Growth method for improving immune stress of broiler chicken in music environment

Abstract

The invention relates to the technical field of animal science, and discloses a growth method of broiler chickens capable of improving immune stress in a music environment, which comprises the following steps: S1, randomly dividing a plurality of male broiler chickens with the age of 1 day into a control group (CON group), a music stimulation group (MUC group), an LPS immune stress group (LPS group) and a music + LPS group (MUC + LPS group); s2, the broiler chickens in the music stimulation group and the music + LPS group are subjected to music stimulation for 4-10 hours every day; s3, injecting an LPS solution into the abdominal cavities of the broilers of the LPS immune stress group and the music + LPS group to prepare models during the 12-20 days of the test; s4, sample collection and analysis are conducted on the 28th day of the test. Through stimulation of the music, the music has a remarkable relieving effect on the broiler chicken under the immune stress condition and shows a lower corticosterone level, which indicates that the stress response induced by the LPS can be relieved to a certain extent through the stimulation of the music.

Description

Technical Field

[0001] The invention relates to the field of animal science and technology, and in particular to a method for improving the growth of immune stress broiler chickens in a music environment. Background Art

[0002] During the production process of broiler chickens, animals often face various stressors, including immune stress, environmental stress, heat stress, transportation stress, etc. Immune stress, especially stress response caused by diseases or pathogens, can cause physiological, metabolic and immune system disorders in broilers, leading to decreased production performance, poor feed conversion rate, and even increased mortality. These stressors not only pose a serious threat to the health of animals, but also affect the growth and development of broilers, thereby damaging the economic benefits of breeders.

[0003] The stress response of broilers is usually triggered by external environmental factors (such as high temperature, noise, intensive feeding) or endogenous factors (such as disease, vaccination). The stress response will lead to increased levels of stress hormones such as corticosterone in the body, suppression of immune function, and damage to the intestinal barrier function, increasing the risk of intestinal diseases through the interaction of the nervous, endocrine and immune systems. These adverse factors not only affect the growth performance of broilers, but also affect the overall health and survival rate of broilers.

[0004] As animal welfare becomes increasingly important in modern farming, researchers have begun to explore ways to improve the quality of life of animals and reduce stress. In recent years, music has gradually been applied to livestock and poultry farming as a non-drug intervention. Studies have found that music can not only improve the behavioral state of livestock and poultry, reduce anxiety and aggression, but also reduce stress responses by regulating hormone levels in the body. For example, in experiments on rodents, music stimulation has been shown to reduce animals' anxious behavior, improve cognitive function, and reduce the levels of stress hormones such as corticosterone in serum.

[0005] In the study of poultry, music has also shown certain positive effects. For example, patent CN116982596A discloses a method for analyzing the effects of different types of music on caged laying ducks. The results show that different types of music can significantly affect the behavior patterns, physiological indicators and growth performance of laying ducks. Specifically, appropriate music stimulation can reduce the anxiety level of poultry, improve growth efficiency, improve feed utilization, etc. However, this study mainly focused on the behavior and production performance of laying ducks, and did not explore in depth the effect of music on alleviating immune stress and its related physiological damage.

[0006] Although there have been preliminary studies and applications on the effects of music on behavior and stress, the question of whether music can alleviate the damage caused by immune stress in poultry has not been deeply verified. Immune stress originates from the stimulation of pathogens, vaccination or other immune system-related interference, which often leads to increased inflammatory response, immunosuppression and damage to intestinal structure and function in broilers. In this context, it is of great scientific significance and practical application value to explore whether music can alleviate the negative effects of immune stress in broilers by reducing the levels of stress hormones such as corticosterone and thyrotropin, or by regulating intestinal function and immune response.

[0007] Although some studies have revealed the positive effects of music on poultry behavior and growth performance, the mechanism of action of music under stress conditions, especially immune stress conditions, has not been fully studied. In existing literature and patents, music is mainly used in the research field of reducing heat stress, reducing animal anxiety or improving poultry behavior. There is no systematic exploration of the potential mitigating effect of music on stress-induced immunosuppression and intestinal damage.

[0008] To this end, those skilled in the art provide a method for improving the growth of immune-stressed broilers in a music environment to solve the problems raised in the above-mentioned background technology. Summary of the invention

[0009] In view of the deficiencies of the prior art, the present invention provides a method for improving the growth of immune-stressed broilers in a music environment, which solves the problem that the prior art is blank in research on the effect of alleviating body damage caused by immune stress.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for improving the growth of immune stress broilers in a music environment, comprising the following steps:

[0011] S1: Multiple one-day-old male broilers were randomly divided into a control group (CON group), a music stimulation group (MUC group), an LPS immune stress group (LPS group) and a music + LPS group (MUC + LPS group), with 10 replicates in each group and 5 broilers in each replicate;

[0012] S2: The broiler chickens in the music stimulation group and the music + LPS group were given 4-10 hours of music stimulation every day;

[0013] S3: During the 12th to 20th day of the experiment, the broilers in the LPS immune stress group and the music + LPS group were intraperitoneally injected with LPS solution to prepare the immune stress model. LPS was dissolved in physiological saline.

[0014] S4: On the 28th day of the experiment, blood and intestinal samples were collected and analyzed from broiler chickens in each group.

[0015] Preferably, the music stimulation in step S2 uses a classical sonata, and the volume is controlled between 60-80 decibels.

[0016] Preferably, the LPS dose injected in step S3 is 450-550 μg / kg, and the mass ratio of LPS to physiological saline is 1:100 to 1:250.

[0017] Preferably, in step S3, the broilers in the control group and the music stimulation group are injected with equal amounts of physiological saline at the same time point.

[0018] Preferably, the blood sample in step S4 is collected through the jugular vein, with a collection volume of 3-5 ml. After standing for 25-35 minutes, it is centrifuged at 1500-2500 rpm for 10-15 minutes, and the serum is frozen and stored at -40°C to -80°C for further analysis.

[0019] Preferably, in step S4, the feed consumption and weight change of each repeated broiler chicken are recorded, and the average daily weight gain, average daily feed intake and feed-to-weight ratio are calculated.

[0020] Preferably, after the small intestine sample collected in step S4 is flushed with phosphate buffer or physiological saline, the jejunum and ileum segments are taken, and part of the intestinal segments are fixed in 4% paraformaldehyde solution for HE staining and immunofluorescence analysis.

[0021] Preferably, the partial intestinal segment of the small intestine sample in step S4 is placed in an electron microscopic fixative containing 2.5% glutaraldehyde for observation under a scanning electron microscope and a transmission electron microscope.

[0022] Preferably, the remaining small intestine segment in step S4 is cut longitudinally, washed with sterile PBS (1×), and the jejunal and ileal mucosa are scraped with a sterile glass slide and collected into sterile cryopreservation tubes. The samples are quickly frozen and stored at -80°C for subsequent analysis.

[0023] Preferably, in step S4, the broiler chickens injected with LPS are killed by bleeding from the jugular vein, and the gastrointestinal tract is removed after dissection, and the small intestine segment is taken for histological and pathological analysis to evaluate the changes in the morphology of the small intestinal villi.

[0024] The present invention provides a method for improving the growth of immune stress broiler chickens in a music environment.

[0025] Beneficial effects:

[0026] 1. The present invention randomly divides 200 one-day-old broilers into 4 groups, and gives music stimulation every day from the first day of the experiment until the end of the experiment on the 28th day. On the 14th, 16th and 18th days of the experiment, each chicken in the LPS immune stress group and the music + LPS group is intraperitoneally injected with LPS, and the control group and the music stimulation group are injected with an equal amount of physiological saline. During the experiment, the production indicators of broilers in each group of 1-14 days and 15-28 days of age are measured. The broilers are killed on the 28th day of the experiment, and serum, small intestinal tissue, mucosa, and cecal contents are collected.

[0027] LPS stimulation induces immune stress in broilers, reduces broiler production performance, destroys intestinal morphology and flora structure, causes disordered expression of intestinal mucosal barrier-related genes and proteins, reduces the activity of antioxidant enzymes, and causes intestinal inflammatory response by activating the TLR4 / NF-κB signaling pathway. Music stimulation can enhance the expression of intestinal barrier proteins to a certain extent, reduce the body's oxidative stress level, inhibit the activation of the TLR4 / NF-κB signaling pathway and thus reduce the overexpression of inflammatory-related factors. At the same time, it protects intestinal health by regulating intestinal flora homeostasis and increasing the number of beneficial bacteria, which provides a theoretical basis for effectively using music stimulation as an environmental enrichment method to improve the anti-stress ability of broilers in production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the steps of the present invention;

[0029] Figure 2 The figure is a result of measuring the levels of serum corticosterone (CORT) and adrenaline stimulating hormone (ACTH) in broiler chickens of each group of the present invention;

[0030] Figure 3 This is a schematic diagram of the morphology of broiler intestinal villi of the present invention;

[0031] Figure 4 This is a schematic diagram of the growth performance results of broiler chickens in each group of the present invention;

[0032] Figure 5 Schematic diagram of cytokines and immunoglobulins in serum of broiler chickens in each group of the present invention;

[0033] Figure 6 It is a schematic diagram of the expression of tight junction proteins in the small intestinal mucosa of broiler chickens in each group of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Embodiment 1:

[0036] Please refer to the attached Figure 1 , an embodiment of the present invention provides a method for improving the growth of immune stress broilers in a music environment, comprising the following steps.

[0037] (1) Two hundred one-day-old male broiler chickens of similar body weight were randomly divided into four treatment groups: control group (CON group), music stimulation group (MUC group), LPS immune stress group (LPS group) and music + LPS group (MUC + LPS group). Each treatment group had 10 replicates, with 5 chickens in each replicate. The music group and the music + LPS group were given 6 hours of music stimulation (Mozart K.448) every day from the first day of age, and the sound was controlled at 65 decibels until the end of the experiment.

[0038] (2) On the 14th, 16th, and 18th days of the experiment, each chicken in the LPS immune stress group and the music + LPS group was intraperitoneally injected with 480 μg / kg LPS dissolved in saline to prepare the immune stress model, where the mass ratio of LPS to saline was 1:150. The control group and the music stimulation group were injected with an equal amount of saline;

[0039] (3) On the 28th day of the experiment, one chicken was randomly picked from each replicate in each group for sample collection. 3 ml of blood was collected from the jugular vein and placed in a serum tube. After being placed under serum for 28 minutes, the sample was processed by centrifugation at a speed of 1600 rpm and stored frozen until further analysis. After death by jugular bleeding, the chickens were dissected, the gastrointestinal tract was removed, the small intestine was separated from the mesentery and connective tissue, and about 2 cm of the jejunum and the middle ileum were rinsed with phosphate buffer to remove feces. Part of the intestinal segment was fixed in paraformaldehyde solution for HE staining and immunofluorescence analysis, and part was placed in electron microscopy fixative for scanning and transmission electron microscopy observation. The remaining small intestinal segment was cut longitudinally and washed with PBS (1×), and then the jejunal and ileal mucosa were scraped with a sterile slide, collected in a sterile cryotube, quickly frozen in liquid nitrogen and stored at -80°C until further analysis;

[0040] (4) The broilers of each replicate were weighed on an empty stomach after a 12-h feeding stop on days 0, 14, and 28 of the experiment, and the feed consumption of each replicate on days 14 and 28 of the experiment was recorded to calculate the average daily weight gain, average daily feed intake, and feed-to-weight ratio for each period.

[0041] Embodiment 2:

[0042] The embodiment of the present invention provides a method for improving the growth of immune-stressed broilers in a music environment, comprising the following steps.

[0043] (1) Two hundred one-day-old male broiler chickens of similar body weight were randomly divided into four treatment groups: control group (CON group), music stimulation group (MUC group), LPS immune stress group (LPS group) and music + LPS group (MUC + LPS group). Each treatment group had 10 replicates, with 5 chickens in each replicate. The music group and music + LPS group were given 8 hours of music stimulation (Mozart K.448) every day from the first day of age, and the sound was controlled at 75 decibels until the end of the experiment.

[0044] (2) On the 14th, 16th, and 18th days of the experiment, each chicken in the LPS immune stress group and the music + LPS group was intraperitoneally injected with 500 μg / kg LPS dissolved in saline to prepare the immune stress model, where the mass ratio of LPS to saline was 1:200. The control group and the music stimulation group were injected with an equal amount of saline;

[0045] (3) On the 28th day of the experiment, one chicken was randomly picked from each replicate in each group for sample collection. 5 ml of blood was collected from the jugular vein and placed in a serum tube. After being placed under serum for 30 min, the sample was processed by centrifugation at a speed of 2000 rpm and stored frozen until further analysis. After death by jugular bleeding, the chickens were dissected, the gastrointestinal tract was removed, the small intestine was separated from the mesentery and connective tissue, and about 2 cm of the jejunum and the middle ileum were rinsed with phosphate buffer to remove feces. Part of the intestinal segment was fixed in paraformaldehyde solution for HE staining and immunofluorescence analysis, and part was placed in electron microscopy fixative for scanning and transmission electron microscopy observation. The remaining small intestinal segment was cut longitudinally and washed with PBS (1×), and then the jejunal and ileal mucosa were scraped with a sterile slide, collected in a sterile cryotube, quickly frozen in liquid nitrogen and stored at -80°C until further analysis;

[0046] (4) The broilers of each replicate were weighed on an empty stomach after a 14-h feeding stop on days 0, 14, and 28 of the experiment, and the feed consumption of each replicate on days 14 and 28 of the experiment was recorded to calculate the average daily weight gain, average daily feed intake, and feed-to-weight ratio for each period.

[0047] Embodiment 3:

[0048] The embodiment of the present invention provides a method for improving the growth of immune stress broiler chickens in a music environment, comprising the following steps:

[0049] (1) Two hundred one-day-old male broiler chickens of similar body weight were randomly divided into four treatment groups: control group (CON group), music stimulation group (MUC group), LPS immune stress group (LPS group) and music + LPS group (MUC + LPS group). Each treatment group had 10 replicates, with 5 chickens in each replicate. The music group and music + LPS group were given 7 hours of music stimulation (Mozart K.448) every day from the first day of age, and the sound was controlled at 70 decibels until the end of the experiment.

[0050] (2) On the 14th, 16th, and 18th days of the experiment, each chicken in the LPS immune stress group and the music + LPS group was intraperitoneally injected with 490 μg / kg LPS dissolved in saline to prepare the immune stress model, where the mass ratio of LPS to saline was 1:175. The control group and the music stimulation group were injected with an equal amount of saline;

[0051] (3) On the 28th day of the experiment, one chicken was randomly picked from each replicate in each group for sample collection. 4 ml of blood was collected from the jugular vein and placed in a serum tube. After being placed under serum for 29 minutes, the sample was processed by centrifugation at a speed of 1800 rpm and stored frozen until further analysis. After death by jugular bleeding, the chickens were dissected, the gastrointestinal tract was removed, the small intestine was separated from the mesentery and connective tissue, and about 2 cm of the jejunum and the middle ileum were rinsed with phosphate buffer to remove feces. Part of the intestinal segment was fixed in 4% paraformaldehyde solution for HE staining and immunofluorescence analysis, and part was placed in electron microscopy fixative for scanning and transmission electron microscopy observation. The remaining small intestinal segment was cut longitudinally and washed with PBS (1×), and then the jejunal and ileal mucosa were scraped with a sterile slide, collected in a sterile cryotube, quickly frozen in liquid nitrogen and stored at -80°C until further analysis;

[0052] (4) The broilers of each replicate were weighed on an empty stomach after a 13-h feeding stoppage on days 0, 14, and 28 of the experiment, and the feed consumption of each replicate on days 14 and 28 of the experiment was recorded to calculate the average daily weight gain, average daily feed intake, and feed-to-weight ratio for each period.

[0053] Embodiment 4:

[0054] The embodiment of the present invention provides a method for improving the growth of immune stress broiler chickens in a music environment, comprising the following steps:

[0055] (1) Two hundred one-day-old male broiler chickens of similar body weight were randomly divided into four treatment groups: control group (CON group), music stimulation group (MUC group), LPS immune stress group (LPS group) and music + LPS group (MUC + LPS group). Each treatment group had 10 replicates, with 5 chickens in each replicate. The music group and the music + LPS group were given 6 hours of music stimulation (Mozart K.448) every day from the first day of age, and the sound was controlled at 65 decibels until the end of the experiment.

[0056] (2) On the 14th, 16th, and 18th days of the experiment, each chicken in the LPS immune stress group and the music + LPS group was intraperitoneally injected with 480 μg / kg LPS dissolved in saline to prepare the immune stress model, where the mass ratio of LPS to saline was 1:150. The control group and the music stimulation group were injected with an equal amount of saline;

[0057] (3) On the 28th day of the experiment, one chicken was randomly picked from each replicate in each group for sample collection. 5 ml of blood was collected from the jugular vein and placed in a serum tube. After being placed under serum for 30 min, the sample was processed by centrifugation at a speed of 2000 rpm and stored frozen until further analysis. After death by jugular bleeding, the chickens were dissected, the gastrointestinal tract was removed, the small intestine was separated from the mesentery and connective tissue, and about 2 cm of the jejunum and the middle ileum were rinsed with phosphate buffer to remove feces. Part of the intestinal segment was fixed in paraformaldehyde solution for HE staining and immunofluorescence analysis, and part was placed in electron microscopy fixative for scanning and transmission electron microscopy observation. The remaining small intestinal segment was cut longitudinally and washed with PBS (1×), and then the jejunal and ileal mucosa were scraped with a sterile slide, collected in a sterile cryotube, quickly frozen in liquid nitrogen and stored at -80°C until further analysis;

[0058] (4) The broilers of each replicate were weighed on an empty stomach after a 13-h feeding stoppage on days 0, 14, and 28 of the experiment, and the feed consumption of each replicate on days 14 and 28 of the experiment was recorded to calculate the average daily weight gain, average daily feed intake, and feed-to-weight ratio for each period.

[0059] Embodiment 5:

[0060] The embodiment of the present invention provides a method for improving the growth of immune stress broiler chickens in a music environment, comprising the following steps:

[0061] (1) Two hundred one-day-old male broiler chickens of similar body weight were randomly divided into four treatment groups: control group (CON group), music stimulation group (MUC group), LPS immune stress group (LPS group) and music + LPS group (MUC + LPS group). Each treatment group had 10 replicates, with 5 chickens in each replicate. The music group and music + LPS group were given 8 hours of music stimulation (Mozart K.448) every day from the first day of age, and the sound was controlled at 75 decibels until the end of the experiment.

[0062] (2) On the 14th, 16th, and 18th days of the experiment, each chicken in the LPS immune stress group and the music + LPS group was intraperitoneally injected with 500 μg / kg LPS dissolved in saline to prepare the immune stress model, where the mass ratio of LPS to saline was 1:200. The control group and the music stimulation group were injected with an equal amount of saline;

[0063] (3) On the 28th day of the experiment, one chicken was randomly picked from each replicate in each group for sample collection. 4 ml of blood was collected from the jugular vein and placed in a serum tube. After being placed under serum for 30 min, the sample was processed by centrifugation at a speed of 1800 rpm and stored frozen until further analysis. After death by jugular bleeding, the chickens were dissected, the gastrointestinal tract was removed, the small intestine was separated from the mesentery and connective tissue, and about 2 cm of the jejunum and the middle ileum were rinsed with phosphate buffer to remove feces. Part of the intestinal segment was fixed in paraformaldehyde solution for HE staining and immunofluorescence analysis, and part was placed in electron microscopy fixative for scanning and transmission electron microscopy observation. The remaining small intestinal segment was cut longitudinally and washed with PBS (1×), and then the jejunal and ileal mucosa were scraped with a sterile slide, collected in a sterile cryotube, quickly frozen in liquid nitrogen and stored at -80°C until further analysis;

[0064] (4) The broilers of each replicate were weighed on an empty stomach after a 12-h feeding stop on days 0, 14, and 28 of the experiment, and the feed consumption of each replicate on days 14 and 28 of the experiment was recorded to calculate the average daily weight gain, average daily feed intake, and feed-to-weight ratio for each period.

[0065] Please refer to the attached Figure 2 The results of the determination of serum corticosterone (ACTH) and pro-epinephrine (CORT) levels in broiler chickens in each group were given. Compared with the control group, the content of CORT in serum of the music stimulation group was significantly reduced (P<0.05); the content of CORT and ACTH in serum of the LPS immune stress group was significantly increased (P<0.05); there was no significant difference in CORT and ACTH in serum of the music+LPS group (P<0.05). Compared with the LPS immune stress group, the content of CORT in serum of the music+LPS group was significantly reduced (P>0.05), and the content of ACTH had a downward trend but no significant difference (P>0.05).

[0066] Please refer to the attached Figure 3 The morphology of the intestinal villi of broilers observed by scanning electron microscopy showed that the villi of the jejunum and ileum of the control group and the music stimulation group were closely and neatly arranged, while the villi of the jejunum and ileum of the broilers in the LPS immune stress group and the music + LPS group were scattered and irregular.

[0067] Please refer to the attached Figure 4 The results of broiler production performance test showed that there were no significant differences in the average daily feed intake, average daily weight gain and feed-to-weight ratio among the treatments from 1 to 14 days of age (before LPS stimulation) (P>0.05).

[0068] At 14-28 days of age, compared with the control group, the average daily feed intake of broilers in the music stimulation group tended to increase, but there was no significant difference (P>0.05); the average daily feed intake and average daily weight gain of broilers in the LPS immune stress group were significantly reduced, and the feed-to-weight ratio was significantly increased (P<0.05); the average daily weight gain and feed-to-weight ratio of the music+LPS group were significantly reduced (P<0.05).

[0069] Compared with the LPS immune stress group, the average daily feed intake and average daily weight gain of broilers in the music + LPS group were significantly increased (P<0.05).

[0070] Please refer to the attached Figure 5 The results of serum cytokines and immunoglobulins of broiler chickens showed that compared with the control group, the contents of interleukin-4 (IL-4) and interleukin-10 (IL-10) in the serum of broiler chickens in the music stimulation group were significantly increased (P<0.05), immunoglobulin M (IgM) showed an upward trend but with no significant difference (P>0.05), and the contents of interleukin-1β (IL-1β), interleukin-2 (IL-2) and interleukin-4 (IL-6) were significantly decreased (P<0.05); the contents of IL-1β, IL-2, IL-6 and tumor necrosis factor α (TNF-α) in the serum of the LPS immune stress group were significantly increased, and the contents of IL-4, IL-10, immunoglobulin G (IgG) and IgM were significantly decreased (P<0.05); the content of TNF-α in the serum of the music + LPS group was significantly increased (P<0.05), and there was no significant difference in other cytokines (P>0.05).

[0071] Compared with the LPS immune stress group, the levels of IL-4, IL-10, IgG, and IgM in the serum of the music + LPS group were significantly increased (P<0.05), and IL-1β, IL-2, IL-6, and TNF-α were significantly decreased (P<0.05).

[0072] Please refer to the attached Figure 6, as shown by the expression of tight junction proteins in the small intestinal mucosa of broiler chickens, the results showed that in the jejunum, compared with the control group, the protein expression level of occludin in the jejunal mucosa of the music stimulation group was significantly increased (P<0.05); the protein expression level of claudin2 in the jejunal mucosa of the LPS immune stress group was significantly increased, and the protein expression levels of occludin, tight junction protein (ZO-1), and mucin 2 (Mucin2) were significantly decreased (P<0.05); the mRNA expression level of Claudin2 in the jejunal mucosa of the music + LPS group was significantly increased (P<0.05).

[0073] Compared with the LPS immune stress group, the protein expression levels of Occludin, ZO-1, and Mucin2 in the jejunal mucosa of the music+LPS group were significantly increased (P<0.05), and the mRNA expression level of Claudin2 was significantly decreased (P<0.05).

[0074] In the ileum, compared with the control group, the protein expression level of Occludin in the ileal mucosa of the music stimulation group was significantly increased (P<0.05); the protein expression level of Claudin2 in the ileal mucosa of the LPS immune stress group was significantly increased, and the protein expression level of Occludin was significantly decreased (P<0.05); there was no significant difference in the expression levels of Claudin2, Occludin, ZO-1, and Mucin2 in the ileal mucosa of the music+LPS group (P>0.05).

[0075] Compared with the LPS immune stress group, the protein expression level of Occludin in the ileal mucosa of the music+LPS group was significantly increased (P<0.05), and the protein expression level of Claudin2 was significantly decreased (P<0.05).

[0076] Note:

[0077] Attached Figure 3 The middle figure (A) - figure (D) are scanning electron microscopy images of the jejunum of broiler chickens in the control group, music stimulation group, LPS immune stress group, and music + LPS group, respectively.

[0078] Figures (E) and (H) are scanning electron microscopic images of the ileum of broiler chickens in the control group, music stimulation group, LPS immune stress group, and music + LPS group, respectively. The magnification in the figure is 120×, and the scale bar is 400μm.

[0079] Attached Figure 4The middle figures (A), (B), and (C) show the average daily feed intake, average daily weight gain, and feed-to-weight ratio of broilers from 1 to 14 days of age and 14 to 28 days of age, respectively. CON, control group; MUC, music stimulation group; LPS, LPS immune stress group; MUC+LPS, music+LPS group. Data are expressed as mean ± standard error. Different superscript letters a, b, and c in the bar graph indicate significant differences between groups (P<0.05), and no letters or the same letters indicate no significant differences (P>0.05).

[0080] Attached Figure 5 Figures (A)-(J) in the middle represent the levels of interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum of broilers in each group. CON, control group; MUC, music stimulation group; LPS, LPS immune stress group; MUC+LPS, music+LPS group. Data are expressed as mean ± standard error. Different superscript letters a, b, c in the bar graph indicate significant differences among the groups (P<0.05), and no letters or the same letters indicate no significant differences (P>0.05).

[0081] Attached Figure 6 Figures (A) and (B) represent the immunofluorescence images of Claudin2, Occludin, ZO-1 and Mucin2 in the jejunum and ileum of broilers, respectively. Figures (C) and (D) represent the statistical results of the average optical density values ​​of tight junction proteins Claudin2, Occludin, ZO-1 and Mucin2 in the jejunum and ileum of broilers, respectively. CON, control group; MUC, music stimulation group; LPS, LPS immune stress group; MUC+LPS, music+LPS group. Data are expressed as mean ± standard error. Different superscript letters a, b, c in the bar graph indicate significant differences between groups (P<0.05), and no letters or the same letters indicate no significant differences (P>0.05).

[0082] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the growth of immune stress broiler chickens in a music environment, characterized in that: The following steps are involved: S1: Multiple one-day-old male broilers were randomly divided into a control group (CON group), a music stimulation group (MUC group), an LPS immune stress group (LPS group) and a music + LPS group (MUC + LPS group), with 10 replicates in each group and 6 broilers in each replicate; S2: The broiler chickens in the music stimulation group and the music + LPS group were given 4-10 hours of music stimulation every day; S3: During the 12th to 20th day of the experiment, the broilers in the LPS immune stress group and the music + LPS group were intraperitoneally injected with LPS solution to prepare the immune stress model. LPS was dissolved in physiological saline. S4: On the 28th day of the experiment, blood and intestinal samples were collected and analyzed from broiler chickens in each group.

2. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: The music stimulus in step S2 is a classical sonata, and the volume is controlled between 60-80 decibels.

3. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: The LPS dose injected in step S3 is 450-550 μg / kg, and the mass ratio of LPS to physiological saline is 1:100 to 1:

250.

4. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: In step S3, the broilers in the control group and the music stimulation group were injected with the same amount of physiological saline at the same time point.

5. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: The blood sample in step S4 is collected from the jugular vein in a volume of 3-5 ml. After standing for 25-35 minutes, the blood sample is centrifuged at 1500-2500 rpm for 10-15 minutes, and the serum is frozen and stored at -80°C for further analysis.

6. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: In step S4, the feed consumption and weight change of each repeated broiler chicken are recorded, and the average daily weight gain, average daily feed intake and feed-to-weight ratio are calculated.

7. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: After the small intestine sample collected in step S4 is rinsed with phosphate buffer or physiological saline, the jejunum and ileum segments are taken, and part of the intestinal segments are fixed in 4% paraformaldehyde solution for HE staining and immunofluorescence analysis.

8. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: In the step S4, the partial intestinal segment of the small intestine sample is placed in 2.5% glutaraldehyde electron microscope fixative for scanning electron microscope and transmission electron microscope observation.

9. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: The remaining small intestine segment in step S4 was cut longitudinally, washed with sterile PBS (1×), and the jejunal and ileal mucosa were scraped with a sterile glass slide and collected into sterile cryotubes. The samples were quickly frozen and stored at -80°C for subsequent analysis.

10. The method for improving the growth of immune stress broiler chickens in a music environment according to claim 1, characterized in that: In step S4, the LPS-injected broiler chickens are killed by bleeding from the jugular vein, and the gastrointestinal tract is removed after dissection, and the small intestine segment is taken for histological and pathological analysis to evaluate the changes in the morphology of the small intestinal villi.

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