Ammonia acts as a key environmental pollutant in fish farming. Elevated ammonia concentrations can induce stress responses in fish, threatening their survival and growth. As a vital metabolic organ, the fish liver plays key roles in detoxification and immune defense. In the present study, juvenile hybrid groupers (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) were subjected to ammonia stress at 4mg/L (A4) and 8mg/L (A8) over a 7-day period. Subsequently, multiple physiological aspects of the liver were assessed across several biological levels, encompassing histomorphology, oxidative stress, inflammatory factors, and metabolic profiles. The results showed that ammonia stress caused hepatic morphological alterations and triggered oxidative stress and inflammatory responses. In detail, MDA content and T-AOC and SOD activities were increased in the A4 and A8 groups (P < 0.05), and LPO content was increased in the A8 group (P < 0.05). Meanwhile, the mRNA expression levels of nrf2, trx, tnfα, and il1β genes were upregulated in the A4 and A8 groups (P < 0.05); gpx and il8 genes were upregulated but il10 gene was downregulated in the A8 group (P < 0.05); nqo1 gene was downregulated in the A4 group (P < 0.05). Furthermore, the hepatic metabolite profiles were perturbed, particularly "histidine metabolism" and "biosynthesis of valine, leucine, and isoleucine" pathways. Additionally, several functional metabolites, including L-tryptophan, indole, carnosine, citric acid, sphingosine, pantothenic acid, and inositol, were emerged as potential candidate biomarkers. These findings demonstrated that ammonia stress could induce physiological dysfunction in the liver of groupers through a cascade of tissue damage, activated oxidative stress, disrupted inflammatory responses and altered metabolic profiles, and such adverse effects were markedly aggravated with the increase in ammonia concentration. Future research should focus on long-term ammonia exposure, concentration-time dose effects, and multi-omics integration to clarify the underlying response mechanisms.

Adipose tissue functions as a metabolic energy source, supporting fish growth, reproduction, embryonic development, and swimming. However, the metabolic changes of adipose tissue in cold-water fish under cold acclimation is still limited. In this study, we found that long-term (30days) cold acclimation reduced adipocytes size and visceral fat mass. In addition, cold acclimation-induced lipolysis was faster than lipogenesis. Furthermore, liquid chromatograph-mass spectrometer (LC-MS) technology was used to provide a comprehensive lipidomic profiles of visceral adipose tissue induced by cold acclimation in S. prenanti. We found that a total of 1651 lipid species were significantly altered in visceral adipose tissue due to cold acclimation, of which 1030 were up-regulated and 621 were downregulated. Notably, cold acclimation substantially altered both the acyl chain composition of triacylglycerols (TG) and the metabolism of glycerophospholipids in visceral adipose tissue. Furthermore, cold acclimation promotes the fatty acid desaturation and glycerophospholipid metabolism of the visceral adipose tissue in the S. prenanti. This study offers evidence for the impact of long-term cold acclimation on the adjustment changes in lipid composition and lipid metabolic pathways in the visceral adipose tissue of S. prenanti.

To evaluate the effects of substituting soybean oil (SO) with soy lecithin (SL) and specialty lipid (CL) on the growth, body composition, and glucose-lipid metabolism of juvenile largemouth bass (Micropterus salmoides). The CL is a composite lipid source formulated by mixing coconut oil, linseed oil, soy lecithin oil, and palm oil in a ratio of 2:2:3:3, with the addition of antioxidants and emulsifiers as fillers. A total of six experimental groups were established: an iso-nitrogenous and iso-lipidic soybean oil (SO) (control group), a 1% soy lecithin supplementation group (1% SL), a 2% soy lecithin supplementation group (2% SL), a 2.5% specialty lipid supplementation group (2.5% CL), a 5% specialty lipid supplementation group (5% CL), and a 7.5% specialty lipid supplementation group (7.5% CL). After a 10-week feeding trial (initial body weight: 12.64 ± 0.02g), the results showed that final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) in the 2.5% CL and 5% CL groups were significantly higher than those in the SO group. The 5% CL group exhibited the lowest feed conversion ratio (FCR) numerically, though no significant differences were observed among groups. Whole-body crude lipid content was significantly reduced in the 7.5% CL. Compared to other groups, the 5% CL and 7.5% CL showed significantly increased levels of saturated fatty acid (SFA), docosahexaenoic acid (DHA), and n-3/n-6 polyunsaturated fatty acid (PUFA), while PUFA levels markedly decreased. Compared to the control, the 7.5% CL showed significant decreases in plasma serum albumin (ALB), total protein (TP), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Regarding lipid metabolism gene expression, the 5% CL exhibited a significantly lower expression level of the peroxisome proliferator-activated receptor-γ (pparγ) in contrast to SO. The expression levels of peroxisome proliferators-activated receptor-α (pparα) were significantly upregulated in the 5% CL and 7.5% CL compared to other groups. Furthermore, the carnitine palmitoyltransferase (cpt1) expression level in the 5% CL was significantly higher than in all other groups. In conclusion, CL enhanced growth performance, effectively reduced whole-body lipid deposition, improved fatty acid composition, and promoted lipid metabolism in juvenile largemouth bass, without adversely affecting liver function. Based on the comprehensive results, the recommended optimal inclusion level for specialty lipid was 5%.

In order to explore the role of piwil1 (Piwi-like RNA-mediated gene silencing 1) in sex differentiation and gonadal development of largemouth bass (Micropterus salmoides). To this end, we established a 17β-estradiol (E2)-treated pseudo-female group (XY-F, sex reversal rate 100%), a control female group (XX-F), and a control male group (XY-M). The full-length cDNA of piwil1 was obtained using the RACE technique, and its expression pattern, response to E2 treatment, and spatial localization were analyzed. The piwil1 cDNA is 3091bp in length and encodes a protein of 855 amino acids. qRT-PCR showed that piwil1 was highly expressed in the gonads, with higher expression in mature testes than in ovaries (P < 0.05). During early gonadal differentiation, piwil1 expression increased in all groups (XY-M, XX-F, and XY-F). At 2 months old, piwil1 expression was significantly suppressed in E2-treated XY-F fish compared to XY-M and XX-F (P < 0.05). By 12 months old, piwil1 expression was highest in XY-M testes. Fluorescence in situ hybridization (FISH) results confirmed the differential expression pattern of the piwil1 gene, and strong signals were observed in oogonia (OG), primary oocytes (OC1), growing oocytes (GOC), vitellogenic oocytes (VO), primary spermatogonia (PSG), secondary spermatogonia (SSG), primary spermatocytes (PSC), and sperm (SP). In conclusion, piwil1 plays an important role in testis development and spermatogenesis, which exhibits a male-biased expression pattern in largemouth bass. These findings provide a potential molecular target for monosex male breeding in largemouth bass.

Phytic acid (PA) is a common anti-nutritional factor in plant-based aquafeeds that can affect nutrient utilization and physiological homeostasis in fish. However, its dose-dependent physiological effects on Nile tilapia (Oreochromis niloticus) remain poorly characterized. This study investigated the effects of graded dietary levels of PA (0, 10, 15, 20, and 25 g/kg) on growth performance, antioxidant status, hepatic metabolism, and immune regulation in Nile tilapia. Results showed that dietary PA significantly reduced body weight gain and increased liver somatic index in a dose-dependent manner. Antioxidant capacity was impaired, hepatic function was disrupted, and immune-related gene expression was altered. No significant differences were observed in survival rate among treatments. Collectively, these findings demonstrate that excessive dietary PA induces metabolic stress in Nile tilapia, characterized by oxidative imbalance, hepatic dysfunction, and immune dysregulation. These results highlight the importance of controlling PA levels or applying targeted mitigation strategies in aquafeed formulations to maintain fish health and optimize aquaculture productivity.

Menthol, a natural monoterpene extracted from Mentha species, has gained attention as an alternative sedative and anesthetic in aquaculture. However, its physiological safety range and potential sublethal toxicity remain insufficiently defined. This study investigated the cardiac and behavioral effects of menthol sedation in Oreochromis niloticus, aiming to identify a therapeutic window and elucidate its underlying mechanisms of action. Juvenile fish were exposed to graded concentrations of menthol (100-250mg/L) to evaluate induction and recovery times, cardiac rhythm stability, and electrocardiographic parameters, including heart rate, QRS amplitude, and PQ and QT intervals. Additional assays with the GABAA receptor antagonist flumazenil (1mgkg⁻1, i.p.) were performed to determine neurophysiological mediation. Menthol induced a concentration-dependent decrease in heart rate and prolongation of ventricular conduction intervals, characterizing sinus bradycardia without arrhythmia. Concentrations between 100 and 200mg/L provided effective sedation and rapid recovery while maintaining hemodynamic stability, defining a safe therapeutic window. Above 200mg/L, pronounced bradycardia and delayed recovery were observed, indicating sublethal cardiac depression. Flumazenil significantly prolonged anesthesia induction, confirming GABAA-mediated mechanisms in menthol sedation. These findings establish the physiological safety limits for menthol use in Nile tilapia and highlight its ecotoxicological significance, as excessive or environmental exposure may impair cardiac homeostasis in aquatic organisms. The results contribute to defining safe anesthetic practices and to understanding the toxicodynamic implications of natural sedatives in aquatic ecosystems.

This study investigated the protective effects of glycyrrhetinic acid (GA) supplementation on metabolic disturbances induced by a high-fat diet (HFD) in juvenile channel catfish (Ictalurus punctatus). Fish were fed a control diet, an HFD, or an HFD supplemented with 0.4, 0.8, and 1.2mg/kg GA, respectively, for 8weeks. Hepatic function, mitochondrial integrity, and metabolic gene expression were assessed. HFD significantly impaired protein efficiency ratio (PER) and relative feed intake (RFI) while promoting excessive lipid deposition and energy storage. These changes were accompanied by elevated plasma levels of liver enzymes (GOT, GPT), non-esterified fatty acids (NEFA), and pro-inflammatory cytokines (TNF-α, IL-6, MCP-1). Mitochondrial dysfunction was evident from reduced activities of succinate dehydrogenase, Na⁺, K⁺-ATPase, and complexes I and II, alongside increased cytosolic cytochrome c, indicating mitochondrial injury and apoptosis. HFD also suppressed expressions of mitochondrial genes (nd1, cytb) and disrupted glucose and lipid metabolism by altering key gene expression patterns. GA supplementation significantly reversed these pathological changes. It improved mitochondrial function, restored respiratory complex activities (particularly complex III), normalized gene expression related to mitochondrial biogenesis, glucose utilization (e.g., ampkα2, glut2), and lipid oxidation (e.g., cpt1, pparα), and reduced inflammation and hepatic damage. Overall, GA redirected energy utilization from lipid storage toward growth, suggesting its potential as a functional feed additive to mitigate high-fat diet-induced metabolic dysfunction in aquaculture species.

The accumulation of metal toxicants in aquatic systems has been shown to create a harmful environment for marine life. This study examined the toxic effects of zirconium oxychloride (ZR) using Nile tilapia (Oreochromis niloticus) as a model organism. The fish were exposed to 10ppm, 20ppm, 40ppm, 60ppm, 80ppm, 100ppm, and 200ppm concentrations of ZR for 45days. Mortality rates in the treated groups were recorded through survival analysis, revealing a dose-dependent increase in mortality among Nile tilapia exposed to ZR. Behavioural changes were observed across all treated groups compared to the control. Histopathological analysis of gills, liver, intestine, and eye showed altered tissue structure in fish exposed to ZR. Biochemical evaluation of oxidative markers in the liver and intestine was also conducted for the first time in Nile tilapia exposed to ZR. Oxidative damage was detected through lipid peroxidation (LPX) levels, and the response of Nile tilapia to oxidative stress induced by ZR was assessed by examining superoxide dismutase (SOD) activity, catalase (CAT), and reduced glutathione (GSH) levels. Elevated LPX levels, along with increased activity of SOD, CAT, and GSH, were detected in the liver and intestines of all treated groups in a dose-dependent manner. These findings may help evaluate ZR toxicity in freshwater fish, especially in mining areas, and underscore the need for further research into the mechanisms of ZR-induced oxidative stress.

A 60-day feeding trial was conducted to evaluate the effects of iron-porphyrin (FePh) supplementation (0, 50, 100, 150, and 200mg/kg Fe3⁺) compared with 50mg/kg FeSO4 in juvenile Epinephelus cyanopodus (16.12 ± 0.15g). The results demonstrated that FePh at 100mg/kg maximized the growth performance (P < 0.05), while 50mg/kg FePh surpassed FeSO4 in enhancing intestinal barrier integrity and growth. FePh improved systemic iron metabolism by elevating elevated transferrin and hepcidin levels (P < 0.05), with 50mg/kg FePh significantly increasing hemoglobin and serum iron (P < 0.05). Antioxidant capacity was significantly enhanced (T-AOC and SOD in serum and gills), alongside the upregulation of immune markers (C3, C4, and IgM). FePh also improved intestinal morphology (GC, MP, VL) and intestinal microbiota (P < 0.05). Serum aminotransferase (ALT and AST) levels decreased with elevated activity of antioxidant enzymes. Transcriptomic analysis revealed that FePh suppressed ferroptosis-related genes acsl4 and fth1, while activating iron-regulation genes slc7a11, slc3a2, and hmox1. In conclusion, FePh effectively improved the growth of juvenile E. cyanopodus; enhanced antioxidant capacity, non-specific immunity, and iron metabolism; maintained the stability of the intestinal mucosal barrier to promote intestinal health; and regulated the expression of key genes in the ferroptosis pathway to protect liver health. The optimal FePh inclusion level was 50-100mg/kg, demonstrating superior efficacy over inorganic iron for sustainable grouper aquaculture.

The search for safe and effective anaesthetic agents for aquaculture is essential to improve fish welfare and management practices. This study evaluated the anaesthetic efficacy of Origanum vulgare essential oil (OVEO) in juvenile tambaqui (Colossoma macropomum), an important Amazonian species. A total of 108 fish were exposed to immersion baths with increasing OVEO concentrations (19.04–57.12 mg L⁻1). Behavioral endpoints, electrocardiographic activity, opercular movements, and plasma glucose were assessed to determine induction, maintenance, and recovery profiles. At 19.04–38.08 mg L⁻1, OVEO induced rapid loss of equilibrium with full recovery, establishing a safe anaesthetic window. Higher concentrations (47.60–57.12 mg.L⁻1) produced deep anesthesia but also concentration-dependent bradycardia, reduced opercular frequency, increased glycaemia, and prolonged recovery times. Electrophysiological analysis revealed a progressive reduction in heart rate (up to 48.5%) and prolongation of RR and PQ intervals, without QRS morphology alteration, indicating reversible sinus bradycardia rather than conduction block. Opercular electromyography also demonstrated concentration-dependent respiratory depression with complete reversibility at lower concentrations. These results provide the first detailed electrophysiological evidence of OVEO-induced anesthesia in C. macropomum, supporting its use as a natural, effective, and sustainable anaesthetic for Amazonian aquaculture, contributing to improved animal welfare and management strategies.