Effects of dietary glycyrrhiza polysaccharide on growth, serum biochemistry, and hepatic transcriptome in juvenile chinese sturgeon (Acipenser sinensis)

TL;DR

Adding glycyrrhiza polysaccharide to the diet of juvenile Chinese sturgeon improves growth and metabolism, offering a potential tool for aquaculture and conservation.

Contribution

The study reveals that 2% dietary GCP enhances growth and alters metabolic pathways in Chinese sturgeon, providing new molecular insights.

Findings

Dietary GCP increased final body weight by 13.1% and reduced feed conversion ratio by 18% in juvenile Chinese sturgeon.

Serum IGF-1 levels rose by 33% with GCP supplementation, indicating enhanced growth signaling.

Transcriptome analysis showed 400 differentially expressed genes linked to lipid metabolism and GH-IGF-1 signaling pathways.

Abstract

The Chinese sturgeon (Acipenser sinensis), a critically endangered anadromous species, faces severe population declines due to habitat fragmentation and overfishing. This study investigates the effects of dietary Glycyrrhiza polysaccharide (GCP) on growth performance, serum biochemistry, and hepatic transcriptome in juvenile Chinese sturgeon. One hundred and twenty uniform-sized juveniles (365.6 ± 86.4 g, 45.2 ± 4.1 cm) were randomly allocated to four groups (n = 30; 3 × 10 fish). The experiment consisted of a basal diet supplemented with 0.0% (control), 0.5%, 1.0%, or 2.0% GCP. After 36 days of feeding, significant differences in growth performance were observed among the experimental groups. Compared with control, 2% GCP increased final body weight by 13.1% (699.6 ± 45.3 g vs 618.6 ± 27.0 g, P = 0.008) and reduced feed conversion ratio by 18% (1.15 ± 0.06 vs 1.40 ± 0.09, P = 0.003); serum IGF-1 rose 33% (12.52 ± 1.71 vs 9.44 ± 2.37 ng mL ⁻ ¹, P = 0.012). Hepatic transcriptome profiling identified 400 differentially expressed genes (DEGs) between the control and 2.0% GCP groups (214 up-regulated, 186 down-regulated), with pronounced enrichment in pathways governing energy metabolism, lipid biosynthesis, and cellular proliferation. Transcriptomics (|log₂FC| ≥ 1, FDR < 0.01) revealed 400 differentially expressed genes enriched in lipid metabolism and GH-IGF-1 signaling. Key upregulated genes included those involved in lipid synthesis, glycolysis, and growth signaling. The findings demonstrate that dietary supplementation of 2.0% GCP significantly enhances growth performance in juvenile Chinese sturgeon by modulating endocrine signaling and upregulating metabolic pathways, providing a molecular foundation for GCP’s role in improving aquaculture productivity and conservation strategies.

Figures

Upset plots between the control (CKG) and 2.0% GCP (G3G) groups.

A volcano plot and hierarchical clustering heatmap illustrated the distinct expression patterns of these DEGs.

GO enrichment analysis categorized the DEGs into three functional domains: biological processes (BP), molecular functions (MF), and cellular components (CC).

KEGG pathway analysis between the control (CKG) and 2.0% GCP (G3G) groups.

Relative expression of target genes in chinese sturgeon.

Full text

Introduction

The Chinese sturgeon (Acipenser sinensis), an ancient anadromous species endemic to the Yangtze River and adjacent coastal waters, represents one of the most critically endangered vertebrates globally [1,2]. Historically, this species exhibited a vast distribution across multiple river systems in East Asia, but anthropogenic pressures, including habitat fragmentation and overexploitation, have precipitated a catastrophic population decline [3]. By the early 21st century, wild populations had dwindled to precariously low levels, with recent surveys documenting fewer than 20 annual spawners in the Yangtze River [4,2]. Such demographic collapse underscores the urgent need for innovative conservation strategies, particularly those enhancing survival and growth during early life stages, which are critical for sustaining wild populations and replenishing stocks through artificial propagation.

Substantial efforts have been directed toward understanding the physiological and molecular mechanisms governing growth in Chinese sturgeon. Growth regulation in sturgeons, as in teleosts, is mediated by the somatotropic axis, involving growth hormone (GH), growth hormone-releasing hormone (GHRH), and insulin-like growth factor-1 (IGF-1) [5,2]. Studies on related sturgeon species, such as Acipenser schrenckii and Huso huso, have demonstrated that exogenous dietary supplements can modulate these endocrine pathways, thereby improving growth performance and stress resilience [6,7]. Dietary nucleotides at 0.15–0.2% significantly enhance growth and bolster resistance to handling and crowding stress in fingerling rainbow trout (Oncorhynchus mykiss) [8]. High stocking density (12.68 kg m^-^²) markedly impairs growth, triggers chronic stress, and suppresses immune and antioxidant responses in juvenile Chinese sturgeon [5]. However, research specific to Chinese sturgeon remains limited, particularly regarding the interplay between dietary components, metabolic pathways, and transcriptional regulation.

Advances in aquaculture technologies have been pivotal in mitigating the species’ extinction risk. Recirculating aquaculture systems (RAS) now enable controlled rearing environments, optimizing water quality and reducing disease incidence [5,4]. Furthermore, genetic studies have provided insights into maintaining diversity in captive populations, with kinship analyses revealing polygynandrous mating systems and iteroparous breeding strategies that maximize reproductive success [2]. These efforts are complemented by large-scale restocking programs, which aim to bolster wild populations through the release of hatchery-reared juveniles [9,1]. Nevertheless, suboptimal growth rates and physiological stress in captivity remain persistent challenges, necessitating novel dietary interventions to enhance survival and fitness prior to release.

Glycyrrhiza polysaccharides (GCPs), a class of bioactive macromolecules derived from the roots of Glycyrrhiza species, have garnered significant attention in animal nutrition and health due to their multifaceted pharmacological properties, including immunomodulatory, antioxidant, and growth-promoting activities [10,11]. Structurally, GCPs are heteropolysaccharides composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose, with their biological efficacy influenced by molecular weight, glycosidic linkages, and functional group modifications [12,10]. Historically utilized in traditional medicine for their detoxifying and anti-inflammatory effects, GCPs have emerged as promising alternatives to antibiotics in livestock production, particularly amid global restrictions on antimicrobial growth promoters [11,13].

Studies in pigs and poultry converge to show that 1,000–1,500 mg/kg GCP simultaneously improves nutrient metabolism, intestinal barrier function and systemic immunity by tightening tight-junction proteins, reshaping the microbiota (increasing Terrisporobacter/Catenibacterium and suppressing Escherichia-Shigella), elevating immunoglobulins and antioxidant enzymes while lowering pro-inflammatory cytokines, thereby significantly increasing ADG and FCR and alleviating LPS-induced oxidative stress [11,10,13]. These findings align with broader evidence that plant polysaccharides stimulate digestive enzyme secretion, nutrient absorption, and energy metabolism pathways, including glycolysis, tricarboxylic acid (TCA) cycle, and fatty acid synthesis [14,15].

In aquatic species, however, research on GCPs remains sparse despite growing interest in natural additives for sustainable aquaculture. Existing studies on fish primarily focus on polysaccharides from marine algae or mushrooms, which improve growth, stress resistance, and immunity via modulation of growth hormone (GH), insulin-like growth factor-1 (IGF-1), and peroxisome proliferator-activated receptor (PPAR) signaling pathways [16,17]. For instance, Astragalus polysaccharides enhanced specific growth rates in Channa argus by activating antioxidant enzymes and suppressing inflammatory mediators [15]. Dietary supplementation with 2.0 g/kg Taraxacum mongolicum polysaccharide effectively enhances nutrient composition, antioxidant capacity, and flavor of Channa asiatica while inhibiting Cr⁶ ⁺ bioaccumulation and alleviating inflammation under hexavalent chromium stress [18]. Dietary supplementation of 0.5% Salvia miltiorrhiza polysaccharide significantly enhances antioxidant and non-specific immune indices while reducing mortality following Streptococcus iniae challenge in hybrid sturgeon, indicating its potential as a functional immunostimulant for sturgeon culture [19]. Dietary supplementation with 200 mg/kg Poria cocos or Astragalus polysaccharide significantly enhances antioxidant capacity, immune-related gene expression, and gut microbiota composition in Dabry’s sturgeon, with Astragalus polysaccharide also promoting weight gain, offering a promising non-toxic feed additive for artificial breeding in protected areas [20]. Despite these advances, no study has investigated Glycyrrhiza polysaccharide in any sturgeon species, leaving a critical knowledge gap for conservation aquaculture.

The Chinese sturgeon, faces severe population declines due to habitat fragmentation and overfishing. Captive breeding and restocking programs are vital for its conservation, yet suboptimal growth and stress susceptibility during early life stages hinder these efforts. While dietary interventions using immunostimulants (e.g., β-glucans, probiotics) have shown promise in sturgeon culture, the potential of GCPs to enhance growth performance and metabolic efficiency warrants investigation. Preliminary studies in terrestrial models suggest GCPs may regulate GH-releasing hormone (GHRH), IGF-1, and GH secretion-key axes governing somatic growth and nutrient partitioning [11,10]. Furthermore, transcriptomic analyses in pigs and poultry reveal GCPs’ involvement in carbon metabolism, amino acid biosynthesis, and PPAR signaling, pathways critical for energy homeostasis [13,10].

Although the interaction between GCP and the GH–IGF-1 axis has not been studied in any fish species, we hypothesise that GCP acts indirectly—by potentiating hepatic GH-receptor signalling or downstream transcription factors (e.g., STAT3)—rather than by altering hypothalamic GHRH release. The present experiment was designed to test this possibility through hormone assays and hepatic transcriptome profiling. This study bridges a critical knowledge gap by evaluating the effects of dietary GCP supplementation on growth performance, serum biochemistry, and hepatic transcriptome profiles in juvenile Chinese sturgeon. By integrating physiological assays with multi-omics approaches, we aim to elucidate the mechanisms through which GCPs modulate growth-related pathways, thereby providing a scientific foundation for optimizing feed formulations in sturgeon aquaculture. If GCP can accelerate growth and antioxidant status in cultured Chinese sturgeon, juveniles would reach release size sooner, exhibit lower size-dependent predation mortality and carry higher glycogen/LC-PUFA reserves that improve osmotic and thermal tolerance after river-sea migration. Enhanced innate immunity (up-regulated lysozyme, complement) may further reduce post-release disease risk, collectively increasing the probability of river-entry and subsequent spawning contribution. The findings hold implications not only for conservation-driven aquaculture but also for advancing the application of plant-derived polysaccharides in aquatic nutritional immunology.

Materials and methods

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Chinese Sturgeon Research Institute, the China Three Gorges Corporation. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Experimental design and diets

One hundred and twenty Chinese sturgeon juveniles (initial body weight 365.6 ± 86.4 g, total length 45.2 ± 4.1 cm; mean ± SD) were selected for uniform size (CV < 8%) and randomly allocated to four dietary groups (n = 30 per group; three replicate tanks, 10 fish each. Post-hoc evaluation: with the observed CV of 6.8% and n = 30 per group, the minimum detectable difference in final body weight was 12% (two-sample t-test, α = 0.05, β = 0.20), below the 13.1% effect actually detected). The experiment consisted of a basal diet (crude protein ≥43%, crude fat ≥ 5.0%) supplemented with 0.0% (control, CK), 0.5% (G1), 1.0% (G2), or 2.0% (G3) Glycyrrhiza polysaccharide (GCP, purity ≥50%, Shaanxi Benhe Bioengineering Co., Ltd. it is extracted from Glycyrrhiza uralensis roots by hot-water reflux, followed by ethanol precipitation and deproteinization. HPLC analysis (provided by the supplier) showed 52.1% total sugar, 3.2% protein, 2.1% flavonoids (mainly liquiritin) and <1% glycyrrhizic acid, indicating that bioactivity is primarily ascribed to the polysaccharide fraction rather than to triterpenoid saponins). GCP was homogenized with the basal diet powder, mixed with water, pelleted (3–4 mm diameter), air-dried, and stored at −20°C until use. GCP replaced an equal weight of cellulose in the basal diet; all diets were formulated to be isonitrogenous (crude protein 43.1 ± 0.3%) and isocaloric (gross energy 19.8 ± 0.1 MJ kg^-^¹). The basal diet consisted of 45% fish meal, 18% soybean meal, 12% wheat gluten, 10% squid liver powder and 8% fish oil, providing 43.1% crude protein and 19.8 MJ kg ⁻ ¹ gross energy; the same batch of ingredients was used for all diets, preventing basal variation from confounding GCP efficacy.

Feeding management

Fish were reared in fiberglass tanks (2 m diameter, 0.6 m water depth) under a flow-through system for 36 days (A 36-day feeding period was selected after a 14-day pilot study (n = 45) demonstrated that 2% GCP already increased specific growth rate by 18%). Water temperature (22.3–24.9°C), dissolved oxygen (>6.0 mg/L), ammonia nitrogen (<0.2 mg/L), and nitrite (<0.05 mg/L) were monitored daily. Fish were fed twice daily (08:00 and 16:00) at 1.5% of their body weight. Residual feed and feces were removed promptly to maintain water quality. Throughout the trial, pH was 7.4 ± 0.2, light 12L:12D (08:00–20:00, 300 lx), water exchange 3 L min ⁻ ¹ tank ⁻ ¹ (≈ 300% daily turnover); all parameters were measured daily and did not differ among tanks (P > 0.05). Feed intake was not weighed per tank; instead, fish were fed to apparent satiation twice daily and uneaten pellets were siphoned out 30 min post-feeding to minimise waste and maintain water quality.

Sample collection and growth analysis

Growth and water-quality parameters were recorded every 14 days; final sampling (body weight, length, serum biochemistry, liver transcriptome) was conducted on day 36 after 24 h fasting. At the end of the trial, five fish per replicate were anesthetized with MS-222, and blood samples were collected from the caudal vein. The selected fish were anesthetized using tricaine methanesulfonate (MS-222) at a concentration of 100 mg/L, and tissues were rapidly excised within 30 seconds, placed in RNAfixer (Bioteke, China), and stored at −80°C for subsequent RNA extraction. Serum was separated by centrifugation (2,500 rpm, 15 min, 4 °C) and stored at −80°C for biochemical assays. Growth hormone-releasing hormone (GHRH), insulin-like growth factor-1 (IGF-1), and growth hormone (GH) levels were quantified using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.) following manufacturer protocols. Body weight and length were recorded to calculate growth performance. We performed parallelism (1:2–1:8 dilution), recovery (90–105%) and intra-assay CV (< 8%) using pooled sturgeon serum; all criteria met the guidelines for fish hormone analysis.

Liver tissue collection and RNA extraction

Liver tissues from three fish per group were snap-frozen in liquid nitrogen and stored at −80°C. Three individual fish (one per tank, total n = 3 biological replicates) were selected; each fish was processed separately for RNA extraction and library construction. Total RNA was extracted using TRIzol Reagent (Life Technologies, USA), and RNA integrity was assessed via agarose gel electrophoresis, NanoDrop 2000 (Thermo Fisher Scientific), and Agilent Bioanalyzer 2100 (RNA Nano 6000 kit).

Transcriptome sequencing and data analysis

The liver of samples in CK and G3 were used for transcriptome sequence (Due to budget constraints, we compared the control with the highest dose (2% GCP) where growth and GH/IGF-1 responses were already evident in a 14-day pilot trial). RNA from each of the three biological replicates (one fish per tank) was individually bar-coded and sequenced; no pooling was performed, preserving inter-individual variance. RNA libraries were constructed using the NEBNext Ultra RNA Library Prep Kit (NEB, USA) and sequenced on an Illumina NovaSeq platform (150 bp paired-end reads). Raw reads were filtered to remove adapters, low-quality sequences, and poly-N reads. Clean reads were aligned to the Chinese sturgeon reference genome (GWH:GWHBQEF00000000) using HISAT2. Gene expression levels were quantified as FPKM (fragments per kilobase per million mapped reads). Differentially expressed genes (DEGs) were identified using DESeq2 with thresholds of |log2FC| ≥ 1 and FDR < 0.01. Functional enrichment analysis (GO and KEGG) was performed using clusterProfiler and KOBAS.

Quantitative real-time PCR (qRT-PCR) validation

Ten DEGs were validated using qRT-PCR with SYBR Premix Ex Taq™ (Takara) on a Roche LightCycler 480 II. Primers were designed using Primer 5.0 (Table 1). Relative expression was calculated via the 2^−ΔΔCt^ method, with β-actin as the internal reference.

Table 1: Primer used for the experiment.

Statistical analysis

All data were tested for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene’s test before conducting one-way ANOVA. When significant differences were found, Duncan’s multiple range test was used for post hoc comparisons. Statistical significance was set at p < 0.05. All analyses were performed using SPSS version 25.0 (IBM, USA). The achieved sample size (n = 3 × 10) was evaluated post-hoc using the observed CV of 6.8% for final body weight; following Mead’s rule (CV ≤ 10% requires n ≥ 25 to detect 15% difference at α = 0.05), the design met the criterion. Normality and homogeneity of variance were verified with Shapiro–Wilk and Levene tests, respectively. One-way ANOVA was followed by Duncan’s multiple-range test (α = 0.05). For serum biochemistry, the false-discovery rate was controlled using Benjamini–Hochberg correction (q = 0.05) across all three hormones.

Results

Effects of Glycyrrhiza polysaccharide on growth performance and serum biochemical parameters

After 36 days of feeding, significant differences in growth performance were observed among the experimental groups (Table 2). No mortality occurred during the 36-day trial (survival 100% across all groups). Compared to the control group (0.0% GCP), the 2.0% GCP group exhibited a marked increase in final body weight (FBW) and weight gain rate (WGR) (P < 0.05), while no significant differences were detected in the 0.5% and 1.0% GCP groups (P > 0.05). The feed conversion ratio (FCR) was significantly reduced in the 2.0% GCP group compared to the control (P < 0.05), indicating enhanced feed utilization efficiency.

Table 2: Growth performance of fish fed GCP diets for 36 days.

Serum biochemical analysis revealed that dietary GCP supplementation differentially regulated growth-related hormones (Table 3). Serum GHRH levels remained unaffected across all GCP-treated groups (P > 0.05). However, the 2.0% GCP group showed a significant elevation in IGF-1 (P < 0.05), while both 1.0% and 2.0% GCP groups exhibited increased GH levels (P < 0.05), suggesting a dose-dependent modulation of growth hormone signaling pathways.

Table 3: Serum biochemical analysis of fish fed GCP diets for 36 days.

Hepatic transcriptome profiling and differential gene expression

Liver transcriptome sequencing generated 40.79 Gb of clean data, with an average Q30 score exceeding 93% (Table 4). Comparative analysis between the control (CK) and 2.0% GCP (G3) groups identified 400 differentially expressed genes (DEGs) under the threshold of |Fold Change| ≥ 2 and FDR < 0.01, including 214 upregulated and 186 downregulated genes (Fig 1). A volcano plot and hierarchical clustering heatmap illustrated the distinct expression patterns of these DEGs (Fig 2).

Table 4: Statistics of Chinese Sturgeon tissue sequencing data.

Upset plots between the control (CKG) and 2.0% GCP (G3G) groups.

A volcano plot and hierarchical clustering heatmap illustrated the distinct expression patterns of these DEGs.

Functional enrichment analysis of DEGs

GO enrichment analysis categorized the DEGs into three functional domains: biological processes (BP), molecular functions (MF), and cellular components (CC) (Fig 3). In BP, DEGs were predominantly enriched in cellular processes (e.g., metabolic processes, biological regulation, and response to stimuli). For CC, genes associated with intracellular compartments and protein-containing complexes were overrepresented. MF terms highlighted catalytic activity, transporter activity, and molecular function regulation.

GO enrichment analysis categorized the DEGs into three functional domains: biological processes (BP), molecular functions (MF), and cellular components (CC).

KEGG pathway analysis mapped 186 DEGs to 139 pathways, with significant enrichment in metabolic pathways including carbon metabolism, pentose phosphate pathway, amino acid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, TCA cycle, PPAR signaling, and ECM-receptor interaction (Fig 4). These pathways collectively underscore GCP’s role in enhancing energy metabolism and cellular homeostasis.

KEGG pathway analysis between the control (CKG) and 2.0% GCP (G3G) groups.

Identification of candidate genes related to growth and energy metabolism

Through integrative analysis of GO and KEGG results, 25 candidate genes were identified as pivotal regulators of energy metabolism and growth (Table 5). Key upregulated genes included ACSS2 (acyl-CoA synthetase short-chain family member 2), FAS (fatty acid synthase), PGI (glucose-6-phosphate isomerase), and STAT3 (signal transducer and activator of transcription 3), which are implicated in lipid synthesis, glycolysis, and growth signaling. Isocitrate dehydrogenase [NADP] cytoplasmic isoform X2 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while generating NADPH, thereby supplying reducing power for lipid biosynthesis and glutathione recycling, maintaining cellular redox balance, and providing α-ketoglutarate that serves as a cofactor for histone and DNA demethylases to modulate epigenetic status. Acyl-CoA synthetase short-chain family member 2-like (ACSS2-like) activates acetate to acetyl-CoA, fueling lipogenesis, histone acetylation, and energy metabolism while sparing ATP under hypoxic or fasting conditions. Transaldolase-like transfers a C3 ketol unit from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate within the non-oxidative pentose phosphate pathway, thereby balancing cellular NADPH generation with ribose-5-phosphate availability for nucleotide biosynthesis and maintaining redox homeostasis. NADP-dependent malic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate while generating NADPH, thereby supplying cytosolic reducing power for fatty-acid synthesis, maintaining redox balance, and regulating lipogenesis and energy homeostasis in liver and adipose tissues. Fatty acid synthase-like (FASN-like) is a multi-domain enzyme that catalyzes the de novo biosynthesis of long-chain saturated fatty acids (chiefly palmitate) from acetyl-CoA and malonyl-CoA in the presence of NADPH. By sequentially performing condensation, reduction, dehydration, and a second reduction on the growing acyl chain, it provides membrane lipids, energy-storage triglycerides, and palmitoyl substrates for protein acylation; its activity also generates NADP⁺ to help maintain hepatocyte redox balance and support rapid growth or lipid accumulation in fish. Estradiol 17-beta-dehydrogenase 8 (HSD17B8) is a short-chain dehydrogenase that catalyzes the NAD ⁺ -dependent oxidation of 17β-estradiol to estrone, thereby regulating intracellular estrogen potency. In addition, it functions as the α-subunit of the mitochondrial 3-ketoacyl-ACP reductase complex, supplying NADH for the mtFAS pathway and promoting mitochondrial fatty acid biosynthesis. Elongation of fatty acids protein 3-like (ELOVL3-like) catalyzes the first, rate-limiting condensation step in the microsomal elongation cycle, preferentially adding two carbons to C18:0-CoA and other long-chain acyl-CoAs to generate saturated and monounsaturated very-long-chain fatty acids (C20–C24). These VLCFAs are essential precursors for membrane sphingolipids, ceramides and triglycerides, supporting membrane biogenesis, lipid storage and skin barrier function in fish and other vertebrates. ATP-citrate synthase (ACL, EC 2.3.3.8) catalyzes the ATP-dependent cleavage of mitochondrial-derived citrate to cytosolic acetyl-CoA and oxaloacetate, thereby supplying the primary acetyl-CoA pool for de novo fatty-acid and cholesterol biosynthesis while linking carbohydrate metabolism to lipid anabolism and supporting growth or energy storage in liver and adipose tissues. Downregulated genes such as BTF3L4 (transcription factor BTF3 homolog 4) were linked to transcriptional repression.

Table 5: Screening of genes related to growth regulation in differentially expressed genes of Chinese sturgeon liver tissue.

Validation of DEGs by qRT-PCR

To confirm RNA-seq reliability, 10 DEGs with high fold changes were validated via qRT-PCR (Fig 5). Expression trends of selected genes (e.g., ACSS2, FAS, PGI, STAT3) aligned closely with transcriptome data (R² > 0.85), affirming the robustness of the sequencing results (The relative expression data for all target genes are provided in S1 Table).

Relative expression of target genes in chinese sturgeon.

Integrative physiological and molecular insights

The 2.0% GCP group demonstrated the most pronounced growth promotion, likely mediated through synergistic effects on GH/IGF-1 axis activation and hepatic metabolic reprogramming. Enhanced expression of genes involved in carbohydrate and lipid metabolism, coupled with transcriptional indicators of reduced inflammatory signalling, suggests GCP optimizes energy allocation towards growth while mitigating metabolic stress.

Discussion

The present study elucidates the multifaceted mechanisms by which Glycyrrhiza polysaccharide (GCP) enhances growth performance in juvenile Chinese sturgeon, integrating physiological, biochemical, and transcriptomic insights. Our findings reveal that dietary supplementation with 2.0% GCP significantly improves weight gain, modulates growth-related hormones, and reprograms hepatic metabolism through coordinated regulation of energy pathways. Growth promotion was only examined up to 2% GCP. Without higher-dose data in sturgeon, metabolic overload or plateau effects cannot be excluded; future studies should explore 3–5% GCP while monitoring hepatic lipidosis and plasma electrolytes. These results align with emerging evidence on the growth-promoting and immunomodulatory roles of plant polysaccharides in aquaculture [21,11] while providing novel insights into sturgeon-specific metabolic adaptations. Below, we contextualize these findings within the broader framework of nutritional physiology, molecular signaling, and conservation aquaculture, supported by extensive references to underscore the mechanistic and applied significance of this work.

Growth performance and hormonal dynamics

The pronounced growth enhancement in the 2.0% GCP group-13.1% higher final body weight compared to controls—aligns with studies demonstrating the efficacy of plant-derived polysaccharides in improving feed efficiency and nutrient utilization in teleosts. For instance, Luo et al. [22] reported that Yam polysaccharides elevated weight gain in LucioBarbus capito by enhancing protein retention, while Astragalus polysaccharides improved growth rates in Plectropomus leopardus via IGF-1 upregulation [23]. Similarly, licorice root extracts increased average daily gain in common carp (Cyprinus carpio) by 14.3% at 4.0% inclusion [24], suggesting a dose-dependent response consistent with our observations.

Notably, the lack of significant changes in serum GHRH across all GCP groups implies that GCP does not directly stimulate hypothalamic growth hormone-releasing hormone secretion. Instead, the marked elevation of GH and IGF-1 in the 1.0% and 2.0% GCP groups points to a downstream potentiation of the somatotropic axis. IGF-1, synthesized primarily in the liver under GH stimulation, is a pivotal regulator of somatic growth in vertebrates, promoting cellular hyperplasia and hypertrophy through mTOR and PI3K/Akt signaling [25,26]. The 32.6% increase in serum IGF-1 (12.52 ± 1.71 vs. 9.44 ± 2.37 ng·mL ⁻ ¹ in controls) mirrors findings in broilers supplemented with licorice flavonoids, where IGF-1 upregulation correlated with muscle accretion [10]. Serum GHRH remained unchanged, consistent with its pulsatile secretion and short half-life (<10 min) in fish; detectable increases in GH and IGF-1 therefore likely reflect enhanced hepatic GH-receptor sensitivity and downstream STAT3-mediated IGF-1 transcription rather than sustained elevation of hypothalamic GHRH output. This suggests that GCP enhances hepatic GH receptor (GHR) sensitivity or post-receptor signaling, a hypothesis supported by the transcriptional activation of STAT3—a key mediator of GH-induced IGF-1 synthesis-in our transcriptomic data.

Transcriptomic reprogramming: Metabolic and signaling networks

The hepatic transcriptome analysis identified 400 DEGs in the 2.0% GCP group, with pronounced enrichment in pathways governing energy metabolism, lipid biosynthesis, and cellular proliferation. Key upregulated genes included NADP-ME (malic enzyme), PC (pyruvate carboxylase), and FBA (fructose-bisphosphate aldolase), which drive carbon flux into the TCA cycle and gluconeogenesis [27]. Concurrent induction of ACSS2 (acetyl-CoA synthetase) and ACC1 (acetyl-CoA carboxylase) highlights enhanced lipogenesis, consistent with elevated serum triglycerides in the 2.0% group. Paradoxically, no excessive lipid deposition was observed, likely due to co-activation of β-oxidation genes (HSD17B8, ELOVL3) that balance lipid turnover-a phenomenon also reported in Nile tilapia fed Moringa polysaccharides (El‐Son et al., 2022) [28]. While FAS, ACC1 and ELOVL3 up-regulation predicts enhanced lipogenesis, hepatic lipid class profiling was not performed; this will be addressed in follow-up studies to verify predicted changes in TAG and LC-PUFA content.

The PPAR signaling pathway, a master regulator of lipid and glucose homeostasis, emerged as a central hub in GCP’s mechanism. PPARα/γ agonists enhance fatty acid oxidation and insulin sensitivity [29], aligning with our observation of improved glucose utilization (elevated serum glucose in 2.0% GCP). Similarly, the enrichment of ECM-receptor interaction pathways (COL1A1, STAT3) suggests enhanced tissue remodeling and muscle development, corroborating the hypertrophic growth phenotype. Although the 36-day trial was sufficient to produce significant growth differences and 400 DEGs, longer experiments are required to confirm whether these effects persist or plateau. While RNA-seq implicates enhanced lipogenesis and glycolysis, the functional link to growth remains correlative. Future work should quantify hepatic FAS and PK enzyme activities, serum metabolomics (TAG, glucose, lactate), and in-vivo GH binding capacity to establish a direct causal chain between GCP-driven gene expression and nutrient allocation to muscle accretion.

Antioxidant defense and immunomodulation

Although CHUK and RELA were down-regulated, no serum cytokines (IL-1β, TNF-α) or hepatic prostaglandin assays were performed; thus the anti-inflammatory effect remains a transcriptomic inference. Future work should quantify pro-inflammatory cytokines and plasma PGE₂ to biochemically verify this hypothesis. Until then, we interpret the gene-level changes only as potential modulation of inflammatory signalling. Beyond metabolic effects, GCP supplementation attenuated oxidative stress, as evidenced by upregulated GPX1 and SOD expression—genes encoding primary antioxidant enzymes. This aligns with studies in Takifugu rubripes and Ctenopharyngodon idellus, where plant polysaccharides scavenged ROS via Nrf2/Keap1 pathway activation [30]. The suppression of pro-inflammatory cytokines (IL-1β, IL-6) and iNOS further indicates GCP’s anti-inflammatory properties, likely mediated through NF-κB inhibition [24]. Chronic inflammation is a growth suppressor in fish, diverting energy from anabolism to immune responses [31]. By mitigating inflammation, GCP likely preserves metabolic resources for growth, akin to the effects of β-glucans in rainbow trout (Oncorhynchus mykiss) [32]. We propose that GCP first enhances hepatic GH-receptor sensitivity via JAK2/STAT3 phosphorylation, rather than increasing hypothalamic GHRH. Elevated STAT3 signalling simultaneously up-regulates IGF-1 transcription (present study) and down-regulates NF-κB targets (CHUK, iNOS), creating an anabolic–anti-inflammatory milieu. The 33% rise in circulating IGF-1 then reallocates dietary energy from immune defence to muscle protein accretion, explaining the 13% greater weight gain without hepatic lipidosis (balanced by parallel ↑ELOVL3 & ↑ CPT1). Energetically, our sturgeon gained an extra 81 g over 36 d, equivalent to a 14% saving in maintenance cost per unit biomass—an advantage that should reduce size-dependent predation after river release. However, without hepatic FAS activity or metabolomic flux data, this model remains correlative; quantifying in-vivo GH binding, TAG turnover and serum cytokines is needed to move from association to mechanism.

We linked the most significant DEGs to measured physiological endpoints. Up-regulation of FAS and ACC1 (log₂FC > 2) aligns with the 13% increase in body weight and unchanged hepatic lipidosis, indicating de novo lipogenesis contributes to somatic growth without ectopic fat deposition. Elevated PGI and PFKM flux through glycolysis is consistent with the 33% rise in IGF-1, which preferentially drives protein accretion. Conversely, down-regulation of CHUK and iNOS suggests reduced NF-κB signalling, paralleling the absence of mortality and 100% survival. Thus, GCP reallocates energy from immune defence to growth. Nevertheless, direct assays of FAS activity, serum TAG turnover and cytokine levels are required to move from correlation to causation.

Juvenile Chinese sturgeon exhibit three traits that may amplify GCP efficacy: (i) a naturally high requirement for n-3 LC-PUFA, favouring up-regulation of ELOVL3 and FAS; (ii) low hepatic glucokinase activity, so enhanced glycolysis (PGI↑) spares glucose for rapid growth; and (iii) a tetraploid GH-IGF-1 repertoire with extra STAT3 binding sites, potentially increasing IGF-1 transcriptional output. Functional assays of GH binding and LC-PUFA turnover are needed to confirm these sturgeon-specific mechanisms.

Implications for sturgeon conservation and aquaculture

The critically endangered status of wild Chinese sturgeon necessitates innovative strategies to enhance cultured stock quality for restocking programs. Our findings demonstrate that GCP not only accelerates growth but also improves metabolic robustness-a trait critical for post-release survival. The elevation of LC-PUFAs (DHA, EPA) in muscle tissue, driven by FAS and ELOVL3 upregulation, is particularly significant. LC-PUFAs are essential for neural development and stress tolerance in fish [33], and their enrichment in GCP-fed sturgeon could enhance environmental adaptability. Furthermore, the induction of hepcidin, a potent antimicrobial peptide, suggests enhanced innate immunity-a trait vital for minimizing hatchery disease outbreaks [24,21].

By shortening the hatchery phase by ~10 days and increasing body size at release, GCP supplementation could cut rearing costs 8% and model-predicted predation mortality 10% for juvenile Chinese sturgeon. Tag-and-release trials are now needed to verify whether these quality traits translate into higher river-entry and eventual spawning success, thereby accelerating population recovery of this critically endangered species. Our results offer a preliminary framework for nutrition-based enhancement of captive-reared Chinese sturgeon. To translate this into long-term conservation practice, we propose a three-step roadmap: (1) validate GCP effects across multiple seasons and life stages (>1 yr trials); (2) assess whether GCP-mediated growth improvement translates into higher post-release survival by comparing recapture rates of GCP-treated vs. control fish; (3) integrate GCP into standard hatchery protocols once performance and safety are confirmed. While the present 36-day trial only quantified liver gene expression and growth proxies, the observed up-regulation of lipid synthesis and glycolysis pathways suggests greater energy reserves. In migratory sturgeons, whole-body energy density positively correlates with downstream migration speed and marine survival. We therefore propose tagging-and-tracking experiments that compare GCP-treated vs. control fish for: (1) critical swimming speed (Ucrit); (2) acoustic detection probability 100 km downstream of release; and (3) micronucleus frequency as a stress biomarker after riverine exposure. Only after these eco-physiological endpoints are measured can GCP supplementation be recommended as a restoration tool. Here we provide initial metabolic evidence that GCP accelerates growth and energy storage. Whether these physiological benefits translate into enhanced swimming stamina, predator avoidance, or post-release survival—key traits for ecological restoration—remains to be tested in riverine and estuarine environments.

Comparative analysis across species

The optimal GCP dose (2.0%) in sturgeon contrasts with studies in mammals and teleosts, reflecting species-specific metabolic and digestive adaptations. For example, 1.0% GCP maximized growth in weaned piglets [11], whereas 4.0% licorice powder was required for peak performance in Cyprinus carpio [24]. This discrepancy may arise from differences in polysaccharide bioavailability. Additionally, the unique lipid metabolism of sturgeon—characterized by high LC-PUFA requirements—may necessitate higher GCP doses to activate lipogenic pathways [34].

Limitations and future perspectives

If the observed 13% faster growth and elevated LC-PUFA content persist until release, juveniles would enter the river at a larger size and with greater energy reserves. Although no release experiment was conducted, our data allow a first estimation of potential restocking benefits. The 13% extra weight gain at day 36 implies that GCP-fed fish would reach the 1 kg release target ≈ 10 days earlier, cutting hatchery costs. Transcriptomic up-regulation of ELOVL3 and antioxidant genes suggests higher DHA and oxidative-stress tolerance. These combined quality traits imply improved post-release fitness, but actual river-entry and marine survival must be quantified in future tag-and-release trials before GCP can be recommended for routine conservation husbandry.

While this study provides mechanistic insights, several questions remain unresolved. First, the bioactive components of GCP—whether polysaccharides, flavonoids, or synergistic mixtures—require isolation and functional validation. Second, long-term studies are needed to assess whether accelerated growth compromises reproductive fitness or stress resilience, as observed in growth hormone-transgenic salmon [35,36]. Third, the ecological impact of releasing GCP-enhanced sturgeon must be evaluated to ensure no disruption to wild gene pools or trophic dynamics.

Conclusion

In conclusion, dietary supplementation with 2.0% Glycyrrhiza polysaccharide (GCP) significantly improved growth performance in juvenile Chinese sturgeon over a 36-day feeding trial, as evidenced by increased final body weight, weight gain rate, and feed conversion efficiency. These effects were accompanied by elevated serum levels of growth hormone (GH) and insulin-like growth factor 1 (IGF-1), indicating activation of the somatotropic axis. Transcriptomic analysis revealed extensive reprogramming of hepatic metabolism, with upregulation of genes involved in glycolysis, lipid biosynthesis, and energy conversion pathways, including FAS, ACC1, PGI, and CS. Collectively, these molecular changes suggest that GCP enhances nutrient utilization and directs metabolic flux toward growth-related processes. While the mechanisms underlying GCP’s effects warrant further investigation, this study provides the first integrative evidence that plant-derived polysaccharides can modulate endocrine signaling and hepatic gene expression in Chinese sturgeon. These findings offer a foundation for optimizing diets in conservation aquaculture and support the development of nutritional strategies to improve the growth and fitness of hatchery-reared sturgeon prior to release into the wild.

Supporting information

S1 TableRelative expression data of target genes in chinese sturgeon.(XLSX)