression levels of dorsal protein ( dorsal ), heat shock protein 60 ( hsp60 ), and myeloid differentiation factor 88 ( myd88 ) in the hepatopancreas in the KM10 group exhibited the lowest level. Additionally, compared to the FM group, the expression levels of fatty acid synthase ( fas ) and acetyl CoA carboxylase ( acc ) were significantly upregulated ( P < 0.05), while the acyl CoA bindingprotein ( acbp ), carnitine palmitoyltransferase I ( cpt I ), and scavenger receptor B I ( sr -b I ) were significantly downregulated in the KM10 group ( P < 0.05). In addition, the results of histopathology sections showed that 5 - 15 % KM could improve the health status of hepatopancreas and was a safe alternative to fishmeal. Overall, the optimal KM inclusion level in feed was 12.96 % (substituting for 34.60 % fishmeal) based on the quadratic fitting curve calculations of WGR and SGR.

Antarctic krill (Euphausia superba) products are a valuable resource with abundant nutritional value, positioning it as a crucial market in the aquafeed industry. Aims to assess the effects of dietary inclusion of Antarctic krill meal (KM), five experimental diets supplemented with graded levels of KM (0%, FM; 5%, KM5; 10%, KM10; 15%, KM15; 20%, KM20; 0%, 13.3%, 26.7%, 40.0%, and 56.7% replacement fishmeal levels, respectively), were prepared to feed oriental river prawn (Macro-brachium nipponense) (0.20 ± 0.01 g) for 8 weeks. The results revealed significant improvements in weight gain rate (WGR) and specific growth rate (SGR) at the inclusion of 5%-20% KM (P < 0.05). In KM10 and KM15 groups, concentrations of hemolymph total protein (TP), albumin (ALB), tri-glycerides (TG), high-density lipoprotein (HDL), and hepatopancreatic glutathione (GSH) in the KM10 and KM15 groups showed significantly higher levels than the FM group (P < 0.05), conversely, contents of hepatopancreatic malondialdehyde (MDA), total nitric oxide synthase (TNOS), and inducible nitric oxide synthase (iNOS) were significantly lower compared to the FM group (P < 0.05). The mRNA expression levels of dorsal, hsp60, and myd88 in the hepatopancreas in the KM10 group exhibited the lowest level. Additionally, compared to the FM group, the expression levels of fas and acc were significantly upregulated (P < 0.05), while the acbp, cpt I, and sr-b I were significantly downregulated in the KM10 group (P < 0.05), with no adverse effects on hepatopancreatic tissue structure. Overall, the optimal inclusion of KM in the diet was found to be 10%-15% (replacing 26.7%-40.0% fishmeal). The findings derived from this study are expected to provide a basis for the potential application of KM as a feed ingredient in oriental river prawn feed.

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H2O2)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 μM of H2O2 for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 μg/mL) for 3 h, and then stimulated with 100 μM of H2O2 for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs' antioxidant capacities. The 200 μg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H2O2-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (p < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca2+ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca2+ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p < 0.05), which rebounded to normal levels in the NC group (p > 0.05). These results indicated that MLP pre-treatment attenuated H2O2-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage.

To evaluate the efficiency of activated charcoal (AC) in cottonseed meal-based feed (CSM; 38.46 % CSM, 1275 mg/kg free gossypol), experimental diets supplemented with 0 % AC, 1.5 % AC and 3.0 % AC were prepared and used to feed grass carp juveniles ( Ctenopharyngodon idellus, initial weight of 5.0 +/- 0.5 g) for 8 weeks. The results showed that the 1.5 % AC group significantly increased final body weight (FBW), weight growth rate (WGR), specific growth rate (SGR), and activated the expressions of intestinal barrier-associated genes zonula occludens-1 (zo-1), zo-2 and claudin-c compared with 0 % AC group (P < 0.05). Additionally, the 1.5 % AC and the 3.0 % AC groups significantly promoted the expression of claudin-12 (P < 0.05). Compared with the 0 % AC group, the 1.5 % AC group had significantly higher total protein (TP) concentration and significantly lower glucose (GLU) concentration and aspartate aminotransferase (AST) activity (P < 0.05). Liver antioxidant results showed that supplementation with 1.5 % AC and 3.0 % AC groups significantly reduced malondialdehyde (MDA) content and increased glutathione reductase (GR) activity (P < 0.05). Total-superoxide dismutase (T-SOD) and glutathione-S-transferase (GST) activities were significantly higher in the 1.5 % AC group than those in the other groups (P < 0.05). The antioxidant gene results showed that gr and GST omega 1 ( gsto1 ) levels were significantly higher in the 3.0 % AC group than in the 0 % AC group (P < 0.05). The 1.5 % AC group significantly promoted nuclear factor erythroid 2-related factor 2 ( nrf2 ) expression, and the 1.5 % AC and 3.0 % AC groups significantly inhibited the expressions of kelch-like ECH-associated protein 1 ( keap1a ) and keap1b (P < 0.05). Liver apoptotic and inflammatory genes showed that the expressions of caspase 9 ( casp9 ), BCL2 associated X a (baxa), nuclear factor-kappa B ( nf kappa b ) and inhibitor of kappa B (i kappa b) were significantly lower in the 1.5 % AC group than those in the other groups (P < 0.05). The expression of casp8 in the 1.5 % AC group was significantly lower than in the 0 % AC group (P < 0.05). Additionally, the expressions of p38 mitogen-activated protein kinase ( p38MAPK ) and i kappa b kinase-beta ( ikk beta ) were significantly suppressed in the 1.5 % AC group and 3.0 % AC group (P < 0.05). In summary, 1.5 % AC supplementation in CSM-based feed can enhance intestinal barrier function, alleviate CSMbased feed-induced apoptosis, improve liver antioxidant, and thus promote grass carp weight growth rate.