In the modern poultry industry, high density is a common phenomenon in broiler rearing, which enables producers to reduce rearing costs and gain more profit. Regarding the breeding standard of broiler stocking density, it varies widely among different countries. For example, the welfare guidelines of the National Chicken Council advocate an ideal rearing density of 41.5 kg/m² for broilers over 2 kg (1), the stocking densities in Netherlands, the United Kingdom, and Switzerland are 45–54 kg/m², 40 kg/m², and 30–36 kg/m², respectively (2). It is well known that high-density stocking is a major contributor to broiler stress. Intensive stocking not only increases corticosterone levels in broilers to induce physiological stress (3) but also decreases average daily feed intake and average daily gain and increases feed-to-gain ratio in broilers (4). Li et al. (5) indicated that serum immunoglobulins (immunoglobulin G and immunoglobulin A) decreased significantly in broilers as stocking density increased from 15 to 18 birds/m², which also led to an imbalance in serum antioxidant levels (decreased superoxide dismutase and glutathione peroxidase activities and increased malondialdehyde levels). Simsek et al. (6) also indicated that when broiler stocking density reached 47.3 kg/m² may lead to oxidative stress and a decrease in bursal weight in broilers. Therefore, how to mitigate some of the adverse effects of increased stocking density on broilers has become an urgent issue in large-scale broiler breeding.

In humans, music is widely used to enhance well-being, reduce stress, and distract patients from unpleasant symptoms (7). Music therapy is now being used to improve the environment of farm animals, which is considered an effective method to improve animal welfare. Playing music can not only cover up the background noise in the house environment, but also reduce anxiety, stress, and aggressive behavior by providing auditory enrichment. For example, cows exposed to classical music showed better performance in terms of milking time, milking speed, cortisol hormone and behavioral parameters (reduction in stereotyped behavior, etc.) (8). Listening to classical music has a calming effect on pet dogs in the stressful environment of veterinary hospitals (9). Short-term classical music stimulation increased activity, tail-dragging and playful behavior in fattening pigs, while long-term music stimulation increased serum interleukin-2, interferon-γ and immunoglobulin levels and decreased serum interleukin-4 levels (10). Early studies suggested that music altered hypothalamic–pituitary–adrenal axis activity by modulating the hypothalamus (11, 12), inducing an increase in dopamine levels in the brains of chicks and hypertensive rats, and inhibiting sympathetic activity (13, 14). The well-developed cochlea of birds has been reported to guide the recognition of audio and thus can process relative tones as in the primary auditory cortex of most mammals (15). African grey parrots and sulfur-crested cockatoos spontaneously move in sync with the music (head bobbing and foot lifting) (16), suggesting that birds also have the ability to perceive external rhythms like mammals. Furthermore, according to our previous study, classical music was found to have better effects on broiler health compared to popular and ethnic music (17).

Applying music to the production of animal farming is simple and easy. However, to the best of our knowledge, no studies have been reported on the effects of classical music on broilers, not to mention at high densities. Therefore, in our study, the effects of classical music on growth performance, stress level, antioxidant index, immune function and meat quality of broiler chickens at different densities were investigated.

Under the high-stocking density, broilers become extremely sensitive to the environment and are susceptible to stress, which may lead to impaired growth and development. Mohammed et al. (20) reported that, compared with the stocking density of 14 and 18 birds/m², the stocking density of 20 birds/m² significantly reduced the ADFI, ADG, and F/G of broilers. Similar to the above results, this study showed that a stocking density of 20.3 birds/m² significantly reduced broiler feed intake and body weight, and increased F/G and mortality. Stress induced by high-stocking density may be the main reason for the decreased growth performance of broilers. Moreover, the high mortality of broilers in this experiment at two densities of 17.9 and 20.3 birds/m² may be due to the high number of broilers in the cages, and severe competition for food among the flock, resulting in sudden death due to severe stress (21, 22). In this study, body weight gain and F/G of broilers were improved by playing classical music. Similar to this result, Gvaryahu et al. (23) found that music increased daily weight gain in broilers, and Tolun et al. (24) showed that music significantly improved the F/G of Ross 308 broilers. The musical environment improved the growth performance of broilers, probably due to the exposure of broilers to music starting at 1 day of age, which enabled them to adapt to the audible environment, thus reducing various stresses and fears during growth, reducing the corresponding energy expenditure and improving feed conversion. This is in agreement with Hoffman (25) and Gaioni et al. (26) who suggested that music may be beneficial in reducing fear in young chicks. On the other hand, the overall mortality rate of broiler chickens in the experimental group dropped by 44%, which may be attributed to classical music modulating the animals’ nervous systems through sound waves to relieve stress (27, 28), this may also contribute to the reduction of mortality in broilers. In this study, there was an interaction effect between classical music and stocking density on broiler F/G. At the same stocking density, the F/G of broilers in the 17.9 and 20.3 birds/m² groups playing classical music was significantly lower than that of the group without music, and the F/G of both groups decreased to the level of low stocking density without music intervention. This suggests that classical music can reverse the growth inhibition caused by increased stocking density and improve broiler growth performance.

When birds experience stress, their HPA axis is activated, and the pituitary gland is stimulated to secrete ACTH and glucocorticoids synthesized by adrenal cortical cells (29). Puvadolpirod et al. (30) found that the injection of high doses of exogenous ACTH increased corticosterone levels in the blood of broilers and increased stress levels by establishing a physiological stress model. In our study, serum CORT and ACTH levels were significantly higher in broilers in the 20.3 birds/m² group than in broilers in the 15.5 birds/m² group. Another study also showed that serum CORT levels were significantly increased when the stocking density of broilers was increased from 30 to 40 kg/m² (31). This means that broilers at high stocking densities are more susceptible to physiological stress. Many researchers have reported a positive effect of classical music on alleviating stress in animals (8). We found that playing classical music significantly reduced these two indicators in broiler serum at stocking densities of 17.9 and 20.3 birds/m². And there was no significant difference in serum CORT and ACTH levels between 17.9 and 15.5 birds/m² and between the 20.3 birds/m² group (playing classical music) and the 17.9 birds/m² group (no music). This suggests that classical music can be useful in alleviating the stress caused by increasing stocking density in broilers. Similarly, Dávila et al. (28) indicated that the auditory enrichment obtained from classical music can reduce stress in chicks. It may be that music can reduce the stress response in the HPA axis by inducing activity in the hippocampus of the brain (11, 12), therefore, the stress-relieving effect of classical music on broiler chicks may be related to the ability to alter neuronal activity within these brain structures, however, the exact mechanisms involved need further investigation.

Oxidative stress can lead to biological damage and a variety of physiological disorders that can reduce the growth performance of broilers (32). Important antioxidant enzymes such as GSH-PX and SOD play an important role in combating oxidative damage. MDA is a product of lipid peroxidation and its concentration reflects the state of oxidative stress (33). Simsek et al. (6) reported that overcrowded rearing environments led to elevated blood MDA levels and increased oxidative damage in broilers. Li et al. (5) suggested that stress-induced lipid peroxidation effects may be due to decreased antioxidant enzyme activity at high stocking densities (18 birds/m²). The above-mentioned reports were similar to our results, with increased serum MDA levels and decreased CAT and GSH-PX activities in the 20.3 birds/m² group compared with the other two densities. It is possible that the crowded space led to increased fighting behavior in broilers, resulting in metabolic disturbances that contributed to high lipid peroxidation and reactive oxygen species production in vivo, increased oxidative damage and MDA production in broilers, and decreased antioxidant enzyme activities (34, 35). In addition, we found that serum MDA concentration decreased, while serum SOD and GSH-PX activities tended to increase in broiler chickens by playing classical music. Previous studies have shown that classical music can increase the growth rate of human cells and increase the level of GSH (36). This implied that classical music may reduce oxidative stress damage by increasing the antioxidant enzyme activity of the body. On the other hand, classical music stimulation can increase positive behavior, reduce aggressive behavior, and relieve stress and anxiety in livestock (9, 37). In this study, it was hypothesized that classical music may serve to alleviate lipid peroxidation damage by reducing stress levels and increasing antioxidant enzyme activity in broilers.

Serum biochemical indicators reflect the nutritional metabolism, stress, and health status of the body (38–41). Mohammed et al. (20) showed that serum TP and ALB levels in broilers were significantly reduced at a stocking density of 20 birds/m². In this study, with the increase of stocking density, serum TP and ALB levels of broilers decreased, while serum TC and GLU levels increased. The decrease in serum protein levels suggested that high stocking densities had a negative impact on nutrient absorption and metabolic function in broilers. Elevated TC levels may be related to the fact that overproduction of CORT levels upregulated gene expression for cholesterol synthesis and uptake in broiler muscle, inducing the accumulation of cholesterol in muscle (42). In addition, stress-induced increases in glucocorticoid levels promote the breakdown of proteins and fats in the body, providing raw material for gluconeogenesis, and resulting in increased GLU levels in the blood (43). In this study, serum GLU levels in broilers were significantly reduced after stimulation by classical music. Similar to the results of this trial, Tabrizi et al. (44) showed that playing music not only reduced blood GLU levels in patients after surgery but also maintained relatively stable cortisol levels compared to the control group (no interference). Finn et al. (45) also reported that listening to music reduced cortisol and blood GLU levels in humans. In our study, we hypothesized that classical music may reduce the secretion of CORT and ACTH in broilers by alleviating the stress response, thus lowering blood GLU.

The relative development of the thymus, bursa, and spleen can be used to assess the immune function and health status of poultry (46). Simitzis et al. (47) found that increased stress levels in broilers at high stocking density resulted in lower bursal weights. Sanchez et al. (48) showed that reduced lymphoid organ weight in poultry was associated with stress-induced high serum corticosterone levels. In this study, the spleen and bursa weight of broilers decreased with increasing stocking density. Elevated concentrations of corticosterone cause degeneration of the lymphoid organs spleen and bursa phalloides and dysregulate the immune response by depleting lymphocytes in germ cells (49–51). Musical stimulation promotes T-cell proliferation in stressed mice (52). This is similar to our results that playing classical music increased the relative weight of the spleen and bursa of broilers. Serum immunoglobulins are important indicators of humoral immunity in animals and are a direct reflection of the immune function of the organism. Li et al. (5) showed that high-stocking density (18 birds/m²) significantly reduced serum IgG and IgA levels in broilers. Similarly, Hafez et al. (53) reported that serum IgA, IgG and IgM levels were significantly higher in the normal density group (10 birds/m²) than in the high-stocking density group (20 birds/m²). In the present study, increased stocking density significantly reduced serum IgA, IgM, and IgG levels in broilers, probably because high-stocking density induced stress increased glucocorticoid levels and inhibited the body’s immune function (54). Li et al. (10) reported that classical music increased serum IgG levels and improved cytokine levels in piglets. We found that classical music increased the serum IgA and IgG levels in broilers. Classical music may affect immune function by stimulating the nervous system of broiler chickens and the autonomic signals emanating from it interact with immune cells to control their function and response (55). However, the exact mechanism is unclear and further research is needed. The results of this study showed that there was an interactive effect between classical music and stocking density on the serum IgG content of broilers, and the IgG content of the 17.9 birds/m² group recovered to the same level as that of the 15.5 birds/m² group after classical music intervention, which indicated that classical music could alleviate the immunosuppression triggered by increased stocking density to some extent.

The quality of the muscle is usually assessed by color, tethering power, and shear force (56). Wu et al. (57) showed that 24 h after slaughter, high-stocking density (18.6 birds/m²) significantly increased L* and decreased a* values of breast muscle in broilers compared to low stocking density (12.9 birds/m²) and also resulted in higher drip losses. Hosseini et al. (58) reported that water loss of breast muscle in broilers was significantly lower in the high-stocking density group (20 birds/m²) than in the stocking density group (10 birds/m²). Similar to these results, in this study, it was also found that L*_{24 h} value and Cooking loss of breast muscle in broiler increased with the increase of stocking density, whereas a*_{24 h} value of breast muscle decreased. It has been reported that the high brightness values of chicken meat may be related to oxidative stress caused by high-stocking density (59). The strength of muscle oxidative capacity is related to high red values (57). High-stocking density caused high levels of nitric oxide production in broilers, increasing the accumulation of reactive oxygen and reactive nitrogen in the muscle, leading to massive oxidation of proteins, which reduced the strength of the myogenic fiber gel and increased the Cooking loss of chicken meat (60). Therefore, the high stocking density in this experiment led to a decrease in meat quality by inducing oxidative stress in broilers, while playing classical music enhanced the antioxidant capacity of broilers and improved the breast muscle a*_24h values.

Our results suggested that high-stocking density (20.3 birds/m²) can reduce growth performance, induce a stress response, decrease antioxidant and immune function, and reduce the meat quality of broilers. However classical music stimulus can indeed alleviate the negative effects of high-stocking density on broilers to a certain extent. Therefore, this was an effective measure to relieve the stressful conditions and increase the growth performance and immune status of broilers raised at the high stocking density condition.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors. The tracks used in this study are searchable on Himalayan Audio Platform (https://www.ximalaya.com/).

The animal study was reviewed and approved by the protocols for the animal study were approved by the Institutional Animal Care and Use Committee of Hebei Agricultural University and carried out under the Guidelines for the Care and Use of Laboratory Animals of China.

XG and JG performed the experiments and drafted the manuscript. XG, ZF, and JG carried out the statistical analysis. BY, YL, HX, YH, JJ, and LL helped the revision of this manuscript. ZF and LL contributed to the supervision and guidance of the present study. All authors contributed to the article and approved the submitted version.

This study was supported by the Hebei Provincial Key R&D Project (20326617D, Shijiazhuang, China) and the Hebei Modern Agricultural Industry Technology System Laying Hens & Brolier Innovation Team Construction Project (HBCT2018150203, Shijiazhuang, China).

JJ was employed by Hebei Jiuxing Agriculture and Animal Husbandry Development Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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