A method for evaluating the resistance of egg ducks by detecting serum components

Abstract

This invention relates to the field of poultry farming technology, specifically to a method for scoring the stress resistance of laying ducks by detecting serum components. Under set farming conditions, a batch of laying ducks is selected; the ducks are divided into two groups through behavioral tests; an equal number of ducks are randomly selected from both groups as the first target laying ducks and the second target laying ducks; serum antioxidant indicators and adrenaline levels of the first and second target laying ducks are obtained, and the corresponding first representative value is determined to be catalase activity and the second representative value to be adrenaline level; differential metabolites between the first and second target laying ducks are obtained, and gabafentin is determined as the third representative value; a multiple linear regression model is established based on the first, second, and third representative values ​​to score the stress resistance of the laying ducks. This invention, through the analytical method of simulating the stress resistance score of laying ducks by detecting serum components, can effectively help farmers breed new breeds of high-quality laying ducks.

Description

Technical Field

[0001] This invention relates to the field of poultry farming technology, and specifically to a method for scoring the stress resistance of laying ducks by detecting serum components. Background Technology

[0002] In China, the cage-raising method for laying ducks is gradually developing. However, ducks are easily frightened under such conditions, and even small disturbances can seriously affect their performance. Among these, heat stress can lead to stunted growth, reduced reproduction and egg production, and decreased meat quality. In extreme cases, prolonged exposure to high temperatures can lead to a decline in poultry egg production performance and even death in severe cases. In addition, in intensive farming models, poultry also face stress from oxidative stress and changes in the microenvironment.

[0003] Animals typically exhibit two states when faced with fear: High Active Avoidance (HAA) and Low Active Avoidance (LAA), often referred to as the "fight or flight" response. Poultry resilience to stress is crucial for their ability to respond appropriately to their social environment. Conversely, extreme aggression in poultry can lead to cross-aggression, thereby reducing productivity. Currently, there is a lack of effective research on the stress resistance of laying ducks during egg production. Therefore, a method for analyzing the stress resistance of laying ducks is urgently needed to help adjust stress conditions that may occur during laying duck farming in a timely manner, thereby enabling the selection of high-quality breeds of laying ducks. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for scoring the stress resistance of laying ducks by detecting serum components. The method includes: selecting a batch of laying ducks under set breeding conditions; dividing the ducks into two groups through behavioral tests; randomly selecting the same number of ducks from both groups as the first target laying ducks and the second target laying ducks; obtaining serum antioxidant indicators and adrenaline levels of the first and second target laying ducks, and determining the corresponding first representative value as catalase activity and the second representative value as adrenaline; obtaining the differential metabolites between the first and second target laying ducks, and determining gabafentin as the third representative value; and establishing a multiple linear regression model based on the first, second, and third representative values ​​to score the stress resistance of the laying ducks. This invention, through the analytical method of simulating the stress resistance score of laying ducks by detecting serum components, can effectively help farmers breed new breeds of high-quality laying ducks.

[0005] This invention employs the following technical solution: a method for scoring the stress resistance of laying ducks by detecting serum components, comprising:

[0006] Select a batch of laying ducks of the same breed under set breeding conditions;

[0007] Behavioral tests were conducted on each selected duck, and the ducks were divided into two groups based on the results of the behavioral tests.

[0008] Select the same number of ducks from any two groups of ducks as the first target ducks and the second target ducks, respectively.

[0009] Blood samples were collected from the first and second target egg-laying ducks to obtain serum antioxidant indicators and adrenal hormone levels.

[0010] The representative factors of serum antioxidant indicators and adrenaline levels were determined by multiple linear regression, and the first representative value corresponding to catalase activity and the second representative value corresponding to adrenaline level were obtained.

[0011] Differential metabolite analysis was performed on the blood of the first target laying duck and the second target laying duck to obtain the differential metabolites between the two ducks, and gabafentin was determined as the third representative value of the differential metabolites.

[0012] A multiple linear regression model is established based on the first representative value, the second representative value, and the third representative value.

[0013] The stress resistance of laying ducks was scored using the aforementioned multiple linear regression model.

[0014] Furthermore, the established aquaculture conditions include:

[0015] Each laying duck is placed in a duck cage measuring 28cm×40cm×40cm;

[0016] The temperature in the duck house for laying ducks should be 20-25℃, and the relative humidity should be 65%-75%.

[0017] The lighting cycle in the duck house for laying ducks is 16 hours of light and 8 hours of darkness.

[0018] Furthermore, based on the behavioral test results, the laying ducks were divided into two groups, specifically:

[0019] The laying ducks were sorted from highest to lowest according to their behavioral test scores. The ducks with the highest scores were assigned to the LAA group, and the ducks with the lowest scores were assigned to the HAA group, resulting in two groups of the same number of ducks.

[0020] In the LAA group, select the highest-scoring ducks as the first target ducks, and in the HAA group, select the lowest-scoring ducks as the second target ducks.

[0021] Furthermore, the serum antioxidant indicators include: total antioxidant capacity, superoxide dismutase activity, catalase activity, and malondialdehyde content.

[0022] Furthermore, the adrenal hormone levels include: adrenocorticotropic hormone, norepinephrine, epinephrine, and dopamine.

[0023] Furthermore, based on the first representative value, the second representative value, and the third representative value, a multiple linear regression model is established, including:

[0024] SPSS software was used to generate a multiple linear regression model for the first representative value, the second representative value, and the third representative value.

[0025] In the SPSS software, the Shapiro-Wilke test was used for phenotypic analysis.

[0026] In the SPSS software, the Student's t-test was used for data comparison.

[0027] The beneficial effects of this invention are as follows: The analytical method proposed in this invention, which simulates the stress resistance score of laying ducks by detecting serum components, first classifies laying ducks through behavioral science, which can initially screen out laying ducks with a certain degree of good adaptability to the environment. Then, by collecting relevant data from the blood of laying ducks through technical means, and combining serum catalase activity, adrenaline and differential metabolites plus bafenidine to establish a multiple linear regression model, an accurate and reasonable stress resistance score of laying ducks can be obtained, thereby effectively helping farmers to breed new breeds of high-quality laying ducks. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of a method for scoring the stress resistance of laying ducks by detecting serum components according to an embodiment of the present invention;

[0030] Figure 2 This is a volcanic map of the differential metabolites in the LAA group and HAA group in this embodiment of the invention;

[0031] Figure 3 This is a normal PP plot of the regression standardized residuals in an embodiment of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] A schematic flowchart of a method for scoring the stress resistance of laying ducks by detecting serum components according to an embodiment of the present invention is shown below. Figure 1 As shown, it includes:

[0034] Select a batch of laying ducks under the established breeding conditions;

[0035] Specifically, the laying ducks are placed individually in three-layer cages, each 28 cm long, with each cage measuring 28 cm × 40 cm × 40 cm. The temperature in the duck house is controlled at 20-25℃, the relative humidity at 65%-75%, and the light cycle is 16 hours of light and 8 hours of darkness (4 a.m. to 8 p.m.). The feed composition is formulated according to the Chinese laying duck standard (GB / T 41189-2021).

[0036] During the breeding process, this invention measures various parameters, including average daily egg weight (ADEW), feed intake (FI), body weight (BW), average egg weight (AEW), and weight gain (ΔW), records the number of eggs laid and the feed weight for each replicate, and records the amount of leftover feed for each replicate to determine the average daily feed intake (ADFI) and feed / egg ratio (F / E).

[0037] Behavioral tests were conducted on 300 healthy laying ducks with no difference in initial weight. Based on the results of the behavioral tests, the laying ducks were divided into two groups.

[0038] Specifically, within one experimental period, a fluorescent pen was fixed behind the cage wall for 30 seconds, 1 cm away from the ducks, and the response states corresponding to different scores were recorded. It should be noted that this invention involves independent behavioral testing to prevent ducks in adjacent cages from seeing these objects prematurely. The ducks with the highest and lowest scores were selected for subsequent analysis.

[0039] In one specific embodiment, after behavioral testing, the present invention divides the 12 ducks with the highest and lowest scores (a total of 24 ducks) into the LAA group and the HAA group, respectively.

[0040] Select the same number of ducks from any two groups of ducks as the first target ducks and the second target ducks, respectively.

[0041] In this invention, blood is collected from 6 out of 12 ducks in each group (using venous blood collection method) and stored at -80°C.

[0042] Blood samples were collected from the first and second target egg-laying ducks to obtain serum antioxidant indicators and adrenal hormone levels.

[0043] The representative factors of serum antioxidant indicators and adrenaline levels were determined by multiple linear regression, and the first representative value corresponding to catalase activity and the second representative value corresponding to adrenaline were obtained.

[0044] In this embodiment of the invention, serum antioxidant indicators include the determination of total antioxidant capacity (T-AOC, Total Antioxidant Capacity Kit, No. HY-60021), superoxide dismutase activity (SOD, Superoxide Dismutase Kit, No. HY-M0001), catalase activity (CAT, Catalase Kit, No. HY-M0018), and malondialdehyde content (MDA, Malondialdehyde Kit, No. HY-M0003).

[0045] Serum adrenal hormone levels (ACTH), norepinephrine (NOR), adrenaline (ADR), and dopamine (DA) were extracted using an adrenaline extraction kit. All levels were determined by measuring absorbance at 450 nm using an ELISA kit, and the concentrations of immunoglobulins and cytokines were calculated using a standard curve analysis method.

[0046] Differential metabolite analysis was performed on the blood of the first and second target laying ducks to obtain the differential metabolites between the two target laying ducks, and gabafentin was determined as the third representative value.

[0047] In this embodiment of the invention, serum samples are thawed at -80°C and vortexed for 1 minute. The samples are mixed with methanol at a ratio of 1:4 and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant is extracted, concentrated, and dried. A 2-chloro-1-phenylalanine solution is added to 80% methanol, the extract is filtered, and analyzed using LC-MS. In order to correct analytical biases caused by mixed samples and instrument errors, a portion of the quality control (QC) sample is mixed with the final sample.

[0048] LC-mass spectrometry analysis was performed using an ultra-high performance liquid chromatography (UHPLC) system, which is equipped with a data acquisition system. HSST3 columns were used, and raw XCMS data were converted to mzXML format using the XCMS wizard. Metabolites were identified based on accurate quality and mass spectrometry / mass spectrometry data and compared with various existing databases. The contribution of variables to classification was determined using p-value, predictor variable (VIP), and fold difference (FC), with significance thresholds of 0.05 for p < and 1.0 for VIP >. This yielded differential metabolites between the first and second target laying ducks, and gabafentin was identified as the third representative value.

[0049] A multiple linear regression model was established based on the first, second, and third representative values; the stress resistance of laying ducks was scored using the multiple linear regression model.

[0050] The statistical analysis in this embodiment of the invention was performed using SPSS software (version 22.0); the phenotypic analysis was performed using the Shapiro-Wilk test, and the data comparison was performed using the Student's t-test.

[0051] Multiple linear regression simulations performed using the SPSS software package, such as Figure 3 As shown, in this embodiment of the invention, the relationship between the stress resistance score and the obtained serum antioxidant index corresponding to the first representative value, the adrenaline hormone corresponding to the second representative value, and the differential metabolite is assumed to be linear. That is, the stress resistance score can be predicted by detecting a linear combination of serum components. In this embodiment of the invention, based on the R-squared value in the multiple linear regression method, the first representative value is catalase (CAT), the second representative value is adrenaline (ADR), and the third representative value is the differential metabolite gabapentin. The mathematical expression of the multiple linear regression model is thus established as follows:

[0052] Y = β0 + β1X1 + β2X2 + ... + β n X n

[0053] Where Y is the score value, X1, X2, ..., X n This invention is for detecting serum components, β0, β1, β2, ..., β n It is the regression coefficient.

[0054] In another embodiment of the present invention:

[0055] In this invention, 300 healthy 42-week-old Muscovy ducks were randomly selected and subjected to a 60-day experiment and behavioral test. Scores were assigned based on different behavioral performances, as shown in Table 1. In this embodiment of the invention, these ducks were divided into two groups, each group including 12 extreme individuals (24 in total) exhibiting two different behaviors. The group with the highest score (LAA) included 12 ducks, while the group with the lowest score (HAA) also included 12 ducks.

[0056] Table 1. Description and distribution of different behavioral responses in the ducks during the behavioral test.

[0057]

[0058] In this embodiment of the invention, during the 60-day trial, a behavioral test was conducted on the 30th day. A fluorescent pen was fixed behind the cage wall for 30 seconds, 1 cm away from the ducks, and the response methods corresponding to different scores were recorded. Five consecutive tests were conducted over a week, and the behavioral tests were conducted independently to prevent ducks in adjacent cages from seeing these objects in advance. The ducks with the highest and lowest scores were selected for subsequent analysis. All tests were conducted by the same person at the same time every day.

[0059] After behavioral testing, in this embodiment of the invention, the 12 ducks with the highest scores and the 12 ducks with the lowest scores (a total of 24 ducks) were divided into the LAA group and the HAA group, respectively. At the end of the experiment, blood was collected from 6 of the 12 ducks in each group (using venous blood collection method) and stored at -80°C.

[0060] In this embodiment of the invention, various parameters were recorded during the experiment, including: average daily egg weight (ADEW), feed intake (FI), body weight (BW), average egg weight (AEW), and weight gain (ΔW). The number of eggs laid and the feed weight for each replicate were recorded daily. Furthermore, the amount of remaining feed for each replicate was recorded weekly to determine the average daily feed intake (ADFI) and feed / egg ratio (F / E). The phenotypic differences among different actively avoiding laying ducks are shown in Table 2.

[0061] Table 2. Phenotypic differences among ducks actively avoiding egg production.

[0062]

[0063]

[0064] Wherein, BW represents body weight, ΔW represents body weight gain, ADEW represents average daily egg weight, ADFI represents average daily feed intake, F / E represents feed conversion ratio, AEW represents average egg weight, and Score corresponds to the score in Table 1; HAA represents the high active avoidance group, and LAA represents the low active avoidance group. P-values ​​were obtained through a t-test between HAA and LAA. The results showed that the ΔW and ADEW of the HAA group were significantly lower than those of the LAA group (P<0.05), and the F / E was significantly higher than that of the LAA group (P<0.05). There were no significant differences in BW, ADFI, and AEW between the two groups (P>0.05).

[0065] Under intensive farming conditions, the environment has a substantial impact on laying ducks. However, research on the resistance of laying ducks to caged environments is limited. This invention shows that, compared to the LAA group, the HAA group of laying ducks exhibited significantly lower ΔW and ADEW, and a significantly higher F / E ratio, indicating that the level of active avoidance affects egg production. In this invention, serum antioxidant indicators included the determination of total antioxidant capacity (T-AOC, total antioxidant capacity kit), superoxide dismutase activity (SOD, superoxide dismutase kit), catalase activity (CAT, catalase kit), and malondialdehyde (MDA, malondialdehyde kit). Table 3 shows a schematic table illustrating the detection of serum antioxidant and hormone levels in laying ducks under different active avoidance conditions.

[0066] Table 3. Serum antioxidant and hormone levels in laying ducks under different active avoidance conditions.

[0067]

[0068] Among them, T-AOC represents total antioxidant capacity; CAT represents catalase; SOD represents superoxide dismutase; MDA represents malondialdehyde; HAA represents high active avoidance group; LAA represents low active avoidance group; DA represents dopamine; ADR represents adrenaline; NOR represents norepinephrine; and ACTH represents adrenaline. The P value is obtained by t-test between HAA and LAA. It can be seen that the serum SOD, CAT activities and T-AOC in the HAA group are significantly lower than those in the LAA group (P<0.05). In addition, the serum MDA levels in the two groups are comparable (P>0.05). The results show that the serum DA, ADR and NOR levels in the HAA group are significantly lower than those in the LAA group (P<0.05), while the serum ACTH level in the HAA group is significantly higher than that in the LAA group (P<0.05).

[0069] As can be seen, the ACTH level of the HAA group of laying ducks was higher than that of the LAA group, while the NOR level was lower. This is because ACTH promotes the release of adrenal glucocorticoids, enabling ducks to remember the fear and participate in the HAA, while the LAA group of laying ducks were not afraid because the NOR level improved the fear condition.

[0070] Serum adrenal hormone levels (ACTH), norepinephrine (NOR), adrenaline (ADR), and dopamine (DA) were extracted using an adrenaline extraction kit. All levels were determined by measuring absorbance at 450 nm using an ELISA kit, and the concentrations of immunoglobulins and cytokines were calculated using a standard curve analysis method.

[0071] In the presence of fear, animals may experience oxidative stress, leading to the production and accumulation of reactive oxygen species (ROS). The brain is highly susceptible to ROS-induced oxidative damage because, due to higher aerobic metabolism and blood perfusion rates, as well as relatively poor antioxidant enzyme activity, the brain produces excessive ROS. Therefore, oxidative stress may lead to neuronal damage and memory impairment. The serum SOD, CAT activity, and T-AOC levels of HAA ducks were significantly lower than those of LAA group laying ducks, suggesting that they have strong antioxidant capacity and can resist fear. When animals encounter stressful situations, they respond to environmental changes by releasing large amounts of hormones, neurotransmitters, and peptides. The experimental results of this invention also showed higher serum NOR and ACTH levels. In addition, NOR and glucocorticoids have opposite effects. Although glucocorticoids impair memory recovery and sensory recovery, resulting in less specific fear memories generated in subsequent tests, norepinephrine can awaken and may promote certain retrieval processes, thereby preventing generalization. Furthermore, glucocorticoids enhance memory after encoding, while norepinephrine stimulates long-term specificity, thereby promoting memory.

[0072] In this embodiment of the invention, the results of serum metabolomics analysis of laying ducks under different active avoidance conditions were as follows: a total of 6077 serum metabolites were detected in the two groups, of which 314 were annotated according to molecular weight and matching annotations in the database.

[0073] like Figure 2As shown, when comparing HAA and LAA, the LAA group had 4 upregulated metabolites and 7 downregulated metabolites (P<0.05). Metabolomics is the analysis of metabolite levels in vivo to capture changes in the metabolic responses of an organism. In this invention, non-targeted LC-MS detected 6074 metabolites in all samples, of which 11 showed differential expression. Compared with HAA, the LAA group had 4 upregulated metabolites and 7 downregulated metabolites. In particular, gabafentin was significantly upregulated in the serum of the LAA group ducks. Gabafentin is a drug approved in several countries for the treatment of anxiety disorders, and its main function is as a sedative. It inhibits neuronal voltage-gated calcium channels by binding to accessory subunits that regulate channel transport and function. In this invention, the LAA group ducks recovered to a normal state when faced with fear due to the upregulated serum gabafentin levels; in addition, the 3-oxalate level was downregulated in the LAA group ducks compared with the HAA group ducks. 3-Gantrenic acid is associated with the tricarboxylic acid cycle (TCA) and may induce oxidative cell death, explaining the increased oxidative stress in the HAA group of laying ducks. In the LAA group, the serum level of γ-linolenic acid (GLA) was significantly downregulated. Low GLA levels are usually found in inflammatory cells because they are converted into diiso-γ-linolenic acid (DGLA) in vivo. DGLA acts as a substrate for cyclooxygenase, producing an inflammatory-suppressive arachidonic acid.

[0074] In this embodiment of the invention, R-squared means that the independent variables catalase (CAT), adrenaline (ADR), and the differential metabolite gabapentin can explain 87.7% of the score changes. That is, 87.7% of the score changes are related to the catalase activity, adrenaline level, and gabapentin expression level in the serum of laying ducks. The summary table of the multiple linear regression model of this invention is shown in Table 4:

[0075] Table 4 Summary of Multiple Linear Regression Models

[0076]

[0077] In this table, the predictor variables are CAT, ADR, and Gabapentin; the dependent variable is the score. Furthermore, the coefficient table of the multiple linear regression model of this invention is shown in Table 5.

[0078] Table 5. Coefficients of the Multiple Linear Regression Model

[0079]

[0080] It can be seen that a significance level less than 0.05 indicates that the independent variable can significantly affect the change in score. B is the influence coefficient. Therefore, the scoring model result obtained from this embodiment of the invention is:

[0081] Y=-5.579+0.020×X1+0.025×X2+5.58×10 -7 ×X3

[0082] Where X1 is CAT; X2 is ADR; and X3 is gabafentin.

[0083] In summary, the analytical method proposed in this invention for simulating the stress resistance score of laying ducks by detecting serum components first classifies laying ducks through behavioral analysis, which can initially screen out laying ducks with a certain degree of good adaptability to the environment. Then, by collecting relevant data from the blood of laying ducks using technical means, and combining this data with serum catalase activity, adrenaline hormones, and the differential metabolite plus bafenidine to establish a multiple linear regression model, an accurate and reasonable stress resistance score for laying ducks can be obtained, thus effectively helping farmers to breed new high-quality laying duck varieties.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for scoring the stress resistance of laying ducks by detecting serum components, characterized in that, include: Select a batch of laying ducks of the same breed under set breeding conditions; Behavioral tests were conducted on each selected duck, and the ducks were divided into two groups based on the results. The behavioral tests were conducted by fixing a fluorescent pen behind the cage wall for 30 seconds, 1 cm away from the duck, and recording the response corresponding to different scores. Five consecutive independent behavioral tests were conducted over a week, and the ducks with the highest and lowest scores were selected for further analysis. The laying ducks were sorted from highest to lowest according to their behavioral test scores. The ducks with the highest scores were assigned to the LAA group, and the ducks with the lowest scores were assigned to the HAA group, resulting in two groups of the same number of ducks. In the LAA group, select the highest-scoring ducks as the first target ducks, and in the HAA group, select the lowest-scoring ducks as the second target ducks. Select the same number of ducks from any two groups of ducks as the first target ducks and the second target ducks, respectively. Blood samples were collected from the first and second target egg-laying ducks to obtain serum antioxidant indicators and adrenal hormone levels. The representative factors of serum antioxidant indicators and adrenaline levels were determined by multiple linear regression, and the first representative value corresponding to catalase activity and the second representative value corresponding to adrenaline level were obtained. Differential metabolite analysis was performed on the blood of the first target laying duck and the second target laying duck to obtain the differential metabolites between the two ducks, and gabafentin was determined as the third representative value of the differential metabolites. A multiple linear regression model is established based on the first representative value, the second representative value, and the third representative value. The stress resistance of laying ducks was scored using the aforementioned multiple linear regression model.

2. The method for scoring the stress resistance of laying ducks by detecting serum components according to claim 1, characterized in that: The established aquaculture conditions include: Each laying duck is placed in a duck cage measuring 28 cm × 40 cm × 40 cm; The temperature in the duck house for laying ducks should be 20-25℃, and the relative humidity should be 65%-75%. The lighting cycle in the duck house for laying ducks is 16 hours of light and 8 hours of darkness.

3. The method for scoring the stress resistance of laying ducks by detecting serum components according to claim 1, characterized in that: The serum antioxidant indicators include: total antioxidant capacity, superoxide dismutase activity, catalase activity, and malondialdehyde content.

4. The method for scoring the stress resistance of laying ducks by detecting serum components according to claim 1, characterized in that: The adrenal hormone levels include: adrenocorticotropic hormone, norepinephrine, epinephrine, and dopamine.

5. The method for scoring the stress resistance of laying ducks by detecting serum components according to claim 1, characterized in that: Based on the first representative value, the second representative value, and the third representative value, a multiple linear regression model is established, including: SPSS software was used to generate a multiple linear regression model for the first representative value, the second representative value, and the third representative value. In the SPSS software, the Shapiro-Wilke test was used for phenotypic analysis. In the SPSS software, the t-test was used for data comparison.

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