CN115005160B

CN115005160B - Method for evaluating influence of different sound stimuli on behavior, welfare, cognition and physiology of laying hens

Info

Publication number: CN115005160B
Application number: CN202210707288.0A
Authority: CN
Inventors: 赵帅; 尹国安; 杨春雪
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-03-14
Anticipated expiration: 2042-06-21
Also published as: CN115005160A

Abstract

The invention provides a method for evaluating the influence of different sound stimuli on the behavior, the welfare, the cognition and the physiology of laying hens. The method comprises the steps of layer feeding, pretreatment before testing, sample collection and treatment, behavior observation, behavior detection, egg quality detection, layer welfare evaluation, index detection and statistical analysis. The 65-75dB music stimulation increases the modification behavior and the comfort behavior of the laying hens, relieves the stress of the laying hens, enhances the immunity and the oxidation resistance of organisms and improves the welfare level of the laying hens. The noise stimulation of 65-75dB causes the continuous inflammatory reaction of the laying hens, reduces the immunity and the antioxidant capacity of the organism, and leads to the reduction of the welfare level of the laying hens.

Description

Method for evaluating influences of different sound stimuli on behavior, welfare, cognition and physiology of laying hens

Technical Field

The invention belongs to the technical field of animal welfare, and particularly relates to a method for evaluating the behavior, welfare, cognition and physiological influences of different sound stimuli on laying hens.

Background

With the widespread use of music in the human medical field, more and more scholars are beginning to focus on its application in animal husbandry production. The uethake study found that playing classical music on cows in a milking parlor could increase the milk production of cows. Liujiajiajia finds that simple-rhythm and mellow-tone light music has positive effects on lactation, energy metabolism and neuroendocrine of cows, and the music possibly transfers information and energy through changes of rhythm, tone and the like to cause harmonious resonance of various organs of an animal body, so that the physiological rhythm of the organs is promoted to be consistent, the disordered state of the organs is improved, the emotion and physiological response are regulated, and further the positive effects are generated on the lactation capacity of the cows. Papoutsogou found that mozart music can promote carp growth and improve carcass and fatty acid composition by way of stress relief. The research also finds that the feed conversion rate and the weight can be improved by playing the cry of the hens in the feeding stage from the birth to the 9 th day of the broiler chicken. Gvaryahu found that the number of times of feeding of broilers contacted with intermittent classical music was significantly increased and the rate of weight gain was significantly increased, compared to broilers not contacted with music. However, music was found to have no significant effect on piglet growth in the plum aromas study.

Captive animals are exposed to a variety of environmental stresses during the rearing process, such as temperature changes, ammonia, shipping, rearing density, and the like, which can adversely affect animal production and management. Noise, which is one of the environmental stressors, also adversely affects animal productivity. It was found that prolonged noise stimulation can lead to slow growth in pigs. An exemplary study in wu finds that long-term noise affects the development of the ovary of a laying hen, which can lead to the reduction of the productivity of the laying hen. In the study of Zhouzuhua, the 73dB noise can cause the irritability of the dairy cows, reduce the milk yield, increase the acidity of the milk and other consequences. And 87dB of noise can reduce the laying rate of the laying hens and increase the egg breaking rate. However, no method for evaluating the behavioral, welfare, cognitive and physiological influences of the laying hens according to different sound stimuli exists in the prior art.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a method for evaluating the influence of different sound stimuli on the behavior, the welfare, the cognition and the physiology of laying hens.

The invention is realized by the following technical scheme, and provides a method for evaluating the influence of different sound stimuli on the behavior, the welfare, the cognition and the physiology of laying hens, which specifically comprises the following steps:

feeding the laying hens: purchasing 360 chicks of 1 day old with the weight of 43 +/-4 g, randomly dividing the chicks into 3 groups of 120 chicks, respectively feeding the chicks in 3 artificial climate chambers, wherein each group has 8 repetitions, each repetition has 15, and each repetition internally marks 3 target animals; the test time was 42 weeks, during the whole test period, the chickens had free access to food and water;

treatment in the early stage of the test: the 3 test groups were: group C, group LM and group LN;

group C: control group, fed under normal conditions without additional sound addition;

and (3) LM group: in the low intensity music group, the chicken is exposed to classical music with sound intensity of 65-75 dB;

LN group: a low-intensity noise group, wherein the chickens are only exposed to noise with the sound intensity of 65-75dB, and the noise is background sound recorded in a feeding farm in advance;

the sound stimulation is continued from day 1 to 16 weeks of age, during which the test group performs sound stimulation every day, the sound playing time is 7-00;

collecting and processing samples: randomly selecting 1 laying hen in each treatment group repeatedly in each treatment group respectively in six weeks of 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks, killing 8 laying hens in total per group while avoiding marked animals for behavior observation, immediately collecting blood, collecting the blood in a centrifuge tube, placing the centrifuge tube in a4 ℃ refrigerator for coagulation for 24 hours, collecting the blood in an EP tube, placing the EP tube in the 4 ℃ refrigerator for coagulation for 24 hours, then centrifuging for 15 minutes at 4000 revolutions per minute, preparing serum, transferring the prepared serum to a new 1.5mL EP tube, and placing the new EP tube in a-80 ℃ refrigerator for storage for subsequent ELISA, oxidation and oxidation resistance detection; cutting open the chicken after blood collection, rapidly collecting brain, hypothalamus and thymus tissue, and storing in a refrigerator at-80 deg.C;

and (4) observing behaviors: counting the state behaviors in each behavior observation time period by taking 5s as 1 time unit, and converting the behavior data into the percentage of the total observation time; the method comprises the following steps that (1) a target animal sampling continuous observation method is adopted for completely recording the behaviors of observed laying hens once, the occurrence of each event behavior is recorded once, and the total occurrence times of each event in behavior observation time are used for representing;

and (3) behavior detection: carrying out a one-time avoidance test and an open field test on the laying hens;

and (3) detecting the quality of the eggs: carrying out statistics on the egg yield, the egg laying rate and the egg breaking rate in four stages of 18-24 weeks, 24-30 weeks, 30-36 weeks and 36-42 weeks; randomly selecting 10 eggs for each treatment group at 23 weeks, 29 weeks, 35 weeks and 41 weeks to detect the egg quality;

evaluating the welfare of the laying hens: evaluating welfare indexes of the laying hens;

index detection: oxidation and oxidation resistance indexes, physiological indexes and immune indexes in serum and PCR detection are carried out on the laying hens;

statistical analysis: firstly, carrying out normal distribution test on data by Shapiro-Wilk, carrying out lg and ln conversion on the data which is not subjected to normal distribution, then, all the data are subjected to normal distribution and show homogeneity of variance, analyzing an ELISA result, an oxidation and oxidation resistance result, a real-time fluorescence quantitative PCR result and a protein expression result by adopting single-variable variance analysis in a general linear model, analyzing behavior data, welfare evaluation and production performance by using single-factor variance analysis and Duncan method for multiple comparison, and carrying out Fisher accurate test on the avoidance rate of a disposable avoidance test; all results were plotted by GraphPadPrism7, with data expressed as mean ± standard error, and P <0.05 indicating significant differences.

Further, the temperature, humidity, ventilation and illumination in the artificial climate chamber are controlled by a central controller, the temperature is set to be 33-35 ℃ in the first week, the temperature is reduced by 2 ℃ every week till the temperature is 21-23 ℃ when the artificial climate chamber is 8 weeks old, and the temperature is controlled to be 18-24 ℃; during the test period, an artificial illumination system is adopted, the illumination is carried out for 1-3 days old and 23 hours every day, the illumination is carried out for 1 hour in darkness, the illumination is carried out for 22 hours for 4 days old, the illumination is reduced for 1 hour every day till 14 days old, the illumination is carried out for 12 hours and the darkness is carried out for 12 hours, the illumination is kept unchanged until 8 weeks old and 8-18 weeks old maintain the illumination for 8 hours, the illumination is increased for 1 hour every week till 26 weeks old and the illumination is carried out for 16 hours, and the illumination is kept till the test is finished; the illumination intensity is 10-15lux, and the humidity is 60-70%.

Further, the stateful behaviors include eating, lying on the stomach, walking, and standing behaviors.

Further, the incident behavior includes drinking, pecking, grooming, comfort and feather pecking, and aggressive pecking.

Further, the disposable avoidance test specifically comprises:

before pre-experimental training, putting a piece of white A4 paper on the ground in a test box, putting the chicks in the test box for adaptive training for 3 hours, and fasting and water prohibition; after the adaptive training is finished, removing A4 white paper, and performing pre-training for 3 times; in the pre-training, a piece of red A4 paper is used for containing normal food and is placed in a test box to attract the attention of the chicks and peck the food, the normal food is displayed to the chicks for 1min in the 1 st and 2 nd pre-experiments, the normal food is displayed to the chicks for 2min in the 3 rd pre-experiments, the interval of each pre-experiment is 5min, and the training test is carried out after 5min of the last pre-training is finished;

during the training trial, the chicks were shown with a blue A4 paper holding the 99% mea soaked feed for 2min; the chicks have aversion reaction after pecking and wipe the mouth on the ground of the cage; and when the chicks peck the food soaked with the MeA and have obvious aversion reaction, taking out the blue paper and the feed, starting timing, and detecting the learning and memory ability after 120 min.

Furthermore, the test of the detection capability of the learning and memory capability is carried out for 2 times, in the first test, the normal food for 1min is firstly displayed for the chicks, but the food is put on a blue paper which has the same color as the food containing MeA, and after the test is finished, the paper and the food are taken away; after 5min intervals, carrying out a second test to show the normal food for 1min for the chicks, and using red paper with the same color as the red paper for containing the normal food in the previous pre-test; during each trial the target chicken pecked and did not peck, and during the trial the layer would relate the color of the paper to the taste of the food and would show avoidance behavior for paper of the color associated with MeA food, but would not avoid paper of the color associated with normal food; after the test was completed, the test chickens were returned to the cages.

Further, the open field test is specifically as follows: the chicken is raised to 23 days for the open field test, 8 chickens are randomly selected from each treatment group, and the open field test is carried out in a single room; in the open field test, 1.5m multiplied by 1.5m square wood boards are used as an open field area, 1.5m high wood boards are used for surrounding the open field area, and white masking paper is used for dividing the open field area into 25 0.3m multiplied by 0.3m square areas; during the test, a chick was held in the open field by the observer, placed in the central grid area of the field, and covered with a 20cm × 30cm carton, which was lifted when the observer left the open field, and began to record the following behavior in 10 minutes by a video recording device placed above the open field, and recorded the following behavior: rigor time, total cry, number of walking steps, number of excretions, and total number of grid walks.

Further, 1 chicken per group was repeatedly selected on 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks of day, respectively, for a total of 8 chickens per group, and 1 chicken per group was repeatedly selected on 23 weeks, 29 weeks, 35 weeks and 41 weeks of day of the laying period, for a total of 12 chickens per group, and the welfare index was evaluated; the welfare index evaluation comprises body weight, duration of tension immobility and feather quality score in the egg producing period.

Further, the feather quality score specifically comprises: evaluating the feather quality of eight parts of the body of the laying hen, namely the head, the neck, the back, the wings, the chest, the legs, the tail and the anus; the feather quality score of each part can be divided into 0-3 points according to the damage degree, namely 0 point-the feather is complete and has no damage; score 1-feather is slightly broken and completely covers the skin; 2 min-feather breakage and skin part exposure, exposure area < 1cm x 1 cm; 3 minutes-feather damaged and skin exposed area > 1cm x 1 cm; finally, the scores for each site are summed, i.e., the highest score is 24 points and the lowest score is 0.

Drawings

FIG. 1 is a flow chart of a one-time avoidance test;

fig. 2 is a schematic diagram of the effect of different sound stimuli on the laying behavior of a chicken;

FIG. 3 is a schematic illustration of the effect of different sound stimuli on the standing behavior of a chicken;

FIG. 4 is a schematic illustration of the effect of different sound stimuli on the walking behavior of chicks;

FIG. 5 is a schematic illustration of the effect of different sound stimuli on the feeding behavior of chicks;

FIG. 6 is a schematic illustration of the effect of different sound stimuli on chick grooming behavior;

FIG. 7 is a schematic illustration of the effect of different sound stimuli on the drinking behavior of chicks;

FIG. 8 is a schematic illustration of the effect of different sound stimuli on the comfort behaviour of a chick;

fig. 9 is a schematic illustration of the effect of different sound stimuli on the cage pecking behavior of a chicken;

fig. 10 is a schematic diagram of the effect of different sound stimuli on the feather picking behavior of chicks.

Detailed Description

The technical solutions in the embodiments of the present invention will be described below clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Laboratory animals and breeding management

Roman white layer chickens were used as test animals. 360 1-day-old chicks weighing 43 ± 4g were purchased from commercial breeder houses, randomly divided into 3 groups of 120, and individually housed in 3 climatic chambers, each group having 8 replicates, each replicate having 15, each replicate internally labeled with 3 target animals (three-color red, blue, and green markers). Cage culture, cage size of 98cm 92cm 45cm, and breeding density of 600cm 2/cage. Chickens were raised to 17 weeks of age for herding. Each treatment group randomly selected 36 laying hens, randomly divided into 12 repetitions, each repetition 3, and transferred into a traditional egg laying cage. The cage size was 192cm 31cm, 12 animals per cage, and the feeding density was 496cm2 animals per animal.

The test period was 42 weeks, and during the whole test period, the chickens were fed with water and fed with standard chicken feed (18.0% crude protein, 7.0% crude fiber, 8.0% crude ash, 0.4-1.5% calcium, 0.3% total phosphorus, 0.3-1.2% sodium chloride, 14% water, 0.4-0.9% methionine), compound feed for the growing period (16.0% crude protein, 7.0% crude fiber, 8.0% crude ash, 0.4-1.5% calcium, 0.3% total phosphorus, 0.3-1.2% sodium chloride, 14% water, 0.3-0.9% methionine), compound feed for the laying period (16.0% crude protein, 7.0% crude fiber, 18.0% crude ash, 2.0-5.0% calcium, 0.3% total phosphorus, 0.3-1.2% sodium chloride, 14% water, 0.35-0.9% methionine). The temperature, humidity, ventilation and illumination in the climatic chamber are controlled by a central controller, the temperature is set to 33-35 ℃ in the first week, then the temperature is reduced by 2 ℃ every week until the temperature is 21-23 ℃ when the climate is 8 weeks old, and then the temperature is controlled at 18-24 ℃. During the test period, an artificial illumination system is adopted, the illumination is carried out for 1-3 days old and 23 hours every day, the illumination is carried out for 1 hour in darkness, the illumination is carried out for 22 hours every 4 days old, the illumination is reduced for 1 hour every day till 14 days old, the illumination is carried out for 12 hours and the darkness is carried out for 12 hours, the illumination is kept unchanged until 8 weeks old and 8-18 weeks old maintain the illumination for 8 hours, the illumination is increased for 1 hour every week till 26 weeks old and the illumination is carried out for 16 hours, and the illumination is kept till the test is finished. The illumination intensity is 10-15lux, and the humidity is 60-70%. The chickens were immunized according to the routine procedure during the experimental period.

Pretreatment of test

The 3 test groups were: group C, group LM and group LN.

Group C: control group, fed under normal conditions, without additional sound addition.

LM group: in the low intensity music group, the chickens were exposed only to classical music with a sound intensity of 65-75dB (Mozart's String questions, K428, K525, K458).

LN group: in the low-intensity noise group, the chicken is exposed to the noise with the sound intensity of 65-75dB, and the noise is the background sound (namely mechanical sound such as a fan and the like) recorded in the breeding farm in advance.

The sound stimulation was continued from day 1 until 16 weeks of age. During this period, the test group was subjected to sound stimulation every day for a period of 7. Within 12h of sound playing, the player plays the sound cyclically (1 h on/1 h off). The sound playing is automatically played by connecting two loudspeakers (SPA 311, philips (China) investment Limited, shanghai, china) in the middle of the artificial climate chamber to the notebook computer according to a preset program. The background sound intensity for all test groups was 40dB, which cannot be excluded, but is much lower than the sound playback intensity, thus excluding the possibility of acoustic interference.

Sample collection and processing

Laying hens were randomly picked per repetition (avoiding animals marked for behavioral observation) in each treatment group for 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks on saturday, respectively, for a total of 8 hens/group were slaughtered and blood was immediately collected. Collecting blood in a centrifuge tube, placing the centrifuge tube in a refrigerator at 4 ℃ for coagulation for 24h, collecting blood in an EP tube, placing the EP tube in the refrigerator at 4 ℃ for coagulation for 24h, then centrifuging the centrifuge tube for 15min at 4000 rpm, preparing serum, transferring the serum into a new EP tube with the volume of 1.5mL, placing the EP tube in a refrigerator at-80 ℃ for storage, and using the EP tube for subsequent ELISA, oxidation and oxidation resistance detection. The collected chicken is cut open, and brain, hypothalamus and thymus tissue are collected rapidly and stored in a refrigerator at-80 deg.C for use.

Detection of behavioral indicators

1. Behavioral observations

The behavior data is recorded by video recording through monitoring equipment (DS-2 CD3T10D, hangzhou Haokiwei digital technology GmbH, hangzhou China), a camera is fixed opposite to a coop in the test, and the angle is based on observing the behaviors of all laying hens in the coop. The behavior video Recording time is 1, 2, 4, 10, 16 weeks, friday morning (9-10. The event behaviors (drinking, pecking, dressing, comfort and feather pecking and aggressive pecking) are recorded once by Sampling (Focal Sampling) Continuous observation (Continuous Recording) of target animals, and the event behaviors are recorded once every occurrence and expressed by the total times of the occurrences of the behaviors in the behavior observation time.

2. Behavior detection

One-time avoidance test:

when the laying hens grow for 21 days, the chicks are subjected to a one-time avoidance test. Due to time and space limitations, the experiments were performed in two days. The chicks were fasted for 7h in advance and 24 chicks were randomly selected per treatment group and divided into 12 pairs. The test chamber (60 × 50 × 40cm) was placed in the arm by the observer. The box is trilateral paper, and a plastic net is convenient for the researcher to observe from a distance. The top of the box body is covered by a paperboard, and an LED lamp with 25 watts is selected for illumination in the box. Each pair of chicks was marked with a black mark pen to distinguish when recording data. The overall test is shown in FIG. 1.

Before pre-experimental training, a piece of white A4 paper is placed on the ground in a test box, and the chicks are placed in the test box for adaptive training for 3 hours and are fasted and forbidden to drink. After the adaptive training is finished, the A4 white paper is removed and pre-training is performed for 3 times. In the pre-training, a piece of red A4 paper was placed in the test chamber to attract the attention of the chicks and peck, and normal food was shown to the chicks for 1min in the 1 st and 2 nd pre-experiments and for 2min in the 3 rd pre-experiment. The interval of each pre-experiment is 5min, and the training experiment is carried out 5min after the last pre-training is finished.

During the training trial, chicks were shown with a blue A4 paper holding 99% MeA soaked feed for a 2min duration. The chicks have aversion reactions after pecking, such as: shake head, close eyes, wipe mouth on the floor of cage (prepare fresh MeA feed every day, ensure each feed is moist, dry naturally during pre-training). When the chicks peck the food soaked with the MeA and obvious aversion reaction occurs, taking out the blue paper and the feed, starting timing, and detecting the learning and memory ability after 120 min.

The testing of the learning and memory ability is carried out for 2 times, in the first test, the normal food for 1min is firstly displayed for the chicks, but the food is put on a piece of paper (blue) with the same color as the MeA food which is put in the first test, and the paper and the food are taken away after the end; after 5min intervals, a second trial was performed showing the chicks with 1min of normal food using paper (red) of the same colour as the normal food contained in the previous pre-trial. Both the target chicken pecked and did not peck at each trial. In the experiment, the layer chicken will correlate the color of the paper with the taste of the food and will show avoidance of the paper of the color associated with the MeA food, but will not avoid the paper of the color associated with the normal food. After the test, the test chickens were returned to the cages.

The learning and memory ability of the chicks was calculated using the resolution (total number of chicks pecking red without pecking feed on blue paper x 100%/total number of test chicks per treatment group). Each chick was used only once in training and resolution tests. During the entire PAL test, chicks that did not peck at any of the three stages of the three pre-training, training and memory testing were excluded from the statistics.

Open field test:

the chicks were raised to 23 days for the open field test, with 8 chicks randomly selected per treatment group and subjected to the open field test in a separate room. The open field test uses 1.5m square boards as the open field area, and the boards are surrounded by 1.5m high boards. The open field area was divided into 25 square areas of 0.3m x 0.3m using white textured paper. During the test, a chick was held in the open field by the observer, placed in the central grid area of the field, and covered with a 20cm × 30cm carton, which was lifted when the observer left the open field, and began to record the following behavior in 10 minutes by a video recording device placed above the open field, and recorded the following behavior: the number of the walking steps (the total number of the chicks walking in the open field), the number of the excretion times (the total number of the chicks excreting in the open field within 10 min), and the number of the square grid walking times (the total number of the chicks passing through the square grid).

Detection of laying performance and egg quality of laying hens

Carrying out statistics on the egg yield, the egg laying rate and the egg breaking rate in four stages of 18-24 weeks, 24-30 weeks, 30-36 weeks and 36-42 weeks; the egg quality test is carried out by randomly selecting 10 eggs for each treatment group of 23 weeks, 29 weeks, 35 weeks and 41 weeks, and the egg quality test mainly comprises egg shape index, egg weight, eggshell strength, eggshell thickness, egg white height and Haugh unit. The specific calculation method is shown in table 1:

TABLE 1 egg quality inspection index and calculation mode

Evaluation of welfare of laying hens

For each group, 1 chicken was repeatedly selected on 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks of the day, and 8 chickens were counted for each group, and for each group, 1 chicken was repeatedly selected on 23 weeks, 29 weeks, 35 weeks and 41 weeks of the day of the laying period, and 12 chickens were counted for each group, and the welfare index was evaluated. Mainly comprises body weight, duration of tension immobility and feather quality score in the egg producing period.

Tension immobility:

after the layers were held for a few seconds, the layers were placed in a U-shaped wooden structure with the back facing down. The chick was then gently pressed on the breast with one hand and on the head and neck with the other hand to initiate catatonic induction. The induction time was 15s, and after the testers released their hands slowly, the time required for the chicks to stand up while turning over was recorded, i.e. the duration of tension immobility (no other movements than breathing and light shivering). Then the testers quit the sight of the chicks and observe the behavior of the chicks, if the chicks turn over and stand for less than 10s, the tension immobility induction fails, the induction needs to be started again, and the induction times are 3 times at most. Conversely, if the chicks did not stand up with a turn over within a 15min test period, the duration of tension immobility was recorded as 900s.

Feather quality scoring:

and (4) performing feather quality scoring on the laying hens in the egg producing period. Feather quality evaluation is mainly carried out on eight parts of the body of the laying hen, namely the head, the neck, the back, the wings, the chest, the legs, the tail and the anus. The feather quality score of each part can be divided into 0-3 points according to the damage degree, and the specific scoring standard is shown in table 2. I.e. 0 min-feather is intact and not damaged; score 1-feathers are slightly broken and completely cover the skin; 2 min-feather breakage and skin part exposure, exposure area < 1cm x 1 cm; 3 min-feathers damaged and skin exposed area > 1cm x 1 cm. Finally, the scores for each site are summed, i.e., the highest (worst) score is 24 and the lowest (best) score is 0.

TABLE 2 feather quality score criteria

Detection of oxidation and anti-oxidation indexes

The oxidation and oxidation resistance indexes in serum of each group of chickens with the age of 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks are respectively detected. The measurement indexes include: superoxide dismutase (SOD), peroxidase (CAT), and hydrogen peroxide (H) ₂ O ₂ ) Nitrogen monoxide (NO), nitric oxide synthase (iNOS), and Malondialdehyde (MDA).

Detection of physiological index and immunological index in serum

The concentrations of immunoglobulin (IgA, igG), cytokine (IL-2, IL-6, IL10, TNF-alpha, IFN-gamma), dopamine (DA) and Corticosterone (CORT) in the serum of each group of chickens aged 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks were measured by enzyme-linked immunosorbent assay (ELISA).

Detection of real-time fluorescent quantitative PCR

Total RNA was extracted from the brains, hypothalamus and thymus of each group of laying hens aged 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks, respectively, using RNAiso Plus (Takara, da., china). The RR047 reverse transcription kit (Takara, dalian, china) was chosen and the instructions were followed to reverse transcribe mRNA into cDNA. Configuring reverse transcription system on ice block. And (3) after the reaction solution is mixed softly and uniformly, placing the mixture in a water bath kettle at 37 ℃ for water bath for 15min, then transferring the mixture into a water bath kettle at 85 ℃ for water bath for 5sec, finally placing the mixture in a4 ℃ environment to finish reverse transcription reaction, and storing the cDNA product obtained by the reaction at-20 ℃ for subsequent real-time fluorescent quantitative PCR reaction.

Real-time fluorescent quantitative PCR use

(Roche, switzerland) was analyzed. A10. Mu.l reaction system for qRT-PCR was used. The qRT-PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, followed by denaturation at 95 ℃ for 15s, extension at 60 ℃ for 60s,40 cycles. Melting curve analysis showed a single peak for each PCR product. GAPDH as reference gene, according to 2 ^-△△CT The relative expression level of mRNA was calculated.

Statistical analysis of data

All data were statistically analyzed using the software SPSS 21. Firstly, carrying out normal distribution test on data by using Shapiro-Wilk, and after carrying out lg and ln conversion on data which is not subjected to normal distribution, all data are subjected to normal distribution and show homogeneity of variance. The ELISA result, the oxidation and oxidation resistance result, the real-time fluorescence quantitative PCR result and the protein expression result are analyzed by adopting univariate variance analysis in a general linear model, behavior data, welfare evaluation and production performance are analyzed by carrying out multiple comparisons by using a one-way ANOVA (one-way ANOVA) method and a Duncan method, and Fisher's Exact Test (Fisher's Exact Test) is carried out on the avoidance rate of a disposable avoidance Test. All results were plotted by GraphPad Prism7, data are expressed as mean. + -. Standard error (mean. + -. SEM), and P <0.05 indicates significant differences.

Influence of different sound stimuli on laying hen behavior

As can be seen from table 3 and fig. 2, the groveling behavior of the chicks in each group tended to increase from week 2 (P < 0.05) with increasing test time. On week 1, the lying time of group C was significantly higher than that of LM and LN groups (P < 0.05); at week 10, the lying time of the LM group is obviously higher than that of the LN group (P < 0.05), and the difference of the lying time of the C group and the lying time of the LM group and the LN group is not obvious (P > 0.05); in other different experimental stages, the differences in lying behavior between chicks in group C, LM and LN were not significant (P > 0.05).

TABLE 3 influence of different sound stimuli on the chick lying on stomach behavior

As can be seen from table 4 and fig. 3, the standing behavior of the chicks of each group tended to decrease with increasing test time (P < 0.05). At week 1, the standing behavior was significantly higher in the LM versus LN group chicks than in the C group (P < 0.05), but the LM versus LN group was not significantly different (P > 0.05). In the other different experimental stages, the standing behavior of chicks from group C, LM and LN was not significantly different (P > 0.05).

Table 4 influence of different sound stimuli on the standing behaviour of chicks

As can be seen from table 5 and fig. 4, the walking behavior of the groups of chicks exhibited a first significant decrease (P < 0.05) and then remained unchanged (P > 0.05) from week 2 as the test time increased. The proportion of the walking behavior of the chicks in each group was not significantly different (P > 0.05) at each experimental stage.

TABLE 5 Effect of different Sound stimuli on chick Walking behavior

As can be seen from table 6 and fig. 5, the feeding behavior of the chicks in group C showed a tendency to increase significantly (P < 0.05), then decrease significantly (P < 0.05) and then increase significantly (P < 0.05) with increasing test time. The LM group and the LN group showed a significantly increased trend (P < 0.05). The differences in the ratio of the feeding behavior of the chicks of each group were not significant (P > 0.05) at each experimental stage.

TABLE 6 influence of different sound stimuli on chick feeding behavior

As can be seen from table 7 and fig. 6, the modification behavior of the chicks of each group showed a tendency to increase significantly (P < 0.05), then decrease significantly (P < 0.05), and then increase significantly (P < 0.05) with increasing test time. At week 1, LM group modification behavior was significantly higher than C and LN groups (P < 0.05), C group was significantly higher than LN group (P < 0.05); at 2 weeks, the LM group modified behavior was significantly higher than the C and LN groups (P < 0.05), with no significant difference between the C and LN groups (P > 0.05); at 4 weeks, the modification behaviors of the LM group and the C group are obviously higher than those of the LN group (P < 0.05), and the difference between the LM group and the C group is not obvious (P > 0.05); in other experimental stages, the modification behavior was not significantly different in chicks of group C, LM and LN (P > 0.05).

TABLE 7 Effect of different Sound stimuli on chick modification behavior

As can be seen from table 8 and fig. 7, the drinking behavior of the chicks of each group showed a trend of significantly increasing (P < 0.05) with increasing test time. The drinking behavior of the chicks in each group was not significantly different at each experimental stage (P > 0.05).

TABLE 8 influence of different sound stimuli on the drinking behavior of chicks

As can be seen from table 9 and fig. 8, the comfort behavior of the chicks of each group showed a significant trend of increasing first and then decreasing (P < 0.05) with increasing test time. At weeks 1 and 2, the comfort behavior of the LM group was significantly higher than that of the C group and the LN group (P < 0.05), and the difference between the C group and the LN group was not significant (P > 0.05); in the other experimental stages, the differences in comfort behaviour were not significant for group C, LM and LN chicks (P > 0.05).

TABLE 9 Effect of different Sound stimuli on the comfort behavior of chicks

As can be seen from table 10 and fig. 9, the pecking behavior of the chicks of each group showed a tendency to increase and decrease significantly and repeatedly with increasing test time (P < 0.05). The cage pecking behavior of chicks from group C, LM and LN was not significantly different (P > 0.05) at each experimental stage.

TABLE 10 Effect of different Sound stimuli on chick cage pecking behavior

As can be seen from table 11 and fig. 10, the feather pecking behavior of the chicks of each group showed a tendency to be significantly increased (P < 0.05) with increasing test time. The pecking behavior of the chicks of each group was not significantly different at each experimental stage (P > 0.05).

TABLE 11 Effect of different Sound stimuli on feather picking behavior of chicks

Influence of different sound environments on learning and memory abilities of laying hens

The avoidance rate differences in the one-time avoidance test were not significant for the 3 week old control group (C), low decibel music group (LM), and low decibel noise group (LN) chicks (P > 0.05). As shown in table 12.

TABLE 12 Effect of different Sound stimuli on the Once-through avoidance task in 3 week old chicks

Influence of different sound stimuli on oxidation and antioxidation functions in serum of laying hens

The CAT activity in the serum of the hens at different time points in the same treatment group showed a trend of significantly and repeatedly decreasing and increasing with the increase of the test time (P < 0.05). At 1 week of age, the CAT activity in the serum of LM group is obviously higher than that of C group and LN group (P < 0.05), and the CAT activity in the serum of C group and LN group is not obviously different (P > 0.05); at the age of 2 weeks, the CAT activity in the LM group serum is obviously higher than that in the C group and the LN group (P < 0.05), and the CAT activity in the C group serum is obviously higher than that in the LN group (P < 0.05); in other experimental stages, the differences in CAT activity were not significant in the three groups of sera (P > 0.05).

With increasing test time, the SOD activity in the serum of the hens in the C group and LN group at different time points was maintained at the same level at 1, 2 and 4 weeks, and then was decreased significantly (P < 0.05), while the LM group showed a trend of increasing significantly and then decreasing (P < 0.05). The SOD activity in the serum of the C group is obviously higher than that of the LM group and the LN group (P < 0.05) at the age of 1 week, and the SOD activity difference in the serum of the LM group and the LN group is not obvious (P > 0.05); at the age of 2 weeks, the SOD activity in the blood serum of the LM group is obviously higher than that of the blood serum of the C group and the LN group (P is less than 0.05), and the SOD activity in the blood serum of the C group is obviously higher than that of the blood serum of the LN group (P is less than 0.05); in other experimental stages, the differences of the SOD activities of the three groups of serum are not significant (P > 0.05).

With the increase of the test time, H in the serum of the layer chicken at different time points in the same treatment group ₂ O ₂ The content showed a significant tendency to increase first and then decrease (P)<0.05). H in LN group serum at 1 week old ₂ O ₂ The content is obviously higher than that of C group and LM group (P)<0.05 H in group C and LM sera ₂ O ₂ The content difference is not significant (P)>0.05 ); h in LN group sera at 4 weeks of age ₂ O ₂ The content is obviously higher than that of C group and LM group (P)<0.05 Group C and LM group serum H) ₂ O ₂ The content difference is not significant (P)>0.05 ); other test phases, three groups of serum H ₂ O ₂ The content difference is not significant (P)>0.05)。

With increasing test time, the serum iNOS content of the hens in the C group and the LM group at different time points showed a marked increase and then a decrease at week 4 (P < 0.05), while the LN group showed a marked increase in repeated decrease (P < 0.05). At 1 week of age, iNOS activity in LN group serum was significantly higher than that in C group and LM group (P < 0.05), and iNOS activity difference in C group and LM group serum was not significant (P > 0.05); in other experimental stages, iNOS activity was not significantly different in the three sera (P > 0.05).

With increasing test time, the MDA content in the serum of the hens in the C group and the same treatment group in the LM group at different time points showed a significant increase and then a decrease at week 4 (P < 0.05), while the LN group showed a significant decrease from week 10 (P < 0.05). When the patients are aged for 1 week and 2 weeks, the content of MDA in the serum of the LN group is remarkably higher than that of the serum of the C group and that of the LM group (P < 0.05), and the difference of the content of MDA in the serum of the C group and that of the LM group is not remarkable (P > 0.05); in other experimental stages, the differences of MDA content in three groups of blood serum are not significant (P > 0.05).

With increasing test time, the NO content in the serum of the hens in the C group and the same treatment group in the LM group at different time points showed a significant increase and then a subsequent decrease at week 4 (P < 0.05), while the LN group showed a significant repeated decrease and increase (P < 0.05). At 1 week of age, the NO content in LN group serum is significantly higher than that in C group and LM group (P < 0.05), and the NO content difference in C group and LM group serum is not significant (P > 0.05); at the age of 2 weeks, the NO content in the serum of the LN group is obviously higher than that of the group C, and the NO content difference between the LM group and the serum of the group C and the LN group is not obvious (P < 0.05); in other experimental stages, the differences in NO content in the three groups of sera were not significant (P > 0.05).

Influence of different sound stimuli on physiological indexes in serum of laying hens

At the age of 2 weeks, the serum CORT concentration of the LN group is obviously higher than that of the C group and the LM group (P < 0.05), and the serum CORT concentration of the C group and the LM group is not obviously different (P > 0.05); the CORT concentration of the serum of the C group and the LN group is obviously higher than that of the LM group (P < 0.05) at the age of 4 weeks, and the CORT concentration of the serum of the C group and the LN group is not significantly different (P > 0.05); the serum CORT concentration differences were not significant between 1 week, 10 and 16 weeks of age (P > 0.05).

At 1 week of age, the DA concentration in the sera of the LM group and the LN group is obviously higher than that in the C group (P < 0.05), and the DA concentration in the sera of the LM group and the LN group is not obviously different (P > 0.05); at the age of 2 weeks, the concentration of the LM group serum DA is obviously higher than that of the C group and the LN group (P < 0.05), and the difference between the C group serum DA and the LN group serum DA is not obvious (P > 0.05); the differences in DA concentration among the three groups were not significant at 4, 10 and 16 weeks of age (P > 0.05).

The serum IgA concentration of LN group is obviously higher than that of C group and LM group (P < 0.05) at 1 week of age, and the difference between the serum IgA concentration of C group and LM group is not obvious (P > 0.05); at the age of 2 weeks, the IgA concentration of the LM group serum is obviously higher than that of the C group and the LN group (P is less than 0.05), and the IgA concentration difference of the C group serum and the LN group serum is not obvious (P is more than 0.05); at 4 weeks of age, the serum IgA concentrations of LM group and LN group were significantly higher than those of C group (P < 0.05), and the difference between the serum IgA concentrations of LM group and LN group was not significant (P > 0.05). The differences in serum IgA concentrations were not significant in the three groups at 10 and 16 weeks of age (P > 0.05).

At 1 week of age, the serum IgG concentration of LM group and LN group is obviously higher than that of C group (P < 0.05), and the difference between the serum IgG concentration of LN group and LM group is not obvious (P > 0.05); at the age of 2 weeks, the LM group serum IgG concentration is obviously higher than that of the C group and the LN group (P < 0.05), and the LN group and the C group serum IgG concentration are not obviously different (P > 0.05); three groups were not significantly different in serum IgG concentration at 4, 10 and 16 weeks of age (P > 0.05).

At 1 week of age, the IL-2 concentration in LN group is significantly higher than that in C group and LM group (P < 0.05), and the IL-2 concentration in C group and LM group is not significantly different (P > 0.05); at the age of 2 weeks, the IL-2 concentration in the LM group and LN group is obviously higher than that in the C group (P < 0.05), and the IL-2 concentration in the LM group is obviously higher than that in the LN group (P < 0.05); the IL-2 concentration in LN group serum is obviously higher than that in LM group and C group (P < 0.05) at 4 weeks of age, and the IL-2 concentration in LM group and C group serum is not significantly different (P > 0.05); three groups had insignificant differences in serum IL-2 concentrations between 10 and 16 weeks of age (P > 0.05).

When the patient is 1 week old, the IL-6 concentration in the LN group serum is obviously higher than that in the C group and the LM group (P < 0.05), and the IL-6 concentration in the C group serum is not obviously different from that in the LM group serum (P > 0.05); when the patient is 2 weeks old, the IL-6 concentration in the LN group and the LM group is obviously higher than that in the C group (P < 0.05), and the IL-6 concentration in the LM group is obviously higher than that in the LN group (P < 0.05); when the patient is 4 weeks old, the IL-6 concentration in LN group serum is obviously higher than that in LM group and C group (P < 0.05), and the IL-6 concentration difference in LM group serum is not obvious (P > 0.05); at the age of 10 weeks, the IL-6 concentration in LN group serum is significantly higher than that in C group and LM group (P < 0.05), and the difference between LM group and C group is not significant (P > 0.05); at 16 weeks of age, the differences in serum IL-6 concentrations were not significant in the three groups (P > 0.05).

At 1 week of age, the IL-10 concentration in LN group is significantly higher than that in LM group and C group (P < 0.05), and the IL-10 concentration in LM group and C group is not significantly different (P > 0.05); at the age of 2 weeks, the IL-10 concentration of the LN group and the LM group is obviously higher than that of the C group (P < 0.05), and the IL-10 concentration of the LM group and the LN group is not significantly different (P > 0.05); when the patient is at 4 weeks of age, the IL-10 concentration of the LN group and the C group serum is obviously higher than that of the LM group (P < 0.05), and the IL-10 concentration of the LN group serum is obviously higher than that of the C group (P < 0.05); the differences in serum IL-10 concentrations were not significant between the 10 and 16 weeks of age (P > 0.05).

In the whole test period, when the patients are aged for 1 week, 4 weeks, 10 weeks and 16 weeks, the serum TNF-alpha concentration of the LN group is obviously higher than that of the C group and the LM group (P < 0.05), and the serum TNF-alpha concentration difference between the C group and the LM group is not obvious (P > 0.05); at 2 weeks of age, the serum TNF-alpha concentrations in LM and LN groups were significantly higher than those in C group (P < 0.05), and the differences between the serum TNF-alpha concentrations in LM and LN groups were not significant (P > 0.05).

When the patient is 1 week old, the IFN-gamma concentration in the serum of the LN group is obviously higher than that of the C group and the LM group (P < 0.05), and the difference between the IFN-gamma concentration in the serum of the C group and that of the LM group is not obvious (P > 0.05); at the age of 2 weeks, the C group serum IFN-gamma concentration is obviously lower than that of LM group and LN group (P < 0.05), and the LM group serum IFN-gamma concentration is obviously higher than that of LN group (P < 0.05); the IFN-gamma concentration of LN group serum is obviously higher than that of C group and LM group (P < 0.05) at 4 weeks of age, and the difference between the IFN-gamma concentration of LM group and C group serum is not significant (P > 0.05); the differences in serum IFN-gamma concentrations were not significant in the three groups at 10 and 16 weeks of age (P > 0.05).

Influence of different sound stimuli on thymus immunity and cytokine mRNA expression of laying hens

At 1 week of age, the expression level of thymus IgAmRNA in the LM group is obviously higher than that in the C group and the LN group (P < 0.05), and the expression level of thymus IgAmRNA in the LN group is obviously higher than that in the C group (P < 0.05); at the age of 2 weeks, the expression level of thymus IgAmRNA of the LM group is obviously higher than that of the thymus IgAmRNA of the C group and the thymus IgAmRNA of the LN group (P < 0.05), and the expression level difference between the thymus IgAmRNA of the LN group and the thymus IgAmRNA of the C group is not obvious (P > 0.05); at 4 weeks, 10 weeks and 16 weeks of age, the difference in expression level of thymic IgA mRNA among the three groups was not significant (P > 0.05).

When the patients are 1 week old and 4 weeks old, the expression level of thymus IgG mRNA in the LM group is remarkably higher than that in the C group and the LN group (P < 0.05), and the difference between the expression levels of thymus IgG mRNA in the C group and the LN group is not remarkable (P > 0.05); when the mice were aged 2 weeks, 10 weeks and 16 weeks, the expression level of IgA mRNA was not significantly different among the three groups (P > 0.05).

At 4 weeks of age, the expression level of thymus IL-2mRNA of the LN group is obviously lower than that of thymus IL-2mRNA of the C group and the LM group (P < 0.05), and the difference between the expression level of thymus IL-2mRNA of the LM group and that of thymus IL-2mRNA of the C group is not obvious (P > 0.05); the difference of IL-2mRNA expression level of three groups of thymus is not significant when the mice are aged for 1 week, 2 weeks, 10 weeks and 16 weeks (P is more than 0.05).

At 1 week of age, the IL-6mRNA expression level of thymus in LN group is significantly higher than that in LN group and C group (P < 0.05), and the IL-6mRNA expression level of thymus in LM group is significantly higher than that in C group (P < 0.05); the differences of IL-6mRNA expression levels of three groups of thymuses are not significant when the mice are aged for 2 weeks, 4 weeks, 10 weeks and 16 weeks (P > 0.05).

When the thymus is aged for 1 week and 10 weeks, the expression level of thymus TNF-alpha mRNA in the LN group is obviously higher than that in the C group and the LM group (P < 0.05), and the expression level difference of the thymus TNF-alpha mRNA in the C group and the LM group is not obvious (P > 0.05); when the thymus is aged 2 weeks, 4 weeks and 16 weeks, the expression level of the thymus TNF-alpha mRNA of the three groups is not significant (P > 0.05).

At 4 weeks of age, the expression level of thymus IFN-gamma mRNA of the C group and the LM group is obviously higher than that of the LN group (P < 0.05), and the difference between the expression level of thymus IFN-gamma mRNA of the C group and that of the LM group is not obvious (P > 0.05); when the thymus is aged for 1 week, 2 weeks, 10 weeks and 16 weeks, the expression quantity difference of the thymus IFN-gamma mRNA in the three groups is not significant (P > 0.05).

Influence of different sound stimuli on expression level of heat shock protein and cytokine mRNA of hypothalamus of laying hen

The expression level of hypothalamic HSP60 mRNA in the LN group is obviously higher than that in the C group and the LM group (P < 0.05) at the age of 1 week and 10 weeks, and the difference between the expression levels of hypothalamic HSP60 mRNA in the C group and the LM group is not obvious (P > 0.05); at 2 weeks, 4 weeks and 16 weeks of age, the differences in the expression levels of hypothalamic HSP60 mRNA were not significant (P > 0.05).

When the mouse is aged 10 weeks, the expression level of hypothalamic HSP70mRNA of the LN group is obviously higher than that of the LM group and the C group (P < 0.05), and the difference of the expression level of hypothalamic HSP70mRNA of the C group and the LM group is not obvious (P > 0.05); three groups of hypothalamic HSP70mRNA expression levels were not significantly different at 1 week, 2 weeks, 4 weeks and 16 weeks of age (P > 0.05).

When the mouse is aged 10 weeks, the expression level of hypothalamic HSP90 mRNA of the LN group is obviously higher than that of the C group and the LM group (P < 0.05), and the difference of the expression level of hypothalamic HSP90 mRNA of the C group and the LM group is not obvious (P > 0.05); at 1 week, 2 weeks, 4 weeks and 16 weeks of age, the differences in expression levels of hypothalamic HSP90 mRNA were not significant (P > 0.05).

Throughout the experimental period, the expression level of IL-2mRNA in the hypothalamus of the three groups was not significantly different (P > 0.05). Throughout the experimental period, the expression level of IL-6mRNA in the hypothalamus of the three groups was not significantly different (P > 0.05).

When the mouse is aged 4 weeks, the expression level of the hypothalamus TNF-alpha mRNA in the LN group is obviously higher than that in the C group and the LM group (P < 0.05), and the expression level of the hypothalamus TNF-alpha mRNA in the C group and the LM group is not obviously different (P > 0.05); three groups of hypothalamus TNF-alpha mRNA expression level differences were not significant at 1 week, 2 weeks, 10 weeks and 16 weeks of age (P > 0.05).

Effect of different Sound stimuli on the brain transcriptome of 4-week-old chicks

The invention compares the gene expression difference between brains of a group C (DN 1), a group LM (DN 2) and a group LN (DN 3) pairwise, and the DN1 vs. DN2, the DN1 vs. DN3 and the DN2 vs. DN3 respectively have 93, 76 and 57 difference genes, wherein 63, 40 and 41 genes are up-regulated difference genes. In other words, DN1 vs. dn2 has more differential genes than DN1 vs. dn3 and DN2 vs. dn3. DN1 vs. dn2, DN1 vs. dn3 and DN2 vs. dn3 have 70, 39 and 37 unique difference genes, respectively.

The results of the PCA analysis were: DN2, DN1 vs. DN3 and DN2 vs. DN3 have different transcription profiles, and experiments prove that noise and music can affect the expression of partial genes of the brain of the laying hens and each has a unique expression pattern. And (3) carrying out cluster analysis on the samples by utilizing PCA (principal component analysis), and finding that the DN3 gene expression dispersion degree is large and the distribution range of the three samples is wide.

Clustering analysis results: common genes exist in three groups of DN1, DN2 and DN3, but the genes which are differentially expressed exist in certain biological processes, physiology, metabolism, development and cell signal paths among the three groups.

As can be seen from the genome circled diagram, the noise and music stimulation genes are basically enriched on the same position of the same chromosome as the control group, and only the up-regulation tendency and the down-regulation tendency are slightly different, and meanwhile, the difference genes on the 3, 7 and Z chromosomes are very few, which indicates that the 3, 7 and Z chromosomes are less involved in the regulation of the sound stimulation by the laying hens. The genes are differentially distributed throughout each chromosome between different groups.

The first 20 GO Term entries with the smallest FDR value, i.e. most significantly enriched, were selected for display by the present invention. And (3) carrying out GO classification on GO enrichment analysis results of the differentially expressed genes according to Molecular Functions (MF), biological Processes (BP) and Cell Components (CC), and selecting the top 10 GO term items with the smallest p value, namely the top 10 GO term items with the most remarkable enrichment in each GO classification for display.

GO enrichment analysis is carried out on 93 differential expression genes between DN1 and DN2, and the result shows that 2 differential expression genes are related to a chaperone complex in the aspect of cell components, and 1 differential expression gene is related to a Tle3-Aes complex; in the molecular function, 14 differentially expressed genes participate in the activity of RNA polymerase II transcription factors, and 14 differentially expressed genes participate in the combination of DNA double strands; biological process analysis shows that 2 differentially expressed genes act on positive regulation and control of phagocytosis behavior, and 2 differentially expressed genes act on negative regulation and control of T cell differentiation. GO enrichment analysis is carried out on 57 differential expression genes between DN2 and DN3, and the result shows that 6 differential expression genes are related to cilia and 3 differential expression genes are related to the moving cilia in the aspect of cell components; in the molecular function, 2 differential expression genes participate in thyroid hormone combination, 2 differential expression genes are related to the activity process of neuropeptide hormone, biological process analysis shows that the 2 differential expression genes are involved in the detection of temperature stimulation in a sensor, and the 2 differential expression genes are involved in the detection of the temperature stimulation in a sensor. GO enrichment analysis is carried out on 76 differential expression genes between DN1 and DN3, the result shows that 2 differential expression genes in the aspect of cell components are related to mast cell particles, 7 differential expression genes are related to cilia, 9 differential expression genes in the molecular function are related to the activity of RNA polymerase II transcription factors, 10 differential expression genes participate in sequence-specific double-strand-combination, and biological process analysis shows that 2 differential expression genes are secretion regulation.

The minimal P value in the KEGG enrichment analysis result of the differential genes is the most significant first 30 metabolic pathways. In DN1 vs. dn2, 1 of the significantly different pathways is a sulfur metabolic pathway; dn3, among 1 significantly different pathway, 1 is a neuroleptic receptor interaction pathway; dn3, there are 4 distinct pathways, histidine, phenylalanine, alanine and tyrosine, respectively.

From the enrichment analysis bubble analysis results, it can be seen that in DN1 vs. dn2, there is gene enrichment difference in the MAPK signaling pathway, the endogenous ligand signaling pathway, and the calcium signaling pathway; dn3, there is differential gene enrichment in tyrosine metabolism, phenylalanine metabolism, alanine metabolism and histidine metabolism; dn3, the neuroligand receptor interaction pathway and the MAPK signaling pathway are differentially gene enriched.

Influence of different sound stimuli on weight and fear level of chicks

As can be seen from table 13, the body weight differences of the three groups of layers were not significant (P > 0.05) at each experimental stage. At week 1, the duration of catatonic inactivity in LN group was significantly higher than that in C and LM groups (P < 0.05), which were not significantly different (P > 0.05); in other experimental stages, the duration of catatonic immobility was not significantly different in the three groups (P > 0.05).

TABLE 13 influence of different sound stimuli on weight and tension immobility of chicks

As can be seen from table 14, at 3 weeks of age, the stiffness time of the LN group laying hens was significantly higher than that of the C group and the LM group (P < 0.05), and the difference between the C group and the LM group was not significant (P > 0.05); the number of steps of the LM group is obviously higher than that of the LN group (P < 0.05), and the difference between the number of steps of the C group and the LM group and the number of steps of the LN group is not obvious (P > 0.05); the total number of times that the LM group walks through the squares is obviously higher than that of the LN group (P < 0.05), and the walking number difference between the C group and the LM group and the LN group is not obvious (P > 0.05); the total sound and excretion times were not significant (P > 0.05).

TABLE 14 Effect of different Sound stimuli on the open field test in 3-week-old chicks

Influence of different sound stimuli on feather scoring and fear level of laying hens in egg producing period

As can be seen from Table 15, the differences between the body weights, feather mass fractions and duration of catatonic immobility of three groups of laying hens were not significant (P > 0.05) at each experimental stage of the laying period.

TABLE 15 influence of different sound stimuli on the weight and tension immobility of laying hens in laying period

Effect of different sound stimuli on the laying behavior and the performance of laying hens during the laying period

The difference between the state behavior and the event behavior of the three groups of laying hens in the laying period is not significant (P > 0.05), and the difference between the quality and the production performance of the eggs of the three groups of laying hens in the laying period is not significant (P > 0.05).

The invention discovers that music and noise stimulation at 1 week old obviously reduces the lying behavior of the laying hens and increases the standing behavior, but has no influence on the ingestion behavior and the walking behavior. Some studies found that animals decreased prone behavior and increased standing behavior in enriched environments. Music and noise stimulation can reduce the lying behavior of the piglets and increase the standing and walking behaviors. This indicates that short term exposure to music and noise can make the layer chicken more active, probably because music and noise attract their attention, resulting in reduced groveling behavior and increased standing behavior of the layer chicken. Studies have shown that classical music is beneficial to animals, making them more relaxed. The study of mariju finds that music can eliminate the tension of rats and enable the rats to be more relaxed. In this experiment, at 10 weeks of age, the music group laying hens had more prone behavior than the noise group laying hens, probably because the music stimulation made the laying hens more relaxed in mood.

The modified behaviour of an avian represents to some extent its relaxed state. Laying hens living in the enriched environment have more natural behavior expressions compared with the barren environment, which shows that the rich environmental conditions can make the laying hens feel more comfortable. In this trial, musical stimulation increased the grooming and comfort behavior of layers at 1 and 2 weeks of age. Therefore, music is a beneficial environmental stimulus for laying hens. Researches find that the noise environment in the henhouse of the commercial chicken farm can influence the sleep and rest of the broilers, so that the broilers are irritated, tense and frightened. Female rats develop anxiety in a sustained 65dB noise (sound with all frequencies) environment, while the same decibel environment of mozart piano curve k.448 reduces anxiety in female rats. Wuyi research also finds that the noise environment can increase the alertness response of the chicken. These anxiety, stress fear, and alertness may affect the expression of normal behavior in birds, and may result in a reduction in modified behavior at weeks 1 and 4 of the laying hen stimulated by noise from cost trials.

In the experiment, it is found that the lying behavior and the ingestion of the laying hens are obviously increased and the standing behavior and the walking behavior are reduced along with the extension of the music and noise stimulation time. Studies have shown that prone time increases with age of the animal. It has also been found in chicken studies that as time increases, layers spend more time lying on their stomach, while under the same conditions an increase in one behaviour necessarily results in a decrease in the performance of the other behaviour, which may be the main reason why layers spend less time in standing and walking behaviour. Research shows that the drinking behavior and the eating behavior of the chickens are in positive correlation. Environmental abundance is time-efficient, i.e., an environmental abundance effect may need to be effective at a particular time period in an animal. For example, in a study of the effect of music on horse race heart activity, it was found that in the first month, horse race Heart Rate Variability (HRV) changed significantly under music stimulation and remained increased at

months

2 and 3, but that in the following time horse race HRV began to return to the original level. It has also been found that the environment is enriched by adding some devices which can provide pecking during the raising process of laying hens, but these devices can only keep the interest of laying hens for 10 days. Thus, the effect of sound on the animal may diminish as the sound stimulation time increases. This is probably the main reason why the difference in behavior between the music group and the noise group was not significant under long-term acoustic stimulation in this experiment.

When an animal is exposed to emotional, physiological or environmental stress, the body will activate various biological response systems in order to deal with the potential threat posed by the stress. The response to stress usually involves activation of the hypothalamic-pituitary-adrenal cortical axis (HPA). Cortisone and cortisol are both the most prominent products at the end of the HPA axis. Therefore, the detection of the levels of corticosterone and cortisol in the body becomes an important index for researching the stress degree suffered by livestock and poultry. The study of Liujiajiajia finds that the mozart music has the effect of relieving the stress of the dairy cows and can reduce the content of cortisol in the blood of the dairy cows. In the test, the CORT level in the serum of the laying hens aged 4 weeks is obviously lower than that of the control group under the music stimulation of 65-75dB, which shows that the music stimulation has the function of relieving the stress of the poultry. The serum CORT content of the laying hens was not significantly different from that of the control group at weeks 10 and 16 with the extension of the stimulation time, which indicates that the effect of relieving stress by music is not persistent but short-term and there is a decay or adaptation process. Hunger, heat, fear, stress or poor feeding conditions can all contribute to a significant increase in blood CORT levels in layer production. Noise is a common source of stress in animal production. In the test, the CORT concentration in the serum of the laying hens can be obviously higher than that of a control group by the continuous noise stimulation of 65-75dB for two weeks, which indicates that the short-term (12 days) repeated noise stimulation causes aversion emotion of animals and causes certain physiological stress reaction, but the CORT content in the serum of the laying hens in the 4 th week, the 10 th week and the 16 th week is not obviously different from that of the control group along with the continuous noise stimulation, which indicates that the strong physiological stress of the laying hens cannot be caused by the long-term repeated noise exposure of the laying hens, probably because the laying hens are adaptive to the long-term repeated noise stimulation.

It is generally accepted that good animal welfare should not be an animal alone without negative experiences, but rather positive emotional expressions such as happiness. Assuming that a decrease in negative emotions means an increase in animal welfare levels, most physiological indicators used to assess animal welfare are often negative indicators, ignoring some positive physiological indicators. It has been suggested that Dopamine (DA) is a positive indicator that can be used to evaluate animal welfare. Such as music exposure, can significantly increase dopamine levels in the chicken brain. In the test, the DA level in the serum of the laying hens at 1 week old and 2 weeks old which are raised under 65-75dB music stimulation is obviously higher than that of the control group, and the DA level in the serum of the laying hens at 1 week old is also obviously higher than that of the control group under 65-75dB noise environment, and the difference from the music group is not obvious. The possible reason for this is that the noise of this fan in the short term is of interest to the layers, thus raising the DA levels in the serum, but the long term repeated exposure to this noise causes the body to adapt so that the DA levels in the serum of subsequent layers tend to be consistent and not significantly different from the control group.

Immunoglobulins are important components of the immune system and play an important role in maintaining the immune function of the body. Studies have shown that the body is exposed to various types of music and that immunoglobulins increase and have a positive effect on the immune system. In the test, the music stimulation obviously improves the IgG level in the serum of the laying hens aged 1 week and 2 weeks and the IgA level in the serum of the laying hens aged 2 weeks and 4 weeks, and along with the continuous music stimulation, the contents of two immunoglobulins are gradually kept equal to those of a control group. Furthermore, in this experiment, the expression of IgA and IgG mRNAs in the thymus under music stimulation was substantially consistent with the levels of both immunoglobulins in serum. Music stimulation obviously improves the mRNA expression quantity of IgA in thymus of laying hens of 1 week and 2 weeks and the mRNA expression quantity of IgG in thymus of laying hens of 1 week and 4 weeks, and along with the continuous music stimulation, the mRNA expression quantity of two immunoglobulins gradually has no obvious difference from a control group. All the results show that music stimulation in a certain time can have positive influence on the immune system of the laying hens. Noise can adversely affect the immune system of the body, e.g., exposure to high levels of noise can result in significant reductions in immunoglobulin levels in the blood of the offspring of mice. In the test, the noise stimulation improves the content of IgA in the serum of the laying hens at the age of 1 week and 4 weeks and the content of IgG in the serum of the laying hens at the age of 1 week, and the content of IgA and IgG in the serum of the laying hens is not obviously different from that of a control group along with the continuation of the noise stimulation. Furthermore, in this experiment, it was found that the mRNA expression of IgA in the thymus under noise stimulation was substantially consistent with the two immunoglobulin levels in serum. Noise stimulation obviously improves the mRNA expression quantity of IgA in the thymus of the laying hens at the age of 1 week, and along with the continuous music stimulation, the mRNA expression quantity of IgA in the thymus of the laying hens is gradually and obviously different from that of a control group. However, in this experiment, noise stimulation did not significantly affect the amount of IgG mRNA expression in thymus. The reason for this is probably that the repeated noise stimulation in a short period is a new environmental stimulation for the laying hens, which temporarily increases the immunoglobulin content in the serum of the laying hens and the expression level of the immunoglobulin mRNA in organs, but as the stimulation continues, the organism generates adaptability, so that the immunoglobulin content and the expression level are not obviously different from those of a control group.

It has been found that interleukins are critical to maintaining immune homeostasis. IL-2 can induce T cell maturation, promote B cell proliferation and differentiation, and mediate immunoglobulin synthesis. IL-6 also has an effect on neurosecretion, regulating the hypothalamic-pituitary-adrenal (HPA) axis and the release of various neurotransmitters and hormones, and promoting inflammatory responses. IL-10 is a pleiotropic cytokine with broad anti-inflammatory effects. IFN-gamma can activate the immune activity of immune-related cells so as to enhance the immune response capability, and TNF-alpha is the earliest and most important inflammatory mediator in the inflammatory reaction process and is a main cytokine participating in the inflammatory reaction. The study finds that IL-2, IL-6, IL-10, TNF-alpha and IFN-gamma in the serum of the laying hens at the age of 2 weeks are transiently increased in the continuous music stimulation, but the long-term music stimulation does not affect the level of the cytokines in the serum of the laying hens along with the continuous stimulation time. It is likely that short-term musical stimulation activates the body's immune system, resulting in a transient increase in the inflammation index. It has been found that adverse environmental stimuli can lead to increased body IL-2, IL-6, and TNF- α, such as noise exposure increases IL-6 expression in rat blood vessels. In experiments it was found that prolonged continuous noise stimulation resulted in increased levels of IL-2, IL-10, IFN- γ in the serum of laying hens aged 1, 2 and 4 weeks, and IL-6 in the serum of laying hens aged 1, 2, 4 and 10 weeks, with significant increases in TNF- α levels throughout the stimulation period. This is probably because the long-lasting noise, as an adverse stimulus, causes a continuous increase in the levels of body IL-2, IL-6, and TNF- α, and causes inflammation, which in turn increases the levels of IL-10 and IFN- γ, which are anti-inflammatory factors. Therefore, the influence of sound stimulation on the cytokine is complicated, and the change mechanism of the cytokine in serum is still under intensive study.

Thymus is the central immune organ in chicken body, and plays an important role in the body immune and neuroendocrine network, if the physiological function of thymus is destroyed, the immune balance and normal physiological function of body can be destroyed. The research shows that the stress can lead the expression level of IL-2 and IFN-gamma of the body to be down-regulated in thymus tissue and the expression level of TNF-alpha to be up-regulated. The research shows that short-term or long-term music stimulation does not affect the expression quantity of IL-2, IL-6, TNF-alpha and IFN-gamma in thymus, while continuous noise stimulation causes the laying hens to down regulate the expression quantity of IL-2 and IFN-gamma at the age of 4 weeks, increase the expression quantity of TNF-alpha in thymus at the ages of 1 week and 10 weeks, and also cause the expression quantity of thymus IL-6 at the age of 1 week to increase. Then, the laying hens gradually adapt to the noise environment along with the time, so that the expression level of the thymocyte factor is not obviously different from that of the control group. The immune system is a complex physiological system, and is regulated by various factors including the nervous system, the endocrine system, environmental factors and the like, and the immune system regulates and influences the nervous system and the endocrine system. It has been shown that there is also an immune response in the CNS, and that cytokines such as IL-2, IL-4, IL-6, IL-10, and TNF are found in the CNS, and that some CNS-mediated diseases are attenuated by the decrease of proinflammatory cytokines. In this experiment, the expression level of cytokine mRNA in the hypothalamus of egg-laying hens was not affected by the continuous music stimulation. However, the noise stimulation causes the expression level of TNF-alpha in the hypothalamus of the laying hens at the age of 4 weeks to be up-regulated.

Oxidative stress is caused by disturbances in the homeostasis of pro-oxidants and antioxidants, resulting in an overproduction of free radicals and reactive oxygen species, damage to cells and causing inflammatory reactions (e.g. by activating NADPH oxidase and eNOS uncoupling), lowering the defense against oxidation and causing a decline in immune function of the body. The music has a fixed rhythm, and the internal organs of the animal body also have a fixed rhythm, when the rhythms of the internal tissues and organs of the animal body are similar to or consistent with the rhythms of the music in the environment, physiological resonance can be generated, and the resonance can have beneficial effects on the body, such as correcting microseismic disorder of the rhythms of the tissues and organs of the body, strengthening the inherent microseismic frequency of the body, preventing cell damage of the body, and improving the disease resistance of the tissues and organs, including immunity and oxidation resistance. Superoxide dismutase is an important component of the enzymatic defense system of free radicals of the body, can effectively remove active oxygen and can terminate chain reaction of the free radicals, and catalase plays a role in removing excessive hydrogen peroxide of the body so as to prevent cells from being toxic by the hydrogen peroxide. Therefore, the immunity of the body is proportional to the activities of superoxide dismutase and catalase in the blood. In tests, the continuous music stimulation obviously improves the catalase activity in the serum of the laying hens at 1-week and 2-week ages and the superoxide dismutase activity in the serum of the laying hens at 1-week age, which shows that the short-term music stimulation can improve the antioxidant capacity of the laying hens, prevent cell damage and improve the immunity of organisms.

Oxidative damage to the body caused by noise is mainly caused by impaired antioxidant defense or activation of endogenous antioxidant pathways. Exposure of the animal body to noise activates the sympathetic nervous system and HPA system, resulting in an increase in stress hormones, such as catecholamines, angiotensin-ii, endothelin-1, and cortisol. These changes lead to direct activation of phagocytic NADPH oxidase, causing oxidative stress, resulting in NO changes, such as noise exposure can reduce NO levels in rat blood vessels. Likewise, noise can also cause damage to the body by increasing lipid peroxidation levels, such as increasing Malondialdehyde (MDA) content and H ₂ O ₂ And (4) generating. Noise-induced oxidative stress can lead to nNOS down-regulation and uncoupling, with the consequences of immune cell activation/infiltration, increased iNOS content, and thus inflammation. In this test, the continuous noise stimulus caused the serum of the hensThe radical content tended to be significantly higher than in the control group. This may be due to the continuous noise stimulation causing the production of a large amount of free radicals in the layers, breaking the balance between oxidation/anti-oxidation systems, which in turn leads to oxidative damage of the body.

After the brain is damaged, the body can activate the heat shock protein gene to protect the brain from further damage. The heat shock protein can regulate and control the different actions of the aggregated protein through ways of preventing folding, degrading and denaturing and the like so as to participate in neuroprotection, thereby effectively reducing the damage of stress to the brain of an organism. In addition, heat shock proteins are involved in the regulation of antioxidant enzyme activity in the body and reduce the production of Reactive Oxygen Species (ROS) by inhibiting the production of free radicals. Therefore, the occurrence of oxidative stress inevitably causes the expression of large amounts of heat shock proteins, so that heat shock proteins can be used as typical tissue damage markers to some extent. It was found that noise pollution can raise the level of HSP70 in rat blood. In the research, short-term and long-term music stimulation does not influence the expression of heat shock protein in the hypothalamus of the laying hens. In the early stage of noise stimulation, the expression of heat shock protein in the hypothalamus of the laying hens is not obviously different from that of a control group, noise is used as chronic stress, and the expression levels of HSP70 and HSP90 in the hypothalamus are obviously regulated by the laying hens at the age of 10 weeks with the continuous noise stimulation, but the expression level of HSP60 is not influenced, which shows that chronic stress is generated by long-term (10 weeks) noise stimulation on the organism, and the brain of the laying hens needs to generate more heat shock protein to protect the nervous system.

Egg yield and weight gain are important production performance indexes of the laying hens, and the subsequent production performance of the laying hens is determined by the weight of the laying hens before laying. The problems of delayed laying of the laying hens, short laying peak period or no laying peak period, small egg weight, poor disease resistance of the laying hens, low feed conversion efficiency and the like can be caused by the unqualified body weight. In the experiment, continuous music and noise stimulation of 65-75dB does not have obvious influence on the production performance of the laying hens. The sound intensity adopted in the test is not high enough, and the stimulation time is 6 hours per day, so that the production performance of the laying hens cannot be obviously influenced by the sound stimulation of the test.

Egg quality mainly includes eggshell quality and internal egg quality. The quality of the eggshell in turn includes eggshell thickness, eggshell weight, and eggshell strength. An ideal shell of an egg should have sufficient thickness and strength to withstand impact during transportation and handling. If the eggshell is too thin, the eggshell is fragile and pathogenic bacteria can easily invade the interior of the egg, so that the quality of the interior egg is influenced, and if the eggshell is too thick, the chick can be prevented from going out of the shell. The inner egg quality is evaluated primarily in terms of protein height and harderian units, with higher protein height and harderian units indicating better inner egg quality. It was found in experiments that prolonged music and noise stimulation did not have a significant effect on the eggshell quality, egg white height and haugh units of the eggs. This is probably because the mild sound stimulation for 6 hours per day does not affect the feeding behavior and the feed intake of the layer chicken, and further causes no difference in egg quality.

In a modern commercial layer feeding mode, feather quality score is an important index of the welfare level of the layer. The poor feather quality is generally caused by feather pecking behaviors among the laying hens, the serious feather pecking behavior in a laying hen population often causes reduction of feather amount of laying hens, damage and reduction of the feathers can reduce heat insulation capability of the feathers, further the feed conversion rate of the laying hens is reduced, the laying hens are damaged, and even the laying hens die in serious cases. Therefore, the feather quality can directly reflect the welfare level of the laying hens. In the experiment, it was found that continuous music and noise stimulation did not have a significant effect on the feather score of the layers, indicating that the sound stimulation did not affect the feather quality of the layers in the caged system.

Noise is used as a stress source and is found to have adverse effects on the cognitive abilities of animals in many studies, for example, environmental noise can cause damage to the spatial memory ability of rats, and in the study, the performance of the PAL test of a music treatment group and a noise treatment group is not significantly different from that of a control group, which indicates that the stimulation intensity and duration of sound in the test cannot have a significant effect on the learning memory ability of laying hens.

Fear level is an effective indicator for assessing the welfare status of birds. Both the catatonic immobility (TI) test and the Open Field (OF) test have been demonstrated in several tests as effective methods for detecting fear in birds. In the catatonic test, the longer the duration of the animal's rigor is indicative of the fear of the animal, and the expression of some behaviors in the open field test, such as excretion, the number of chirps, the number of crossing squares, etc., are all related to the intensity of the fear suffered by the animal. In the research of the tension immobility of the sound stimulation to the poultry, the poultry respond to the sound stimulation in different breeds and different feeding stages differently, and in the research, the continuous music stimulation does not have obvious influence on the tension immobility of the laying hens. However, the continuous noise stimulation obviously increases the duration of tension immobility of the laying hens only at the age of 1 week, and the laying hens do not show fear level along with the continuous stimulation of the noise. The research shows that the fear level has a phenomenon of attenuation along with the duration of the stimulation time, which can explain that the noise stimulation repeated for a long time in the test does not cause the fear behavior of the laying hens. The study found that, under continuous music and noise stimulation, 3-week-old laying hens showed significant differences in the open field test, noise group laying hens showed more rigor time relative to the music group and the control group, and the music group showed more walking steps and total number of squares relative to the noise group, but had no significant differences in the number of excretions and total cry. This indicates that the noise stimulation caused the laying hens to be more frightened about the new environment, and the walking steps and the total number of the squares walked also indicate that the noise group caused the decline of the athletic ability due to the frightening relative to the music group. It was found that the level of fear tested in both the catatonic and open field tests was essentially different. Duration of nervousness is considered an evasive strategy for predators, and the locomotor and vocalizing behavior exhibited by birds in open field trials is considered a conflict between fear of new circumstances (reduced locomotor ability) and craving of returning flocks (increased vocalizing ability). Open field trials have shown that layers may have reduced mobility and increased rigidity indicating that they are afraid of a new environment and that they will be immobilized and remain quiet in order to avoid the discovery of predators. The catatonic test is a reaction against predators and therefore may be less sensitive to environmental changes, which also indicates that a fear test alone is not sufficient to define changes in the level of fear.

Many studies have demonstrated that music and noise stimuli can affect the physiology, behavior and welfare levels of animal organisms, but these studies rarely explain these phenomena at the genetic level. The GO enrichment analysis result in the test shows that music and noise can generate certain influence on behavioral expression, stress response, immunity and neural activity, the effect of continuous music stimulation on the brains of laying hens at the age of 4 weeks is mainly concentrated on the stress response and neural development, and the noise is mainly concentrated on inflammation and neural development. The KEGG results show that the significant difference of the music group and the noise group is only 1 compared with the control group, but the significant difference of the music group and the noise group is 4 compared with the control group. It is speculated that the possible reason is that the pathway regulation pattern of the noise group versus the music group is complex and completely different in terms of the differential pathways of the KEGG enrichment analysis.

In the research, 90 genes with larger difference multiples are firstly screened out preliminarily, and as a plurality of genes are new genes and the functions of the genes are not completely clear, 12 different genes with more perfect function research are further screened out as candidate genes which are respectively up-regulated genes of BMP4, ZIC1, ZIC3, ZIC4, SOCS1, SOD3, rasGRP1 and Egr4 and down-regulated genes of HSPB8, BAG3, PRDM12 and Ascl1.

The small heat shock protein B8 (HSPB 8) is a chaperone protein induced by adverse events such as proteasome inhibition, which is abundantly expressed in the brain. HSPB8 is significantly upregulated under stress conditions and acts by promoting autophagy, thereby preventing the accumulation of misfolded proteins that have a deleterious effect on the cell. There is new evidence that HSPB8 shows protective effects in both neurophysiological and neuropathological conditions. BAG3 is one of the members of the BAG chaperone family, which interacts with the atpase domain of heat shock proteins through a conserved BAG domain. BAG3 has a wide range of biological functions including regulation of stress response, autophagy, apoptosis, and cell survival. Oxidative stress leads to massive expression of BAG3, which in turn protects the cells from damage. In the experiment, the gene expression levels of HSPB8 and BAG3 in the brains of the 4-week-old laying hens are remarkably regulated under music stimulation, which shows that the anti-stress capability of the organism can be improved through music stimulation.

BMP4 is an important regulator of neural development, and as a member of the bone morphogenetic protein (BMP 4) family, it plays an important role in the neural stem cell differentiation pathway. In addition, the increased expression levels of BMP4 protein and mRNA also promote differentiation of neural stem cells into astrocytes. The ZIC gene codes for a zinc finger transcription factor family, and 5 typical ZIC genes (ZIC 1-5) are contained in vertebrates, and play an important role in the formation of nerves and neural crest of animals. Wherein the reduced expression of ZIC3 results in a neural tube defect in the animal, and the reduced expression of ZIC1 and ZIC4 results in a cerebellar abnormality in the animal. SOCS1 belongs to the SOCS protein family, is a group of inhibitory regulators of cytokine signal pathways, plays an important role in maintaining organ homeostasis, and can inhibit the expression of inflammatory cytokines by inhibiting P65 (one of the strongest activators of NF-kB family). Superoxide dismutase (SOD 3) is the major antioxidant enzyme that scavenges superoxide anions in cells. The increased expression level of SOD3 can protect the brain from stress injury. In the experiment, the gene expression levels of BMP4, ZIC1, ZIC3, ZIC4, SOCS1 and SOD3 in the brain of the laying hen are remarkably up-regulated under music stimulation, which shows that the music stimulation can promote the neural development in the brain of the laying hen, improve the anti-stress capability and protect the brain from inflammation.

Ras guanine releasing protein 1 (RasGRP 1) is a member of the guanine nucleotide exchange factor that activates Ras proteins. RasGRP1 is critical for signaling of mature T cells, differentiation of thymocytes, and proliferation of B cells. Furthermore, rasGRP1mRNA and protein can be expressed in large amounts in the brain. Research shows that RasGRP1 can change the expression level of protein related to various nervous system diseases, for example, rasGRP1 expression level can be obviously up-regulated in the brain of a Parkinson patient, and the RasGRP1 expression level is positively correlated with the expression of neuroinflammation cell factors. In the test, the continuous noise stimulation enables the brains of the laying hens aged 4 weeks to obviously up-regulate the expression levels of RasGRP1 and Egr4, which shows that the noise stimulation can cause neuroinflammatory reaction of the brains of the bodies, and further influences the development of nervous systems. Early growth response protein 4 (Egr 4) is an Egr family transcription factor that regulates peripheral immune responses. The up-regulation of Egr4 can reduce the activation of a brain injury rat JNK/c-JUN pathway and the expression of proinflammatory cytokines, reduce the damage of stress to the brain, and reduce the damage of stress to brain tissues and neurons. Continuous noise stimulation in the experiment enables the expression level of Egr4 in the brains of the laying hens aged 4 weeks to be up-regulated, and presumably the laying hens are stressed when aged 4 weeks under continuous noise stimulation, so that the brains of the laying hens are damaged, and a large amount of expression of Egr4 is stimulated.

PRDM12 belongs to the PRDM family of genes encoding epigenetic regulators, which are important regulators in cell proliferation and differentiation. PRDM12 plays an important role in the development of sensory and pain neurons. Pain sensation is essential to maintain the integrity of the animal's body function. The absence of PRDM12 results in the complete inability of specific neuronal progenitor cells to maintain neuronal protein expression, to activate pre-neuronal gene expression, and to develop into nociceptive neurons. In the experiment, the long-term continuous noise stimulation can reduce the gene expression level of PRDM12, which is unfavorable for the neural development of the laying hens. Ascl1 is a basic helix-loop-helix transcription factor that binds to the E-box motif in DNA. It is considered to be an pioneer transcription factor, on the one hand, a pioneer or pan-generic neurogenic factor, but on the other hand also has strong re-editing properties. It has been found that Ascl1 can directly convert mouse fibroblasts into excitatory neurons. In addition, the preneural gene Ascl1 can effectively transform astrocytes into induced neuronal cells in vivo, and thus is important for the development of the central nervous system. In this experiment, continuous noise stimulation down-regulated the expression of Ascl1 in the brains of 4-week-old layers, which also indicates that continuous noise stimulation is detrimental to the development of the nervous system of animals.

The method for evaluating the effect of different sound stimuli on the behavior, welfare, cognition and physiology of the laying hens is described in detail, a specific example is applied to explain the principle and the implementation mode of the method, and the description of the embodiment is only used for helping to understand the method and the core idea of the method; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Claims

1. A method for evaluating the effects of different sound stimuli on the behavior, welfare, cognition and physiology of laying hens is characterized by comprising the following steps:

feeding the laying hens: purchasing 360 1-day-old chicks with the weight of 43 +/-4 g, randomly dividing the chicks into 3 groups of 120 chicks, respectively feeding the chicks in 3 artificial climate chambers, wherein each group has 8 repetitions, each repetition has 15, and each repetition internally marks 3 target animals as marked animals; the test period was 42 weeks, and the chickens were fed and drunk freely throughout the test period;

LM group: low intensity music group, chickens exposed to classical music with sound intensity of 65-75 dB;

the sound stimulation is continued from day 1 to 16 weeks, during the period, the test group carries out sound stimulation every day, the sound playing time is 7-00;

collecting and processing samples: randomly selecting 1 laying hen for each test group repeatedly in each test group in six weeks of 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks respectively, killing 8 laying hens per group in total while avoiding marked animals for behavior observation, immediately collecting blood, collecting the blood in an EP (European patent) tube, placing the EP tube in a refrigerator at 4 ℃ for coagulation for 24 hours, then centrifuging for 15 minutes at 4000 revolutions per minute, preparing serum, transferring the prepared serum into a new 1.5mLEP tube, and placing the tube in the refrigerator at-80 ℃ for storage for subsequent ELISA, oxidation and oxidation resistance detection; cutting open the chicken after blood collection, rapidly collecting cerebrum, hypothalamus and thymus tissue, and storing in a refrigerator at-80 deg.C for use;

and (4) behavior observation: counting the state behaviors in each behavior observation time period by taking 5s as 1 time unit, and converting the behavior data into the percentage of the total observation time; the method comprises the following steps that (1) a target animal sampling continuous observation method is adopted for completely recording the behaviors of observed laying hens once, the occurrence of each event behavior is recorded once, and the total occurrence times of each event in behavior observation time are used for representing;

detecting the quality of the eggs: carrying out statistics on the egg yield, the egg laying rate and the egg breaking rate in four stages of 18-24 weeks, 24-30 weeks, 30-36 weeks and 36-42 weeks; randomly selecting 10 eggs from each test group for 23 weeks, 29 weeks, 35 weeks and 41 weeks to detect the egg quality;

statistical analysis: firstly, carrying out normal distribution test on data by Shapiro-Wilk, carrying out lg and ln conversion on the data which is not subjected to normal distribution, then, carrying out normal distribution on all the data, displaying the homogeneity of variance, analyzing an ELISA result, an oxidation and oxidation resistance result, a real-time fluorescence quantitative PCR result and a protein expression result by adopting single-variable variance analysis in a general linear model, carrying out multiple comparison analysis on behavior data, welfare evaluation and production performance by using a single-factor variance analysis and a Duncan method, and carrying out Fisher precise test on the avoidance rate of a one-time avoidance test; all results were plotted by GraphPadPrism7, with data expressed as mean ± standard error, and P <0.05 indicating significant differences.

2. The method according to claim 1, characterized in that the climatic chamber temperature, humidity, ventilation and lighting are controlled by a central controller, the temperature is set at 33-35 ℃ for the first week, thereafter the temperature is reduced by 2 ℃ per week until 21-23 ℃ at 8 weeks of age, after which the temperature is controlled at 18-24 ℃; during the test period, an artificial illumination system is adopted, the illumination is carried out for 1-3 days old and 23 hours every day, the illumination is carried out for 1 hour in darkness, the illumination is carried out for 22 hours for 4 days old, the illumination is reduced for 1 hour every day till 14 days old, the illumination is carried out for 12 hours and the darkness is carried out for 12 hours, the illumination is kept unchanged until 8 weeks old and 8-18 weeks old maintain the illumination for 8 hours, the illumination is increased for 1 hour every week till 26 weeks old and the illumination is carried out for 16 hours, and the illumination is kept till the test is finished; the illumination intensity is 10-15lux, and the humidity is 60-70%.

3. The method of claim 2, wherein the stateful behaviors include eating, lying prone, walking, and standing behaviors.

4. The method of claim 2, wherein the incident behavior comprises drinking, pecking, grooming, comfort and feather pecking, and aggressive pecking.

5. The method according to claim 4, wherein the one-time avoidance test is specifically:

before pre-experimental training, putting a piece of white A4 paper on the ground in a test box, putting the chicks in the test box for adaptive training for 3 hours, and fasting and water prohibition; after the adaptive training is finished, removing A4 white paper, and performing pre-training for 3 times; in the pre-training, a piece of red A4 paper is used for containing normal food and is placed in a test box to attract the attention of the chicks and peck the food, the normal food is displayed to the chicks for 1min in the 1 st and 2 nd pre-experiments, the normal food is displayed to the chicks for 2min in the 3 rd pre-experiments, the interval of each pre-experiment is 5min, and the training test is carried out after the last pre-training is finished for 5 min;

during the training trial, the chicks were shown with a blue A4 paper holding the 99% mea soaked feed for 2min; the chicks have aversion reaction after pecking and wipe the mouth on the ground of the cage; when the chicks peck the food soaked with the MeA and obvious aversion reaction occurs, taking out the blue paper and the feed, starting timing, and detecting the learning and memory ability after 120 min.

6. The method of claim 5, wherein the learning and memory ability test is performed 2 times, and in the first test, the chicken are given 1min of normal food, but the food is placed on a blue paper of the same color as the food containing MeA before, and after completion, the paper and food are removed; after 5min intervals, carrying out a second test to show the normal food for 1min for the chicks, and using red paper with the same color as the red paper for containing the normal food in the previous pre-test; during each trial the target chicken pecked and did not peck, and during the trial the layer would relate the color of the paper to the taste of the food and would show avoidance behavior for paper of the color associated with MeA food, but would not avoid paper of the color associated with normal food; after the test was completed, the test chickens were returned to the cages.

7. The method according to claim 1, characterized in that the open field test is in particular: the method comprises the following steps of (1) carrying out an open field test when the chicks are raised for 23 days, randomly selecting 8 chicks in each test group, and carrying out the open field test in a single room; in the open field test, 1.5m × 1.5m square boards are used as an open field area, 1.5m high boards are used for surrounding the open field area, and white masking paper is used for dividing the open field area into 25 0.3m × 0.3m square areas; during the test, a chick was held in the open field by the observer, placed in the central grid area of the field and covered with a box of 20cm × 30cm, and when the observer left the open field, the box was lifted and the video recording of the following behavior in the next 10 minutes was started by a video recording device placed above the open field and the following behaviors were recorded: rigor mortis time, total cry, number of steps walked, number of excretions, and total number of squares walked.

8. The method according to claim 1, wherein evaluation of the welfare index is carried out by selecting 1 out of a total of 8 out of each group for each repetition on 1 week, 2 weeks, 4 weeks, 10 weeks and 16 weeks of the day, respectively, and selecting 1 out of a total of 8 out of each group on 23 weeks, 29 weeks, 35 weeks and 41 weeks of the day of the laying period; the welfare index evaluation comprises body weight, duration of tension immobility and feather quality score in the egg producing period.

9. The method of claim 8, wherein the feather quality score is specifically: evaluating the feather quality of eight parts of the body of the laying hen, namely the head, the neck, the back, the wings, the chest, the legs, the tail and the anus; the feather quality score of each part can be divided into 0-3 points according to the damage degree, namely 0 point-the feather is complete and has no damage; score 1-feathers are slightly broken and completely cover the skin; 2 min-feather breakage and skin part exposure, exposure area < 1cm x 1 cm; 3 minutes-feather damaged and skin exposed area > 1cm x 1 cm; finally, the scores for each site are summed, i.e., the highest score is 24 points and the lowest score is 0.