Effects of dietary histamine on gastrointestinal morphology, antioxidant capacity, and muscle quality in striped catfish (Pangasianodon hypophthalmus)

Abstract

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.

Highlights

• •Dietary histamine reduced intestinal digestive enzyme activities in striped catfish.
• •Excess histamine impaired gastric and intestinal morphology in striped catfish.
• •Histamine compromised intestinal barrier integrity via tight junction downregulation.
• •Dietary histamine deteriorated fillet color and texture quality in striped catfish.

1. Introduction

Striped catfish (Pangasianodon hypophthalmus) is a globally significant farmed species, originating from Southern Vietnam's Mekong River Delta (De Silva & Phuong, 2011). Owing to its strong adaptability, desirable flesh quality, and rapid growth, this species is widely farmed across Asia and plays a pivotal role in the regional and international aquaculture industry (Dao Minh et al., 2022). In the last twenty years, striped catfish production has expanded substantially, reaching 2.8 million tonnes in 2022, thereby underscoring its increasing significance in global trade (FAO, 2025). However, intensified aquaculture has also led to the prevalent use of low-quality fish meal in an effort to reduce production costs (Edwards et al., 2004). Such fish meal, particularly aged or coarsely processed red fish meals, typically exhibit reduced freshness, higher levels of lipid oxidation, and elevated histamine accumulation compared with freshly processed meals (Opstvedt et al., 2000). Moreover, the warm and humid conditions characteristic of subtropical and tropical aquaculture environments promote feed oxidation and spoilage, further accelerating histamine accumulation (Feng et al., 2016). Collectively, these factors make histamine a practical and recurring risk factor in aquafeeds, especially under tropical farming and storage conditions.

Histamine, a biogenic amine (C₅H₉N₃; molecular weight 111.15 g/mol), is formed in vitro by the L-histidine decarboxylase-catalyzed decarboxylation of histidine (Chu et al., 2017; Francis et al., 2013). In aquatic animals, increasing evidence suggests that excessive dietary histamine can impair digestive tract structure and function, and alter intestinal microbiota composition (Qiu et al., 2024; Zhang et al., 2023). For example, in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus♂), dietary histamine elicited dose-dependent intestinal immunosuppression and inflammation, accompanied by microbiota dysbiosis (Cheng et al., 2026). In vivo, histamine predominantly exists in an inactive, bound form within various cell types, including mast cells, basophils, platelets, and specialized neurons such as histaminergic and enterochromaffin cells (Mulero et al., 2007). Upon stimulation or during allergic responses, free histamine is released into the extracellular milieu, where it initiates a broad range of physiological effects. However, most available studies have focused on individual physiological responses or growth-related parameters, whereas integrated evidence linking gastrointestinal morphology, intestinal barrier function, antioxidant capacity, and muscle quality remains limited. In previous studies examining dietary histamine exposure in aquatic animals, adverse outcomes have generally been evaluated using growth performance and feed utilization, together with a limited set of physiological indicators/endpoints (Qiu et al., 2024; Tapia-Salazar et al., 2001). These commonly include digestive enzyme activities, histological assessment of gastrointestinal morphology, and indicators of barrier integrity, as well as oxidative status assessed by antioxidant enzymes and lipid peroxidation indices; in some cases, fillet quality traits are also considered (Zhang et al., 2023). However, these endpoints are often assessed separately rather than within an integrated framework, thereby limiting a dose-dependent interpretation linking gastrointestinal impairment and oxidative dysregulation with downstream changes in muscle quality. As a tropical species, striped catfish appears particularly susceptible to dietary histamine toxicity; however, this susceptibility remains insufficiently understood (Liu et al., 2022).

Based on these considerations, we hypothesized that excessive dietary histamine would impair gastrointestinal morphology and antioxidant defenses, thereby compromising muscle quality in striped catfish. Therefore, the present study aimed to systematically evaluate the dose-dependent effects of dietary histamine on antioxidant capacity, gastrointestinal morphology, and muscle quality in striped catfish.

2. Materials and methods

2.1. Ethical approval

This study was approved by Guangdong Ocean University Research Ethics Committee (reference GDOU-IACUC-2023-A0121), and all experimental procedures complied with the Guidance for the Care and Use of Laboratory Animals in China (GB/T 35892–2018).

2.2. Experimental diets

To reduce basal histamine concentrations, white fish meal (≤ 40 mg/kg histamine) was used. Seven isonitrogenous and isolipidic diets (34.0% protein, 10.5% lipid) were formulated to contain histamine concentrations of 0, 15, 30, 60, 120, 240, and 480 mg/kg. The highest histamine concentration (480 mg/kg) was included to represent a worst-case exposure scenario associated with severely deteriorated aquafeeds, rather than typical commercial feeding conditions. After being crushed, sieved through a 60-mesh screen, and accurately weighed, all raw ingredients were thoroughly blended in a V-type mixer. After adding soybean oil and water, the mixture was kneaded into a dough and extruded into 2.0 mm pellets using a twin-screw extruder (School of Chemical Engineering, South China University of Technology, Guangdong, China). These wet pellets were then dried in a temperature-controlled, dehumidified room, packaged, and stored at −20 °C until use. Detailed dietary formulations and nutrient compositions are presented in Table 1.

Table 1.

Ingredients and nutritional composition of experimental diets (g/kg, dry matter basis).

Ingredients	H0	H15	H30	H60	H120	H240	H480
White fish meal	150.0	150.0	150.0	150.0	150.0	150.0	150.0
Rapeseed meal	200.0	200.0	200.0	200.0	200.0	200.0	200.0
Soybean meal	200.0	200.0	200.0	200.0	200.0	200.0	200.0
Wheat flour	150.0	150.0	150.0	150.0	150.0	150.0	150.0
Rice bran meal	252.99	252.99	252.99	252.99	252.99	252.99	252.99
Soybean oil	20.0	20.0	20.0	20.0	20.0	20.0	20.0
Ca(H2PO4)2	12.0	12.0	12.0	12.0	12.0	12.0	12.0
Choline chloride (50%)	4.0	4.0	4.0	4.0	4.0	4.0	4.0
Vitamin C	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Compound premixa	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Histamine dihydrochloride	0.00	0.025	0.05	0.10	0.20	0.41	0.81
Cellulose microcrystalline	0.81	0.785	0.76	0.71	0.61	0.40	0.00
Proximate composition
Dry matter	895.2	890.2	894.9	895.5	894.5	902.8	894.2
Crude protein	338.0	342.9	342.4	338.5	347.5	353.8	348.9
Crude lipid	106.5	104.6	103.9	109.8	107.3	105.2	104.4
Ash	104.5	107.8	101.8	103.9	99.0	97.0	101.6
Gross energy (MJ/kg)b	18.3	18.2	18.3	18.4	18.5	18.7	18.4
Histamine (mg/kg)	8.5	23.6	38.6	68.5	128.7	248.9	488.8

^aCompound premix (g/kg mixture): vitamin A, 0.20 g; vitamin D₃, 0.003 g; vitamin E, 4.40 g; vitamin K₃, 0.66 g; vitamin B₁, 0.33 g; vitamin B₂, 0.88 g; vitamin B₆, 0.73 g; vitamin B₁₂, 0.001 g; nicotinic acid, 2.89 g; calcium pantothenate, 1.64 g; folic acid, 0.07 g; biotin, 0.003 g; vitamin C, 10.01 g; FeSO₄·7H₂O, 52.87 g; H₃ClCu₂O₃, 0.65 g; ZnSO₄·7H₂O, 43.15 g; MnSO₄·7H₂O, 31.56 g; MgSO₄·H₂O, 44.65 g; Ca(IO₃)₂, 0.42 g; Na₂SeO₃, 0.11 g; CoCl₂·6H₂O, 0.14 g.

2.3. Experimental fish

An 8-week feeding trial was conducted in a cement pond (approximately 5.5 m × 5.5 m × 1.5 m) equipped with an aerated continuous groundwater flow-through system at Guangdong Ocean University. Fish were reared in floating cages (0.7 m × 0.7 m × 1 m), with a total of 21 cages and three replicate cages per dietary treatment. All cages were placed within the same pond and shared the same water source. Healthy fish (initial weight: 31.38 ± 0.09 g) were randomly distributed into the cages, with three replicate cages per treatment. A total of 630 striped catfish were used in the experiment. Each cage contained 30 fish, and 21 cages were used in total. Fish sex was not determined, as sexual dimorphism is not evident at this developmental stage. Fish were randomly allocated to cages using a computer-generated random number sequence. Cage positions within the pond were also randomly assigned to minimise potential positional effects. Daily feed consumption per cage was recorded, and fish mortality was monitored throughout the trial. The experimental fish were fed twice daily at fixed time intervals (08:00 and 17:00). Water temperature was maintained between 29 and 32 °C, dissolved oxygen was kept above 4 mg/L, and pH was monitored daily. Ammonia nitrogen concentration was measured twice per week and maintained below 0.04 mg/L.

2.4. Sample collection

The striped catfish were fasted for 24 h, anaesthetized with a eugenol solution (1:10,000; Macklin, Shanghai, China), and then sampled. All sampling procedures were completed within the morning to minimise potential circadian effects. From each cage, caudal vein blood was collected from four fish and centrifuged at 3500 rpm for 10 min, after which the serum was preserved at −80 °C until further analysis. Posterior intestines from four fish per cage were fixed overnight in 4% formaldehyde at 4 °C for morphological analysis. Additionally, posterior intestines from another four randomly selected fish per cage were used for subsequent relative mRNA expression analysis. Blinding was implemented during sample analysis. All samples were coded with anonymized IDs, and the analyst was unaware of dietary treatments until all measurements and data recording were completed.

2.5. Proximate component analyses

The nutritional components of experimental diets were analyzed following the official standard protocols of the AOAC (2005). Moisture content was assessed through oven drying at 105 °C until no further change in sample mass was detected, with moisture expressed as the resulting weight loss. Crude protein levels were determined using a Kjeltec™ 8400 analyzer (FOSS, Hillerød, Denmark), while crude lipid content was analyzed by Soxhlet extraction employing petroleum ether as the solvent. Ash content was measured following incineration at 550 °C for 6 h.

2.6. Biochemical index analysis

Stomach and intestinal samples were weighed and homogenized in nine volumes (v/m) of phosphate-buffered saline (pH 7.4) to obtain crude enzyme extracts. The homogenates were centrifuged at 875 ×g for 10 min at 4 °C. The supernatant was used for subsequent analyses. Biochemical assays were performed primarily using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Total protein concentration was determined by the BCA method (kit A045–3-2). Antioxidant capacity and oxidative stress markers were evaluated as follows: total antioxidant capacity (T-AOC) by the ABTS method (A015–2-1), activities of peroxidase (POD, A084–1-1), catalase (CAT, A007–2-1), superoxide dismutase (SOD, A001–3-2), glutathione peroxidase (GPX, A005–1-2), and glutathione reductase (GR, A062–1-1) by their respective colorimetric or ultraviolet methods, and malondialdehyde (MDA) content by the TBA method (A003–1-2). Digestive enzyme activities (pepsin, A080–1-1; trypsin, A080–2-2; lipase, A054–2-1; amylase, C016–1-1; maltase, A082–3-1) and diamine oxidase (DAO, A088–2-1) activity were also measured using colorimetric, ultraviolet colorimetric, or microplate methods from the same supplier. In a separate assay, lipopolysaccharide (LPS) content was measured using a commercial ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). All biochemical and ELISA assays were conducted strictly in accordance with the manufacturers' instructions. The commercial kits used in this study were validated for fish tissues by the manufacturers or have been widely applied in previous studies involving fish species.

2.7. Hematoxylin and eosin (H&E) staining

For histological analysis, stomach and hindgut tissues were sequentially dehydrated using graded ethanol solutions, cleared with xylene, and subsequently embedded in paraffin to obtain solid blocks. The blocks were sectioned into 5 μm-thick sections using an RM2016 pathological microtome (Leica Microsystems, Wetzlar, Germany). The prepared paraffin sections were subjected to H&E staining and mounted on glass slides using neutral gum for long-term preservation. Intestinal morphology was observed under an inverted fluorescence microscope (Nikon, Tokyo, Japan), and villus height and width, along with muscular layer thickness, were measured using Topview software. For each cage, four fish were randomly selected for histological evaluation. From each fish, three non-consecutive paraffin sections were prepared, and five randomly selected microscopic fields were measured per section. Measurements were first averaged at the individual fish level, and fish-level means were subsequently averaged to obtain a single cage mean for statistical analysis. Thus, the cage was considered the experimental unit (biological replicate), with multiple technical measurements (sections and microscopic fields) averaged to generate one representative value per cage.

2.8. Gene expression analysis

Total RNA was isolated from the foregut, midgut, and hindgut for downstream analysis with the TransZol Up Plus RNA Kit (Beijing TransGen Biotech Co., Ltd., Beijing, China). RNA integrity was assessed using 1% agarose gel electrophoresis, and its purity and concentration were determined via spectrophotometry (A260:280 nm). RNA samples were reverse-transcribed into cDNA using the PrimeScriptTM RT Reagent Kit (Takara, Shiga, Japan) following the manufacturer's protocol. For qPCR, gene-specific primers (Table 2) were designed using Primer 5 software, based on NCBI-annotated reference sequences. qPCR amplification was performed on a LightCycler® 480 System (Roche Diagnostics, Basel, Switzerland) using the SYBR® Premix ExTaqTM II Kit (Takara, Shiga, Japan). The reaction mixture (10 μL) contained 5 μL SYBR® Premix ExTaqTM II, 3.2 μL sterile water, 1 μL cDNA, and 0.4 μL each of the forward and reverse primers. The qPCR thermal cycling protocol comprised an initial denaturation step (95 °C, 30 s) and 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Relative gene expression was then quantified by the 2^−ΔΔCT method (Livak & Schmittgen, 2001). β-actin was used as the reference gene for normalization, as it has been widely validated to exhibit stable expression in fish intestinal tissues. Preliminary analyses confirmed that β-actin expression was stable across dietary histamine treatments, and thus β-actin was used as the reference gene for normalization.

Table 2.

Primer pair sequences used in real-time PCR.

Target gene	Primer sequence (5′ − 3′)	Size (bp)	Accession No.
Claudin 12	F-CTCCAGCGGGTGTTACTACTTTGAC R-GCAGAGCCAAGCCAGAGAAGAAC	116	XM_026924172.3
Claudin 7a	F-CGATGTTCTTGGCGGAGCTTTATTG R-CCTTGGTGCTGCTGGAAGTGC	101	XM_026935848.3
Occludin	F-ATGGAAGGTGTCATCGTGGTGTTG R-TGCCGAGACCGTACCCAGAAC	124	XM_053236246.1
ZO-2	F-TGGGTGTGGTGAGCAGAGAGTC R-TGGTTGGTTGAAGTGTCCGTGTG	128	XM_034306344.2
H2a	F-ATCCTCCACGCACAGCATATTCAG R-CTCCAGCCACACAACAGCCTTAC	130	XM_026919619.3
H2b	F-CGTGAACAGGTGCGTCGTATCC R-CAGCCAGCAGATGATGAAGACTCC	141	XM_026910315.3
H3	F-TGCGTCTGGTTGGCTGCTTTC R-AGTGGAGGCGGTCATCAGGAAG	141	XM_026937662.3
β-actin	F-GGCTACTCCTTCACCACCACA R-ATTGAGTCGGCGTGAAGTGGTAAC	100	XM_026929614.2

ZO-2, zonula occludens-2; H2a, histamine receptor 2a; H2b, histamine receptor 2b; H3, histamine receptor 3.

2.9. Muscle quality assessment

Dorsal muscle color (measured on both left and right sections) was measured via a Minolta Chroma Meter (CR400; Konica Minolta Holdings, Inc., Tokyo, Japan) under standard D65 lighting conditions with a 10° viewing angle. Muscle color was assessed using the CIE system, with parameters L* (lightness), a* (+redness, −greenness), and b* (+yellowness, −blueness). The dorsal muscle was sectioned into standard dimensions (length: 2 cm; width: 1 cm; height: 1 cm) for texture analysis, performed using a texture analyzer (TA.XTplusC, Stable Micro Systems, Godalming, UK).

2.10. Statistical analysis

Data were subjected to one-way ANOVA (SPSS 27.0), with pairwise differences identified using Duncan's test (P < 0.05). Normality and homogeneity of variance were assessed prior to ANOVA. Duncan's multiple range test was selected as a post hoc procedure because the present study was designed as an exploratory dose–response feeding trial aimed at identifying potential differences among dietary histamine concentrations. The cage was considered the experimental unit for all statistical analyses, as diets were applied and environmental conditions were shared at the cage level. For each cage, measurements obtained from multiple sampled fish were averaged to generate a single cage mean, which was used for subsequent statistical comparisons. Results were presented as mean ± SEM (n = 3 cages per treatment). No predefined exclusion criteria were applied, and no experimental units were excluded from the analysis. The sample size was selected based on previous dose–dependent feeding trials in fish nutrition that have demonstrated sufficient sensitivity to detect biologically relevant differences under comparable experimental conditions. Orthogonal polynomial contrasts (linear and quadratic) were applied to assess response patterns across the histamine concentration gradient. Orthogonal polynomial contrasts were predefined a priori and applied under the assumption of equally spaced dietary histamine concentrations to evaluate linear and quadratic dose–response trends. The adjusted coefficient of determination (Adj. R²) was calculated according to Kvålseth (1985). Each group of response variables (digestive enzyme activities, gastrointestinal morphology, tight junction gene expression, antioxidant parameters, and muscle quality traits) was analyzed independently based on predefined biological hypotheses. Therefore, no global multiple testing correction was applied across different endpoint categories, and statistical significance was interpreted within each functional category rather than across all measured variables.

3. Results

3.1. Digestive enzyme activity

Dietary histamine inclusion had no significant effect on pepsin or intestinal amylase activities (P > 0.05; Table 3). As dietary histamine concentration increased, the activities of intestinal trypsin, lipase, and maltase exhibited a linear decline. Intestinal trypsin activity was significantly decreased in the H30-H480 groups compared to the H0-H15 groups, lipase activity was lower in the H480 group compared to the H0 group, and maltase activity was lower in the H240-H480 groups than in the H0-H15 groups (P < 0.05).

Table 3.

Effect of dietary histamine on the intestinal digestive enzyme activities in striped catfish.

	Groups								Regression
	H0	H15	H30	H60	H120	H240	H480		Model	P value	Adj. R2
Pepsin (U/mg protein)	27.2 ± 3.5	26.5 ± 5.4	26.9 ± 2.2	23.5 ± 5.6	24.1 ± 2.0	20.1 ± 2.4	21.8 ± 2.6		N/A
Trypsin (U/mg protein)	501.6 ± 1.0b	499.6 ± 21.4b	393.1 ± 47.1a	392.1 ± 12.0a	337.9 ± 18.8a	378.4 ± 38.6a	388.8 ± 25.0a		L	0.002	0.461
Lipase (U/g protein)	123.3 ± 13.4b	113.9 ± 19.4ab	116.9 ± 1.6ab	99.7 ± 7.2ab	101.4 ± 13.1ab	99.1 ± 11.7ab	87.8 ± 3.6a		L	0.001	0.559
Amylase (U/mg protein)	0.2 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.1 ± 0.0		N/A
Maltase (U/mg protein)	233.2 ± 18.3c	214.3 ± 36.3c	203.9 ± 5.6bc	199.5 ± 10.2bc	192.2 ± 8.3bc	137.3 ± 21.6ab	107.9 ± 4.4a		L	<0.001	0.737

Values are presented as means ± SEM (n = 3 cages per treatment). Means in the same row with different superscripts indicate significant difference (P < 0.05). N/A, not applicable; L, linear trend; Adj. R², adjusted R square.

3.2. Gastrointestinal morphological structure

H&E staining observations of the stomach and anterior intestine are presented in Fig. 1, Fig. 2, respectively. In the stomach, as the dietary histamine concentration increased, villus widths were significantly reduced starting from the H15 group, and muscular thickness was also significantly reduced starting from the H60 group (P < 0.05; Fig. 1). In the anterior intestine, as the dietary histamine concentration increased, villus height was significantly reduced in the H480 group, and muscular layer thickness was significantly lower in the H15, H60, H120, H240, and H480 groups compared with the H0 group (P < 0.05; Fig. 2).

Fig. 1.

Effect of dietary histamine on gastric histology of striped catfish. (A): The gastric hematoxylin-eosin (H&E) staining analysis. a, villi height; b, villi width; c, muscular thickness (× 40). (B): The effect of dietary histamine on gastric villi and muscular layer of striped catfish. Values are means with their standard errors represented by vertical bars (n = 3). ^a,b,c Means with different letters indicate significant difference (P < 0.05).

Fig. 2.

Effect of dietary histamine on intestinal histology of striped catfish. (A): The intestinal hematoxylin-eosin (H&E) staining analysis. a, villi height; b, villi width; c, muscular thickness (× 40). (B): The effect of dietary histamine on intestinal villi and muscular layer of striped catfish. Values are means with their standard errors represented by vertical bars (n = 3). ^a,b,c Means with different letters indicate significant difference (P < 0.05).

3.3. Intestinal permeability indicators

As shown in Fig. 3, dietary histamine had no significant influence on serum DAO activity (P > 0.05). However, as the dietary histamine concentration increased, serum LPS concentration exhibited an upward trend, and when histamine inclusion exceeded 30 mg/kg, LPS level increased significantly (P < 0.05).

Fig. 3.

Effect of dietary histamine on intestinal mucosal permeability in striped catfish. Values are means with their standard errors represented by vertical bars (n = 3). ^a,b,c Means with different letters indicate significant difference (P < 0.05). DAO, diamine oxidase; LPS, lipopolysaccharide.

3.4. Intestinal tight junction and histamine receptor gene expression

The relative mRNA expression levels of intestinal tight junction-related genes are shown in Fig. 4. As the dietary histamine concentration increased, the relative expression of these genes exhibited varying degrees of downregulation. Specifically, the intestinal expression of zo-2 in the H240 and H480 groups, occludin in the H30-H480 groups, claudin7a in the H60-H480 groups, and claudin12 in the H120-H480 groups were significantly lower than those in the H0 group (P < 0.05).

Fig. 4.

Effect of dietary histamine on the relative expression of intestinal tight junction protein in striped catfish. Values are means with their standard errors represented by vertical bars (n = 6). ^a,b,c Means with different letters indicate significant difference (P < 0.05). ZO-2, zonula occludens-2.

As illustrated in Fig. 5, dietary histamine inclusion above 120 mg/kg significantly upregulated the relative expression levels of intestinal histamine receptor genes, namely histamine receptor 2a, histamine receptor 2b, and histamine receptor 3 (P < 0.05).

Fig. 5.

Effect of dietary histamine on the relative expression of intestinal histamine receptor in striped catfish. Values are means with their standard errors represented by vertical bars (n = 6). ^a,b,c Means with different letters indicate significant difference (P < 0.05). H2a, histamine receptor 2a; H2b, histamine receptor 2b; H3, histamine receptor 3.

3.5. Serum antioxidative capacity

Dietary histamine supplementation did not significantly influence serum MDA levels. (P > 0.05). In contrast, it significantly reduced the activities of serum T-AOC, POD, and CAT in the H480 group (P < 0.05; Table 4). In addition, serum SOD, GPX, and GR activities in the H120-H480 groups were significantly reduced compared with the H0 group (P < 0.05). Furthermore, as the dietary histamine concentration increased, the activities of POD, SOD, GPX, and GR displayed a clear linear downward trend.

Table 4.

Effect of dietary histamine on antioxidant-related parameters in serum of striped catfish.

	Groups								Regression
	H0	H15	H30	H60	H120	H240	H480		Model	P value	Adj. R2
T-AOC (U/mL)	0.9 ± 0.3b	0.8 ± 0.1ab	0.7 ± 0.2ab	0.7 ± 0.1ab	0.8 ± 0.1ab	0.6 ± 0.1ab	0.6 ± 0.1a		NR
POD (U/L)	71.2 ± 10.8b	65.8 ± 3.2ab	61.7 ± 15.2ab	62.3 ± 10.3ab	57.8 ± 12.8ab	54.1 ± 3.3ab	44.8 ± 4.3a		L	0.003	0.490
CAT (U/mL)	15.7 ± 3.7b	13.6 ± 2.4ab	13.0 ± 4.6ab	12.2 ± 0.3ab	11.7 ± 0.3ab	12.7 ± 1.0ab	8.5 ± 2.1a		NR
SOD (U/mL)	63.6 ± 2.9c	60.5 ± 1.1c	55.7 ± 5.0bc	55.5 ± 1.7bc	42.6 ± 15.3ab	44.1 ± 4.8ab	36.6 ± 7.9a		L	<0.001	0.702
GPX (U/L)	36.5 ± 0.8b	33.3 ± 5.7ab	29.5 ± 5.8ab	29.5 ± 1.8ab	25.7 ± 1.7a	26.5 ± 2.2a	26.7 ± 1.7a		L	0.003	0.489
GR (U/L)	28.2 ± 0.6b	27.9 ± 4.2b	26.7 ± 1.0ab	29.4 ± 0.8b	21.7 ± 0.9a	21.1 ± 3.1a	20.7 ± 2.3a		L	0.002	0.533
MDA (nmol/mL)	14.7 ± 1.3	14.3 ± 2.5	15.5 ± 1.3	14.1 ± 1.6	15.4 ± 2.0	15.3 ± 1.5	13.9 ± 1.4		N/A

Values are presented as means ± SEM (n = 3). Means in the same row with different superscripts indicate significant difference (P < 0.05). T-AOC, total antioxidant capacity; POD, peroxidase; CAT, catalase; SOD, superoxide dismutase; GPX, glutathione peroxidase; GR, glutathione reductase; MDA, malondialdehyde. NR, no relationship; N/A, not applicable; L, linear trend; Adj. R², adjusted R square.

3.6. Muscle quality

Dietary histamine inclusion had no significant effects on the a* value, hardness, cohesiveness, or resilience of the dorsal muscle (P > 0.05; Table 5). The L* value of the dorsal muscle exhibited a decreasing trend, with significantly lower value observed in the H120-H480 groups than in the H0 group. Conversely, the b* value showed an opposite pattern, with significantly higher value detected in the H30-H480 groups compared with the H0 group (P < 0.05). Furthermore, muscle chewiness and springiness were significantly elevated in the H240 and H480 groups relative to the H0 group (P < 0.05), while an increase in gumminess was observed exclusively in the H480 group (P < 0.05).

Table 5.

Effect of dietary histamine on muscle quality of striped catfish.

	Groups								Regression
	H0	H15	H30	H60	H120	H240	H480		Model	P value	Adj. R2
L*	63.00 ± 3.15b	61.31 ± 3.89b	61.17 ± 2.23b	59.40 ± 6.10ab	56.08 ± 4.18a	54.74 ± 3.70a	55.10 ± 2.83a		L	0.003	0.387
a*	−1.09 ± 1.27	−0.65 ± 1.06	−0.78 ± 1.31	−0.96 ± 1.10	−1.60 ± 0.85	−1.24 ± 1.12	−1.40 ± 1.32		N/A
b*	6.30 ± 1.18a	7.69 ± 1.57ab	9.37 ± 0.79bc	9.54 ± 0.88bc	10.55 ± 1.57c	10.56 ± 0.83c	11.13 ± 2.98c		L	<0.001	0.556
Hardness(kg)	2.24 ± 0.11	2.07 ± 0.10	2.12 ± 0.24	2.09 ± 0.11	2.18 ± 0.15	2.05 ± 0.06	2.12 ± 0.17		N/A
Springiness	0.23 ± 0.04a	0.23 ± 0.03a	0.23 ± 0.03a	0.24 ± 0.03a	0.24 ± 0.03a	0.49 ± 0.02b	0.52 ± 0.06b		L	0.005	0.662
Cohesiveness	0.10 ± 0.00	0.10 ± 0.03	0.12 ± 0.03	0.11 ± 0.02	0.12 ± 0.02	0.12 ± 0.00	0.13 ± 0.01		N/A
Gumminess	203.07 ± 12.56a	207.41 ± 14.80a	214.92 ± 10.52a	241.59 ± 41.37ab	252.32 ± 29.82ab	247.35 ± 14.66ab	268.24 ± 33.20b		NR
Chewiness	64.92 ± 17.52a	67.44 ± 21.57a	62.64 ± 14.32a	79.21 ± 18.31a	67.24 ± 6.25a	117.72 ± 15.52b	129.22 ± 23.24b		L	0.002	0.722
Resilience	0.05 ± 0.00	0.05 ± 0.01	0.06 ± 0.01	0.05 ± 0.00	0.05 ± 0.00	0.05 ± 0.00	0.06 ± 0.00		N/A

Values are presented as means ± SEM (n = 3). Means in the same row with different superscripts indicate significant difference (P < 0.05). N/A, not applicable; NR, no relationship; L, linear trend; Adj. R², adjusted R square.

4. Discussion

Histamine has been well documented to exert detrimental effects on the digestive tract of various aquatic organisms (Opstvedt et al., 2000; Shiozaki et al., 2004; Tapia-Salazar et al., 2001). Comprising the stomach and intestine, the digestive tract is essential for the key physiological function of nutrient acquisition in aquatic animals. Before nutrients can be assimilated by fish, they must first be hydrolyzed by digestive enzymes, the activity of which is a key determinant of feed nutrient utilization efficiency (Deng et al., 2021). Our findings demonstrate that elevated dietary histamine concentrations are associated with significantly reduced activities of intestinal trypsin, lipase, and maltase in striped catfish, suggesting a potential decrease in nutrient utilizaiton efficiency. This observation provides a plausible explanation for the decline in growth performance, as high histamine concentrations in feed likely diminish digestive enzyme activity and thereby impair nutrient utilization. Studies in terrestrial species, such as pigs and chickens, have shown that histamine modulates digestive enzyme activity by altering luminal pH, a mechanism that may also operate in striped catfish (Sugahara et al., 1988). Determining whether a similar pH-mediated mechanism operates in striped catfish could provide valuable insights into feed safety and digestive efficiency, particularly under conditions of feed spoilage or elevated histamine concentrations.

Strongly influenced by dietary constituents, the morphological characteristics of the digestive tract are closely linked to its physiological efficacy (Fang et al., 2019). The structural integrity of the digestive tract, a key indicator of intestinal health (Lin et al., 2020), is associated with changes in villi count, height, and width-factors that critically impact digestive function (Liu et al., 2022). An increased mucosal contact area between the digestive tract and feed is generally considered beneficial for digestion. In this study, elevated histamine concentrations led to a decrease in gastric villus width and gastric muscular thickness. Additionally, reductions were observed in both the height of intestinal villi and the thickness of the intestinal muscular layer. These changes indicate a reduction in both digestive and absorptive surface areas, as well as diminished peristaltic efficiency, ultimately compromising the overall digestive and absorptive capacities of striped catfish. The suboptimal morphology of the digestive tract likely contributes to the observed reduction in muscular thickness.

Intestinal integrity is closely related to the architecture of tight junctions, which are formed by the scaffolding protein family (zonulae occludens) and transmembrane proteins (Buckley & Turner, 2018). Our study revealed a significant downregulation of the relative expression of key intestinal tight junction proteins, including zo-2, occludin, claudin7a, and claudin12, under high dietary histamine conditions. This structural disruption of tight junctions likely contributes to increased intestinal permeability, a phenomenon further supported by the elevated serum LPS concentration observed in this study. The modulation of tight junction protein gene expression provides a potential mechanism through which elevated dietary histamine concentrations may compromise intestinal barrier integrity, as indicated by these findings.

The antioxidant defense system, comprising enzymes such as SOD, CAT, GPX, and GR, plays a crucial role in scavenging reactive oxygen species and mitigating oxidative stress (Abdel-Latif et al., 2023). As a result of lipid peroxidation, MDA is widely utilized as a biomarker to indicate oxidative stress levels (Pan et al., 2022). Previous studies have reported that dietary histamine significantly depresses antioxidant levels in the serum of hybrid grouper (Qiu et al., 2024; Zhang et al., 2023). Similarly, our results showed that high dietary histamine markedly suppressed serum SOD, CAT, GPX, and GR activities, while simultaneously elevating MDA level, suggesting compromised antioxidant capacity in striped catfish. This compromised antioxidant status may result from histamine-induced structural damage to the digestive tract. Similar outcomes have been observed in Pacific white shrimp (Litopenaeus vannamei) (Lin et al., 2022) and orange-spotted grouper (Epinephelus coioides) (Liu et al., 2021). Liu et al. (2021) further proposed that prolonged histamine intake disrupts antioxidant defenses by impairing the organism's capacity to neutralize reactive oxygen species.

Food color, an essential sensory attribute, significantly influences consumer food choices and thereby serves as a key determinant of product quality (Li et al., 2022). In striped catfish, the dorsal muscle is the primary edible portion and is typically processed into fillets for market distribution (Phan et al., 2021). Consequently, muscle color plays a critical role in determining commercial value. Our findings revealed that when dietary histamine exceeded 60 mg/kg, dorsal muscle yellowness (b* value) increased markedly, whereas brightness (L* value) declined. These color alterations may cause consumers to perceive the fillets as less fresh, thereby reducing their market appeal.

In addition to color, muscle texture represents a key determinant of consumer acceptance and is typically evaluated using attributes such as hardness, elasticity, cohesiveness, adhesiveness, chewiness, and resilience (Li et al., 2022). In this study, muscle springiness and chewiness were significantly elevated in the H240 and H480 groups, whereas gumminess was significantly higher in the H480 group compared with the control group. Collectively, these changes suggest that high dietary histamine modifies muscle texture, leading to increased cohesiveness, chewiness, and adhesiveness. Such modifications may negatively affect muscle palatability and potentially reduce consumer purchasing intent. Accordingly, responses observed at the highest dietary histamine concentration should be interpreted as toxicological effects under extreme exposure conditions rather than physiological regulation relevant to routine aquafeed formulations.

5. Conclusion

High dietary histamine concentrations were found to reduce digestive enzyme activity and to impair the structural integrity of the digestive tract in striped catfish, thereby ultimately compromising nutrient utilization. Additionally, histamine significantly downregulated the relative expression of tight junction-related genes, leading to enhanced intestinal permeability. As dietary histamine content increased, antioxidant capacity progressively declined. Notably, this study also demonstrated that histamine adversely affects the flesh quality of striped catfish, characterized by a significant increase in yellowness and a decrease in dorsal muscle brightness; these changes may negatively influence consumer purchasing decisions.

CRediT authorship contribution statement

Cong Wang: Writing – original draft, Investigation, Conceptualization. Langji Fuxin: Formal analysis. Chunfeng Yao: Supervision, Methodology. Beiping Tan: Supervision, Methodology. Shiwei Xie: Supervision, Methodology. Junming Deng: Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.