In this experiment, the primary intestinal epithelial cells (IECs) inflammation model was constructed to investigate the intestinal injury mechanism under high level addition of plant-derived proteins in hybrid grouper (Epinephelus fuscoguttatus female x E. lanceolatus male), and citric acid (CA) addition was used to investigate the protective effect in the primary IECs inflammation model. The vitro experiment was fed 30 fishes to three replicates of each group for 8 weeks. In the inflammation model of IECs, the addition of CA (1 mM) remarkably enhanced cell survival, and significantly reduced the secretion of inflammatory cytokines TNF-alpha and IL-1 beta (P < 0.05). CA activated phosphorylation of S6K and 4EBP-1 in the mTOR pathway and promoted the protein expression of tight junction proteins (ZO-1, E-cadherin, occludin and claudin-1) (P < 0.05). The supplementation of CA in the diet also could improve the expression of tight junction in intestine of hybrid grouper (P < 0.05). CA attenuated the protein expression of inflammatory cytokines (IL-1 beta, IL-6 and TNF-alpha) in inflammation models by regulating ERK and NF-kappa B pathways (P < 0.05). These findings suggest that citric acid enhances cell survival, strengthens the intestinal tight junction barrier and enhances intestinal immune function in the LPS-stimulated inflammatory model of IECs. Citric acid could serve as an effective means to counteract growth inhibition and reduced digestibility caused by high proportions of plant protein in fish feed.

The persistent use of enrofloxacin (ENR) in aquaculture poses significant risks to food safety. This study examined the interaction between dietary lipid levels (4.99%, 7.94%, and 10.88%) and ENR (0.04%) in Litopenaeus vannamei. A high-lipid diet significantly increased ENR residues, upregulated antibiotic resistance genes, and disrupted the intestinal microbiota by promoting pathogenic Vibrio. Transcriptomic analysis showed that under ENR exposure, a high-fat diet suppressed V-type proton ATPase and key structural genes in shrimp, thereby weakening the physical defenses of the hepatopancreas and resulting in impaired immunity and increased susceptibility to Vibrio. In addition, risk assessment indicated increased health concerns associated with hepatopancreas consumption. Overall, these findings demonstrate that excessive dietary lipids exacerbate ENR persistence, induce microbial and immune dysfunction, and intensify food safety risks.

Muscle yellowing is an emerging quality defect in farmed grass carp, yet its chemical and molecular regulation remain insufficiently understood. Here, we integrated transcriptomics, proteomics and metabolomics with targeted quality assessments to compare normal (N) and yellowish (Y) dorsal muscles in grass carp. Yellowish muscle showed marked discoloration, with a significant decreases in lightness (L*: 57.57 vs. 39.76) and an increase in yellowness (b*: 1.53 vs. 3.84), accompanied by an overall decline in eating quality. Multi-omics profiling identified 4601 differentially expressed genes, 937 differentially abundant proteins, and 120 differential metabolites, and the three datasets consistently converged on pathways related to oxidative stress, ferroptosis, and dysregulated lipid metabolism. Biochemical indices further supported intensified oxidative damage and weakened antioxidant capacity in yellowish muscle, indicating disrupted redox homeostasis that favors lipid peroxidation. Odor- and taste-related fingerprints, assessed using an electronic nose and electronic tongue, clearly separated the N and Y groups, suggesting systematic alterations in volatile and taste-active compounds during yellowing. Moreover, amino acid profiling indicated reduced nutritional value in yellowish muscle, reflected by decreased levels of multiple amino acids. Together, these findings link muscle yellowing with oxidative stress–driven lipid peroxidation and ferroptosis-associated regulation, providing mechanistic insight and potential biomarker candidates for quality monitoring and targeted mitigation in grass carp production.

Concentrated cottonseed protein (CPC) serves as an ideal protein replacement for fish meal in diets, though excessive use can adversely affect aquatic life. Organic trace element replacements can sustain the growth and metabolic functions of Litopenaeus vannamei, aiding in the conservation of high-quality protein resources such as fish meal. The purpose of this experiment is to explore the impact of substituting fish meal with CPC as a protein source, along with substituting amino acid trace element chelates, on the growth, immune antioxidant capacity, and intestinal microbiota of Litopenaeus vannamei. Four experimental diets were formulated: the control group FM (no CPC substitution, supplemented with inorganic trace elements), the inorganic trace element group C0 (CPC substituted for 40 % fish meal, supplemented with inorganic trace elements), and the experimental group C1 (CPC substituted for 40 % fish meal, amino acid trace element chelates substituted for 40 % inorganic trace elements). and C2 (CPC replacing 40 % fish meal, amino acid chelated trace minerals replacing 60 % inorganic trace minerals). Each group had three replicates. At the end of a 10-week feeding trial, the research results are as follows: Group C2 had significantly higher crude protein and crude lipid contents compared to Group C0 (P < 0.05). In comparison to Group C0, the FM group demonstrated much higher activities of CuZn-SOD, GPX, and CAT (P < 0.01), group C0 exhibited notably lower activities compared to Groups C1 and C2 (P < 0.05). Relative abundances of major intestinal microbiota species in Groups C1 and C2 underwent significant changes. Compared to Groups C0 and C2, the expression levels of lipid metabolism genes were much higher in Group C1 (P < 0.05). In fish meal replacement diets with CPC, using 40 % amino acid chelates can mitigate adverse effects of plant protein and enhance shrimp lipid metabolism, immune antioxidants, and intestinal flora.

High-starch diets are increasingly used in aquafeeds to reduce feed costs, but carnivorous fish such as largemouth bass (Micropterus salmoides) have limited capacity to utilize dietary starch and are prone to hepatic metabolic disorders. In the present study, we evaluated whether dietary N-carbamylglutamate (NCG) could alleviate high-starch-induced hepatic oxidative stress and liver injury in largemouth bass. Fish were fed a control diet containing 11.50% starch, a high-starch diet containing 18.00% starch, or a high-starch diet supplemented with 0.15%, 0.20%, or 0.25% NCG for 8 weeks. Compared with the high-starch group, dietary NCG supplementation significantly reduced serum glucose and triglyceride levels, decreased hepatic glycogen and malondialdehyde contents, and increased hepatic superoxide dismutase and glutathione peroxidase activities. NCG also reduced serum alanine aminotransferase and aspartate aminotransferase activities and alleviated hepatic histopathological damage. At the transcriptional level, NCG upregulated genes related to insulin signaling, glycolysis, lipid catabolism, and antioxidant regulation, including insr, irs, gk, pk, atgl, hsl, ampk, and nrf2, while downregulating the expression of keap1, nf-κB, mtor, and multiple inflammation- and apoptosis-related genes. These changes were accompanied by increased serum nitric oxide levels and improved survival and growth performance under high-starch feeding conditions. Collectively, these results indicate that dietary NCG supplementation attenuates high-starch-induced hepatic oxidative stress and redox-associated liver injury in largemouth bass, which may be associated with the transcriptional modulation of genes related to the AMPK/Nrf2/Keap1 and mTOR/NF-κB signaling pathways.

In response to current restrictions on antibiotic use in aquaculture, developing eco-friendly feed additives as alternatives is essential to sustain industry growth. This study evaluated the effects of dietary sodium fulvate (SF) on growth, intestinal health, antioxidant capacity, and immune responses in largemouth bass (Micropterus salmoides). Six experimental diets containing 0%, 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% SF were formulated and fed to fish maintained in three replicate cages over 9 weeks. Dietary SF inclusion did not significantly affect growth performance (weight gain rate, specific growth rate) or feed utilization (feed conversion rate, protein efficiency ratio) (P > 0.05). However, second-order polynomial regression indicated that dietary inclusion of 0.17% SF maximized intestinal trypsin activity (P < 0.05), whereas supplementation with 0.30%–0.58% SF reduced plasma diamine oxidase, endothelin-1, and lipopolysaccharide concentrations. Furthermore, supplementation with 0.44%–0.56% SF significantly enhanced the Shannon diversity index and increased the relative abundance of Fusobacteria, Ralstonia, and Cetobacterium. Dietary inclusion of 0.06%–0.46% SF enhanced intestinal superoxide dismutase and catalase activities; increased serum complement 3, complement 4, immunoglobulin M levels, and the relative expression levels of TNF-α, IL-10, and TGF-β1; and reduced intestinal malondialdehyde level. Collectively, these findings indicate that dietary supplementation with 0.06%–0.58% SF promoted fish health by improving intestinal digestive capacity, reinforcing mucosal barrier integrity, and enhancing antioxidant and immune responses, underscoring its potential as a functional feed additive for antibiotic-free aquaculture.

Feeding low fishmeal diets (LF) in aquaculture can induce oxidative stress and impair intestinal and immune homeostasis of shrimp. This study explored the impact of dietary phytosterol on intestinal health, antioxidant capacity, immune-related responses, sterol metabolism, and microbiota composition in juvenile Litopenaeus vannamei fed an LF diet. A total of 800 shrimp (0.29 ± 0.03 g) were randomly allocated to 20 tanks and were fed one of five diets for 7 weeks: a high fishmeal diet (HF), a LF diet, or the LF diet enrinched with phytosterol at 0.02% (LP1), 0.04% (LP2), or 0.08% (LP3). Growth performance did not differ significantly among treatments. However, dietary phytosterol (0.02-0.08%) improved antioxidant status by increasing superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) and decreasing malondialdehyde (MDA), while also enhancing the hemolymph lipid profile via reduced triglycerides (TG) and total cholesterol (T-CHO) and increased high-density lipoprotein (HDL-C). Histologically, 0.08% phytosterol enhanced hepatopancreatic tubule integrity and intestinal microvillus structure. Phytosterol supplementation modulated the expression of genes involved in fatty acid oxidation, sterol metabolism, immune responses, and apoptosis regulation. Phytosterol supplementation enriched beneficial gut microbiota (Bacillus) while reducing potential pathogens. In conclusion, the results indicate that dietary phytosterol provides protective effect against oxidative and intestinal stress induced by LF diet and enhances immune-related physiological homeostasis for juvenile L. vannamei, thereby supporting its use as a functional immunonutritional additive in LF feeds.

This study evaluated the effects of replacing fishmeal with Antarctic krill meal (AKM) on the growth and reproductive performance of Litopenaeus vannamei broodstock. Six isonitrogenous and isolipidic diets were formulated by replacing fishmeal with different proportions of AKM (0% (M1, control), 20% (M2), 40% (M3), 60% (M4), 80% (M5), and 100% (M6)) and fed to L. vannamei broodstock (initial weight: 27.95 ± 0.23 g) for 70 days. Results showed: (1) The weight gain rate (WGR) and specific growth rate (SGR) of the M6 group were significantly lower than the control group ( P < 0.05). (2) The gonadosomatic index (GSI) of the M2 and M3 groups was significantly higher than the control group ( P < 0.05). (3) AKM significantly increased triglyceride (TG), total cholesterol (TC), and estradiol (E 2 ) levels in hemolymph, hepatopancreas, and gonads, elevated progesterone (PROG) in hemolymph, and increased vitellogenin (VTG) in hemolymph and gonads while decreasing VTG in the hepatopancreas ( P < 0.05). (4) An appropriate replacement level of krill meal significantly reduced gonad-inhibiting hormone (GIH) in the eyestalk ( P < 0.05), with no significant effect on molting-inhibiting hormone (MIH) ( P > 0.05). (5) Gene expression analysis revealed that krill meal significantly upregulated ovarian development-related genes ( vtg , vtgr , vasa , e75 , rxr ) and steroidogenesis pathway genes ( star , 3β-hsd , 17β-hsd ) ( P < 0.05). Polynomial regression analysis of WGR and GSI indicated that the optimal dietary replacement level of fishmeal by AKM was 27.22%-41.78% under the present experimental conditions.

This study investigated the effects of taurine supplementation in low-fishmeal diets on growth, antioxidant capacity and hepato-intestinal health of spotted sea bass (Lateolabrax maculatus). A high-fishmeal diet (HF, 30% fish meal) and four low-fishmeal diets (60% fish meal replaced by CAP) supplemented with 0%, 0.2%, 0.4% and 0.6% taurine were used. Each treatment had four replicates with 20 fish per tank (initial weight: 65.35±0.07 g), and the trial lasted 8 weeks. Results showed that weight gain rate (WGR) of the LF group was significantly lower than those of HF, T2 and T3 groups, while WGR in HF and T3 was significantly higher than in LF and T1 (P<0.05). Feed conversion ratio (FCR) of LF was significantly higher, and specific growth rate (SGR) and protein efficiency ratio (PER) were significantly lower than in other groups (P<0.05). Protein deposition rate (PDR) in HF was significantly higher than in LF and T2 (P<0.05). Taurine improved growth, feed efficiency and antioxidant status, reduced lipid peroxidation, and enhanced expression of immune-related genes (il-10, tlr2, myd88) in liver and intestine. Hepatic triglyceride content decreased with increasing taurine. Expression of srebp1 and fas was downregulated, and lpl was upregulated in T3 (P<0.05). Histology showed taurine alleviated hepatocyte vacuolization and inflammatory damage. In summary, 0.4%–0.6% taurine effectively mitigates low-fishmeal diet adverse effects and improves growth, antioxidant capacity, immunity and hepato-intestinal health in L. maculatus.

Chlorogenic acid (CGA) is a natural polyphenolic compound with antioxidant activity. We hypothesised that dietary CGA supplementation could improve the growth performance and selected muscle-related traits of Procambarus clarkii, possibly in association with changes in antioxidant-related indices and the expression of selected genes. To test this hypothesis, a 6-week feeding trial was conducted using five diets containing 0, 200, 400, 600, and 800 mg/kg CGA. Dietary supplementation with 400-600 mg/kg CGA improved growth performance and feed conversion ratio, and was associated with higher collagen-related indices, better texture in some parameters, lower freezing loss, and higher amino acid contents in some treatment groups. CGA supplementation was also associated with higher antioxidant enzyme activities, lower reactive oxygen species and malondialdehyde levels, and changes in the expression of genes related to antioxidant defence and muscle development. Overall, these results suggest that dietary CGA supplementation, particularly at 400-600 mg/kg, may improve growth performance and selected muscle-related traits of P. clarkii under the present experimental conditions. These findings support further evaluation of CGA as a dietary supplement in P. clarkii.

Exposure to low temperatures greatly affects shrimp reproduction, development, and growth, potentially causing high mortality rates and leading to significant economic losses in the global shrimp farming industry. The aim of this study was to investigate the potential protective effects of compound feed attractants (including yeast extracts and enzymatically hydrolyzed plant proteins) against cold stress in Penaeus monodon (P. monodon). Specifically, under low-temperature conditions (19-21 degrees C), three levels of compound feed attractants addition (0, 0.05 %, and 0.1 %) were tested, designated as A0, A0.05, and A0.1, respectively. A total of 360 healthy shrimp (initial weight: 0.49 +/- 0.01 g) were allocated into three groups, with four replicates per group, and fed for eight weeks. The results indicated that compared to the A0 group, the final body weight (FBW), weight gain (WG), specific growth rate (SGR), survival rate (SR), and whole-body crude lipid content were significantly higher in the A0.05 and A0.1 groups, while the feed conversion ratio (FCR) was significantly lower. Higher activities of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM), along with a lower malondialdehyde (MDA) content, were observed in the A0.05 group compared to the A0.1 group. Meanwhile, the activities of digestive enzymes (lipase, protease, and amylase) were significantly higher in the A0.05 and A0.1 groups than in the A0 group. Compared to the A0 group, the A0.05 group optimally preserved hepatopancreatic architecture and improve the integrity of intestinal structure in P. monodon. Gene expression analysis revealed that the A0.05 and A0.1 groups upregulated the expression of genes related to PI3K-AKT (igfr, pi3k, raptor, ppar, akt, 4e-bp), IMD (relish, atg8), AMPs (alf, crustin-2, penaeidin-3), and lipid metabolism (fas, fabp4, ech, elovl4, lcfatp1). Additionally, the A0.05 group had the highest expression levels of gsh-px, Mnsod, cat, and the lowest expression levels of hsp70, hsp90, ldlr. Compared to the A0.05 group, the A0.1 group upregulated the expression of p53 and caspase-3. Overall, 0.05 % compound feed attractants significantly enhances the growth performance, antioxidant capacity, and intestinal health of P. monodon under low-temperature stress, while a 0.1 % dosage may trigger stress responses.

Excessive dietary cottonseed protein concentrate (CPC) impairs growth and health in pearl gentian grouper (Epinephelus lanceolatus male & times; E. fuscoguttatus female), but the underlying molecular mechanisms remain unclear. This study thus aimed to investigate the molecular mechanisms by which dietary CPC affects the growth performance and physiological status of grouper. 375 juveniles (15.00 +/- 0.05 g) were randomly distributed into 15 tanks (25 fish/tank). Five isonitrogenous and isolipidic diets were formulated, in which CPC replaced 0% (control diet, C0), 20% (C20), 40% (C40), 60% (C60), and 80% (C80) of fishmeal. Each diet was assigned to triplicate tanks and fed to juveniles for 49 days. Growth showed a linear decreasing trend with increasing dietary CPC content (P < 0.05). With increasing dietary CPC content, malondialdehyde (MDA), iron (Fe), and oxidized glutathione (GSSG) levels significantly increased (P < 0.05) in both intestinal and hepatic tissues. In contrast, glutathione peroxidase (GSH-PX) activity, total glutathione (T-GSH), and the ratio of GSH and GSSG markedly decreased (P < 0.05). Transcriptomic profiling of intestine and metabolomic analysis of liver were conducted for the C0, C20, and C60 groups to elucidate the molecular mechanisms underlying CPC-induced health impairments. In the intestinal transcriptome, pathway enrichment analysis of cellular processes revealed that low-level CPC substitution elicited significant enrichment (P < 0.05) in cell cycle, p53 signaling pathway, and apoptosis. Conversely, excessive dietary CPC content was associated with enrichment in the ferroptosis, phagosome, and peroxisome pathways (P < 0.05). Trend analysis revealed that the ferroptosis pathway (P < 0.05) was significantly enriched by the shared DEGs. Among these, SLC3A2, GSS, and GPX4 expression was significantly down-regulated, while transferrin expression was up-regulated (P < 0.05). The protein expression levels of glutathione synthetase (GSS) and glutathione peroxidase 4 (GPX4) mutually validated their transcriptional profiles (P < 0.05). Hepatic metabolomics indicated significantly elevated glutamate and depleted glutathione levels in the C60 group versus controls. Collectively, escalating dietary CPC levels promoted significant iron accumulation in hepatic and intestinal tissues, disrupted glutathione metabolic networks, and suppressed GPX4 expression. These alterations potentiated intracellular peroxide accumulation, culminating in ferroptotic cell death and consequent physiological impairments.

This study investigated the dose-dependent effects of increasing dietary histamine concentrations on gastrointestinal function, antioxidant status, and muscle quality in striped catfish (Pangasianodon hypophthalmus), to assess potential histamine-related health risks. An eight-week feeding trial was conducted using seven isonitrogenous and isolipidic diets supplemented with 0, 15, 30, 60, 120, 240, and 480 mg/kg histamine, respectively. The findings demonstrated that as the dietary histamine concentration increased, the activities of intestinal trypsin, lipase, and maltase displayed a linear decline. Histological examination of hematoxylin and eosin-stained gastric and intestinal sections showed that gastric villus width, gastric muscular thickness, and intestinal villus height were significantly reduced when dietary histamine inclusion exceeded 15, 60, and 480 mg/kg, respectively. The relative expressions of tight junction-related genes (zonula occludens-2, occludin, claudin 7a, and claudin 12) were progressively downregulated with increasing dietary histamine, accompanied by elevated intestinal permeability. This was supported by a significant increase in serum lipopolysaccharide level at histamine inclusions above 30 mg/kg. Compared with the H0 group, the H480 group exhibited significantly lower serum total antioxidant capacity and peroxidase and catalase activities. Muscle yellowness, elasticity, and chewability increased as dietary histamine increased, with significant elevations observed above 30, 240, and 480 mg/kg, respectively. Collectively, increasing dietary histamine concentrations were associated with pronounced impairments in striped catfish, highlighting the importance of controlling histamine concentrations in aquafeeds and providing evidence to support risk assessment and the development of safety guidelines.

This study investigated how defatted silkworm pupae meal (DSPM) modulates flesh quality in pearl gentian grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). A total of 360 size-uniform fish were randomly assigned to four dietary treatments for 8 weeks: D0, the fishmeal (FM) based control diet; and D1, D2, and D3, in which DSPM substituted 25%, 50%, and 100% of dietary FM, respectively, with three replicates per treatment and 30 fish per replicate. Compared with D0, D1 and D2 improved final body weight, weight gain rate, specific growth rate, and feed conversion ratio, whereas D3 reduced survival and feed utilization. For flesh quality, D2 showed the most favorable phenotype, characterized by higher crude protein, collagen, flavor-associated amino acids, pH, texture attributes, and muscle fiber density, together with lower ether extract, cooking loss, freezing loss, and shear force. DSPM substitution also increased unsaturated and polyunsaturated fatty acid proportions and improved lipid health indices. Moderate substitution, especially D2, enhanced oxidative stability by increasing SOD and CAT activities and reducing MDA accumulation. Non-targeted metabolomics combined with qPCR analysis of selected flesh quality-related genes suggested that these improvements were associated with osmotic regulation, collagen remodeling, purine metabolism, membrane lipid homeostasis, nutrient sensing, myogenesis, and antioxidant defense. Complete FM substitution impaired muscle structure, water retention, and redox balance. Thus, 50% FM substitution with DSPM effectively optimized grouper flesh quality in this study.

This study evaluated the effects of dietary CpG oligodeoxynucleotide (CpG ODNs) on growth performance, immunity, digestive function, intestinal microbiota, and transcriptomic responses in Litopenaeus vannamei. Shrimp with an initial body weight of 0.30 ± 0.02 g were fed diets supplemented with 0, 0.1, 0.4, 1.6, 6.4, or 25.6 mg kg-1 CpG ODNs for 8 weeks. Dietary CpG ODNs did not significantly improve most growth parameters; however, shrimp fed the diet supplemented with 1.6 mg kg-1 CpG ODNs showed a lower feed conversion ratio. After white spot syndrome virus challenge, shrimp fed the diet supplemented with 25.6 mg kg-1 CpG ODNs showed higher survival than those in the control group, whereas survival after Vibrio parahaemolyticus challenge did not differ significantly among treatments. Dietary supplementation with CpG ODNs at levels above 0.4 mg kg-1 increased the activities of several serum immune-related and antioxidant enzymes, reduced malondialdehyde content, and enhanced intestinal trypsin and amylase activities. In addition, this treatment increased microbial richness indices, while reducing the relative abundances of Vibrio and Photobacterium. Transcriptomic analysis showed that several pathways, including the AMPK signaling pathway and JAK-STAT signaling pathway, were significantly enriched in the CpG ODNs (above 0.4 mg kg-1) groups compared with the control group. In conclusion, dietary supplementation with CpG ODNs did not significantly improve the growth performance of L. vannamei. However, dietary supplementation with CpG ODNs at levels above 0.4 mg kg-1 may help enhance immune and digestive functions and modulate the intestinal microbiota composition of L. vannamei.

During lipid metabolism, bile acids are involved in the emulsification and absorption of lipids, and the liver serves as the primary organ responsible for mediating their regulatory functions. Crustaceans lack de novo bile acid synthesis and may differ substantially from vertebrates in bile acid metabolism. To investigate the role of bile acids in the lipid metabolism of crustaceans, this study divided 480 Litopenaeus vannamei (initial weight: 0.640 ± 0.003 g) into three groups. Each group included four replicate tanks, with 40 shrimp per tank. Fed the following diets for an 8-week feeding trial, respectively: high fishmeal diet (HF; 25% fish meal), low fishmeal diet (LF; 12.5% fish meal), or low fishmeal diet supplemented with 400 mg/kg bile acid (LFB). The final body weight, weight gain rate and average daily gain of the LFB group were significantly higher than those of the LF and HF groups (P < 0.05). Hepatopancreatic histomorphological and biochemical analyses revealed that the bile acid supplementation alleviated the accumulation of collagen fibers in the hepatopancreas and reduced the activity of glutamic-pyruvic transaminase (GPT) (P < 0.001). Single-nucleus RNA sequencing (snRNA-Seq) enabled the construction of the first comprehensive hepatopancreatic cell atlas in invertebrates, with 11 major cell types identified via transcriptomic profiling. Bile acid administration significantly increased F cell abundance while reducing the number of R cell and B cell populations (P < 0.05). Intercellular communication analysis demonstrated that bile acid supplementation decreased signal enrichment in neural cell adhesion molecule (NCAM) and collagen pathways, with differential functional pathways between the LFB group and LF group predominantly enriched in non-alcoholic fatty liver disease (NAFLD) pathways. Notably, Maribacter and Tamlana emerged as significantly differentiated genera in the bile acid treatment group, exhibiting functional associations with the mitigation of NAFLD and the catalysis of short-chain fatty acid degradation. Pseudotime trajectory analysis further uncovered potential hepatopancreatic cell differentiation pathways: E cells, as progenitor cells, differentiate into R cells, which subsequently bifurcate into B cell and F cell lineages. Collectively, bile acids alleviate hepatopancreatic fibrosis and inflammation by inhibiting cell junction-related signaling pathways, while concurrently regulating lipid metabolism through the enhancement of F cell proportions.

This study investigated the effects of dietary n-3/n-6 highly unsaturated fatty acid (HUFA) ratios on growth performance, hepatic health, antioxidant capacity, and immune function in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Six isonitrogenous and isolipidic diets were formulated with graded n-3/n-6 HUFA ratios of 25.29 (Y1), 19.54 (Y2), 13.27 (Y3), 7.74 (Y4), 3.96 (Y5), and 1.90 (Y6), and fed for 8 weeks. Optimal specific growth rate and protein efficiency ratio were observed at ratios 7.74–13.27, indicating superior nutrient utilization within this range. Hepatic lipid accumulation was lowest at a ratio of 19.54, as evidenced by minimal triglyceride content and lipid droplet area, whereas both excessively high and low ratios stimulated fatty acid synthase activity and promoted steatosis. The strongest antioxidant defense was observed at 7.74, characterized by upregulated glutathione peroxidase (gpx) and catalase (cat) gene expression, elevated glutathione peroxidase activity, induction of heat shock proteins (hsp70/hsp90), and the lowest malondialdehyde levels. Hepatocellular integrity was best preserved at 13.27, reflected by minimal glutamate pyruvate transaminase activity. Furthermore, this ratio enhanced humoral immunity—marked by the highest serum IgM—and shifted the inflammatory balance toward anti-inflammatory dominance through downregulation of interleukin 6 (il-6) and interleukin 8 (il-8) and upregulation of interleukin 10 (il-10) and transforming growth-factor β (tgf-β). Collectively, dietary n-3/n-6 HUFA ratios modulate growth and liver health in hybrid grouper by coordinately regulating protein utilization, lipid metabolism, redox homeostasis, and immune responses, with a functional optimum between 7.74 and 13.27 for integrated physiological performance.

This study evaluated the effects of olive leaf extract (OLE) on growth, intestinal health, and resistance against Vibrio parahaemolyticus challenge in juvenile Litopenaeus vannamei. To a basal diet containing 12% fishmeal, OLE was added at 0%, 0.10%, 0.20%, 0.40%, and 0.80%, forming diets with equal nitrogen and lipid contents. The diets were divided into five groups and named OLE0 (control group), OLE1, OLE2, OLE4, and OLE8. The findings revealed that relative to OLE0, the weight gain rate (WGR) and specific growth rate (SGR) increased in the OLE1 group, whereas the feed conversion ratio (FCR) declined (p < 0.05). The crude protein (CP) content for whole shrimp in the OLE8 group was significantly higher (p < 0.05), and the crude lipid (CL) content in the OLE1, OLE2 and OLE8 groups significantly decreased compared with OLE0 (p < 0.05). In serum indices, the OLE8 group had notably higher total protein (TP) content than the control (p < 0.05). Compared with the OLE0 group, high-density lipoprotein cholesterol (HDL-C) levels in the OLE1 and OLE2 groups were increased, whereas low-density lipoprotein cholesterol (LDL-C) levels in the OLE4 and OLE8 groups were significantly decreased compared with the control (p < 0.05). Hepatopancreatic malondialdehyde (MDA) in all experimental groups was significantly decreased compared with the control (p < 0.05). The superoxide dismutase (SOD) activity in all experimental groups increased (p < 0.05) and was the largest in the OLE1 group. The OLE1, OLE2 and OLE4 groups had significantly increased glutathione peroxidase (GPX) activity (p < 0.05). Catalase (CAT) activity in the OLE1 group was significantly higher than the control (p < 0.05). The toll, lzm, imd, cru a and propo mRNA expression levels in the hepatopancreas were significantly upregulated in each experimental group (p < 0.05). Intestinal microbial analysis indicated that the relative abundance of Bacteroidetes was elevated in the OLE4 and OLE8 groups, accompanied by a reduction in Vibrio proportion (p < 0.05). Post-challenge assays demonstrated that the OLE4 and OLE8 groups achieved remarkably higher cumulative survival rates than OLE0 (p < 0.05). In summary, OLE in a low-fishmeal diet can promote growth performance, intestinal health and disease resistance for shrimp. According to the broken-line regression model of WGR against OLE dosage, 0.08% was the optimal dietary OLE inclusion level of the OLE additive in juvenile L. vannamei.

This study evaluated the effects of replacing fishmeal with superworm meal (SM, Zophobas morio) on growth performance and the gill, liver, and intestinal health of pearl gentian grouper (Epinephelus lanceolatus ♂ × E. fuscoguttatus ♀). Five isonitrogenous and isolipidic diets were formulated with 0%, 20%, 40%, 60%, and 80% SM replacement and fed to juveniles for 49 days.Results showed that replacement levels ≤20% did not affect growth performance and improved intestinal health by enhancing digestive capacity, increasing goblet cell numbers, and optimizing gut microbiota through reducing potential pathogens and increasing beneficial bacteria (P < 0.05). However, high replacement levels (≥60%) significantly reduced growth and deteriorated water quality. Gill damage was evidenced by oxidative stress, inflammatory responses, and tight junction disruption, accompanied by lamellar fusion and epithelial injury. Excessive SM inclusion also impaired liver function, as indicated by elevated AST and ALT, decreased albumin and cholesterol, hepatic vacuolation, and disturbances in taurine and energy metabolism. Furthermore, intestinal villus height and muscularis thickness decreased at high replacement levels, although transcriptomic analysis suggested compensatory activation of digestion and absorption pathways.Overall, SM can replace up to 20% of fishmeal without adverse effects while improving intestinal health. Higher substitution levels negatively affect growth and organ health. These findings support SM as a sustainable alternative protein source in grouper diets.

This study evaluated the combined phenotypic and transcriptomic responses of Litopenaeus vannamei to graded dietary replacement of fish meal with enzymatic corn gluten meal (ECGM). Six diets contained 0–5% ECGM (ECGM 0–5), with three replicates of 40 shrimp (initial weight: 0.33 ± 0.01 g) cultured for 56 days. Growth, somatic indices, whole-body/muscle composition, serum biochemistry, hepatopancreatic enzyme activity and histology were determined; hepatopancreas RNA-seq was further conducted on ECGM0, ECGM2 and ECGM5 groups. Elevated ECGM linearly reduced FBW, WGR, SGR and PER (P < 0.05), while no growth difference existed between ECGM0 and ECGM2 (P > 0.05). High ECGM decreased muscle crude protein, and low supplementation increased muscle lipid (P < 0.05). ECGM0 had the highest serum T-CHO, TG, HDL-C and MDA, whereas moderate ECGM boosted antioxidant enzyme activities (P < 0.05). Hepatopancreatic PKC, ALT, HL, LPL, CPT I and PFK rose with increasing ECGM, but GK declined up to 4% ECGM (P < 0.05). Hepatopancreatic cytoplasmic vacuolation exhibited a dose-dependent increase; severe lesions only appeared in ECGM5, while ECGM0–2 maintained intact tissue structure. Transcriptomics showed ECGM regulated PPAR signaling, protein digestion and absorption pathways, and enhanced antioxidant capacity via ferroptosis and glutathione metabolism modulation. Notably, merely 11 DEGs were found between ECGM2 and ECGM5, indicating minor transcriptional variation between medium and high substitution groups. Integrated data suggested the appropriate dietary inclusion range of ECGM for L. vannamei was 1–3%.