Construction method of laying hen chronic unpredictable mild stress model

Abstract

The invention discloses a construction method of a laying hen chronic unpredictable mild stress model, and belongs to the technical field of animal husbandry. The invention aims to provide a construction method of a laying hen chronic unpredictable mild stress model. The invention provides a method for constructing a chronic unpredictable mild stress model of laying hens, which comprises the following steps of: selecting more than 300 laying hens at the age of one day, randomly dividing the laying hens into two groups, namely a control group and a chronic unpredictable mild stress group, repeating the same number in each group, and containing the same laying hens; the laying hens in the two test groups are respectively fed in two environment control rooms with the same structure for weeks, the chronic unpredictable mild stress group is selected to be subjected to stress source stimulation and fed with basic ration every day, and the control group is only fed with the basic ration without other stress treatment; after the chronic unpredictable mild stress program is finished, tension immobility and attention deviation tests are carried out, and the influence of chronic unpredictable mild stress on the laying chicks is evaluated. And a theoretical basis is provided for chronic unpredictable mild stress of laying hens.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and particularly relates to a method for constructing a chronic unpredictable mild stress model of laying hens. Background Art

[0002] Problems caused by fear and anxiety are of great concern because they have a significant negative impact on the physiological and psychological health of animals. If these emotions are not relieved for a long time, they may highlight the lack of adaptability of animals to their living environment. Fear is a short-term emotional response, usually a flight or freeze response to an immediate and direct threat to survival. Anxiety is a longer-term emotional state that prompts animals to remain highly alert to perceived potential threats, and this response is often amplified by adverse early life experiences. It has been reported that laying hens are often exposed to a variety of unpredictable and repeated stressors, including capture, transportation, noise and crowding, which can cause great stress to animals. Chronic unpredictable mild stress is a compound stimulus that combines multiple stressors and has a significant negative impact on the physiological and psychological health of animals. Long-term chronic unpredictable mild stress often triggers fear and anxiety in animals, further affecting their behavior and health. Excessive fear and anxiety not only significantly reduce body weight and food intake, but also increase anxiety-like behavior in animals, such as prolonged duration of tense immobility. In addition, chronic, unpredictable, mild stress may also lead to an imbalance in the intestinal microbiota and even cause serious health problems or death.

[0003] In many studies on birds, fear is often assessed by the tonic immobility (TI) test. TI is a freezing response to predation, that is, pretending to be dead, which is performed by prey animals as a last resort when they cannot escape. Animals with higher levels of fear usually show longer durations of TI. In addition, the attention bias test has also been shown to be effective in assessing the anxiety level of animals. Studies have found that fearful laying hens have longer durations of tonic immobility. Another study found that hens receiving anxiogenic drugs showed significantly slower eating speed, faster vocalization speed, and increased motor behavior when exposed to the same alarm calls, and these behaviors were significantly different from hens receiving saline injections.

[0004] In view of the serious impact of chronic unpredictable mild stress on the health of laying hens, there is an urgent need to provide a method for constructing a chronic unpredictable mild stress model for laying hens, develop and optimize a chronic stress model that can better simulate the actual environment of poultry production, so as to study its effects on animal health and behavior. Summary of the invention

[0005] The purpose of the invention is to provide a method for constructing a chronic unpredictable mild stress model for laying hens.

[0006] The present invention provides a method for constructing a chronic unpredictable mild stress model of laying hens, and the specific steps of the method are as follows:

[0007] Step 1: Treatment of laying hens: 300 one-day-old laying hens were randomly divided into two groups, a control group and a chronic unpredictable mild stress group, with the same number of replicates in each group and each replicate containing the same laying hens;

[0008] Step 2: The laying hens in the two experimental groups in step 1 were raised in two identically constructed environmental control rooms for several weeks. The chronic unpredictable mild stress group was given stressor stimulation and fed a basic diet every day, while the control group was only fed a basic diet without other stress treatments.

[0009] Step 3: After the chronic unpredictable mild stress program, the stress immobility and attention bias tests were performed to evaluate the effects of chronic unpredictable mild stress on laying hens.

[0010] It is further defined that the number of chickens selected in step 1 is 300; each group is 15 replicates, and each replicate has 10 laying hens.

[0011] It is further defined that the time in the environmental control room in step 2 is 7 weeks, including 1 week of pre-feeding period, 5 weeks of chronic unpredictable stress, and 1 week of behavioral testing.

[0012] Further defined, the method of feeding the basal diet in step 2 is: feeding once in the morning, noon and evening every day, and the chickens have free access to food and water during the entire experimental period.

[0013] It is further defined that the environmental control room described in step 2 is a soundproof room with a length, width and height of 150cm×150cm×90cm, and the temperature, humidity, ventilation and lighting are controlled by a central controller.

[0014] It is further defined that the same environment in step 2 includes daily treatment, the conditions are a breeding temperature of 16-20°C, a relative humidity of 52%, a light intensity of 20lux, and a light regime of 14L:10D.

[0015] It is further defined that the stressors described in step 2 are catching chickens for 5 minutes, artificial driving, strangers walking around for 10 minutes, fasting for 12 hours, water deprivation for 12 hours, 4°C cold stress for 3 hours, noise interference for 3 hours, transportation, lighting overnight, waving red and yellow flags for 5 minutes, and darkness interference for 4 hours.

[0016] Further defined, the method of the tension immobility test in step 3 is as follows: 15 laying hens are randomly selected from each group, and the laying hens are carefully placed with their abdomens facing upward into a "U"-shaped wooden trough. While applying a slight pressure on the chest of the laying hens, the head of the laying hens is slowly pressed. After 15 seconds, the hands are slowly released and the laying hens are gradually moved away from their sight. After successful induction, a stopwatch is used to record the delay time for the laying hens to turn over. If the laying hens try to get up immediately within 10 seconds, it is deemed that the induction has failed, and the above-mentioned restraint operation is repeated again. Successful induction of laying hens is manifested by the absence of any behavior except trembling and breathing.

[0017] To further define, the method of the attention bias test in step 3 is as follows: laying hens are transported to the test site in a rectangular black plastic basket. The test begins after the laying hens enter the site, and a Bluetooth loudspeaker placed out of the line of sight of the laying hens begins to play an alarm that lasts for 10 seconds. After the alarm ends, all activities of the laying hens within 10 minutes are recorded using video surveillance equipment; the length, width and height of the test site are 150cm×150cm×90cm, and the surface of the surrounding walls is a square metal frame site covered with plastic cloth, and the floor is a white polystyrene foam board; a pile of feed mixed with laying hen diet, oatmeal and dried mealworms is placed in the center of the test site; the alarm is the alarm call of a larger flock of chickens (240) frightened by the horn of a truck near the chicken coop, and the audio data sampling rate is 48kHz and the bit rate is 148kbps.

[0018] It is further defined that during the trial period, the weight and feed consumption of laying hens are recorded, and the average daily weight gain, average daily feed intake and feed-to-weight ratio are calculated.

[0019] Beneficial effects: (1) The present invention proposes a method for constructing a chronic unpredictable mild stress model for laying hens to explore whether chronic unpredictable stress has adverse effects on laying hens, providing a theoretical basis for improving animal welfare and health levels in production practice;

[0020] (2) This evaluation method is simple and produces stable results, which fills the gap in the construction of a method for chronic unpredictable mild stress in laying hens and can be well promoted and applied in research in this field.

[0021] (3) The construction of this model not only helps to gain a deeper understanding of the role of fear and anxiety in animal stress responses, but also provides a theoretical basis and practical guidance for improving animal welfare and formulating effective stress intervention measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The results of the test process, growth performance and behavioral tests (tense immobility, attention bias) are shown in Figure A, where Figure B is the test process, growth performance: including average daily weight gain, average daily feed intake and feed-to-weight ratio, Figure C is the duration of tense immobility, and Figure D is the attention bias test: including the latency of the first vocalization of laying hens, the latency of the first movement, and the latency of the first feeding;

[0023] Figure 2 The results of metagenomic analysis of cecal contents of laying hens under chronic unpredictable mild stress, among which Figure A is a Venn diagram, Figure B is the α diversity result, which consists of chao1, observed_species, Shannon, and Simpson. Figure C shows the changes in β diversity, which are evaluated by principal component analysis (PCA), principal coordinates analysis (PCoA), and nonmetric multidimensional scaling (NMDS). Figure D shows the relative abundance of the top 20 intestinal flora at the phylum level. Figure E shows the relative abundance of the top 20 intestinal flora at the genus level. F shows the comparison of the abundance of differential phyla between the two groups. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1. A method for constructing a chronic unpredictable mild stress model for laying hens

[0026] 1. This experiment is a method to construct a chronic unpredictable mild stress model for laying hens. 300 one-day-old, healthy Hy-Linen laying hens were selected and fed conventionally for 7 days. Then, laying hens with similar body weight were randomly divided into 2 groups, with 15 replicates in each group and 10 chickens in each replicate. The experimental groups are as follows: control group (CON): fed with a basic diet without any stress treatment; chronic unpredictable mild stress group (CUMS): fed with a basic diet and given 5 weeks of stress stimulation.

[0027] 2. The composition and nutritional level of the experimental basic diet are shown in Table 1, which meets the nutritional needs of laying hens. The chickens were kept in an artificial climate house throughout the whole process. The temperature, humidity, ventilation and lighting in the house were controlled by a central controller. The temperature was 16-20℃, the relative humidity was 52%, the light intensity was 20lux, and the lighting system was 14L:10D. During the experiment, the chickens had free access to food and water.

[0028]

[0029] 3. Method and procedure for establishing chronic unpredictable mild stress model in laying hens. During the whole experiment, laying hens in chronic unpredictable mild stress group were randomly treated with 2 stressors every day, and they were not repeated within 3 days: catching chickens for 5 minutes, artificial driving, strangers walking around for 10 minutes, fasting / water deprivation for 12 hours, 4℃ cold stress for 3 hours, noise interference for 3 hours, transportation, lighting overnight, red and yellow flags waving for 5 minutes, and dark interference for 4 hours. The specific implementation plan is described in Table 2.

[0030]

[0031] 4. During the experiment, the effect of chronic unpredictable mild stress stimulation on the production performance of laying hens was detected. That is, during the experiment, the weight of laying hens in each group was recorded every week, the feed intake was counted, and the average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F / G) were calculated as follows:

[0032] Average daily gain g (ADG) = (final weight - initial weight) g / day;

[0033] Average daily feed intake g (ADFI) = feed consumption g / day;

[0034] Feed conversion ratio (F / G) = average daily feed intake g / average daily weight gain g;

[0035] 5. At the end of the experiment, the effects of chronic unpredictable mild stress on the behavior and emotions of laying hens were evaluated. First, 15 laying hens were randomly selected from each group for the tense immobility test (TI). The tester wore cotton gloves and slowly placed the laying hens with their abdomens facing up into a "U"-shaped wooden trough. While applying slight pressure on the chest of the laying hens, he slowly pressed the head of the laying hens. This induced behavior lasted for at least 15 seconds, after which the tester slowly released his hands and gradually moved away from the laying hens' sight, looking elsewhere. A stopwatch was used to record the delay in the laying hens' righting reaction. Successfully induced laying hens showed no other behaviors except trembling and breathing. If the laying hens immediately stood up within 10 seconds, it was considered an induction failure, and the tester repeated the above-mentioned restraint operation again. If a laying hen failed to induce three times in a row, the lowest score was recorded as 0s. If the laying hen remained motionless after 600 seconds, the duration of the maximum latency of 600 seconds was recorded.

[0036] All laying hens were tested for attention bias test. During the attention bias test, all laying hens were fasted for at least 4 h. The laying hens were taken out of their cages one by one and transported to the test site in a rectangular black plastic basket. A small pile of feed consisting of laying hen diet, oatmeal and dried mealworms was placed on the floor in the center of the test site. When the laying hens entered the site, the test began. A Bluetooth speaker (CM-510, Huawei, Shenzhen, China) placed out of sight of the laying hens began to play an alarm for 10 s. The alarm recording was from a warning call from a larger flock of 240 chickens. The reason was that the chickens were frightened by the sound of truck horns on the road near the chicken house. The audio data obtained had a sampling rate of 48 kHz and a bit rate of 148 kbps. After the alarm was played, all the activities of the laying hens were recorded by a video surveillance device DS-7104N-F1 (Hikvision, Hangzhou, China). At the same time, an experimenter recorded the latency of the first vocalization of the laying hens in an area out of sight of the laying hens. After the attention bias test, all videos were observed by the same experimenter to record the latency of the first movement and the first feeding of the laying hen. During the test, each laying hen was assigned a test time of 10 minutes (600 seconds). If the laying hen did not express any behavior until the end of the test, the maximum time was recorded as 600 seconds.

[0037] 5. All data were first tested for normal distribution. SPSS 20.0 software was used to perform independent sample Tukey test analysis for data that conformed to normal distribution. Single factor nonparametric analysis Mann-Whitney U test was performed for TI data that did not conform to normal distribution. GraphPad Prism 8.0 was used to draw patterns. Results are expressed as mean ± standard error. P < 0.05 was significantly different, and P < 0.01 was extremely significantly different.

[0038] Figure 1 The flowchart of this implementation case, the effects of chronic unpredictable mild stress on the growth performance of laying hens, and the results of the stress immobility test and the attention bias test are presented. Figure 1 B The results showed that in the five-week trial, there was no significant difference in the growth performance of laying hens between the CON group and the CUMS group in the 1st and 2nd weeks (P>0.05), while in the 3rd, 4th and 5th weeks, CUMS significantly reduced the ADG and ADFI of laying hens (P<0.05), and the F / G was significantly higher than that of the CON group in the fifth week (P<0.05). The above results showed that CUMS had a negative impact on the growth of laying hens.

[0039] The behavioral test results are as follows Figure 1 As shown, the tension immobility test ( Figure 1 C) The results showed that chronic unpredictable mild stress exposure significantly increased the duration of stress immobility in laying hens compared with the CON group (P<0.05). Figure 1 D) The results showed that the latency of first vocalization, first movement and first feeding of CUMS-stressed laying hens were significantly higher than those of the CON group (P<0.05). The above results suggest that chronic unpredictable mild stress has a negative impact on the welfare of laying hens, leading to fear and anxiety in laying hens.

[0040] The following test was used to verify the effect:

[0041] At the end of the behavioral experiment, the animals were slaughtered and sampled, and fresh cecal contents were collected in 1.5 mL sterile tubes, frozen in liquid nitrogen, and transferred to -80°C for metagenomic analysis.

[0042] Figure 2 The results of the changes in the cecal microbiome of laying hens caused by chronic unpredictable mild stress were presented. Based on the OTU and annotation results, species complexity analysis, species diversity analysis and correlation analysis were performed. Figure 2 A Venn diagram shows that the number of OTUs shared by the two groups of samples is 1,004,281, the number of OUTs in the CON group is 15,121, and the number of OTUs in the CUMS group is 13,032, indicating that CUMS significantly reduces the total number of bacteria in the cecum of laying hens.

[0043] Alpha diversity analysis was used to assess the richness and diversity of the microbial community, and the results were Figure 2 As shown in B, the Shannon and Simpson indexes of the cecum of laying hens in the CUMS group were significantly reduced (P<0.05), indicating that the richness and diversity of the population were significantly reduced.

[0044] The results of principal coordinates analysis (PCA) and principal coordinates analysis (PCoA) based on Bray-Curtis distance ( Figure 2 C) showed that the discreteness of samples in the two groups was similar, indicating that the differences in samples within the groups were small and had certain clustering characteristics; however, the samples between the two groups were clearly distinguished, indicating that there were significant differences in the composition of cecal microbial flora between the CON group and the CUMS group. The results of nonmetric multidimensional scaling (NMDS) analysis showed that the NMDS stress value was <0.2, which verified the reliability of the results.

[0045] At door level ( Figure 2 D), Firmicutes, Bacteroidetes and Proteoacteria were the main dominant bacterial phyla in the cecum of the two groups of laying hens. Figure 2 E), Clostridium, Lachnoclostridium, and Bacteroides were the dominant bacterial genera in laying hens. The results of the different bacterial phyla between the two groups are as follows Figure 2 As shown in F, compared with the CON group, the relative abundance of Firmicutes and F / B (Firmicutes / Bacteroidetes) ratio in the cecum of laying hens in the CUMS group was significantly reduced (P<0.05), while the relative abundance of Bacteroidetes and Proteoacteria was significantly increased (P<0.05). The above results show that chronic unpredictable mild stress leads to intestinal microbiota disorder and microecological damage in laying hens.

Claims

1. A method for constructing a chronic unpredictable mild stress model for laying hens, characterized in that: The specific steps of the method are as follows: Step 1: Layer treatment: 300 one-day-old laying hens were randomly divided into two groups, a control group and a chronic unpredictable mild stress group, with the same number of replicates in each group and each replicate containing the same laying hens; Step 2: The laying hens in the two experimental groups in step 1 were raised in two identically constructed environmental control rooms for several weeks. The chronic unpredictable mild stress group was given stressor stimulation and fed a basic diet every day, while the control group was only fed a basic diet without other stress treatments. Step 3: After the chronic unpredictable mild stress program, the stress immobility and attention bias tests were performed to evaluate the effects of chronic unpredictable mild stress on laying hens.

2. The method according to claim 1, characterized in that The number of chickens selected in step 1 was 300; each group had 15 replicates, with 10 laying hens in each replicate.

3. The method according to claim 1, characterized in that In step 2, the time in the environmental control room is 7 weeks, including 1 week of pre-feeding period, 5 weeks of chronic unpredictable mild stress, and 1 week of behavioral testing.

4. The method according to claim 1, characterized in that Method of feeding the basic diet in step 2: feeding once in the morning, noon and evening every day. During the entire experimental period, the chickens can eat and drink freely.

5. The method according to claim 1, characterized in that The environmental control room described in step 2 is a soundproof room with a length, width and height of 150 cm × 150 cm × 90 cm, including a system where temperature, humidity, ventilation and lighting are controlled by a central controller.

6. The method according to claim 1, characterized in that The same environment as in step 2 includes daily handling, with the conditions of a rearing temperature of 16-20°C, a relative humidity of 52%, a light intensity of 20 lux, and a light regime of 14L:10D.

7. The method according to claim 1, characterized in that The stressors described in step 2 were catching chickens for 5 min, artificial driving, strangers walking around for 10 min, fasting for 12 h, water deprivation for 12 h, 4°C cold stress for 3 h, noise disturbance for 3 h, transportation, lighting overnight, waving red and yellow flags for 5 min, and darkness disturbance for 4 h.

8. The method according to claim 1, characterized in that The method of the stress immobility test in step 3 is as follows: 15 laying hens are randomly selected from each group, and the laying hens are carefully placed with their abdomens facing upwards into a "U"-shaped wooden trough. While applying a slight pressure on the chest of the laying hens, the head of the laying hens is slowly pressed. After 15 seconds, the hands are slowly released and the laying hens are gradually moved away from their sight. After successful induction, a stopwatch is used to record the delay time for the laying hens to turn over. If the laying hens try to get up immediately within 10 seconds, it is considered an induction failure, and the above-mentioned restraint operation is repeated again. Successful induction of laying hens is manifested by the fact that they do not express any behavior except trembling and breathing.

9. The method according to claim 1, characterized in that: The method of the attention bias test in step 3 is as follows: laying hens are transported to the test site in a rectangular black plastic basket. The test starts after the laying hens enter the site. A Bluetooth loudspeaker placed out of the line of sight of the laying hens starts playing an alarm that lasts for 10 seconds. After the alarm ends, all activities of the laying hens within 10 minutes are recorded by video surveillance equipment. The length, width and height of the test site are 150cm×150cm×90cm, and the walls are covered with plastic cloth on the four sides. The floor is a white polystyrene foam board. A pile of feed mixed with laying hen rations, oatmeal and dried mealworms is placed in the center of the test site. The alarm is the alarm call of a flock of 240 chickens frightened by the horn of a truck near the chicken coop. The audio data sampling rate is 48kHz and the bit rate is 148kbps.

10. The method according to claim 1, characterized in that During the trial period, the weight and feed consumption of laying hens were recorded, and the average daily weight gain, average daily feed intake and feed-to-weight ratio were calculated.

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