Effects of Different Stocking Densities on Growth Performance, Stress Resistance, Antioxidant Capacity and Immunity of Grass Carp

TL;DR

This study shows that medium stocking density improves grass carp growth and health, while high density causes stress and weakens immunity.

Contribution

The study identifies 1.13 kg/m³ as the optimal stocking density for grass carp in intensive farming.

Findings

Medium stocking density improved growth performance and physiological health in grass carp.

High stocking density increased stress markers and reduced immune and antioxidant capacities.

Optimal stocking density of 1.13 kg/m³ supports healthy aquaculture practices.

Abstract

This study investigates the effects of different stocking densities on grass carp, aiming to clarify how high-density farming impacts the fish’s growth, stress resistance, antioxidant capacity, and immunity, while also identifying an optimal stocking density for intensive farming regimes. Uniformly sized grass carp were selected and allocated to three groups with distinct stocking densities. After a 75-day pond cage culture period, results indicated that grass carp in the medium-density group exhibited the best growth performance. Conversely, high stocking density increased stress-related substances and reduced immune and antioxidant capacities. This study elucidates the comprehensive impacts of stocking density on grass carp, providing theoretical support for intensive, high-yield grass carp farming. The findings facilitate scientific stocking density planning, enhance grass carp yield and quality, promote the healthy development of aquaculture, and ensure a stable aquatic food supply.

With the rapid development of intensive aquaculture, unreasonable stocking density has become a major factor restricting the healthy growth of grass carp (Ctenopharyngodon idella). This study aimed to evaluate the effects of three stocking densities (0.57, 1.13, and 2.27 kg/m3) on the growth performance, stress response, antioxidant capacity, and immunity of grass carp. Grass carp with an initial body weight of 81.76 ± 17.69 g were randomly assigned to three groups with three replicates. After 75 days of cultivation, we randomly sampled and measured their growth performance. Reagent kits were used to detect serum biochemical indicators, kidney immune enzyme activity, and liver antioxidant indicators in each treatment group. The expression of spleen immune-related genes was detected using real-time quantitative PCR (RT-qPCR). Results showed that the final body weight, weight gain rate, specific growth rate, and condition factor were significantly higher in the medium-stocking-density group (p < 0.05). High stocking density significantly increased serum cortisol, glucose, transaminases, creatinine, and urea nitrogen, and decreased cholesterol and triglyceride levels (p < 0.05). For immune parameters, the activities of immunoglobulin M (IgM), lysozyme (LZM), antimicrobial peptide (AMP), alkaline phosphatase (AKP), and acid phosphatase (ACP) in the kidneys decreased with increasing density. The mRNA levels of IL-1β, IL-6, TNF-α, and IL-10 in the spleen were significantly upregulated, while IgM was downregulated in the high-density group (p < 0.05). Regarding antioxidant capacity, hepatic total antioxidant capacity (T-AOC), catalase (CAT), and glutathione (GSH) levels increased initially and then decreased with increasing density, while malondialdehyde (MDA) content increased continuously. Collectively, these findings suggest that high stocking density induces growth inhibition, oxidative stress, and immune dysfunction in grass carp. The medium density of 1.13 kg/m3 was found to be optimal for the growth and physiological health of grass carp in this study, providing a scientific basis for the optimization of intensive farming strategies.

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1. Introduction

Grass carp (Ctenopharyngodon idella) represents one of the most economically important freshwater finfish species supporting global aquaculture production [1]. Owing to its superior growth performance, strong environmental adaptability, and high feed efficiency, this species has become a pillar of freshwater aquaculture in China and many other regions [2]. With the global expansion of intensive aquaculture systems, increasing stocking density has been widely implemented as a key strategy to maximize production output and economic returns [3]. However, intensive aquaculture inevitably leads to chronic crowding stress, which disrupts physiological homeostasis, suppresses immune competence, and ultimately compromises animal welfare and the sustainability of aquaculture operations [4].

Stocking density is one of the most critical environmental factors affecting physiological status, growth performance, and welfare in cultured fish [5]. In intensive aquaculture systems, excessive stocking density often leads to chronic crowding stress, which induces a wide range of adverse physiological and metabolic responses. These responses include significant inhibition of growth performance, as well as abnormal secretion and dysregulation of stress-related indicators such as cortisol and glucose, which are widely used to evaluate the degree of stress and physiological disturbance in fish [5]. Previous studies in various teleost species have clearly demonstrated that overly high stocking density intensifies intraspecific competition for food, living space, and dissolved oxygen, which in turn reduces feed intake, impairs feed utilization efficiency, and ultimately decreases specific growth rate and body weight gain [6]. Furthermore, persistent crowding stress activates the hypothalamic–pituitary–interrenal axis, leading to a sustained increase in circulating cortisol levels [7]. Elevated cortisol not only reflects the activation of stress pathways but also initiates downstream metabolic and oxidative disturbances, thereby affecting multiple physiological processes closely related to growth and health [8,9]. Therefore, investigating the effects of crowding stress induced by different stocking densities is essential for understanding the physiological adaptation mechanisms of fish and optimizing culture management practices.

The antioxidant and immune systems serve as the primary defensive barriers maintaining physiological stability and disease resistance under environmental stressors [10]. High stocking density typically suppresses the activities of key antioxidant enzymes, including CAT and GSH, increasing malondialdehyde content, indicating severe lipid peroxidation and oxidative injury in tissues [11]. Concurrently, crowding stress impairs innate and adaptive immunity by downregulating the expression of immune-related genes such as IgM and LZM and inhibiting the activities of non-specific immune enzymes, thereby increasing vulnerability to pathogenic infections in fish [12]. Despite these advances, the integrated effects of stocking density on growth, stress physiology, antioxidant capacity, and immune function in grass carp remain incompletely elucidated. In addition, most studies have focused on single or few indicators, lacking a systematic evaluation combining growth performance, serum biochemistry, antioxidant status, and immune function under graded stocking densities. Therefore, identifying an optimal stocking density that reconciles high productivity with fish health is urgently needed for the development of efficient and sustainable intensive aquaculture.

To fill these research gaps, the present study was conducted to systematically evaluate the effects of three graded stocking densities on growth performance, stress response, serum biochemical profiles, antioxidant capacity, and innate immune status of grass carp reared in pond cage systems. The primary objectives were to determine the optimal stocking density for growth and physiological health and to provide a scientific basis for alleviating stress-related immunosuppression and disease risk in intensive grass carp culture. This study found that high stocking density will lead to growth inhibition, stress response and immune disorder of grass carp, and medium stocking density is most suitable for growth and health. These findings will contribute to the development of evidence-based management strategies for sustainable, intensive, and health-oriented grass carp aquaculture.

2. Materials and Methods

2.1. Experimental Design and Feeding Management

This experiment was conducted in pond cages measuring 2 m in length, 1.5 m in width, and 1 m in height, located in Liulihe, Fangshan District, Beijing. A total of 420 healthy grass carp of uniform size were used, with an initial average body weight of 84.87 ± 2.22 g. The selection of three different stocking density points was based on previous research results [13,14], the size of experimental fish, and actual aquaculture conditions in this study. The grass carp were assigned into three stocking densities: 0.57 kg/m^3^ (low stocking density, LSD), 1.13 kg/m^3^ (medium stocking density, MSD), and 2.27 kg/m^3^ (high stocking density, HSD). Each density treatment was replicated in three cages. The fish were fed a commercial expanded feed (Sikaiting Biotechnology Co., Ltd., Zhuhai, China), containing 30% crude protein, 4% crude fat, 15% crude fiber, 13% crude ash, 1.4% lysine, 4.5% calcium, 1.0–1.8% total phosphorus, and 11% moisture. Feeding was conducted three times daily (8:30, 13:00, and 18:00) at a rate of 3% of total biomass. The biomass in each cage was assessed every 15 days, and the feeding amount was adjusted accordingly. The 75-day trial was conducted from August to October 2025. Water quality parameters were monitored daily each morning using a multiparameter water quality analyzer (YT-S11, Yuntang Keqi, Shandong Yuntang Intelligent Technology Co., Ltd., Weifang, China). Each fish farming cage was continuously aerated through an oxygen diffuser connected to a 1.5 kW blower (XGB-1500, Taizhou Baofu, Taizhou Xiangcheng Electromechanical Co., Ltd., Taizhou, China) to ensure dissolved oxygen. All procedures complied with animal ethics guidelines and relevant regulatory standards.

2.2. Sample Collection

Upon completion of the culture period, 24 fish were randomly sampled from cages representing the three stocking density groups. The fish were anesthetized with MS222, and approximately 2 mL of blood was collected from the caudal vein of each individual. Blood samples were allowed to clot at room temperature for 2 h, followed by centrifugation at 3000 rpm for 10 min to separate serum. A caudal fin clip was taken from each fish, and genomic DNA was extracted using an Animal Tissue Genomic DNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China). DNA purity was assessed by measuring the A260/A280 ratio on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and integrity was verified by 1% agarose gel electrophoresis. DNA samples were diluted to a working concentration of 50 ng·μL^−1^ and stored at −20 °C for subsequent analysis. Simultaneously, dorsal muscle, liver, spleen, and kidney tissues were collected, immediately flash-frozen in liquid nitrogen, and transferred to a −80 °C laboratory freezer for long-term storage.

2.3. Measurement of Growth Performance Indicators

At the conclusion of the 75-day experimental period, all cage-cultured grass carp were harvested and individually measured. The following indicators were calculated to evaluate growth performance and survival.

$$[eqn]$$ $$[eqn]$$ $$[eqn]$$ $$[eqn]$$

W_0_(g): Initial body weight,

W_t_(g): Final body weight,

F(g): Feed intake,

L(cm): Body length of fish at the end of the experiment,

t(d): Duration of experiment.

2.4. Determination of Serum Biochemical Indicators

A 2 mL blood sample was collected from each fish and allowed to clot at room temperature for 2 h. Subsequently, the sample was centrifuged at 3000 rpm for 10 min to separate the serum, and the supernatant was transferred to ice for subsequent testing. Cortisol (COR) content was quantified using a competitive enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-linked Biotechnology Co., Shanghai, China) following the manufacturer’s protocol. For tissue analyses, 0.5 g each of kidney and liver samples was homogenized on ice with 5 mL of extraction buffer. The homogenate was centrifuged at 8000× g for 10 min at 4 °C, and the resulting supernatant was transferred to ice for further assays. Total protein (TP) content was determined with a BCA protein assay kit. Glucose (GLU) was measured using a glucose oxidase peroxidase (GOPOD) kit. Serum creatinine (CRE) was analyzed via the sarcosine oxidase method with a commercial assay kit. Lactate (LAC) concentration was assessed using a Lactate Colorimetric Assay Kit. Total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were all measured by enzymatic methods using corresponding assay kits. All kits mentioned above were supplied by Jiangsu Aidisheng Biotechnology Co., Ltd. (Yancheng, China) and were used according to the manufacturer’s instructions. Absorbance readings for all assays were obtained using an Infinite F50 microplate reader (Tecan, Männedorf, Switzerland).

2.5. Antioxidant and Immune Enzyme Activity Assay

The levels of IgM, LZM, AMP, AKP, and ACP were measured using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Shanghai, China). The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), reduced glutathione (GSH), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the protocols provided by the supplier. All results are expressed as units per milligram of protein (U/mg protein). Each assay was performed in triplicate, and absorbance was measured with an Infinite F50 microplate reader (Tecan, Switzerland).

2.6. RNA Extraction and cDNA Synthesis

Total RNA was extracted from spleen tissue using TRIzol reagent (Takara, Dalian, China). RNA integrity was assessed using 1.0% agarose gel electrophoresis, and concentration was quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNAs meeting quality criteria (A260/A280 ≥ 2.0 and A260/A230 ≥ 1.8) were retained as templates and stored at −80 °C. Complementary DNA (cDNA) was synthesized from qualified RNA using the Evo M-MLV Reverse Transcription Premix Kit (Accurate Biology, Changsha, China) and stored at −20 °C for subsequent use.

2.7. Real-Time Quantitative PCR (RT-qPCR)

Gene sequences of spleen immune-related genes in grass carp were obtained from the NCBI database. Primers were designed using Primer Premier 5.0 software, with specific sequences listed in Supplementary Table S1. RT-qPCR was conducted utilizing the SYBR Green Realtime PCR Master Mix kit (TOYOBO, Osaka, Japan). Each reaction contained 10 μL of SYBR Green Master Mix (2×), 2.0 μL of diluted cDNA template, 0.5 μL of forward and reverse primers (10 pmol/μL^−1^), and 7.0 μL of ddH_2_O. The amplification protocol was as follows: initial denaturation at 95 °C for 5 min, followed by 10 cycles of gradient amplification (denaturation at 95 °C for 30 s, gradient annealing from 62 °C to 52 °C for 30 s with a 1 °C decrease per cycle, and extension at 72 °C for 30 s). Subsequently, 25 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s were performed, followed by a final extension at 72 °C for 20 min and storage at 4 °C. The relative expression levels were calculated using the 2^−ΔΔCt^ method [15].

2.8. Statistical Analysis

Experimental data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA and Tukey–Kramer’s post hoc tests were conducted using GraphPad Prism 8 software to analyze significant differences among various indicators under different stocking densities, with p < 0.05 considered statistically significant. Figures were generated using R and GraphPad Prism 9.5 software.

3. Results

3.1. The Impact of Different Stocking Densities on the Growth Performance of Grass Carp

As demonstrated in Table 1, the MSD group exhibited significantly higher final body weight, WGR, and SGR in grass carp compared to the LSD and HSD groups (p < 0.05). Furthermore, the CF of grass carp in the MSD group was significantly greater than that in the LSD and HSD groups (p < 0.05). However, the FCR in MSD group was significantly lower than that of the other two groups (p < 0.05), indicating that the MSD fish group has a better utilization efficiency of feed and the best growth performance. We further compared the actual measured water quality data of each treatment group. The study found that as the stocking density increased, metabolic wastes such as ammonia nitrogen and nitrite also increased. In addition, the dissolved oxygen in the water decreased numerically with the increase in stocking density, indicating that high density leads to water quality deterioration.

3.2. The Impact of Different Stocking Densities on Serum Biochemical Indicators of Grass Carp

The effects of varying stocking densities on serum biochemical parameters in grass carp are illustrated in Figure 1. As the stocking density increased from low to high, serum cortisol concentrations exhibited a progressive rise. Notably, the cortisol level in the HSD group was significantly elevated compared to both the MSD and LSD groups (p < 0.05). Furthermore, the cortisol concentration in the MSD group was significantly higher than that in the LSD group (p < 0.05). Glucose levels demonstrated a similar upward trend, with the LSD group displaying the lowest glucose content and the HSD group the highest. The glucose concentration in the HSD group was significantly increased relative to the LSD group (p < 0.05). Among the indicators of protein and lipid metabolism, serum levels of TC and TG decreased with rising stocking density. Specifically, TC and TG levels in both the LSD and MSD groups were significantly higher than those in the HSD group (p < 0.05), while the serum TG level in the LSD group was also significantly greater than that in the MSD group (p < 0.05). In terms of liver function and tissue damage indicators, serum levels of ALT, AST, CRE, and BUN increased with higher stocking densities. The activities of ALT and AST, along with BUN concentrations in the HSD group, were significantly higher than those in the MSD and LSD groups (p < 0.05). Additionally, the serum CRE concentration in the HSD group was significantly elevated compared to the LSD group (p < 0.05). Moreover, the serum AST activity, as well as the concentrations of CRE and BUN, were significantly higher in the MSD group compared to the LSD group (p < 0.05).

3.3. The Impact of Different Stocking Densities on the Immunity of Grass Carp

The indices of kidney immune enzyme activity in grass carp under varying stocking densities are presented in Figure 2. In the kidneys, the activities of IgM, LZM, AMP, AKP, and ACP declined as stocking density increased. Notably, the activities of IgM, LZM, AMP, AKP, and ACP in the HSD group were significantly lower than those in the MSD and LSD groups (p < 0.05). Furthermore, the activities of these indices in the MSD group were significantly reduced compared to the LSD group (p < 0.05). This study also assessed the expression of immune genes in the spleen using quantitative real-time PCR. As illustrated in Figure 3, the expression levels of three pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were significantly upregulated with increasing stocking density. The gene expression levels of IL-1β, IL-6, and TNF-α in the HSD group were markedly higher than those in the MSD and LSD groups (p < 0.05). Moreover, the expression level of IL-1β in the MSD group was significantly elevated compared to that in the LSD group (p < 0.05). The expression level of the anti-inflammatory cytokine IL-10 also demonstrated an increasing trend with rising stocking density. The IL-10 gene expression in the HSD group was significantly greater than that in the MSD and LSD groups (p < 0.05), while no significant difference was observed between the MSD and LSD groups (p > 0.05). Additionally, the gene expression level of the chemokine IL-8 exhibited an increasing trend with rising stocking density. The expression of the IL-8 gene in the HSD group was significantly elevated compared to the LSD group (p < 0.05). Additionally, the IL-8 gene expression in the MSD group was also significantly higher than that in the LSD group (p < 0.05). However, no significant difference was observed between the HSD and MSD groups (p > 0.05). Conversely, the gene expression level of immunoglobulin IgM decreased significantly with increasing stocking density, with the IgM gene expression in the HSD group being significantly lower than that in the LSD group (p < 0.05).

3.4. The Impact of Different Stocking Densities on the Antioxidant Capacity of Grass Carp

The effects of varying stocking densities on the liver antioxidant capacity of grass carp are illustrated in Figure 4. As the stocking density increased from low to medium, the total antioxidant capacity (T-AOC) of the liver exhibited an upward trend, with a numerical increase from 0.14 ± 0.01 mmol/g protein to 0.16 ± 0.02 mmol/g protein; however, this difference was not statistically significant (p > 0.05). Upon further increasing the stocking density to high density (HSD group), the liver T-AOC activity significantly decreased, reaching its lowest level in the HSD group, which was markedly lower than that in the MSD group (p < 0.05). Additionally, the activities of the antioxidant enzyme catalase (CAT) and reduced glutathione (GSH) initially increased before decreasing with higher stocking density. The CAT and GSH activities in the HSD group were significantly lower than those in the MSD and LSD groups (p < 0.05). The activity of MDA, a key indicator of oxidative stress, exhibited an increasing trend with higher stocking density. The MDA activity in the HSD group was significantly higher than that in the MSD group (p < 0.05). Additionally, there were no significant differences in the activities of the antioxidant enzymes SOD and GSH-Px among the groups (p > 0.05).

4. Discussion

The present study demonstrated that a moderate stocking density (MSD, 1.13 kg/m^3^) significantly improved final body weight, weight gain rate, specific growth rate, and condition factor in grass carp. These findings are consistent with previous studies. For instance, existing evidence indicates that optimal stocking density optimizes growth performance in climbing perch (Anabas testudineus), whereas excessively high or low stocking densities exert negative effects on growth [16]. Similarly, moderate density enhances growth efficiency in rohu (Labeo rohita), while high density leads to growth inhibition [17]. Collectively, these results suggest an optimal density range that balances individual growth space and intraspecific competition. The low-density group (LSD, 0.57 kg/m^3^) likely failed to utilize resources efficiently due to reduced social interaction. In contrast, high density (HSD, 2.27 kg/m^3^) induced growth suppression, consistent with observations in other fish species [18], highlighting the necessity of density control to prevent overcrowding.

Serum biochemical indicators are crucial metrics that reflect the physiological state and stress levels of fish [19]. Cortisol, a key hormone involved in the stress response of fish, serves as an indicator of stress levels, with elevated concentrations signifying increased stress [20]. Chronic crowding stress in intensive aquaculture systems activates the Hypothalamic–Pituitary–Interrenal (HPI) axis, resulting in elevated plasma cortisol levels, which act as the primary glucocorticoid mediator of stress responses in teleost fish [21,22]. This hormonal alteration is accompanied by increased glucose concentrations, indicative of heightened gluconeogenesis to meet energy demands under crowded conditions [23,24]. In addition, cortisol binds to glucocorticoid receptors in hepatocytes [25], directly upregulating key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase [26], which may explain the observed increase in blood glucose under crowding stress. Simultaneously, liver function is compromised, as evidenced by increased activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are clinical markers of hepatic stress and cellular damage [27]. These enzymatic alterations correlate with reduced levels of cholesterol and TG, suggesting a metabolic shift toward catabolism to sustain energy production under density-induced stress [28]. The accumulation of nitrogenous waste products, CRE and BUN, further highlights renal and hepatic overload, as excessive protein metabolism generates toxic byproducts that challenge detoxification pathways [29]. Notably, the decline in serum TG and cholesterol aligns with findings in other teleosts, where stress-induced lipid peroxidation impairs lipid synthesis and storage [24]. This study demonstrates that with increasing breeding density, the serum levels of cortisol, glucose, ALT, AST, CRE, and BUN in grass carp significantly increased, while the levels of cholesterol and TG significantly decreased. The findings indicate that high-density breeding induces a stress response in grass carp, resulting in accelerated energy metabolism and increased protein breakdown as a means to cope with the pressures of a crowded environment. In addition, changes in water quality can cause stress reactions in fish [30]. In this study, the high-quality water quality management of the low-density group was manifested in the reduction in metabolic waste such as ammonia nitrogen and nitrite, which may partially explain the differences in stress indicators such as cortisol among the treatment groups.

The immune function of fish is a critical determinant of their health and survival, particularly in aquaculture settings where environmental stressors such as high stocking density can compromise immunological resilience [31]. This study demonstrates that elevated stocking density significantly suppresses key immune parameters in grass carp (Ctenopharyngodon idella), including reductions in immunoglobulin M (IgM), lysozyme, antimicrobial peptides, alkaline phosphatase (ALP), and acid phosphatase (ACP) activities in the kidneys. These findings are consistent with prior research across diverse fish species, underscoring the universal vulnerability of teleost immune systems to crowding stress [32]. The kidney, as a primary immune organ in teleosts, plays a pivotal role in systemic immunity by housing lymphocytes and macrophages that orchestrate humoral and cellular defenses [33]. The observed decline in IgM suggests impaired antigen recognition and pathogen neutralization capacity, while diminished lysozyme and antimicrobial peptides reflect compromised innate defenses against bacterial infections [34]. Notably, ALP and ACP are integral to phagocytic activity and lysosomal degradation of pathogens; their reduction indicates dysfunctional macrophage responses under high-density conditions [35]. In addition, an increase in the concentrations of total ammonia nitrogen and nitrite nitrogen in water, which are significant sources of stress, can lead to immune suppression in fish [30]. Consequently, the effective management of water quality in the low-density group may partially elucidate the differences in immune activity outcomes observed between the LSD group and other stocking density groups. The observed alterations in cytokine and immunoglobulin gene expression in high-density cultured grass carp (Ctenopharyngodon idella) reveal a complex immunological adaptation to crowded farming conditions. Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory IL-10 were significantly upregulated, suggesting an activated but potentially dysregulated immune response under high-density stress [36]. Concurrently, the downregulation of IgM indicates compromised antibody-mediated defense mechanisms, which may elevate disease susceptibility [37]. As an anti-inflammatory cytokine, IL-10 upregulation under crowding stress induced by high stocking density serves as a compensatory response to excessive pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) production, mitigating uncontrolled tissue inflammatory damage [38]. However, sustained IL-10 overexpression negatively regulates the trade-off between innate and adaptive immunity: it directly suppresses the expression of innate immune-related enzymes (LZM, AKP, ACP), while inhibiting immunoglobulin (e.g., IgM) synthesis critical for adaptive immunity. This dual inhibitory effect of IL-10 mediates the observed trade-off (attenuated innate defense and impaired adaptive response), increasing grass carp susceptibility to pathogenic infections under chronic crowding stress. This aligns with studies showing that high-density conditions disrupt mucosal barriers in grass carp, exacerbating inflammation while impairing pathogen clearance [39]. The upregulation of IL-10 may represent a compensatory anti-inflammatory response aimed at counterbalancing excessive pro-inflammatory signaling, as observed in bacterial infections such as Aeromonas hydrophila [39]. However, prolonged elevation of IL-10 can also suppress effector immune functions, further compromising disease resistance [36]. Therefore, high-density cultivation imposes a multifaceted burden on fish immunity, with the kidney emerging as a critical vulnerability node. The integration of immune monitoring into aquaculture management could transform industry standards, ensuring sustainable production without compromising fish health.

The antioxidant defense system of fish plays a crucial role in protecting cells from environmental stress [40]. This study elucidates a biphasic response in grass carp (Ctenopharyngodon idella), where moderate stocking density initially enhances antioxidant capacity, as evidenced by elevated total antioxidant capacity (T-AOC), catalase (CAT) activity, and glutathione (GSH) levels in the liver. This response serves as an adaptive mechanism to counteract mild oxidative stress [41,42], and its molecular basis lies in the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway—the core regulatory pathway of antioxidant defense in grass carp [10]. Under moderate stocking density, mild stress induces the generation of low levels of reactive oxygen species (ROS), which act as signaling molecules to trigger the dissociation of Nrf2 from its cytoplasmic repressor Keap1 [43]. The activated Nrf2 then translocates to the nucleus, binds to antioxidant response elements (AREs) in the promoter regions of CAT, SOD, and GSH-Px genes, and upregulates their transcription and subsequent enzymatic activities, thereby scavenging excess ROS and maintaining cellular redox homeostasis [44]. However, beyond a threshold density, this defense system becomes overwhelmed, leading to a decline in antioxidant markers and a concomitant rise in malondialdehyde (MDA), a lipid peroxidation byproduct indicative of oxidative damage [45]. At the molecular level, excessive-stocking-density-induced chronic stress results in overproduction of ROS, which trigger Keap1-dependent ubiquitination and degradation of Nrf2 [46], thereby inhibiting the activity of CAT, SOD, and GSH-Px, and then collectively leading to the collapse of the antioxidant defense system. This nonlinear relationship underscores the delicate balance between stress adaptation and system collapse, with significant implications for aquaculture management and fish welfare. The initial upregulation of antioxidant enzymes (e.g., CAT, SOD, GSH-Px) under moderate stocking density reflects a compensatory response to mitigate reactive oxygen species (ROS) generated by metabolic and environmental stressors [47]. For instance, studies on Cherax quadricarinatus and Trachinotus ovatus demonstrated similar adaptive elevations in GSH-Px and T-AOC under high-density conditions, suggesting a conserved evolutionary strategy across aquatic species [48,49]. Conversely, excessive crowding disrupts this equilibrium, depleting GSH reserves and impairing enzyme activities. The resultant oxidative stress manifests as DNA damage, protein oxidation, and apoptosis, exacerbated by the inability to neutralize ROS [50]. The continuous increase in MDA levels under high-density conditions highlights the vulnerability of lipid membranes to oxidative attack [51]. Although grass carp muscle exhibits tolerance to density-induced lipid peroxidation, the accumulation of MDA in the liver indicates systemic oxidative dysfunction [52]. This finding is consistent with research results on largemouth bass (Micropterus salmoides), where metabolism shifts towards lipolysis under chronic stress [5]. This study elucidates the dual role of stocking density in regulating the antioxidant capacity of grass carp. It emphasizes that a moderate stocking density can activate the antioxidant defense system, whereas overcrowding may trigger a collapse of the oxidative system, leading to cascading effects on lipid metabolism and immune function. Future research should investigate species-specific thresholds and the interplay between oxidative stress and epigenetic regulation to enhance mitigation strategies.

5. Conclusions

In conclusion, the present study systematically demonstrates that stocking density exerts a significant regulatory effect on the growth performance, serum biochemical profiles, immune competence, antioxidant capacity, and tissue morphology of grass carp (Ctenopharyngodon idella). Specifically, a medium stocking density was found to optimize the growth performance of grass carp while maintaining their physiological homeostasis and immune integrity, thereby supporting superior health status. Collectively, these findings highlight the critical importance of appropriate stocking density management in practical grass carp aquaculture. Future research should focus on exploring the welfare status and disease resistance of grass carp under varying stocking densities, which will provide a more comprehensive scientific basis for the sustainable and intensive culture of this economically important freshwater fish species.