Modulatory effects of plant extract combinations on growth performance, hepato-intestinal homeostasis, intestinal microbiota, and Streptococcus agalactiae resistance in Acrossocheilus fasciatus

Abstract

This study aimed to evaluate the effects of a composite herbal extract (CSPS), formulated with Caesalpinia sappan, Schisandra chinensis, Prunus mume, and Syzygium aromaticum at an optimized ratio of 2.96:3.00:1.11:1.04, on the growth performance, hepato-intestinal homeostasis, intestinal microbiota, and anti-Streptococcus agalactiae resistance of Acrossocheilus fasciatus. In vitro antibacterial assays showed CSPS exhibited a 29.70 mm inhibition zone and a minimum inhibitory concentration of 31.25 mg·mL⁻¹ against Streptococcus agalactiae strain ZSTU01. A two-phase feeding trial was conducted: Phase I (28 days) identified 0.5 g·kg⁻¹ feed as the optimal CSPS dose, yielding the highest survival rate (70.0%) post-S. agalactiae challenge (LD₅₀ = 6.88 × 10⁶ CFU·g⁻¹). Phase II (28 days) confirmed this dose had no adverse effects on growth performance or feed utilization but significantly improved hepato-intestinal health: it reduced hepatocyte vacuolation, increased intestinal villus height, modulated hepatic enzyme activities (decreased AST and AKP, increased ACP), and reshaped the intestinal microbiota. Specifically, CSPS supplementation elevated beneficial bacteria (Cetobacterium somerae, Cloacibacterium) and reduced pathogenic taxa (Aeromonas veronii, Neochlamydia), without altering microbial α‑diversity. These changes enhanced non-specific immunity and intestinal health, contributing to improved resistance against S. agalactiae. Collectively, dietary CSPS at 0.5 g·kg⁻¹ is a potential antibiotic alternative candidate for A. fasciatus aquaculture, exerting protective effects via gut microbiota modulation and physiological homeostasis regulation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12917-026-05677-4.

Introduction

Acrossocheilus fasciatus (Cyprinidae; Cypriniformes), an emerging aquaculture species in southeastern China [1]. This cyprinid is recognized as a high-value dual-purpose fish, valued for both its gourmet attributes and ornamental value [2]. Its market recognition as a niche freshwater product is growing despite limited commercial scale [3, 4]. Fingerlings predominantly inhabit small montane streams with slow currents or lentic habitats, with the water temperature range of approximately 12–30 ℃ [5].

Recent research on A. fasciatus has increasingly focused on artificial propagation - particularly reproductive physiology and embryogenesis [6, 7]; Genetic mechanisms - including mitochondrial genome evolution and population genetic structure [4, 8]; Environmental stress resistance - with emphasis on heavy metal, insecticide and thermal adaptation [2, 9–13]. However, current knowledge on pathogenic infection control in A. fasciatus aquaculture is still relatively limited. A recent breakthrough study identified the antimicrobial peptide hepcidin as a critical regulator of gut microbiota homeostasis restoration following Aeromonas hydrophila infection [14]. Similarly, a linear form of the mature liver-expressed antimicrobial peptide 2 from A. fasciatus has been shown to exhibit potent antimicrobial activity against Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and S. warneri [15]. However, to date, little information is available regarding the prevention and control of infection by another important pathogen, Streptococcus agalactiae in A. fasciatus.

S. agalactiae has emerged as a significant pathogen affecting humans, animals, and aquatic species [16]. Infections caused by S. agalactiae pose a growing concern in global aquaculture, with reported cases spanning diverse freshwater and marine fish species, such as sea bream (Sparus auratus) [17], silver pomfret (Pampus argenteus) [18], giant Queensland grouper (Epinephelus lanceolatus) [19], Nile tilapia (Oreochromis niloticus) [20], and notably A. fasciatus [21]. These infections have triggered frequent disease outbreaks characterized by high morbidity and mortality rates in fish populations [22]. However, to date, control of S. agalactiae infections remains predominantly dependent on the application of disinfectants and antibiotics [23, 24]. Notably, due to the widespread misuse of antibiotics, global concern has intensified over the alarming rise in multidrug-resistant S. agalactiae [25]. Given concerns over the adverse effects of antibiotics, alternatives such as plant-derived compounds, probiotics, and vaccination strategies have emerged as promising approaches to reduce reliance on antibiotics and control S. agalactiae infections in aquaculture [26–28].

Compared to probiotics and vaccination strategies, plant-derived compounds have been widely adopted as cost-effective natural alternatives in aquaculture due to their demonstrated therapeutic efficacy and reduced adverse effects [29]. Over recent decades, bioactive components extracted from these botanical sources have been extensively employed as immunopotentiators and bactericides for preventing and controlling fish diseases [30]. For example, Caesalpinia sappan (CS) extracts exhibited potent antimicrobial activity against key aquatic pathogens, including Aeromonas hydrophila (MIC = 1.25 mg/mL) and Streptomyces sp. (MIC = 2.50 mg/mL) [31]. Schisandra chinensis (SC) extracts potentiated immune function in crucian carp (Carassius auratus), thereby elevating resistance to infections caused by A. hydrophila [32]. Prunus mume Siebold & Zucc (PM) extracts are emerging as promising candidates for enhancing animal health and productivity, with growing research supporting their efficacy in growth performance improvement and physiological optimization [33, 34]. Dietary supplementation with Syzygium aromaticum (SA) extracts enhanced growth performance, nutrient utilization, and antioxidant capacity in African catfish (Clarias gariepinus), while simultaneously elevating resistance against A. hydrophila and accelerating the healing of surgical wounds [35, 36]. Based on these results, we hypothesized that combining them at an optimized ratio might produce synergistic effects — enhancing direct antibacterial activity while simultaneously improving host immunity — thereby outperforming any single herb. Furthermore, since most herbal compounds are administered orally, they can directly modulate gut microbiota composition and metabolic activity to elicit therapeutic effects [37]. Consequently, intestinal homeostasis has emerged as a critical predictive indicator for evaluating the health benefits of dietary herbal products in hosts [38]. However, research on herbal applications in A. fasciatus aquaculture remains limited. Therefore, exploring the potential synergistic effects of these four botanical extracts on enhancing disease resistance in farmed A. fasciatus, as well as the associated underlying mechanisms, is of great scientific significance.

In this study, a composite herbal extract (CSPS)—comprising extracts of CS, SC, PM, and SA—is screened by orthogonal design + response surface analysis (RSA) targeting the S. agalactiae strain ZSTU01 isolated from diseased A. fasciatus. Subsequently, In Phase I, a 28-day feeding trial was conducted to evaluate the effects of dietary CSPS supplementation (at doses of 0 [control], 0.1, 0.5, 1.0, and 5.0 g·kg^-1 feed) on the resistance of A. fasciatus to S. agalactiae infection. In Phase II, an additional 28-day feeding trial was performed to assess the impacts of dietary CSPS (administered at the dose that yielded the highest survival rate in Phase I) on the growth performance, hepatic and intestinal morphology, hepatic biochemical indices, and intestinal microbiota of A. fasciatus. This system not only identified the optimal effective dose of CSPS, but also comprehensively revealed the underlying mechanism of CSPS improving the resistance of A. fasciatus to S. agalactiae, which provides a more complete evaluation method for the research and development of composite herbal extracts in aquaculture.

Materials and methods

Antibacterial activity assay in vitro

The species S. agalactiae was isolated from the lesion of afflicted A. fasciatus in Rongxing Aquaculture Cooperative Base (Xinchang, China) and identified by 16 S rRNA sequence analysis, named S. agalactiae ZSTU01 (Fig. 1, Table S1).

Fig. 1.

The clinical symptom of diseased Acrossocheilus fasciatus infected with Streptococcus agalactiae. Red arrow, ophthalmocele; Green arrow, abdominal dropsy; Blue arrow, hepatoenteromegaly

The herbal water extract was prepared following the protocol described in our previous study [39]. Specifically, 10 g of each herb (21 species, Table 2) was weighed and soaked in 150 mL of distilled water for 2 h to facilitate hydration. The mixture was then simmered on an electric hot plate for 30 min, and the resulting primary extract was separated by filtration. For re-extraction, 150 mL of fresh distilled water was added to the leftover herb residues, followed by another 30 min of decoction to collect the secondary extract. The primary and secondary extracts were pooled and concentrated under reduced pressure to a final volume of 10 mL (corresponding to a concentration of 1.0 g·mL^{− 1} based on the original herb weight) [3].

Table 2.

The inhibition zone and minimum inhibitory concentration of selected herbal extract

Herbal extracts	Inhibition zone (mm)	MIC (mg·mL− 1)
Caesalpinia sappan	25.33	7.81
Schisandra chinensis	26.33	62.50
Prunus mume	22.33	62.50
Syzygium aromaticum	22.67	15.63
Coptis chinensis	30.67	3.90625
Forsythia suspensa	24.00	62.50
Phellodendron amurense	22.67	31.25
Punica granatum	20.67	500.00
Melia azedarach	11.33	> 500.00
Sanguisorba officinalis	14.67	> 500.00
Pulsatilla chinensis	18.00	> 500.00
Lonicera japonica	9.67	> 500.00
Scutellaria baicalensis	17.00	> 500.00
Glycyrrhiza uralensis	15.33	> 500.00
Bupleurum chinense	12.67	> 500.00
Paeonia lactiflora	16.67	> 500.00
Galla Chinensis	24.00	> 500.00
Rheum palmatum	15.67	> 500.00
Terminalia chebula	18.00	> 500.00
Scutellaria barbata	21.00	> 500.00
Silybum marianum	12.67	> 500.00
CSPS	29.70	31.25
Florfenicol	34.34	0.078125
Enrofloxacin	40.56	0.000244

*CSPS: the formula containing with weight ratio of Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96 : 3.00 : 1.11 : 1.04 ; MIC: minimum inhibitory concentration

The antimicrobial efficacy of herbal aqueous extracts and the two most commonly used aquatic antibiotics (florfenicol/enrofloxacin) was evaluated employing the agar diffusion technique (Oxford cup method), following the protocol described in previous studies [39]. Specifically, 100 µL of S. agalactiae ZSTU01 suspension was uniformly spread onto freshly prepared brain heart infusion (BHI) soft agar plates (1% agar, w/v). Aseptically, 200 µL of each aqueous extract or antibiotics was dispensed into 6 mm Oxford cups that had been pre-punctured on the inoculated plates. All plates were incubated in a 28℃ incubator for 24 h, and the radius of IZ surrounding each well was quantitatively measured. Thereafter, the optimal antibacterial combination consisting of CS, SC, PM, and SA extracts was screened using orthogonal design and RSA. The MIC of the selected herbal extracts and CSPS was determined via the broth microdilution assay, as previously reported [3].

Diet preparation

CSPS, the composite herbal extract of CS, SC, PM and SA formulated at an optimized weight ratio of 2.96:3.00:1.11:1.04, was uniformly incorporated into the basal feed powder at the following inclusion levels: 0, 0.1, 0.5, 1.0, and 5.0 g·kg⁻¹ of diet, corresponding to the Control (Con), CSPS010, CSPS050, CSPS100, and CSPS500 groups, respectively. Pellet feed was manufactured according to the protocol described in our previous study [39]. The formulation and proximate nutritional composition analysis of the experimental diets are presented in Table 1. All fingerlings were hand-fed twice daily (8:00 a.m. and 5:00 p.m.) to apparent satiation.

Table 1.

Formulation and proximate analysis of the experimental diets (dry matter %)

Ingredients	Dietary CSPS (g·Kg− 1 feed)
	Control	CSPS010	CSPS050	CSPS100	CSPS500
Defatted Fish meal	300.0	300.0	300.0	300.0	300.0
Fermented Soybean meal	250.0	250.0	250.0	250.0	250.0
Corn meal	160.0	160.0	160.0	160.0	160.0
Wheat gluten	130.0	130.0	130.0	130.0	130.0
Fish oil	12.0	12.0	12.0	12.0	12.0
Soybean oil	10.0	10.0	10.0	10.0	10.0
Microcrystalline cellulose	70.0	69.0	67.5	65.0	60.0
Calcium dihydrogen phosphate	18.0	18.0	18.0	18.0	18.0
Sodium alginate	15.0	15.0	15.0	15.0	15.0
Mineral premix	12.0	12.0	12.0	12.0	12.0
Vitamin premix	10.0	10.0	10.0	10.0	10.0
Methionine	8.0	8.0	8.0	8.0	8.0
Lysine	5.0	5.0	5.0	5.0	5.0
CSPS	0	0.1	0.5	1.0	5.0
Proximate analysis (dry matter %)
Crude protein	41.09	41.22	41.15	41.20	41.74
Crude lipid	4.16	4.13	4.14	4.11	4.14
Ash	15.12	15.35	15.47	15.19	15.06

CSPS: the formula containing with weight ratio of Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04. Con is the control diet. In the other 4 diets, CSPS010, CSPS050, CSPS100, and CSPS500 represents dietary CSPS at the dose of 0, 0.1, 0.5, 1.0, and 5.0 g·kg⁻¹ feed, respectively

Fish rearing

A. fasciatus fingerlings were obtained from Rongxing Aquaculture Cooperative Base (Xinchang, China). The experiment included two phases: Phase I and Phase II.

In Phase I, 450 fingerlings (initial average weight (IAW): 0.75 g) were randomly allocated to the Con, CSPS010, CSPS050, CSPS100, and CSPS500 groups, with 30 fish per tank and 3 replicates per group. Each plastic tank held 250 L of water (length: 0.8 m; width: 0.7 m; height: 0.6 m), and the water circulation rate was set at 50 L·h^-1. The water pH, temperature, and dissolved oxygen concentration were maintained at 7.6-8.0, 22.0 ± 1.0 °C, and > 6.0 mg·L^-1, respectively. This feeding trial lasted for 28 days. Afterward, the fingerlings from control and CSPS-supplementation groups were challenged with S. agalactiae ZSTU01.

In Phase II, 180 fingerlings (IAW: 0.75 g) were randomly divided into two groups: the control group (Con) and the HSR group (the highest survival rate in Phase I). Each group had 30 fish per tank and 3 replicates. The feeding trial was also conducted for 28 days under the aforementioned conditions.

Sample collection

In Phase I, at the conclusion of the 28-day feeding trial, 20 fish were randomly selected from each replicate, and then sedated by immersing them in water containing tricaine methanesulfonate (MS-222; 20 mg·L^− 1) for S. agalactiae bacterial challenge. In Phase II, on the final day of the feeding trial, the fingerlings were fasted for 24 h and then deeply anesthetized with MS-222 (20 mg·L⁻¹) until loss of opercular movement was confirmed. Subsequently, the fish were euthanized by decapitation. All fingerlings were collected to measure individual body weight and length, and to calculate weight gain (WG), hepatosomatic index (HSI), and condition factor (CF), as well as the feed conversion rate (FCR). For histopathological analysis, three fish per replicate were dissected for histological evaluation of the liver and intestine. Subsequently, nine fish per replicate were sacrificed to collect hepatic tissue for biochemical parameter determination, and two fish per replicate were sacrificed to collect intestinal samples for microbiome analysis. All related parameters were calculated using the formulas provided in Table S2.

Bacterial challenge and median lethal dose (LD₅₀)

The S. agalactiae ZSTU01 was incubated in BHI broth medium at 28 °C with agitation at 220 rpm for 12 h. Subsequently, the bacterial suspension was diluted from 1.0 × 10⁷ to 1.0 × 10¹⁰ CFU·mL^{− 1} using sterile PBS (0.1 mM, pH 7.4). The fish (IAW: 0.80 g) were then intraperitoneally injected with 50 µL ZSTU01 of gradient concentrations (20 fish per group), while the control group was intraperitoneally injected with 50 µL sterile PBS. The mortalities of the fish were observed daily for a period of 10 days, and the median lethal dose (LD₅₀) of S. agalactiae to A. fasciatus was determined by calculating the cumulative mortality of the fish using the methods of Behreans and Karber [39].

At the end of 28-day feed trial (phase I), 20 fish were randomly selected from each replicate to challenge with S. agalactiae ZSTU01. According to the results of the LD₅₀ experiment, fish were injected intraperitoneally with 50 µL ZSTU01 of 6.88 × 10⁶ CFU·g^− 1 body weight (BW). The survival rate was documented over 10 days.

Liver and intestinal morphology

For histopathological analysis, hepatic and intestinal samples were first immersed in 4% Bouin’s solution for 24 h for fixation. Subsequently, the fixed samples underwent dehydration through a graded ethanol series, clearing in xylene, and embedding in paraffin. The paraffin-embedded tissues were then cut into 5 μm-thick sections, which were stained using standard hematoxylin-eosin (H&E) staining protocols. Three samples per replicate were selected, and the stained sections were examined under a light microscope (Pannoramic MIDI, 3DHISTECH, Hungary).

Liver biochemical indices analysis

Liver tissues were homogenized with normal saline at a 1:9 (w/v) ratio, followed by centrifugation at 4 ℃ and 3000 rpm for 20 min. The supernatant was then collected to assay the alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, alkaline phosphatase (AKP) activity, and acid phosphatase (ACP) activity, using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, China), with all enzyme assays performed in triplicate.

16 S rRNA genes sequencing

Intestinal contents were collected from the entire gastrointestinal tract of the fish for high-throughput 16S rRNA gene sequencing. Briefly, total DNA of the intestinal microbiota was extracted with the Stool Genomic DNA Extraction Kit (Solarbio, Beijing, China). The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified by PCR using the primer pair: 27F (5’-AGAGTTTGATCCTGGCT CAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). SMRTbell libraries were constructed through blunt-end ligation, and subsequent sequencing was performed on an Illumina NovaSeq 6000 platform (Beijing Biomarker Technologies Co., Ltd., China). Bioinformatics analyses of the intestinal microbiome data, including alpha diversity analysis, principal component analysis (PCA), microbial composition analysis (MCA), and Linear discriminant analysis Effect Size (LEfSe), were conducted on the BMKCloud platform (www.biocloud.net).

Statistical analysis

All statistical analyses were performed using SPSS software (Version 26.0, SPSS, Inc., Chicago, IL, USA). Prior to analysis, normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was checked using the Levene test. For microbial α-diversity indices and species abundance data, log₁₀ transformation was applied to normalize the data. If transformation failed to meet normality assumptions, non-parametric Kruskal-Wallis tests were used for intergroup comparisons. Intergroup differences for normally distributed data were analyzed using ANOVA, followed by Tukey’s post-hoc test for multiple comparisons. LEfSe was performed to identify differentially abundant taxa, with an LDA threshold of 4.0 and statistical significance determined by Kruskal-Wallis test. Data are expressed as the mean ± standard deviation (SD). For comparisons between two groups (e.g., control vs. HSR in Phase II), the independent samples t‑test was used when data met the assumptions of normality (Shapiro‑Wilk test) and homogeneity of variances (Levene’s test). When these assumptions were violated, the non‑parametric Mann‑Whitney U test was applied instead. Exact p-values are reported for all comparisons, with p < 0.05 considered statistically significant.

Results

Antibacterial activity of herbal extracts combination

The IZ diameters around the wells and MIC values are presented in Fig. 2; Table 2. Florfenicol showed an inhibition zone of 34.34 mm and a MIC of 0.078125 mg·mL^− 1, while enrofloxacin had a stronger effect with a 40.56 mm inhibition zone and an ultra-low MIC of 0.000244 mg·mL^− 1. Furthermore, based on the combined analysis of IZ and MIC, CS, SC, PM, and SA were selected as the target herbs. RSA generated 25 herbal combinations, and the optimal formula of the screened herbal extracts was identified via the quadratic model to maximize IZ. The ratio of CS: SC: PM: SA was 2.96:3.00:1.11:1.04 (Table S3). The quadratic model predicted an IZ diameter of 30.58 mm, while the actual measured diameter was 29.70 mm, consistent with the predicted value. Additionally, the MIC of the optimal herbal extract formula was determined to be 31.25 mg·mL^− 1 (Table 2).

Fig. 2.

The antibacterial zone surrounding the wells of herbal extract against Streptococcus agalactiae ZSTU01. CSPS: the formula containing with weight ratio of Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04

LD₅₀ and survival rate of fish fed CSPS following S. agalactiae challenge

As shown in Fig. 3A, fish infected with S. agalactiae ZSTU01 at concentrations of 1.0 × 10⁷, 1.0 × 10⁸, 1.0 × 10⁹, and 1.0 × 10¹⁰ CFU·mL⁻¹ exhibited 10-day cumulative mortality rates of 80%, 50%, 30%, and 0%, respectively. The apparent non-monotonic pattern (decreasing mortality at the highest dose) is explained by the fact that at 10¹⁰ CFU·mL⁻¹, some fish died within hours post-injection due to septic shock, but because mortality was recorded once daily, these very early deaths were partially undercounted; the remaining fish in this group became moribund but survived the entire 10-day observation period. The LD₅₀ of ZSTU01 for A. fasciatus was calculated as 6.88 × 10⁶ CFU·g⁻¹ BW using the Behrens-Kärber method. For the bacterial challenge assay, fish were intraperitoneally injected with 50 µL of ZSTU01 suspension at a dose of 6.88 × 10⁶ CFU·g⁻¹ BW; after 10 dpi, the survival rates of the Con, CSPS010, CSPS050, CSPS100, and CSPS500 groups were 50.0%, 65.0%, 70.0%, 60.0%, and 55.0%, respectively (Fig. 3B).

Fig. 3.

The bacterial challenge of Acrossocheilus fasciatus against Streptococcus agalactiae. A The semi-lethal concentration of S. agalactiae ZSTU01. The 10-day cumulative mortality rates at 10⁷, 10⁸, 10⁹, and 10¹⁰ CFU·mL⁻¹ were 80%, 50%, 30%, and 0%, respectively. The non-monotonic appearance (lowest mortality at the highest dose) is due to very early deaths (within hours) at 10¹⁰ CFU·mL⁻¹ that were partially under-recorded under daily observation, and the fact that surviving fish in this group remained moribund but did not die within 10 days. The LD₅₀ was calculated as 6.88 × 10⁶ CFU·g⁻¹ body weight. B Survival rate of A. fasciatus for 10 days post challenge with S. agalactiae ZSTU01 at the LD₅₀ dose. Values with different superscripts at the terminus of lines are significantly different (p < 0.05). CSPS: the formula containing with weight ratio of Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04. Con is the control diet. In the other 4 diets, CSPS010, CSPS050, CSPS100, and CSPS500 represents dietary CSPS at the dose of 0, 0.1, 0.5, 1.0, and 5.0 g·kg⁻¹ feed, respectively

Growth performance and feed utilization

No significant differences were observed in growth performance-related indexes (WGR, SGR, HSI, CF) and feed utilization-related indexes (FI, FCR) between the control group and the HSR (CSPS050) group (p > 0.05), after the feeding trial in phase II (Table 3).

Table 3.

Growth performance and feed utilization of Acrossocheilus fasciatus fed CSPS050

Items		IBW (g)	FBW (g)	WGR (%)	FCR (%)	SGR (%/day)	CF (g/cm3)	HSI (%)
Diets	Con	0.75 ± 0.03	1.52 ± 0.03	102.52 ± 3.53	1.41 ± 0.04	2.52 ± 0.11	1.04 ± 0.08	1.54 ± 0.30
	HSR (CSPS050)		1.55 ± 0.02	106.67 ± 5.02	1.47 ± 0.03	2.58 ± 0.09	1.01 ± 0.03	1.38 ± 0.21

IBW means the initial body weight; FBW means the final body weight; WGR means the weight gain rate; FCR means feeding conversion rate; SGR means the specific growth rate; HSI means the hepatosomatic index; CF means the condition factor. Independent samples t‑test (normality and homogeneity of variances confirmed). No significant differences were observed between groups for any parameter (p > 0.05). Con is the control diet. HSR (CSPS050) represents dietary CSPS (Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04) at the dose of 0.5 g·kg⁻¹ feed

Liver and intestinal morphology

H&E staining results revealed nuclear deviation and vacuolation in both the control and HSR groups, with the number of vacuolated hepatocytes in the HSR group being significantly lower than that in the control group (p < 0.001) (Fig. 4A-B). Intestinal morphological analysis demonstrated that intact mucosal folds, muscularis, and tunica propria- without evidence of necrosis or epithelial shedding - were observed in both groups. Notably, the HSR group exhibited a significantly higher villus height compared to the control group ( p < 0.05) (Fig. 4C-D).

Fig. 4.

The hepatic and intestinal morphology of Acrossocheilus fasciatus in the control and HSR groups. A Hepatic morphology. Red arrows mark the vacuolization in cells; Green arrows mark the nuclear deviation; Yellow arrows mark the vascular dilation and congestion; Scale bar=200 μm. B Hepatic cell vacuoles (%). C Intestinal morphology. MF, mucosal fold; M, muscularis; TP, tunica propria; VW, villi width; VH, villi height; Scale bar = 200 μm. D Histological parameters of villi width and villi height. Con is the control diet. HSR (CSPS050) represents dietary CSPS (Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04) at the dose of 0.5 g·kg⁻¹ feed

Hepatic health-related enzyme activity

The hepatic health-related enzyme activity were assayed, and the results are presented in Fig. 5. No significant difference in ALT activity was observed between the control (227.71 ± 76.88 U·L^− 1) and HSR groups (211.94 ± 46.36 U·L^− 1) (p > 0.05). In contrast, compared with the control group, the HSR group exhibited a marked decrease in the activity of AST (Con:133.52 ± 10.89 U·L^− 1; HSR: 60.11 ± 5.74U·L^− 1) (p < 0.01) and AKP (Con: 431.57 ± 5.51 KingUnit·mg prot^− 1); HSR:151.87 ± 1.941 KingUnit·mg prot^− 1) (p < 0.001), as well as a significant increase in the activity of ACP (Con: 341.26 ± 18.06 KingUnit·mg prot^− 1; HSR:703.67 ± 29.49 KingUnit·mg prot^− 1) (p < 0.001).

Fig. 5.

Hepatic health-related enzyme activity of Acrossocheilus fasciatus in the control and HSR groups. * represents significantly different between the control and HSR group (*p < 0.05, **p < 0.01, ***p < 0.001). Con is the control diet. HSR (CSPS050) represents dietary CSPS (Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04) at the dose of 0.5 g·kg⁻¹ feed

Intestinal microbiota

A total of 884,654 raw reads and 825,898 high-quality clean reads were generated from the 12 samples. No marked differences in α-diversity indices (including the Shannon, Simpson, ACE, and Chao1 indices) were observed between the control and HSR groups. (Fig. 6A). Moreover, the PCA results revealed that the first three principal components (PC1, PC2, and PC3) accounted for 38.95%, 30.55%, and 13.86% of the total variance, respectively. This result indicated a significant difference in microbial community composition between the two groups (Fig. 6B). The MCA results at the phylum level revealed that Bacteroidota was the dominant taxon across two groups. In the control group, it was followed by Proteobacteria and Fusobacteriota, whereas in the HSR group, Fusobacteriota replaced Proteobacteria to become the second most abundant taxon (Fig. 7A). At the genus level, Flavobacterium and Cetobacterium were the principal taxon in both groups. While in the HSR group, Citrobacter substituted Aeromonas to become the third most abundant genus (Fig. 7B). Additionally, LEfSe analysis revealed a significant enrichment of Cetobacterium somerae, Citrobacter freundii, and Cloacibacterium, alongside a marked reduction in the abundance of Aeromonas veronii and Neochlamydia in the HSR group compared to the control group (p < 0.05) (Fig. 7C-D).

Fig. 6.

Diversity of intestinal microbiota of Acrossocheilus fasciatus in the control and HSR groups. A α-diversity; (B) Principal component analysis of intestinal microbiota communities. Each point represents an individual sample (n = 6 samples per group). Red points indicate the control group, and bright blue points indicate the HSR group. The first three principal components (PC1, PC2, PC3) accounted for 38.95%, 30.55%, and 13.86% of the total variance, respectively. Con is the control diet. HSR (CSPS050) represents dietary CSPS (Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04) at the dose of 0.5 g·kg⁻¹ feed

Fig. 7.

Analysis of the difference in intestinal microbiota of Acrossocheilus fasciatus in the control and HSR groups. A Microbial community at the phylum level. B Microbial community at the genus level. C Linear discriminant analysis score plot of differentially abundant taxa identified by LEfSe (threshold was set to 4.0). D Comparison of the differentially abundant taxa identified by LEfSe. *represents significantly different between the control and HSR group (*p < 0.05, **p < 0.01, ***p < 0.001). Con is the control diet. HSR (CSPS050) represents dietary CSPS (Caesalpinia sappan: Schisandra chinensis: Prunus mume: Syzygium aromaticum = 2.96:3.00:1.11:1.04) at the dose of 0.5 g·kg⁻¹ feed

Discussion

Composite herbal extracts have emerged as promising antibiotic alternatives in aquaculture, owing to their multi-targeted activity, low toxicity and minimal environmental residue [40, 41]. In this study, the optimized CSPS formula showed clear in vitro antibacterial activity against S. agalactiae ZSTU01, with an inhibition zone of 29.70 mm and MIC of 31.25 mg·mL⁻¹. Although its in vitro bacteriostatic potency was lower than that of florfenicol and enrofloxacin, CSPS exerts protective effects through a dual mechanism: direct pathogen inhibition and indirect host homeostasis regulation. The in vivo challenge test confirmed that dietary supplementation with 0.5 g·kg⁻¹ CSPS significantly improved the survival rate of A. fasciatus after S. agalactiae infection, supporting its potential as a functional feed additive for streptococcosis control.

Growth performance is a core indicator for evaluating the safety and application value of feed additives [42]. In this study, no significant differences in growth parameters (WGR, SGR, HSI, CF) or feed utilization (FI, FCR) were observed between the HSR group and the control group after 28 days of feeding. This growth-neutral result indicates that 0.5 g·kg⁻¹ CSPS has no toxic side effects on A. fasciatus, and verifies the biological safety of the optimal dose. High-dose herbal supplementation often introduces anti-nutritional factors (e.g., tannins, saponins) that reduce feed palatability and impair intestinal function [3, 43], which also explains why high-dose CSPS groups did not show better disease resistance.

Hepato-intestinal health is closely associated with fish immune defense and disease resistance. Histological observations showed that CSPS supplementation significantly reduced hepatocyte vacuolization and increased intestinal villus height. Reduced hepatocyte vacuolization indicates improved hepatic lipid metabolism and alleviated cellular stress, which is consistent with the decreased AST activity in the HSR group. As a sensitive marker of hepatocellular injury, decreased AST directly reflects the hepatoprotective effect of CSPS [44, 45]. For immune-related enzymes, CSPS significantly increased ACP activity and decreased AKP activity in the liver. ACP is a key lysosomal enzyme in macrophages; its elevation indicates enhanced phagocytic activity and pathogen degradation capacity, representing improved non-specific immunity [46, 47]. The decrease in AKP activity, combined with improved hepato-intestinal morphology, reflects restored metabolic homeostasis and reduced basal inflammatory stress, rather than immune suppression. Similar AKP reductions following plant extract supplementation have been reported in A. fasciatus and Monopterus albus [48, 49], which may be a species-specific adaptive response to improved hepato-intestinal health.

Intestinal microbiota is a critical mediator linking dietary components to host health [50]. In this study, CSPS supplementation did not alter the α-diversity of gut microbiota, but significantly shifted the community composition (β-diversity). This finding supports the view that the health benefits of herbal additives depend more on functional restructuring of the microbiota rather than simple changes in diversity [51, 52]. At the taxonomic level, CSPS significantly enriched beneficial taxa represented by Cetobacterium somerae and Cloacibacterium, and reduced the abundance of the conditional pathogen Aeromonas veronii. These compositional changes form the microbial basis for improved intestinal health and enhanced disease resistance through the following mechanisms: First, C. somerae is a core anaerobic probiotic in the intestine of freshwater fish, with vitamin B₁₂ and short-chain fatty acids (SCFAs) as its main functional metabolites [53]. Vitamin B₁₂ strengthens intestinal epithelial tight junctions and enhances the stability of microbial interspecies interactions, forming a biological barrier to block the colonization and invasion of S. agalactiae [54]. Meanwhile, SCFAs produced by C. somerae activate the TLR2-type I IFN signaling axis, systematically upregulating the host’s innate immune response and improving the clearance efficiency of invading pathogens [55]. This is consistent with the increased intestinal villus height and elevated ACP activity observed in this study, jointly confirming that C. somerae enrichment enhances both intestinal barrier function and systemic immunity. Second, Cloacibacterium is a facultative anaerobic carbohydrate-fermenting bacterium that mainly produces acetic acid and butyric acid [56, 57]. Butyric acid is the primary energy source for intestinal epithelial cells, promoting epithelial cell renewal and maintaining the integrity of the intestinal mucosal barrier [58]. Acetic acid enters the systemic circulation through the intestinal wall, regulating macrophage phagocytic function and enhancing the host’s non-specific immune level [59]. The increase in hepatic ACP activity in the HSR group may be partly related to SCFAs produced by Cloacibacterium entering the liver via the portal vein, forming a “gut-liver axis” regulatory pathway. Third, the reduction of A. veronii also contributes to improved disease resistance. A. veronii is a common conditional pathogen in freshwater fish, which secretes virulence factors such as hemolysin and aerolysin to damage intestinal epithelial cells and induce intestinal inflammation [60, 61]. High abundance of A. veronii keeps the intestine in a low-grade inflammatory state, consuming host immune resources and reducing defense capacity against exogenous pathogens. The direct in vitro antibacterial activity of CSPS against aquatic pathogens may inhibit the colonization of A. veronii in the intestine. The reduction of A. veronii not only reduces the risk of secondary infection, but also alleviates basal intestinal inflammation, allowing the host to mobilize more immune resources to resist S. agalactiae infection. Although direct comparisons with other herbal combinations are limited by differences in experimental conditions, the protective effect of CSPS (70.0% survival) is comparable to or better than several recently reported plant‑based additives in fish [40, 41].

Several limitations of this study should be acknowledged. First, the biological replicate size for microbiome analysis and histological detection is relatively limited, which may affect the statistical robustness of partial results. Future studies should expand the sample size to improve the reliability and generalizability of the findings. Second, this study only adopted water extraction to prepare herbal extracts, which mainly enriches polar active components such as polysaccharides and polyphenols, while fat-soluble components such as volatile oils and terpenoids may be lost. Subsequent studies can compare the efficacy of extracts prepared by different processes to identify the core active substances of CSPS. Third, the feeding trial lasted only 28 days, which is a short-term intervention. The long-term effects of CSPS on growth performance, nutrient metabolism and sustained disease resistance of A. fasciatus remain to be verified, and long-term breeding experiments under actual aquaculture conditions are needed to evaluate its application value. In addition, this study only revealed the correlation between gut microbiota changes and improved disease resistance. The causal relationship between specific bacterial taxa and S. agalactiae resistance, as well as the exact role of microbial metabolites, needs to be further verified by germ-free fish colonization assays and targeted metabolomics.

Conclusion

This study demonstrated that dietary supplementation with CSPS effectively enhances resistance of A. fasciatus against S. agalactiae infection. The optimal dose of 0.5 g·kg⁻¹ feed significantly improved survival without compromising growth performance or feed utilization. CSPS promoted hepato-intestinal health by reducing hepatocellular stress, as evidenced by decreased vacuolation and lower AST and AKP activities, while simultaneously boosting non-specific immunity through elevated ACP activity. Moreover, CSPS modulated the gut microbiota composition, enriching beneficial taxa such as C. somerae, and Cloacibacterium, and suppressing potential pathogens like Aeromonas veronii. Nevertheless, the present findings only furnish preliminary experimental evidence for the potential of CSPS as an alternative to antibiotics, without demonstrating comparable efficacy to clinical antibiotics. Further studies are warranted to validate the actual application value of CSPS as an antibiotic alternative in aquaculture.

Authors’ contributions

Leyao Qian: Conducting experiments; Daozhi Yang: Writing - Original draft and data curation; Yilian Yin: Conducting experiments; Mengmeng Huang: Writing-Review and Editing; Shun Yang: Writing-Review and Editing; Hui Fei: Conceptualizatio and Supervision of this paper, Writing-Review and Editing.

Funding

This study was funded by Zhejiang Province Leading Geese Plan (No. 2024C02012).

Data availability

All 16s amplicon sequencing data has also been deposited in NCBI under the accession number PRJNA1381092. All other datasets generated for this study are included in the article. The data that support the findings of this study and not presented in the figures, tables and supplementary files are available upon reasonable request.

Declarations

Ethics approval and consent to participate

All experimental procedures involving Acrossocheilus fasciatus were strictly approved by the Ethics Committee for Animal Care and Experimentation of Zhejiang Sci-Tech University (Approval No. 20250804-01). The study was designed and conducted to minimize animal suffering, reduce the number of experimental animals used, and optimize experimental protocols in line with the 3R principles (Replacement, Reduction, Refinement) for animal experimentation.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.