The Effects of Interactions Between Key Environmental Factors on Non-Specific Indicators in Carassius auratus

Abstract

Carassius auratus exhibits significant physiological and behavioral alterations under the combined stress of temperature and dissolved oxygen (DO) fluctuations, which are common challenges in aquaculture. In this investigation, we employed controlled thermal and DO gradients to characterize the multidimensional response profile of this species. The key findings revealed that thermal elevation profoundly influenced blood glucose and cortisol concentrations. Notably, exposure to hyperoxic conditions markedly attenuated stress responses relative to hypoxia at equivalent temperatures: cortisol levels were significantly suppressed (reductions of 60.11%, 118.06%, and 34.72%), while blood glucose levels exhibited concurrent increases (16.42%, 26.43%, and 26.34%). Distinctive behavioral patterns, including floating head behavior, surface swimming behavior, and rollover behavior, were identified as indicative behaviors of thermal–oxygen stress. Molecular analysis demonstrated the upregulated expression of stress-associated genes (HSP70, HSP90, HIF-1α, and Prdx3), which correlated temporally with elevated cortisol and glucose concentrations and the manifestation of stress behaviors. Furthermore, a muscle texture assessment indicated that increased DO availability mitigated the textural deterioration induced by heat stress. Collectively, this work establishes an authentic biomarker framework, providing crucial threshold parameters essential for the development of intelligent, real-time environmental monitoring and dynamic regulation systems to enhance climate-resilient aquaculture management.

1. Introduction

Global aquaculture production, dominated by China, Indonesia, and India, with China contributing nearly two-thirds of the global yield [1,2], is a vital source of protein and income. However, its sustainability faces significant challenges from water scarcity and pollution. Environmentally controlled intensive systems represent a critical developmental pathway, within which the coupled temperature–dissolved oxygen environment is a fundamental ecological determinant that profoundly influences fish welfare, growth, and productivity [3,4]. While threshold-based environmental control strategies are common [5], they often prove inadequate during seasonal extremes, leading to substantial reductions in fish performance, largely due to their simplistic, single-factor approaches that fail to capture the complex interactions of co-varying stressors, such as temperature and dissolved oxygen. Consequently, advancements in environmental regulation technologies, such as precision aeration and optimized recirculating aquaculture systems (RAS) [6,7,8], fundamentally depend on developing accurate dynamic models mapping environmental parameters to multifaceted biological responses [9,10]. A comprehensive understanding of the integrated effects of thermo-oxygen interactive stress is therefore paramount for intelligent precision aquaculture.

Studies have indicated that the stress cascade in fish triggered by thermo-oxygen imbalance typically follows a “physiological dysregulation–behavioral compensation” axis [11,12], with behavioral signatures increasingly recognized as valuable non-invasive early-warning biomarkers [13]. Technological breakthroughs now enable the quantitative characterization of feeding rhythms and automated recognition of aberrant swimming [14,15,16,17], offering novel real-time monitoring avenues. Nevertheless, a significant knowledge gap persists: the multi-scale response mechanisms (physiological, behavioral, molecular, and tissue-level) of fish under interactive temperature-and-dissolved-oxygen stress remain inadequately systematized and quantified, as current research often focuses on single stressors or isolated response levels.

Addressing this gap is crucial, as exemplified by the focus on the economically pivotal crucian carp. This highly adaptable cyprinid, which is native to East Asia but has spread globally because of its exceptional tolerance to low oxygen levels, pollution, and a wide range of temperatures (0–38 °C), is one of China’s highest-yield farmed fish (>2.8 million tons annually), serving as a vital high-protein, low-fat food source and an excellent model for intensive aquaculture stress studies. Heat shock proteins HSP70 and HSP90 are potential biomarkers reflecting fish responses to thermal changes. They protect cellular components from environmental damage [18]. Additionally, HIF-1α serves as the primary transcription factor responding to hypoxia in vertebrates [19], while Prdx3 plays a key role in antioxidant defense and tissue repair [20]. Changes in these genes can reflect the stress response in fish.

Therefore, in this study, we aim to bridge the identified knowledge gaps by systematically investigating the biological mechanisms underlying dual-factor (temperature and dissolved oxygen) stress in crucian carp. We employed a multidimensional data integration approach encompassing physiological–biochemical indices as primary stress markers, ethological signatures for quantifiable behavioral indicators of acute stress, stress-responsive gene expression profiles to elucidate molecular adaptations, and muscle tissue texture parameters to assess chronic stress impacts on product quality. By establishing relationships and thresholds across these response levels under controlled temperature and dissolved oxygen gradients, we aimed to provide the critical biological response parameters necessary for developing IoT-based intelligent aquaculture regulation systems, enabling precision environmental control strategies optimized for crucian carp welfare and productivity.

2. Materials and Methods

The Animal Ethics Committee of the Shanghai Academy of Agricultural Sciences approved all animal procedures (SAASXM0625022).

2.1. Acquisition and Husbandry of Experimental Fish

A total of 400 crucian carp were obtained from the aquaculture research facility of the Shanghai Academy of Agricultural Sciences. The fish had an average initial body weight of 52.3 ± 0.8 g. Prior to experimentation, the fish were acclimatized in a recirculating aquaculture system for 30 days under controlled conditions: the dissolved oxygen concentration was maintained above 5.0 mg/L, the water temperature was regulated between 22 and 27 °C, the ammonia nitrogen levels were kept below 0.12 mg/L, and the pH was stabilized at 7.00 ± 0.50.

2.2. Experimental Design

2.2.1. Temperature and Oxygen Control Methodology

In this experiment, we employed a dual-factor precision regulation system, comprising the following:

• Temperature control module: A closed-loop temperature regulation system based on a PID algorithm was established, integrating immersion heaters with digital temperature sensors. The compensation mechanism was automatically activated when the detected temperature deviated by >0.5 °C from the setpoint. The power of the heater was 100 W (Sensen Group Co., Ltd., Zhoushan, China). The temperature control range was 15–34 °C.
• Dissolved oxygen regulation module: A micro-aeration device coupled with optical DO probes was utilized, achieving dynamic DO balance through fuzzy-PID control. Specifically, the hypoxic group was maintained through synergistic regulation of biological oxygen consumption and micro-oxygen supplementation, while normoxic and hyperoxic groups were stabilized using gradient aeration strategies.

During this period, the pH of the water was maintained at 7.0 ± 0.5, the ammonia nitrogen content was maintained at 0–0.12 mg/L, and the nitrite content was maintained at 0–0.12 mg/L.

2.2.2. Experiment I: Effects of Thermo-Oxygen Stress on Physiological Parameters and Gene Expression in Crucian Carp

A 30-day experiment was conducted with three temperature levels (10, 20, and 30 °C) and two oxygen concentrations (2.50 and 7.50 mg/L), resulting in six treatments with three replicates each. The treatment groups were designated as low temperature–low oxygen (LT-LO), low temperature–normoxic (LT-CO), control temperature–low oxygen (CT-LO), control temperature–normoxic (CT-CO), high temperature–low oxygen (HT-LO), and high temperature–normoxic (HT-CO). The experimental setup for each independent unit is illustrated in Figure 1.

Based on preliminary single-factor experiments, crucian carp exhibited favorable performance at 20 °C. To investigate their biological responses under temperature stress, low-temperature (10 °C) and high-temperature (30 °C) conditions were selected. Furthermore, the dissolved oxygen (DO) level for normal physiological activity in crucian carp is 5 mg/L. Considering the increased volatilization of dissolved oxygen in water under elevated temperatures, the high-oxygen condition was set at 7.5 mg/L, while the low-oxygen condition was set at 2.5 mg/L.

Eighteen 200 L polyethylene tanks were randomly allocated, each configured as a semi-closed recirculating system. A total of 180 crucian carp were used, with 10 fish per tank. One-third of the water volume was replaced daily, and commercial pellet feed (provided by Binzhou Ruixing Biotechnology Co., Ltd., Binzhou, China) was manually administered at 2% of the total fish biomass per day.

Upon completion of the experiment, serum and liver tissue samples were collected for subsequent analysis.

2.2.3. Experiment II: Effects of Warm Oxygen Stress on Stress Behavior and Cortisol in Crucian Carp

A total of 50 crucian carp with an average initial body weight of 52.3 ± 0.8 g were used in this experiment. Initially, the fish were acclimatized in an 800 L recirculating aquaculture system for two weeks. Following the acclimation period, stress-induced behavioral changes were observed and recorded in response to temperature reduction and oxygen deprivation treatments. During the hypoxia challenge, fish exhibiting pronounced stress-related behavioral signatures were immediately sampled for cortisol level determination. The experimental setup is illustrated in Figure 2. To minimize observer bias, all behavioral observations were conducted by a single trained researcher throughout the experiment.

2.2.4. Experiment III: Effects of Thermo-Oxygen Stress on Muscle Texture Properties in Crucian Carp

Three experimental groups were established using separate tanks: normal temperature–normoxic (S₀), high temperature–normoxic (S₁), and normal temperature–hypoxic (S₂). A total of 30 healthy crucian carp were randomly allocated, with 10 fish per tank, and maintained for 20 days. The experiment was conducted in triplicate. Following the experimental period, muscle texture parameters were measured, including the water loss percentage and morphological characteristics.

2.3. Determination of Stress Parameters

The crucian carp were subjected to anesthetic treatment in a water tank containing 120 mg/L MS-222. Once deeply anesthetized, blood samples were collected via the puncture bleeding method. The collected sera were left to stand at room temperature for 1 h, followed by centrifugation at 2000× g for 10 min to obtain clear supernatant. The serum cortisol levels were determined using competitive immunoassay [21], while the blood glucose concentrations were measured through spectrophotometric analysis. The detection kits for cortisol and glucose were, respectively, purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China) and Nanchang Xibao Biotechnology Co., Ltd. (Nanchang, China).

2.4. Observation of Stress-Related Behavioral Characteristics

2.4.1. Hypoxic Stress-Induced Behavioral Alterations

Fifty crucian carp were placed in the experimental tank (Figure 2) and subjected to oxygen deprivation treatment to observe their stress behavior characteristics. Under hypoxic conditions, fish stress behaviors were generally categorized into four types:

• Sustained surface stress response: Persistent surfacing behavior or surface-proximal swimming for aerial oxygen uptake;
• Transient stress compensation: Temporary surfacing episodes, followed by resumption of normal swimming activity;
• Immobility-mediated stress adaptation: Reduced locomotor activity to minimize metabolic demand;
• Active stress avoidance: Vertical exploration of the water column in search of oxygen-enriched zones (Figure 3).

A hierarchical classification system was established based on the number of stressed individuals: Level 1 (0 fish exhibiting stress behaviors), Level 2 (1–3 fish), Level 3 (4–6 fish), and Level 4 (≥7 fish). Behavioral observations were recorded at 5 min intervals for three consecutive time points during each trial. To ensure experimental reliability, the entire procedure was repeated three times independently to eliminate interference from external factors.

2.4.2. Thermal Stress Behavior

Fifty individuals of crucian carp were randomly allocated to the experimental tanks (Figure 2). A cryogenic thermal stress protocol was implemented by sequentially infusing ice-cold water into the system, resulting in a controlled temperature decline to 10 °C. Simultaneously, a 20 °C reference tank was established to serve as the thermally neutral control. Behavioral observations focused on lateral tilting episodes were conducted using standardized video analysis software. Data collection occurred at 5 min intervals over three consecutive observation windows, with the entire experimental paradigm repeated thrice to account for environmental stochasticity.

2.5. Isolation of Tissue RNA and Quantitative Real-Time PCR Analysis

2.5.1. Isolation of Liver Tissue RNA

Freshly isolated crucian carp liver tissue was placed in a sterile culture dish, minced into uniform fragments, and transferred to a pre-cooled EP tube containing 1 mL of TRIzol^® (Invitrogen, Waltham, MA, USA) reagent. The lysate was subjected to ultrasonication in an ice bath at 40 kHz for 15 min until complete tissue homogenization. Following homogenization, the supernatant was transferred to a new EP tube and mixed with an equal volume of chloroform. The mixture was vigorously vortexed for 30 s and left to stand at 4 °C for 15 min to achieve phase separation. The aqueous phase was carefully aspirated into a fresh EP tube, followed by the addition of 1 mL of isopropyl alcohol. The solution was vortexed thoroughly and incubated at 4 °C for 10 min to precipitate nucleic acids. After centrifugation at 12,000× g for 10 min at 4 °C, the white precipitate was collected by removing the supernatant. The supernatant was carefully aspirated off using a pipette, and the precipitate was transferred to a bio-safety cabinet. The exhaust fan was activated for 5–10 min to dry the precipitate completely, until the white RNA pellet exhibited transparency under visual inspection. Subsequently, 20 μL of DEPC-treated water was added to the tube, followed by gentle vortexing and incubation at 4 °C overnight for complete dissolution of the nucleic acid precipitate.

2.5.2. Reverse Transcription of mRNA

The RNA concentrations of the different treatment groups were determined and recorded using a Nanodrop™ spectrophotometer. cDNA synthesis was performed according to the product manual of the Novozymes Reverse Transcription Kit, with the following procedure: First, the reaction mixture was prepared in an Eppendorf tube containing 2 μL of 10× RT Mix, 2 μL of HiScript III Enzyme Mix, 1 μL of Oligo(dT)20 VN, 1 μL of Random hexamers, and 14 μL of total RNA + RNase-free ddH₂O. The mixture was gently pipetted to ensure homogenization. The PCR thermal cycler was programmed as follows: 37 °C for 15 min; 85 °C for 5 s. The resulting translucent cDNA product could be directly used for quantitative real-time PCR or stored long-term at −80 °C.

2.5.3. Quantitative Real-Time PCR Analysis

The quantitative PCR reaction system was configured in an 8-tube PCR strip according to the protocol, followed by brief centrifugation of the strip. Finally, the reaction conditions were set and initiated on a real-time PCR instrument, and the results were analyzed using the 2^−ΔΔCt method.

This study used β-actin as a housekeeping gene to perform relative quantification of stress response-related genes, including HSP70, HSP90, HIF-1α, and Prdx3, to normalize the results. All primers were designed using Primer BLAST (NCBI), and the gene sequences were sourced from the NCBI database (https://www.ncbi.nlm.nih.gov, accessed on 5 June 2023). The sequences of all genes used in the experiment are listed in

Table 1. Primer sequence of genes.

Gene	Forward Primer	Reverse Primer
HSP70	TGC CAT CCT CTC TGG TGA TAA GT	ACC AGC CGT TTC AAT TCC AA
HSP90	CTC CCC AAC GTT CAC GAA	CGG CTT TGG TCA TCC CAA T
HIF-1α	TAA CCT CCC ACC TGG ACA AAG CCT C	TCG TTC TTG TCC GCT TCA TCA G
Prdx3	ATC AAC ACC CCA CGC AAG ACT G	ACC GTT TGG ATC AAT GAG GAA CAG ACC
β-actin	ATG GTG GGG ATG GGA CAG A	CTG TGA GCA GGA CGG GGT G

Note: HSP70 and HSP90 are heat shock proteins, HIF-1α is the hypoxia-inducible factor, Prdx3 is a peroxiredoxin 3, and β-actin serves as a housekeeping gene.

.

2.6. Determination of Muscle Moisture Loss in Crucian Carp

After a 24 h fasting period following experimental treatment III, six crucian carp were randomly selected from each tank and immersed in water containing MS-222 anesthetic. Once deep anesthesia was confirmed by the absence of opercular movement, the dorsal muscle tissue was dissected. Three specimens were allocated for the determination of drip loss and cooking loss, while the remaining three were allocated for centrifugal loss measurement [22].

• Drip loss

The freshly extracted crucian carp muscle samples (10 ± 0.5 g) were stored in a 4 °C refrigerator for 24 h. Subsequently, the samples were dried using absorbent paper and weighed. The drip loss calculation method used is as follows:

$$L_1=\left(W_1-W_2\right)/W_1\times 100\%,$$

(1)

$\mathrm{w}\mathrm{h}\mathrm{e}\mathrm{r}\mathrm{e} {\mathrm{L}}_1$ represents the drip loss, ${\mathrm{W}}_1$ denotes the initial mass of the muscle sample, and ${\mathrm{W}}_2$ indicates the post-drying mass.

• Cooking loss

After measuring the drip loss, the muscle samples were placed in plastic bags, sealed, and incubated in an 80 °C water bath for 30 min. Subsequently, the samples were cooled to room temperature, dried with absorbent paper, and reweighed. The cooking loss calculation method used is as follows:

$$L_2=\left(W_2-W_3\right)/W_2\times 100\%,$$

(2)

$\mathrm{w}\mathrm{h}\mathrm{e}\mathrm{r}\mathrm{e} {\mathrm{L}}_2$ represents the cooking loss, ${\mathrm{W}}_2$ denotes the post-drying mass, and ${\mathrm{W}}_3$ indicates the cooked mass.

• Centrifugal loss

The freshly extracted crucian carp muscle samples (10 ± 0.5 g) were centrifuged at 4 °C for 30 min at 4000 rpm. Subsequently, the samples were dried using absorbent paper and reweighed. The centrifugal loss calculation method used is as follows:

$$L_3=\left(W_4-W_5\right)/W_4\times 100\%,$$

(3)

$\mathrm{w}\mathrm{h}\mathrm{e}\mathrm{r}\mathrm{e} {\mathrm{L}}_3$ represents the centrifugal loss, ${\mathrm{W}}_4$ denotes the initial mass of the muscle sample, and ${\mathrm{W}}_5$ indicates the post-centrifugation mass.

2.7. Determination of Crucian Carp Muscle Morphology and Texture Characteristics

After a 24 h fasting period following experimental treatment III, three crucian carp were randomly selected from each tank and immersed in water containing MS-222 anesthetic. Once deep anesthesia was confirmed by the absence of opercular movement, the dorsal muscle tissue was dissected and placed into sterile EP tubes. Subsequently, 1 mL of 4% paraformaldehyde was added to each tube to immerse the muscle tissue, which was then fixed in a 4 °C refrigerator for 16–24 h. The fixed samples were sent to Jiangsu Chentong Biotechnology Co., Ltd. (Nantong, China) for subsequent processing, including tissue embedding, sectioning, and HE staining.

2.8. Data Analysis

Data analysis was performed using GraphPad Prism 5.0 software. The qPCR data were evaluated using the 2^−ΔΔCt method. Between-group comparisons were analyzed via an unpaired t-test, while within-group differences were assessed through one-way ANOVA. The data homogeneity of variances was assessed using the F-test, with a significance threshold of p > 0.05 indicating homogeneity. Statistical significance was defined as p < 0.05. All results are presented as mean ± SD (n = 9).

3. Results

3.1. Effects of Thermal and Hypoxic Stress on Cortisol and Blood Glucose Levels in Crucian Carp

The crucian carp exhibited significant alterations in cortisol levels under thermal–oxygen stress. When subjected to hypoxia, the cortisol levels increased by 12.29% at 10 °C and 28.90% at 30 °C relative to the 20 °C reference. Hyperoxic conditions induced cortisol elevations of 21.29% (10 °C) and 85.81% (30 °C) compared to the 20 °C condition. Crucially, hyperoxia substantially reduced cortisol levels by 60.11% (10 °C), 118.06% (20 °C), and 34.72% (30 °C) relative to hypoxia at equivalent temperatures (Figure 4a). The blood glucose levels mirrored these patterns, with hyperoxia significantly elevating glucose concentrations by 16.42% (10 °C), 26.43% (20 °C), and 26.34% (30 °C) compared to hypoxic conditions (Figure 4b). The two-way ANOVA analysis demonstrated significant influences of temperature, oxygen level, and their interaction on the blood glucose concentrations (

Table 2. The p values of two-way ANOVA on the effects of temperature and oxygen stress on changes in serum cortisol and blood glucose parameters in crucian carp.

Treatment	Cortisol’s p-Value	Blood Glucose’s p-Value
Temperature	<0.001	<0.001
Oxygen	<0.001	<0.001
Temperature × Oxygen	0.003	0.020

).

3.2. Behavioral Response Characteristics of Crucian Carp to Thermal and Hypoxic Stress

The behaviors of crucian carp induced by hypoxia were observed and documented in this study. Notably, hypoxic stress elicited distinct abnormal behavioral patterns, specifically: (1) surface swimming behavior, characterized by vertical positioning in the upper water column; (2) air-breathing behavior, manifested by protruding the mouth above water surface. Based on established hypoxia stress behavior grading criteria, we plotted the temporal variation in hypoxic fish counts. As shown in Figure 5, the results demonstrated that behavioral severity escalated gradually, progressing from Level 1 (0–25 min) to Level 4 (85–100 min) as hypoxia intensified.

Furthermore, our observations reveal that the crucian carp exhibited not only obvious air-breathing and surface swimming behaviors under hypoxia but also bottom-dwelling behavior. Subsequently, this study investigated cortisol level variations among four behaviorally characterized groups of crucian carp, with normal individuals serving as the control group:

T₀: Normal crucian carp.

T₁: Crucian carp exhibiting air-breathing behavior.

T₂: Crucian carp displaying surface swimming behavior.

T₃: Crucian carp demonstrating bottom-dwelling behavior.

As shown in Figure 6a, the results indicate that the cortisol levels in the stressed groups were significantly elevated compared to the normal controls (p < 0.05). Notably, T₁ exhibited substantially higher cortisol concentrations than both the T₂ and T₃ groups. However, no significant difference was observed between T₂ and T₃ (p > 0.05).

Under normal circumstances, low-temperature stress similarly alters fish physiological states, thereby inducing stress responses [23]. Our experimental findings demonstrate that the crucian carp exhibited lateral flipping behavior under low-temperature conditions, manifested as a loss of body balance and lateral tilting movements. As illustrated in Figure 6b, during the experimental period, the crucian carp showed fewer lateral flipping instances at 20 °C compared to significantly increased frequencies at 10 °C.

3.3. Gene Expression Response Patterns of Crucian Carp to Thermal and Hypoxic Stress

Molecular responses revealed temperature–oxygen interactions across stress-related genes. HSP70 and HSP90 expression significantly increased at thermal extremes (10 °C/30 °C) versus 20 °C under fixed oxygen (Figure 7a,b). Hypoxia further amplified expression, elevating HSP70 by 29.00% (20 °C) and 48.91% (30 °C) versus hyperoxia. whereas no significant difference in HSP70 was observed between the hypoxic and hyperoxic groups at 10 °C. Furthermore, at constant temperatures, the HSP90 expression levels in the hypoxic groups were significantly elevated compared to those in the hyperoxic groups by 40.94% at 10 °C, 36.00% at 20 °C, and 25.93% at 30 °C.

As shown in Figure 7c, under identical oxygen concentrations, HIF-1α expression levels in crucian carp livers were significantly higher at 30 °C compared to those under lower temperatures. Notably, under hypoxic conditions, HIF-1α expression in the 30 °C treatment exhibited significant increases of 81.38% and 116.43% relative to the 10 °C and 20 °C treatments, respectively, while no significant difference was observed between the 10 °C and 20 °C groups. Consistently, a similar trend was observed under the hyperoxic conditions. Furthermore, at constant temperatures, the HIF-1α expression levels in the hypoxic groups were significantly elevated compared to the hyperoxic groups by 111.11%, 104.95%, and 119.61% at 10, 20, and 30 °C, respectively.

Additionally, as shown in Figure 7d, under identical oxygen concentrations, the Prdx3 expression levels in crucian carp livers were significantly higher at 20 °C compared to other conditions. Notably, under hypoxic conditions, Prdx3 expression in 20 °C treatment exhibited increases of 14.58% and 80.00% relative to the 10 °C and 30 °C treatments, respectively. The hyperoxic conditions similarly demonstrated a consistent effect across parameters. Furthermore, at constant temperatures, the Prdx3 expression levels in the hypoxic groups were significantly lower than in the hyperoxic groups by 40.00%, 45.00%, and 58.33% at 10, 20, and 30 °C, respectively. The results of two-way ANOVA also confirm that the gene expression levels in the crucian carp were significantly affected by temperature, oxygen concentration, and their interaction (

Table 3. The p values of two-way ANOVA on the effect of temperature and oxygen stress on changes in hepatic expression levels of HSP70, HSP90, HIF-1α, and Prdx3.

Treatment	HSP70	HSP90	HIF-1α	Prdx3
Temperature	<0.001	<0.001	<0.001	<0.001
Oxygen	<0.001	<0.001	<0.001	<0.001
Temperature × Oxygen	0.014	0.010	0.029	0.040

Statistical analysis of experimental data was conducted using two-way ANOVA.

).

3.4. Variation Patterns in Crucian Carp Muscle Texture Parameters Under Stress Conditions

Table 4. Analysis results of water loss in crucian carp muscle in different experimental groups.

Index (%)	S0	S1	S2
Drip loss	3.70 ± 0.24 a	5.60 ± 0.41 b	5.47 ± 0.37 b
Cooking loss	18.74 ± 0.25 a	22.31 ± 1.58 b	21.29 ± 0.74 b
Centrifugal loss	15.10 ± 0.14 a	17.87 ± 0.45 b	18.40 ± 0.54 b

Results are presented as mean ± standard deviation. Different lowercase letters denote significant differences between treatments (p < 0.05).

results demonstrate that drip loss, cooking loss, and centrifugal loss in the crucian carp from group S₀ were significantly lower than those in Groups S₁ and S₂, with no significant difference observed between the S₁ and S₂ groups. Additionally, muscle morphology analysis revealed that compared to the control group (20 °C, normoxia), myofibril diameter in Groups S₁ (30 °C, normoxia) and S₂ (20 °C, hypoxia) exhibited significant decreases, while myofibril density showed significant increases (Figure 8). These findings indicate that optimal temperature and oxygen conditions enhance water retention capacity in crucian carp [24,25]. Conversely, high-temperature and hypoxic stresses elevate respiration rates in fish, leading to excessive ion loss and thereby reducing the water retention capacity while compromising muscle texture [26].

4. Discussion

4.1. Response Patterns of Crucian Carp Stress-Induced Parameters to Thermal and Hypoxic Stress

Cortisol is a robust biomarker for fish stress assessment [27]. Our data confirm that elevated temperatures (30 °C) and hypoxia synergistically amplify cortisol levels, whereas hyperoxia significantly mitigates this response, particularly at high temperatures. This suggests that supplemental oxygenation is a viable strategy to alleviate thermal stress in intensive aquaculture systems [28]. Blood glucose is a critical metabolic substrate essential for maintaining normal cellular and physiological functions [29]. The lowest blood glucose levels were observed in the L10 group, which induced reduced feeding appetite in crucian carp. Notably, hyperoxic conditions significantly facilitated glucose recovery compared to hypoxic conditions, potentially through enhanced metabolic rates and accelerated glucose synthesis pathways, thereby promoting normal physiological homeostasis [30]. These findings highlight dissolved oxygen management as a critical lever for maintaining physiological resilience under climate-induced thermal fluctuations, offering a practical approach to optimizing feeding efficiency and growth performance in farmed Carassius auratus.

4.2. Response Patterns in Crucian Carp Stress-Induced Behaviors to Thermal and Hypoxic Stress

Numerous researchers utilize behavioral metrics to monitor environmental stress in fish, representing an active research focus. Although various behavioral traits have been proposed [31,32], we demonstrate that >50% of a population exhibiting these behaviors signifies systemic stress, enabling early intervention [33]. Notably, behavioral hierarchies (e.g., floating vs. bottom-dwelling) correlate with cortisol gradients, validating their utility in automated monitoring systems. During the initial phase when oxygen levels were sufficient in the aquarium, crucian carp exhibited an initial upward movement followed by descent, likely reflecting foraging motivation and food-seeking behavior [33]. After a period of time, all individuals remained at the bottom habitat. Subsequent upward movements may indicate oxygen deficiency in the water column; some individuals temporarily stayed at the surface before returning to the bottom layer after oxygen absorption, possibly due to a preference for the optimal bottom microenvironment. Meanwhile, persistent surface dwellers might maintain elevated oxygen demand. Over time, the number of upward migrations gradually increased as oxygen deficiency intensified in the water body. Ultimately, pronounced floating behavior emerged, with floated individuals exhibiting severe hypoxia symptoms. Crucially, tilting behavior under cold stress (10 °C) revealed temperature fluctuations as a distinct stressor independent of hypoxia. This underscores the need for dual-parameter (temperature and oxygen) surveillance in aquaculture IoT platforms, where behavioral algorithms could trigger adaptive aeration or thermal buffering to preempt chronic stress [34].

4.3. Response Patterns in Crucian Carp Gene Expression to Thermal and Hypoxic Stress

HSP70 and HSP90 are nonspecific cytoprotective proteins [18] that play crucial roles in protecting organisms against various adverse conditions, particularly thermal stress [35,36]. Compared to 20 °C, the expression levels of HSP70 and HSP90 in crucian carp liver were significantly upregulated at 30 °C, indicating that crucian carp can regulate HSP gene expression to adapt to temperature changes and enhance their heat tolerance [37]. Additionally, moderate cold stress can also induce HSP70 and HSP90 expression, as evidenced by the rapid immune response observed in crucian carp at 10 °C [38]. Studies have demonstrated that elevated HSP70 and HSP90 expression correlates with increased glucose and cortisol levels [39], supporting this observation. Furthermore, fish with lower cortisol levels exhibit superior swimming performance [40], suggesting that physiological stress negatively impacts locomotor capabilities and induces abnormal behavioral traits.

HIF-1α is the primary transcription factor responding to hypoxia in vertebrates [19]. Under isothermal conditions, HIF-1α expression in crucian carp liver exhibits significant upregulation under hypoxic stress, as oxygen deprivation serves as the primary inducer of its expression. Furthermore, elevated HIF-1α levels trigger morphological and surface area alterations in gills, affecting respiratory frequency and subsequently inducing cortisol and glucose level changes [41]. Studies have demonstrated that HIF-1α gene knockout reduces fish tolerance to hypoxic environments; under hypoxia, fish migrate to the water–air interface for respiration, thereby alleviating environmental hypoxia impacts [42]. Prdx3 is recognized as a critical factor in antioxidant defense and tissue repair mechanisms [20]. Thermal and hypoxic stress can impair fish antioxidant defense capabilities [43], with studies demonstrating that fish exhibit elevated Prdx3 gene expression and cortisol levels under oxidative stress [44]. Furthermore, in investigations exploring methods to mitigate oxidative stress responses, Prdx3 and other stress-related genes, along with cortisol and glucose levels, have been established as key biomarkers [45], underscoring their intrinsic association with physiological parameters. Notably, oxidative stress directly induces a series of abnormal behavioral responses in fish [46].

In general, the upregulation of HSP70/90 and HIF-1α under combined stressors reflects conserved molecular pathways for cellular protection and oxygen sensing. Importantly, Prdx3 suppression in hypoxic/thermal groups indicates compromised antioxidant capacity, linking oxidative damage to tissue deterioration. These genes constitute a diagnostic panel for stress severity, with HIF-1α as a hypoxia-specific marker and HSP responding dominantly to temperature. In aquaculture, this implies that gene expression profiling could refine stress thresholds in breeding programs.

4.4. Effects of Thermal and Hypoxic Stress on Muscle Texture Parameters in Crucian Carp

Muscle texture degradation (increased water loss, reduced myofiber diameter) under stress directly impacts product quality and economic value [47]. Our data reveal that textural deterioration parallels physiological dysregulation (cortisol/glucose imbalances) and gene expression shifts, establishing a multi-tiered stress signature. Fish selectively regulate essential substance synthesis and structural formation in response to environmental changes, impacting enzyme activity within muscle fibers [48]. This leads to stagnation in muscle fiber length and diameter growth, mild atrophy of myofibers, toughened meat texture, and dull coloration [49]. Additionally, such texture alterations reduce water retention capacity and significantly decrease tenderness. Therefore, thermal and hypoxic stress may modify fish muscle texture through comprehensive effects on muscle composition and myofibrillar structure. This demonstrates the correlation between fish behavioral traits, physiological stress responses, and muscle fiber dynamics [40]. This synergy suggests that muscle quality can serve as a post-mortem indicator of pre-harvest welfare, advocating for environmental controls to preserve market traits.

This study analyzed variation patterns in crucian carp across genetic, physiological, behavioral, and morphological dimensions under the interactive effects of temperature and dissolved oxygen. It elucidated the multiscale biological response mechanisms between crucian carp and environmental changes, establishing a theoretical foundation for intelligent regulation of aquaculture environments.

5. Conclusions

This study elucidates the multi-scale biological response mechanisms of crucian carp to thermal and hypoxic stress across the gene regulation, physiological response, behavioral adaptation, and tissue metabolism dimensions, demonstrating that dissolved oxygen elevation induces significant physiological compensatory effects against high-temperature stress. The findings reveal that (1) HSP70, HSP90, HIF-1α, and Prdx3 constitute a molecular biomarker combination for assessing fish stress; (2) floating and tilting behaviors serve as early-warning indicators of stress in crucian carp; and (3) the muscle water retention capacity and myofiber parameters enable the quantitative characterization of chronic stress-induced damage. These results provide critical control parameters for establishing intelligent aquaculture systems and lay the theoretical foundation for precision environmental regulation. The selection of slice samples and indicators involved in this study, as well as individual differences in crucian carp, may to some extent affect the experimental results. Multiple experiments will be conducted in the future to obtain accurate conclusions. The findings are expected to provide critical biological response thresholds for developing IoT-based intelligent aquaculture regulation systems, thereby establishing precision environmental control strategies.