Evaluation of the impact of music on antioxidant mechanisms and survival in salt-stressed goldfish

1 Introduction

In freshwater fish, short-term exposure to high salt concentrations may have a beneficial effect on reducing diseases; however, prolonged exposure to high-salinity environments can considerably impact their survival, growth, immune system, and antioxidant response [1]. For instance, goldfish (Carassius auratus) tolerate ≤12 ppt salt concentrations for extended periods; however, at higher salt concentrations, they are prone to mortality [2]. Adverse environmental conditions induce stress responses in animals, resulting in an increased production of reactive oxygen species (ROS), which can lead to oxidative damage to lipids and proteins [3]. To mitigate the effects of salt-induced damage, fish have evolved adaptive mechanisms, such as salt excretion and antioxidant defense [4,5]. Freshwater fish use the antioxidant defense system to tolerate salt stress. Activation of the antioxidant system, which includes enzymatic components (e.g., superoxide dismutase [SOD] and catalase [CAT]) and nonenzymatic antioxidants such as glutathione (GSH), is essential for oxidative stress mitigation. For instance, in the common carp (Cyprinus carpio), salt stress can result in a notable reduction in the activities of antioxidant enzymes such as SOD, CAT, and glutathione peroxidase [GPX] in the liver, intestines, and gills, along with exacerbated lipid oxidative damage [5]. Moreover, in animal cells, oxidative stress can substantially influence the contents of long-chain polyunsaturated fatty acids (PUFAs), which are crucial for animal health maintenance [6].

In natural high-salinity settings, elevated salinity can have detrimental effects on freshwater fish growth [7,8]. This may explain the aversion of freshwater fish to certain high-salinity environments, such as those near estuaries [9]. Enhancing fish nutrition can facilitate the survival of freshwater fish species such as tilapia in high-salinity environments [10]. Moreover, marine fish species (e.g., Atlantic salmon) may demonstrate low survival rates in freshwater habitats [11]. Thus, developing cost-effective, straightforward techniques that enhance fish adaptability to varying salinity is warranted. Nevertheless, except for nutritional enhancement [10], strategies to bolster freshwater fish viability and development in saline habitats are lacking.

Goldfish is a model organism commonly used to study physiological adaptations in freshwater fish. Its widespread use stems from several advantages: (1) high environmental plasticity allowing tolerance to various stressors, (2) well-characterized stress response pathways conserved across teleost species, and (3) established genomic resources facilitating molecular investigations. As a member of Cyprinidae, the largest fish family, goldfish provides translational insights for economically important relatives such as carp and zebrafish. Its robust stress responses make it ideal for evaluating novel mitigation strategies, including acoustic interventions.

Music has calming and mood-elevating properties not only in humans but also in various animal species. Emerging evidence also indicates that musical stimuli can modulate fish physiology through multiple pathways. In the common carp, Mozart’s music has been demonstrated to reduce cortisol levels and enhance growth hormone secretion, particularly under optimal light conditions [12,13]. In the rainbow trout (Oncorhynchus mykiss), musical exposure was noted to improve growth performance, along with increased hepatic antioxidant enzyme activities and decreased lipid peroxidation [14]. In contrast, abrupt exposure to loud music has been observed to elevate stress biomarkers such as plasma cortisol in fish [15]. These results suggest that acoustic characteristics particularly determine physiological outcomes. Furthermore, music can mitigate stress-induced responses in dogs within challenging environments [16]. For instance, in dogs, light exposure can hinder growth, whereas playing Beethoven’s Serenade can mitigate the negative effects of light-induced anxiety and promote growth [16]. However, whether music transmission can also enhance freshwater fish survival and growth in saline environments remains unexplored.

In the current study, we assessed the effects of exposure to Beethoven’s Serenade on goldfish survival rates, growth, food uptake, and oxidative damage under high-salinity conditions and explored the potential regulatory mechanisms related to antioxidant defense. Our results may aid in enhancing aquaculture outcomes for freshwater fish in the presence of salt-induced damage.

2 Materials and methods

2.2 Experiment design

In total, 240 healthy goldfish (body weight, 15.4 ± 0.8 g; standard length, 3.5 ± 0.4 cm) were obtained 3 weeks after hatching from Duoduo Ornamental Fish Farm (Luoyang) and acclimated for 7 days in 40 L glass tanks at 25 ± 0.5°C under a 12 h light–dark cycle, pH of 7.2 ± 0.1, and continuous aeration. The experiment included a completely randomized design with four treatment groups, namely, control, 0.36% NaCl, music, 0.36% NaCl+ music, each containing six replicate tanks (10 fish per tank with a total of 60 fish per group). Water quality was rigorously maintained using dual 5-W submersible oxygen pumps (Taoge AP928) per tank [17] to achieve dissolved oxygen levels of 10.5 ± 0.2 mg/L, monitored hourly using a YSI Pro20 oximeter. Additional parameters including temperature (monitored with a mercury thermometer), ammonia (0.02 ± 0.01 mg/L; monitored with Hach DR3900 spectrophotometer), nitrite (0.05 ± 0.02 mg/L; monitored with a colorimetry kit), and hardness (80 ± 5 mg/L CaCO₃; monitored through EDTA titration) were monitored daily; moreover, 30% water changes were performed every 48 h using aged tap water, aerated for ≥24 h.

The fish were fed 400 mg/fish/day (between 9:00 and 9:30 AM) with commercial pellets (50 mg/pellet, containing 42% protein and 8% lipid; Zhongshan Feed) [18]. Unconsumed feed was collected after 10 min of each feeding by using 200 μm mesh nets, oven-dried (60°C, 24 h), and weighed to calculate precise consumption rates. Salt stress was induced with daily 0.05% NaCl increments (dissolved in 1 L of tank water before addition) over 7 days to reach 0.36% salinity (3.6 ppt; confirmed using an Atago S/Mill-E refractometer).

For music treatment [12], Beethoven’s Serenade (K525; 70 ± 2 dB at the water surface, calibrated with Extech SL10) was delivered via an Ouang M11 speaker (frequency response, 100 Hz to 20 kHz) mounted onto a 5 cm thick polyethylene foam platform (20 × 20 cm²; Figure 1) floating centrally in each tank. The platform’s 1 cm diameter aperture (aligned with the speaker diaphragm) was used to optimize sound transmission (underwater sound pressure level, 65 ± 3 dB at 15 cm depth, verified using an Aquarian H2a hydrophone).

Figure 1

Schematic of experimental setup comprising a glass aquarium, dual submersible oxygen pumps, and an acoustic delivery system. The speaker (Ouang M11; frequency response: 100 Hz to 20 kHz) was mounted on a 5 cm thick polyethylene foam platform (20 × 20 cm²) with a 1 cm central aperture to optimize underwater sound transmission (65 ± 3 dB at 15 cm depth).

Behavioral monitoring with a Sony HDR-CX405 confirmed reductions in erratic movements (indicated by the goldfish lining up and swimming back and forth in the same direction in an orderly, uniform manner after playing the music, thus appearing very relaxed) and increased shoaling behavior during 30 min daily music treatment sessions (10:00–10:30 AM). At sampling intervals (weeks 0–4), the fish were netted individually and anesthetized with 150 mg/L clove oil (≥85% eugenol; Sigma-Aldrich; prepared as a 10 g/L stock in ethanol, diluted in tank water) until opercular movement ceased (within 3 min); finally, they were euthanized via spinal transection. Livers were excised within 90 s of euthanasia, weighed (0.01 mg precision; Mettler Toledo XS205), flash-frozen in liquid nitrogen (3 min immersion), and stored at −80°C (Thermo Scientific Forma 900 series) until subsequent analyses for oxidative stress markers (H₂O₂, lipid peroxidation markers, and protein carbonyls), antioxidant parameters (GSH, SOD, CAT, and GPX activities), and oxidative stress-related gene expression (SOD, CAT, and GPX expression). Mortality was recorded twice daily (at 8:00 AM and 6:00 PM), and deceased fish were immediately removed and subjected to necropsy.

3 Results

3.1 Survival rate, body weight, and feed intake

Compared with the control group, NaCl administration significantly reduced the goldfish survival rate, body weight, and feed intake over 4 weeks. Nevertheless, in the presence of music, the adverse effects of salt stress were notably mitigated (Figure 2). In particular, after 4 weeks of treatment, the survival rate, body weight, and feed intake were 82, 38, and 72% lower in the NaCl group than in the control group, respectively. In contrast, within the same period, the survival rate, body weight, and feed intake were 233, 16, and 64% higher in the NaCl+ music group than in the NaCl group, respectively (Figure 2). Compared with the control group, the music group demonstrated only a marginal enhancement in the survival rate, without a substantial impact on weight and feed intake (Figure 2).

Figure 2

Effects of music exposure on (a) survival rate (%), (b) body weight (g), and (c) feed intake (mg/fish/day) in goldfish across four treatments: control, NaCl (0.36%), music, NaCl+ music over 4 weeks. Data are mean values ± SD (n = 3 replicates/treatment). Bars sharing the same lowercase letter within a time point are non-significant (p > 0.05, Duncan’s test).

3.2 Oxidative damage, H₂O₂ content, and GSH/oxidized glutathione ratio

NaCl treatment over 4 weeks significantly increased lipid peroxidation (i.e., TBARS content), protein oxidation (i.e., carbonyl content), and H₂O₂ content but reduced the liver GSH/GSSG ratio in goldfish livers. Nevertheless, exposure to music substantially mitigated these deleterious effects of salt-induced stress (Figure 3). After 4 weeks of NaCl treatment, TBARS, carbonyl, and H₂O₂ contents increased by 98, 82, and 59% compared with the control group, respectively, whereas the GSH/GSSG ratio decreased by 64%. Similarly, within the same duration, treatment with both NaCl and music treatment increased TBARS, carbonyl, and H₂O₂ contents by only 24, 20, and 14% compared with NaCl treatment alone, respectively, whereas the GSH/GSSG ratio decreased by only 45% (Figure 3). Furthermore, compared to the control group, music alone led to minimal impact on the GSH/GSSG ratio and did not affect TBARS, carbonyl, and H₂O₂ contents significantly (Figure 3).

Figure 3

Effects of music exposure on goldfish liver: (a) Lipid peroxidation (TBARS, nmol g ⁻¹ dry weight), (b) protein oxidation (carbonyl content, nmol mg ⁻¹ protein), (c) H₂O₂ content (nmol mg ⁻¹ protein), and (d) GSH/GSSG ratio after 4 weeks. Treatments: Control, NaCl, music, NaCl+ music. Data are mean values ± SD (n = 3). Bars sharing the same letter are non-significant (p > 0.05).

3.3 Enzyme activities and GSH content

Compared with the control group, NaCl treatment over 4 weeks significantly affected SOD, CAT, and GPX activities, as well as the GSH content, in goldfish livers. However, exposure to music notably mitigated the effects of salt stress (Figure 4). In particular, 4 weeks of NaCl treatment reduced SOD activity, CAT activity, and GSH content in goldfish livers by 69, 60, and 75% compared with those in the control group, respectively, but increased GPX activity by 55% (Figure 4). Combined NaCl and music treatment increased SOD activity, CAT activity, and GSH content in goldfish livers by 78, 64, and 110% compared with those in the NaCl group, respectively, but reduced GPX activity by 17% (Figure 4). Nevertheless, compared with the control group, treatment with music alone did not alter the aforementioned enzyme activities or GSH content significantly (Figure 4).

Figure 4

Effects of music exposure on goldfish liver: (a) SOD activity (U mg ⁻¹ protein), (b) CAT activity (μmol min ⁻¹ mg ⁻¹ protein), (c) GPX activity (μmol min ⁻¹ mg ⁻¹ protein), and (d) GSH content (nmol mg ⁻¹ protein) after 4 weeks. Treatments: control, NaCl, music, NaCl+ music. Data are mean values ± SD (n = 3). Bars sharing the same letter are non-significant (p > 0.05).

3.4 Antioxidant enzyme – encoding gene expression

Compared with the control group, both the NaCl and NaCl+ music groups demonstrated fluctuations in SOD, CAT, and GPX expression after 24 and 72 h (Figure 5). In contrast, music treatment alone led to minimal alterations in the expression of the genes. At 24 h after treatment, SOD and CAT expression decreased by 14 and 35% in the NaCl group compared with the control group, respectively, whereas GPX expression increased by 73%. In contrast, the NaCl+ music group demonstrated a 45, 34, and 57% increase in SOD, CAT, and GPX expression compared with the NaCl group, respectively (Figure 5). Furthermore, at 72 h after treatment, SOD and CAT expression increased by 84% and 124% in the NaCl+ music group compared with the NaCl group, respectively, whereas GPX expression decreased by 12% (Figure 5).

Figure 5

Effects of music exposure on hepatic gene expression of (a) SOD, (b) CAT, and (c) GPX (fold change vs control) in goldfish at 0, 24, and 72 h after treatment onset. Treatments: Control, NaCl, music, NaCl+ music. Data are mean values ± SD (n = 3). Bars sharing the same letter are non-significant (p > 0.05).

4 Discussion

In this study, the salt concentration was increased to six times the freshwater level (from 0.06 to 0.36%) and administered at a dosage of 400 mg per fish to exacerbate salt damage in our goldfish [18]. The results indicated that elevated salt levels significantly reduced the survival rate, weight gain, and feed intake in the goldfish (Figure 2). This aligns with previous results demonstrating the negative effects of high salt concentrations on the body condition, feeding behavior, and mortality of freshwater fish [2,7,18,28]. For instance, Sahoo et al. [28] reported that freshwater fish, specifically Clarias batrachus (Linn.), exhibited a significant decrease in weight and feed intake after exposure to >6 ppt of NaCl over 1 week. Wang et al. [29] suggested that diminished feed intake resulting from salt-induced stress and its potential amelioration through the transmission of music may be attributable to alterations in the neural activity of animals. Efforts are being made to enhance fish survival and growth in challenging environments through supplementation of fish food with amino acids and vitamins [10,11,30]. In the current study, the exposure to Beethoven’s Serenade significantly influenced the survival rate, feed intake, and weight loss of goldfish exposed to high-salinity conditions (Figure 2). In contrast, studies have indicated that exposure to light can suppress carp growth, elevating brain neurotransmitter secretion as a stress response. In contrast, playing music for 30 min had been noted to effectively reduce brain neurotransmitter secretion, suggesting its potential in alleviating stress and anxiety, as well as facilitating growth, in carp [13]. Several studies have demonstrated the deleterious effects of salt damage on the antioxidant defense systems of fish organs [5], highlighting the crucial role of these systems in mitigating the impact of stressors such as salt [31,32]. Moreover, the antioxidant system plays a major role in alleviating environmental stress in chicks exposed to stimuli, such as noise and high-density farming [33,34]. Thus, the music-induced reduction of salt damage in goldfish may be associated with antioxidant system activation.

Here we examined the impact of music transmission on lipid and protein damage, H₂O₂ content, GSH level, redox balance (based on the GSH/GSSG ratio), and antioxidant enzyme (SOD, CAT, and GPX) activities in the liver tissues of goldfish subjected to salt stress over 4 weeks. We noted that the elevated salt stress significantly increased liver lipid peroxidation, protein oxidation, and H₂O₂ content but reduced liver GSH/GSSG ratio in the goldfish (Figure 3); moreover, it significantly reduced liver SOD, CAT, and GPX activities and liver GSH content (Figure 4). The GSH content and GSH/GSSG ratio depletions caused by abiotic stress are crucial markers of redox imbalance in animal cells [35]; they highlight the profound impact of high salt stress on the redox status in goldfish livers. This finding corroborates previous results indicating that salinity considerably impacts the oxidative damage and redox state in goldfish, as demonstrated by Yang et al. [18] and Wang et al. [1]. Moreover, López-Olmeda et al. [36] reported that including melatonin in fish diets can mitigate oxidative damage through stimulation of the antioxidant system. The current results demonstrated that music transmission significantly reduced lipid peroxidation, protein oxidation, and H₂O₂ accumulation but increased GSH content, GSH/GSSG ratio, and antioxidant enzyme (SOD, CAT, and GPX) activities in the liver tissues of our goldfish under salt stress (Figures 3 and 4). Moreover, music transmission effectively mitigated salt stress-induced SOD and CAT expression inhibition and GPX expression enhancement in goldfish livers (Figure 5). This finding aligns with previous results demonstrating a reduction in SOD and CAT expression in goldfish after triphenyltin treatment, with subsequent restoration through fructooligosaccharide treatment [37]. Moreover, the observed increase in antioxidant enzyme activity may be partially attributable to music-induced liver SOD, CAT, and GPX expression in goldfish. The protective effects of music against salt-induced oxidative stress in goldfish may operate through multilevel regulatory mechanisms. First, music may attenuate neuroendocrine stress responses, reducing cortisol secretion (as observed in carp [13,14]) and subsequently lowering ROS production. Second, acoustic enrichment may enhance metabolic efficiency – as evidenced by the increased feed intake (Figure 2) – providing substrates (e.g., amino acids and adenosine triphosphate) for GSH synthesis and antioxidant enzyme production [3,38]. Third, music-induced GPX upregulation (Figure 5) suggests direct transcriptional regulation of antioxidant pathways, possibly mediated by stress-responsive transcription factors activated by acoustic stimuli [33]. Notably, the GSH/GSSG ratio restoration (Figure 4) indicates that music may stabilize redox homeostasis, critical for cell survival under stress [34]. Similar studies in chicks have demonstrated that music mitigates NF-κB-mediated inflammation and boosts SOD and CAT activities [33,34], supporting a conserved acoustic–antioxidant crosstalk. Thus, music may act synergistically by (1) reducing initial oxidative insult via stress hormone modulation and (2) augmenting antioxidant capacity through nutritional and genomic pathways. Thus, whether music transmission activates the antioxidant system in goldfish, thereby improving their survival rate under salt stress, warrants investigation.

Numerous studies have demonstrated that long-chain PUFA contents in both animal and human cells considerably decrease under salt stress. In humans, nonalcoholic hepatitis is associated with a substantial reduction in long-chain PUFA contents as a consequence of oxidative stress [38]. Moreover, various human health conditions and diseases related to oxidative stress can result in a major decline in PUFA contents [6,39]. Thus, we hypothesized that salt-induced oxidative stress leads to a considerable reduction in PUFA content in goldfish liver. Our results demonstrated that salt-induced damage considerably led to substantial reductions in long-chain PUFA contents; nevertheless, exposure to music significantly mitigated this reduction (Table 2). Thus, music transmission may activate the antioxidant system, maintaining ROS at low levels; this may reduce oxidative damage to long-chain PUFAs, enhance health status, and lower mortality rates in goldfish under salt stress.

The current findings indicated that high salinity leads to reduced feed intake and increased weight loss in goldfish; nevertheless, exposure to music led to increased feed intake and reduced weight loss (Figure 2). Animals exhibit elevated metabolic and energy requirements to produce antioxidants in response to heightened oxidative stress [3,40]. In this study, we observed that exposure to music increased food consumption by goldfish under salt stress (Figure 2), potentially providing them with additional energy resources for antioxidant synthesis. Furthermore, exposure to music increased liver antioxidant enzyme activities (Figure 4) and expression (Figure 5) in the goldfish. Gao et al. [34] reported that high-density farming of chicks could result in reduced daily feed intake and increased mortality rates; nevertheless, music transmission effectively counteracted these negative effects. Moreover, noise was noted to suppress antioxidant enzyme activities in the immune organs of chicks; nevertheless, music transmission reversed this suppression to some extent [33]. Therefore, music transmission may alleviate anxiety in animals under stressful environments, eventually leading to improved feeding behavior and increased antioxidant synthesis for oxidative stress mitigation [3].

Empirical evidence is increasingly supporting the stress-reducing effects of acoustic interventions in aquaculture. Li et al. [41] highlighted that controlled sound exposure (e.g., music or specific frequencies) can modulate fish stress responses by regulating cortisol levels and enhancing antioxidant capacity – corroborating our findings in goldfish. Notably, acoustic enrichment has been noted to improve growth performance and survival in species such as Nile tilapia (Oreochromis niloticus) under crowding stress, possibly through neural pathways attenuating anxiety-related behaviors. In the current study, the observed upregulation of liver antioxidant enzyme genes (SOD, CAT, and GPX) and improved feeding under salt stress align with the aforementioned mechanisms. Thus, music may function as an acoustic enrichment tool to mitigate environmental challenges. Practical applications of similar noninvasive strategies – ranging from classical music to pulsed low-frequency sounds – are gaining traction in commercial aquaculture because they do not require chemical treatments but reduce production costs. Additional studies optimizing acoustic parameters (e.g., frequency and duration) for species-specific responses, as reviewed by Li et al. [41], to maximize welfare benefits in intensive farming systems are therefore required.

Although our study demonstrated the beneficial effects of music on goldfish under salt stress, it has several limitations. First, individual variability in growth rates and health status among fish (e.g., genetic differences and prior stress exposure) may have influenced responses to salinity and acoustic stimuli, potentially worsening the consistency of our results. Second, the 4 week experimental period may not have fully reflected long-term sustainability; prolonged exposure studies assessing whether music retains its efficacy or leads to habituation effects in aquaculture settings are required. Finally, metabolic variations (e.g., diurnal rhythms and sex-specific differences in stress tolerance) may have modulated antioxidant responses to music, warranting further investigation under controlled conditions. Future studies should address these factors to optimize acoustic enrichment protocols for commercial applications.

Animal resilience in challenging environments is attributable to not only the antioxidant defense mechanisms but also various other protective strategies [42]. Consequently, additional studies delving further into the molecular mechanisms underlying antioxidant defense, as well as exploring the underlying processes from various aspects, including immune response and neurotransmitter signaling [43], are warranted.

Beyond elucidating the physiological mechanisms, our findings suggest potential long-term implications for sustainable aquaculture practices. The demonstrated efficacy of brief, non-invasive acoustic interventions in mitigating salt stress and improving survival in goldfish warrants consideration of its practical application in fish farming. If sustained benefits and absence of detrimental habituation effects can be confirmed over extended production cycles, incorporating specific acoustic enrichment protocols could offer a low-cost, welfare-enhancing strategy to improve resilience in farmed freshwater fish exposed to salinity fluctuations or other stressors. Future research should focus on optimizing parameters (e.g., duration, frequency, sound type) for different species and scaling these interventions to commercial aquaculture settings to assess their true sustainability and economic viability.

5 Conclusion

The current results demonstrated that music, such as Beethoven’s Serenade, may mitigate salt stress-induced mortality and reduced feeding in goldfish, a freshwater fish species. This mitigation may partially be attributable to antioxidant system activation in goldfish liver. Compared with supplementing specific small molecule metabolites with unique functions via fish feed [36], aquatic music exposure can more simply and cost-effectively enhance salt tolerance in freshwater fish. Our study introduces a novel method for fish growth enhancement in challenging environments.