Metabolite marker related to heat stress of broiler chickens and application of metabolite marker

Abstract

The invention provides a metabolite marker related to heat stress of broiler chickens and application of the metabolite marker. The marker comprises adenosine. Based on metabonomics, compared with healthy broilers, the abundance of adenosine in broilers about to be subjected to heat stress or having been subjected to heat stress is obviously reduced, and adenosine can accurately distinguish healthy broilers from broiler individuals about to be subjected to heat stress or having been subjected to heat stress and can be used as a metabolic marker for detecting and / or treating broiler heat stress. And the method can be used for screening heat-stress-resistant broiler individuals, and is of great significance to actual production.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology. Specifically, it relates to a metabolite biomarker related to broiler heat stress and its application. Background Art

[0002] With the intensification of global warming and the popularization of intensive farming models, the problem of broiler heat stress has become a key bottleneck restricting the sustainable development of the poultry industry. Heat stress disrupts the body's energy metabolism homeostasis, induces oxidative damage and neuroendocrine disorders, resulting in a decline in broiler growth performance (for example, reduced feed intake and decreased body weight gain) and an increase in mortality, causing certain economic losses. Although existing technologies evaluate the degree of heat stress by detecting serum corticosterone, heat shock proteins (HSP70 / 90) and enzyme activity indicators (such as lactate dehydrogenase), these biomarkers have significant limitations: firstly, insufficient specificity (such as corticosterone responding to multiple stressors simultaneously, and HSP70 also increasing in inflammatory responses); secondly, relying on invasive tissue sampling (for example, detecting myocardial HSP requires sacrificing animals), making it difficult to achieve in vivo dynamic monitoring.

[0003] The breakthrough of metabolomics technology provides a new idea for this problem. As a "dynamic bridge" connecting genotype and phenotype, metabolomics can systematically analyze the metabolic pathway reconstruction caused by heat stress, such as disturbances in the tricarboxylic acid cycle and increased lipid peroxidation, and accurately capture stress characteristics through serum metabolite profiles. Compared with traditional protein biomarkers, serum metabolites have the outstanding characteristics of strong operability, small sample volume, rapid sensitivity and non-invasiveness. Only a small amount of serum is required to achieve high-throughput detection through LC-MS / MS, which is suitable for in vivo continuous monitoring. Therefore, it is effective and feasible to use serum metabolomics technology to quantitatively analyze the levels of various metabolites in the serum of heat-stressed chickens, find relatively specific and significantly changed metabolite types, and use them as potential heat stress biomarkers, which has important value for actual production. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent.

[0005] Starting from the perspective of metabolism, through metabolomics, the present invention discovers a serum metabolite adenosine, whose molecular formula is C 10 H 13 N5O4, with a molecular weight of 267.24 and a CAS registration number of 58-61-7. Adenosine can predict and monitor broiler heat stress, and can be used as an important serum metabolite to predict and monitor the occurrence of broiler heat stress, and screen heat stress-resistant broilers, providing new ideas for reducing the occurrence of broiler heat stress and screening heat stress-resistant broilers.

[0006] Therefore, in the first aspect of the present invention, the present invention proposes a method for screening metabolic markers for heat-stressed broilers. According to the embodiments of the present invention, the markers include adenosine. According to the specific embodiments of the present invention, the metabolic markers are determined by the inventors through differential comparative analysis of the abundances (levels) of metabolites in serum samples of a large number of heat-stressed broiler individuals and a large number of healthy control broiler individuals, as well as verification. The metabolic markers according to the embodiments of the present invention show significant differences between healthy broilers and heat-stressed broiler populations. The abundance of adenosine in the serum of heat-stressed broilers decreases significantly, which can accurately distinguish healthy broilers from heat-stressed broilers. Furthermore, it is also possible to determine the high or low probability of a broiler experiencing heat stress or being in a healthy state, which can be used to predict, early detect, or screen for broiler heat stress, or screen for heat-stress-resistant broilers that are not prone to heat stress.

[0007] In the second aspect of the present invention, the present invention proposes the use of a reagent for detecting the metabolic marker described in the first aspect in the preparation of a kit for screening heat-stress-resistant broilers. The metabolic markers according to the embodiments of the present invention show significant differences between healthy broilers and heat-stressed broiler populations. Compared with healthy individuals, the abundance of the metabolic marker in the serum of heat-stressed broilers decreases significantly. Therefore, this metabolic marker can accurately distinguish healthy broilers from heat-stressed broilers. Therefore, a kit prepared using a reagent for detecting this metabolite can not only predict or assist in predicting the occurrence of broiler heat stress, but also screen for heat-stress-resistant broilers.

[0008] In the third aspect of the present invention, the present invention proposes a kit. According to the embodiments of the present invention, the kit contains a reagent suitable for detecting the metabolic marker described in the first aspect. The kit can not only detect broilers that are prone to and / or have experienced heat stress, but also screen for heat-stress-resistant broilers.

[0009] It should be noted that the kit can be used in actual production and / or scientific research, such as qualitatively or quantitatively detecting the level of metabolic markers in biological samples, and can also be used to judge the individual state. For example, after obtaining the metabolic marker level of an individual, it can be judged whether its metabolic marker level is significantly higher or lower than the normal level, and whether the individual is a heat-stress-resistant individual. The biological sample can be cells, tissues, blood, serum, etc.

[0010] In the fourth aspect of the present invention, the present invention provides the use of the metabolic marker described in the first aspect in screening heat stress-resistant broilers. According to the embodiments of the present invention, there are significant differences in the metabolic markers between healthy broilers and heat-stressed broilers. Compared with healthy individuals, the abundance of the metabolic marker in the serum of heat-stressed broilers is significantly reduced. Therefore, this metabolic marker can accurately distinguish between healthy broilers and heat-stressed broilers. Therefore, using this metabolite can not only predict or assist in predicting the occurrence of heat stress in broilers, but also screen out heat stress-resistant broilers.

[0011] According to the embodiments of the present invention, the abundance of the metabolic marker of the broiler reaching or higher than a predetermined threshold is an indication that the broiler is a heat stress-resistant broiler.

[0012] According to the embodiments of the present invention, the threshold includes the abundance value of the metabolic marker described in the first aspect in known heat stress-resistant broilers or non-heat stress-resistant broilers.

[0013] According to some specific embodiments of the present invention, the threshold is preset. The abundance (level) values of the metabolic marker in healthy broiler individuals (heat stress-resistant broiler individuals) and heat-stressed broilers (non-heat stress-resistant broiler individuals) are pre-determined and saved for use as the basis for setting the threshold. The threshold can be a numerical value or a numerical range. Based on the average abundance of the metabolic marker in known heat-stressed or healthy-state broiler individuals, the corresponding threshold for this metabolite marker can be set to the 95%, 90%, 85%, 80%, 75% or 70% confidence interval of the average abundance of this metabolite. It should be noted that according to different purposes or requirements, there may be different requirements for the credibility of determining the state results of broiler individuals, and those skilled in the art can select different significance levels or thresholds. According to the specific embodiments of the present invention, the confidence interval is set to 95%. In the present invention, the abundance of the metabolic marker in healthy broiler individuals (heat stress-resistant broiler individuals) is 1577.90 ± 322.74, and the abundance of the metabolic marker in heat-stressed broilers (non-heat stress-resistant broiler individuals) is 1022.38 ± 267.01. This value of the present invention can be used as the threshold. For example, the abundance of the metabolic marker of the broiler reaching or higher than 1577.90 ± 322.74 is an indication that the broiler is a heat stress-resistant broiler. It should be noted that this abundance value is the original abundance value.

[0014] In the fifth aspect of the present invention, the present invention provides a method for screening heat stress-resistant broilers. According to the embodiments of the present invention, the method includes: (1) determining the abundance of the metabolic marker described in the first aspect in the serum sample of the broiler to be tested; (2) comparing the abundance obtained in step (1) with a predetermined threshold to determine whether the broiler to be tested is a heat stress-resistant broiler.

[0015] According to an embodiment of the present invention, the level of the metabolic marker in step (1) is obtained by targeted detection or non-targeted detection. According to a specific embodiment of the present invention, the method suitable for detecting the level of the metabolic marker is not particularly limited, and any reagent that can directly or indirectly detect the level of the metabolic marker is included within the scope of the present invention. For example, targeted metabolomics and non-targeted metabolomics. The former is the quantitative analysis of specific metabolites; the latter is the evaluation of all quantifiable analytes in a specific sample, including unknown biochemical substances. The analysis platform of metabolomics technology consists of separation technology and detection technology. The separation technology includes liquid chromatography (LC) and gas chromatography (GC). Due to the wide availability and continuous development of instruments, liquid chromatography coupled with mass spectrometry (LC-MS), gas chromatography coupled with mass spectrometry (GC-MS), or capillary electrophoresis coupled with mass spectrometry (CE-MS) technologies are now more widely used in metabolomics analysis, and each technology has unique advantages in terms of sensitivity, accuracy, resolution, dynamic range, reproducibility, and throughput.

[0016] According to an embodiment of the present invention, step (1) is achieved by ultra-high performance liquid chromatography-mass spectrometry analysis method.

[0017] According to an embodiment of the present invention, the abundance obtained in step (1) reaching or higher than the abundance value of the metabolic marker described in claim 1 in heat stress-resistant broilers is an indication that the broiler is a heat stress-resistant broiler.

[0018] According to an embodiment of the present invention, the threshold is preset. Exemplarily, it includes the abundance value of the metabolic marker described in the first aspect in known heat stress-resistant broilers or non-heat stress-resistant broilers. The abundance (level) values of the metabolic marker in healthy broiler individuals and heat-stressed broilers are pre-determined and saved to be used as the basis for setting the threshold. The threshold can be a numerical value or a numerical range. Based on the average abundance of the metabolic marker in known heat-stressed or healthy state broiler individuals, the corresponding threshold of this metabolite marker can be set to the confidence interval of 95%, 90%, 85%, 80%, 75% or 70% of the average abundance of this metabolite. It should be noted that according to different purposes or requirements, there may be different requirements for the credibility of the result of determining the state of broiler individuals. Those skilled in the art can select different significance levels or thresholds. According to a specific embodiment of the present invention, the confidence interval is set to 95%. In the present invention, the abundance of the metabolic marker in healthy broiler individuals (heat stress-resistant broiler individuals) is 1577.90±322.74, and the abundance of the metabolic marker in heat-stressed broilers (non-heat stress-resistant broiler individuals) is 1022.38±267.01. This value of the present invention can be used as the threshold. For example, when the abundance of the metabolic marker of the broiler reaches or is higher than 1577.90±322.74, it is an indication that the broiler is a heat stress-resistant broiler. It should be noted that this abundance value is the original abundance value.

[0019] In the sixth aspect of the present invention, the present invention proposes a device for determining whether a to-be-detected broiler is a heat stress-resistant broiler. According to an embodiment of the present invention, the device includes: a metabolic marker abundance determination unit for determining the abundance of the metabolic marker described in the first aspect in the serum sample of the to-be-detected broiler; a comparison unit for comparing the obtained abundance with a predetermined threshold to determine whether the to-be-detected broiler is a heat stress-resistant broiler. The device according to the embodiment of the present invention can accurately determine whether a broiler individual is a high-risk individual of heat stress or has already suffered from heat stress, and screen out heat stress-resistant broiler individuals.

[0020] According to an embodiment of the present invention, the threshold includes the abundance value of the metabolic marker described in the first aspect in known heat stress-resistant broilers or non-heat stress-resistant broilers.

[0021] According to an embodiment of the present invention, when the abundance of the metabolic marker of the to-be-detected broiler reaches or is higher than the predetermined threshold, it is an indication that the broiler is a heat stress-resistant broiler.

[0022] According to an embodiment of the present invention, the threshold is preset. Exemplarily, it includes the abundance value of the metabolic marker described in the first aspect in known heat - stress - resistant broilers or non - heat - stress - resistant broilers. The abundance (level) values of the metabolic marker in healthy broiler individuals and heat - stressed broilers are pre - determined and saved to be used as the basis for setting the threshold. The threshold can be a numerical value or a numerical range. Based on the average abundance of the metabolic marker in known heat - stressed or healthy broiler individuals, the corresponding threshold of this metabolite marker can be set as the 95%, 90%, 85%, 80%, 75% or 70% confidence interval of the average abundance of this metabolite. It should be noted that according to different purposes or requirements, there may be different requirements for the credibility of the result of determining the state of broiler individuals. Those skilled in the art can select different significance levels or thresholds. According to a specific embodiment of the present invention, the confidence interval is set to 95%. In the present invention, the abundance of the metabolic marker in healthy broiler individuals (heat - stress - resistant broiler individuals) is 1577.90 ± 322.74, and the abundance of the metabolic marker in heat - stressed broilers (non - heat - stress - resistant broiler individuals) is 1022.38 ± 267.01. The value of the present invention can be used as the threshold. For example, when the abundance of the metabolic marker of the broiler reaches or is higher than 1577.90 ± 322.74, it is an indication that the broiler is a heat - stress - resistant broiler. It should be noted that this abundance value is the original abundance value.

[0023] In the seventh aspect of the present invention, the present invention provides a device for screening heat - stress - resistant broilers. According to an embodiment of the present invention, the device includes: a computer - readable storage medium storing a computer program thereon, and the program is used to execute the method described in the fifth aspect; and one or more processors for executing the program in the computer - readable storage medium. The device according to the embodiment of the present invention can accurately determine whether a broiler individual is a high - risk individual of heat stress or has already had heat stress, and screen out heat - stress - resistant broiler individuals.

[0024] In the eighth aspect of the present invention, the present invention provides the use of a preparation for increasing the abundance of the metabolic marker described in the first aspect in the preparation of a drug for preventing, alleviating and / or treating heat stress in broilers.

[0025] In the ninth aspect of the present invention, the present invention provides a drug. According to an embodiment of the present invention, the drug contains a preparation for increasing the abundance of the metabolic marker described in the first aspect. As mentioned above, compared with healthy broiler individuals, the abundance of the metabolic marker described in the first aspect in heat - stressed broiler individuals is significantly reduced. Therefore, a preparation that can increase this metabolic marker can effectively prevent, alleviate and / or treat heat stress in broilers.

[0026] The above-mentioned metabolic markers were determined by the inventors through differential comparative analysis of the abundances of various serum metabolites in the serum samples of the broiler leg disease group and the healthy group, and verified through a large number of serum samples with known status. Adenosine showed a significant decrease in abundance in the heat stress group compared to the healthy group. The significant decrease means that the abundances of the above-mentioned metabolites in the heat stress group were statistically lower or significantly and substantially lower than those in the healthy broiler group compared to the abundances in the healthy group; a preparation (substance) that can increase the abundance of adenosine can be used for preventing, alleviating, and / or treating the heat stress of broilers or is beneficial for heat-stressed broilers to take. The preparation (substance) is not particularly limited, and substances that can increase its abundance include, but are not limited to, drugs for treating heat stress.

[0027] According to an embodiment of the present invention, the above-mentioned drug further includes at least one of the following additional technical features.

[0028] According to some embodiments of the present invention, the drug further includes a pharmaceutically acceptable excipient or carrier.

[0029] In the tenth aspect of the present invention, the present invention proposes a method for screening drugs. According to an embodiment of the present invention, the drug is used for preventing, alleviating, and / or treating the heat stress of broilers. The method includes: (1) administering a candidate drug to the heat-stressed broilers; (2) detecting the abundance of the metabolic marker described in the first aspect in the serum of the heat-stressed broilers before and after administration. Among them, after the administration, a candidate drug that increases the abundance of the metabolic marker is suitable for preventing, alleviating, and / or treating the heat stress of broilers. The method according to the embodiment of the present invention can produce or screen drugs that increase the abundance of the metabolic marker, which is of great significance for preventing, alleviating, and / or treating the heat stress of broilers.

[0030] The additional aspects and advantages of the present invention will be partly given in the following description, partly become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a detection result diagram of the Adenosine levels in the sera of the healthy control group broilers and the heat stress group broilers according to Embodiment 1 of the present invention, where C represents the healthy control group and T represents the heat stress group; Figure 2 Detection result graph of differential accumulation of Adenosine in the sera of broilers in the healthy control group (Control) and the heat stress group (HS) according to Example 1 of the present invention; Figure 3 Detection result graph of metabolites with increased or decreased levels in the sera of broilers in the healthy control group (Control) and the heat stress group (HS) according to Example 1 of the present invention; Figure 4 Importance ranking of serum metabolites obtained by random forest screening for broilers in the healthy control group (Control) and the heat stress group (HS) according to Example 1 of the present invention; Figure 5 Graph showing the between-group difference results of Adenosine levels in 30 individual broilers in the second phase according to Example 2 of the present invention, where Content represents the content and Group represents the group (C represents the healthy control group and T represents the heat stress group); Figure 6 Graph showing the AUC value results under the ROC curve for the 30 sample data in the second phase according to Example 2 of the present invention, where the confidence interval is 95%, Specificity represents the specificity, that is, predicted as positive and actually positive, true positive, and the ordinate Sensitivity represents the sensitivity, that is, true negative. Detailed implementation manners

[0033] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as a limitation to the present invention.

[0034] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0035] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0036] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0037] In this text, unless otherwise specified, "a plurality of" means two or more. In this text, unless otherwise clearly defined and limited, terms such as "connected" and "joined" shall be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components.

[0038] In this text, a biological marker is a cellular, biochemical, or molecular alteration that can be detected from a biological medium. Biological media include various body fluids, blood, plasma, serum, tissues, cells, feces, hair, exhaled breath, etc. In the present invention, the biological medium is blood and / or serum.

[0039] In this text, the chemical structure of adenosine is shown as Formula I:

[0040] Formula I.

[0041] In this text, broiler chickens include white - feather broiler chickens. More specifically, broiler chickens include Guangming No. 2 white - feather broiler chickens. Thus, it is possible to more accurately judge the probability of heat stress occurrence and screen heat - stress - resistant broiler chickens.

[0042] In this text, "heat stress" in broiler chickens refers to a series of physiological and behavioral stress responses that occur when chickens are in a high - temperature environment and the environmental temperature exceeds the upper limit of their thermoneutral zone. In this situation, the chicken body cannot effectively dissipate heat through its own regulatory mechanism, resulting in an increase in body temperature. This stress response will have a negative impact on the production performance, health status, and immune function of chickens. In actual farming, when the temperature in the chicken house is ≥27°C, heat stress will pose a threat to chickens and other poultry. Under heat - stress conditions, the feed intake of chickens will decrease, the water intake will increase, and the production performance will decline, such as a decrease in egg production and a reduction in egg quality. At the same time, heat stress will also weaken the immune function of chickens, making their resistance to diseases decline. Exemplarily, the heat stress described in the present invention includes at least one of the following physiological responses: (1) Heat panting: The respiratory movement speeds up, carbon dioxide excretion increases, and respiratory alkalosis occurs. (2) Heart rate acceleration: Heart failure, cerebral congestion, pulmonary edema, hypoxia, etc. (3) Increased oxidative metabolism in the body, increased peroxides, and damage to the membrane system. (4) Enhanced functions of the thyroid and adrenal glands: Accelerated substance metabolism and reduced immunity. (5) Disorders of water and electrolyte balance: Increased urination and increased ion loss.

[0043] In this text, "confidence interval" refers to the estimated interval of the population parameter constructed by the sample statistic. In statistics, the confidence interval of a probability sample is the interval estimate of a certain population parameter of this sample. The confidence interval shows the degree to which the true value of this parameter has a certain probability of falling around the measurement result. The confidence interval gives the credibility of the measured value of the measured parameter, that is, the "certain probability" required above, and this probability is called the confidence level.

[0044] In this text, the term "drug" generally refers to the unit dosage form and can be prepared by any one of the methods well-known in the pharmaceutical field. All methods include the step of combining the active ingredient with a carrier constituting one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0045] In this text, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the avian animals treated therewith. Preferably, the "pharmaceutically acceptable" as described in the present invention refers to those used in animals, especially avian, which are approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopeias.

[0046] In this text, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or their combination, and these carriers are known to those skilled in the art. Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0047] In this text, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. Except within the scope where any conventional excipient is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope contemplated by the present invention.

[0048] In this text, the term "administration" refers to introducing a predetermined amount of a substance into the subject to be treated by a suitable means. The drugs of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous injection, intramuscular injection, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration.

[0049] As used herein, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or an adverse effect caused by the disease. "Treatment" as used herein covers diseases of mammals, particularly broiler chickens, and also includes other avian species, including: (a) preventing the occurrence of a disease or disorder in a subject prone to heat stress but not yet diagnosed; (b) inhibiting a disease, such as arresting the progression of the disease; or (c) alleviating a disease, such as reducing symptoms associated with the disease. "Treatment" as used herein covers any administration of a drug or compound to a subject to treat, cure, alleviate, improve, reduce or inhibit a disease of the subject, including but not limited to administering a drug as described herein to a subject in need thereof.

[0050] The administration of the drug according to the present invention can be carried out by any acceptable administration method. The drugs of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as injections, lyophilized powders. The current methods for preparing these dosage forms are known or will be obvious to those skilled in the art. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, transdermal, buccal, rectal routes. The drugs of the present invention are formulated to allow the bioactive ingredients contained therein to be bioavailable after administration to a subject.

[0051] The effective amount of the drug according to the present invention may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by a person of ordinary skill in the art according to various factors. Such factors include but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the subject, the weight of the subject, the immune status of the subject, the route of administration, etc. For example, due to the urgency of the treatment situation, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0052] The drugs of the present invention can be incorporated into drugs suitable for parenteral administration (such as subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (such as injection solutions and infusion solutions) or lyophilized powders.

[0053] The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0054] Unless otherwise specified, the practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, immunology, genetic breeding, and bioinformatics, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature publicly available in this field, and each such literature is hereby incorporated by reference expressly herein.

[0055] The following embodiments include a first stage and a second stage, namely, the discovery and screening stage and the verification stage of corresponding metabolite markers. The discovery stage includes: determining serum metabolite markers based on the analysis and comparison of the serum metabolite components and functional changes of 28 heat-stressed broilers (heat stress group) and 30 healthy broiler individuals (healthy control group); the verification stage includes: verifying the accuracy of the results of the first stage by using 15 heat-stressed broiler individuals and 15 healthy broiler individuals.

[0056] Example 1 Screening of Serum Metabolite Markers 1.1 Establishment of Heat Stress Animal Model and Sample Collection The experimental population in this example includes 43 heat-stressed broilers (HS group) and 45 healthy control broilers (Control group). Both groups of broilers are 42-day-old Guangming No. 2 white-feathered broiler roosters with a body weight of 2963.35 ± 76.37 g. Among them, the heat stress treatment group is induced by raising the laboratory temperature from 24°C to 38°C within 0.5 hour and continuously treating for 3 hours (the heat stress treatment operation was pre-experimented by the applicant and designed in combination with the following reference: Zhang Minhong. Environmental and Nutritional Regulation of Healthy Broiler Production. Northern Animal Husbandry, 2021, (24): 21-24. Effect of relative humidity at either acute or chronic moderate temperature on growth performance and droppings' corticosterone metabolites of broilers), and the blank control group is treated at 24°C for 3 hours, and the other experimental conditions are the same for both groups. After collecting whole blood from the wing tip veins of the two groups of experimental individuals respectively and standing for 5 hours, centrifuge at 2000 r / min for 8 minutes in a high-speed centrifuge, aspirate the serum into a centrifuge tube, and store it at -80°C after quick-freezing in liquid nitrogen for subsequent analysis.

[0057] The Beckman AU480 automatic biochemical analyzer was used to measure lactate dehydrogenase (LDH) to test whether the heat stress animal model was successfully established. The detection kit was purchased from Nanjing Jiancheng Bioengineering Institute, and the specific operation steps refer to the instruction manual of the kit. The detection results are asFigure 1 As shown in the figure, there was a significant difference in the content of lactate dehydrogenase between the healthy control group and the heat stress treatment group (P<0.001). The content of lactate dehydrogenase in the heat stress treatment group was significantly higher than that in the healthy control group, and the heat stress animal model was successfully established.

[0058] 1.2 Metabolomics analysis 1.2.1 Sample pretreatment (1) Thaw the above serum samples stored at -80°C at room temperature. Take 60 μL of serum samples into a 1.5 mL centrifuge tube, add small steel beads and 600 μL of L-2-chlorophenylalanine (0.3 mg / mL) dissolved in methanol as an internal standard, and vortex for 10 s; (2) Pre-cool in a -40°C refrigerator for 2 min, and then put it into a grinder for grinding (60 Hz, 2 min); (3) Ultrasonic extraction in an ice-water bath for 10 min, and let it stand overnight at -40°C; (4) Centrifuge the product of step (3) at 12000 rpm and 4°C for 10 min, take 200 μL of the supernatant and transfer it into an LC-MS injection vial to evaporate to dryness; (5) Use a mixture of 300 μL of methanol and water (volume ratio 1:4), vortex the sample for 30 s, ultrasonic extract in an ice-water bath for 3 min, and let it stand at -40°C for 2 hours; (6) Centrifuge at 12000 rpm and 4°C for 10 min, suck 150 μL of the supernatant with a syringe, filter it through a 0.22 μm organic phase needle filter, transfer it to an LC vial, and store it at -80°C for subsequent LC-MS analysis.

[0059] (7) The quality control sample (QC) was prepared by mixing equal volumes of the extraction solutions of all samples.

[0060] 1.2.2 Serum untargeted metabolomics detection Performed using an ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry system, which consists of an ultra-high performance liquid chromatography (ACQUITY UPLC I-Class plus system, Waters Corporation, Milford, USA) and a mass spectrometry detection system (Q-Exactive mass spectrometer equipped with heated electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA)), including ESI positive ion mode and ESI negative ion mode. The solvent system contains mobile phase A (ultrapure water containing 0.1% formic acid) and mobile phase B (acetonitrile). The elution gradient is set as shown in Table 1. The flow rate is 0.35 mL / min, the column temperature is 45 °C, and the injection volume is 2 μL. After completing the high performance liquid chromatography analysis, the separated samples are introduced into the high-resolution ion mobility system for mass spectrometry analysis, and the parameters are shown in Table 2. Data acquisition is performed in full scan mode (m / z: 50 - 1000) combined with MSE mode, and two independent scans with different collision energies (CE) are alternately obtained during operation, namely low energy scan (CE 4 eV) and high energy scan (collision energy range of 20 - 45 eV) for ion fragmentation.

[0061] Table 1: Elution gradient of ultra-high performance liquid chromatography Table 2: Detailed parameters of high-resolution ion mobility mass spectrometry system

[0062] 1.2.3 Metabolome data processing and quality control The original LC-MS raw data was processed by Progenesis QI V2.3 (Nonlinear, Dynamics, Newcastle, UK) software for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization. The main parameters were 5 ppm precursor tolerance, 10 ppm product tolerance, and 5% product ion threshold. Compound identification was performed using The HumanMetabolome Database, Lipidmaps (V2.3), Metlin, and a self-built database for qualitative analysis, and compound identification was based on accurate mass-to-charge ratio, secondary fragments, and isotope distribution. Then, any peaks with more than 50% missing values (ion intensity = 0) in the group were removed from the extracted data, zero values were replaced with half of the minimum value, and screening was performed according to the qualitative results of the compounds. Compounds with a database matching score lower than 36 points (total score 80 points) were also considered inaccurate and deleted, and the positive and negative ion data were combined into a data matrix.

[0063] 1.3 Screening of differential metabolites Figure 2 The results showed that orthogonal partial least squares discriminant analysis was used to analyze differential metabolites, and there were significant differences in the serum metabolite accumulation patterns between the heat stress groups. According to statistics, the original abundance value of Adenosine in healthy broiler chickens in this invention was 1577.90 ± 322.74, and the original abundance value of Adenosine in heat-stressed broiler chickens was 1022.38 ± 267.01. The VIP values obtained from the model were used to rank the overall contribution of each variable to population discrimination, and a combined two-tailed Student's t-test was used to identify differential metabolites related to broiler heat stress. The specific results are as Figure 3 shown in Table 3.

[0064] Table 3: Differential metabolites 1.4 Screening of important metabolites for heat stress by random forest The caret package (v4.3.2) was used to train a random forest model to screen important metabolites for heat stress, as Figure 4 shown. It was found that Adenosine had a high importance score. Combining the results of orthogonal partial least squares discriminant analysis and differential metabolite screening, Adenosine was determined as a marker metabolite for heat stress.

[0065] Example 2 Verification of candidate metabolite markers 2.1 Collection of experimental samples The experimental individuals selected in this example are other individuals with the same growth conditions as those selected in Example 1. In this example, 15 additional healthy control broiler chickens and 15 broiler chickens subjected to heat stress treatment were selected. Both groups of individuals were 42-day-old male Guangming No. 2 white - feather broiler chickens with a body weight of 2963.35 ± 76.37 g. Serum was collected by referring to the method of Example 1, and metabolites were detected using an ultra - high - performance liquid chromatography - tandem high - resolution mass spectrometry system. The quality control process and parameters of the downloaded data were the same as those in 1.2.3 of Example 1.

[0066] 2.2 Experimental methods The AUC evaluation results of the biomarker metabolite Adenosine as a diagnostic indicator, where Specificity represents the specificity, that is, predicted as positive and actually positive, true positive, and the ordinate Sensitivity represents the sensitivity, that is, true negative, and the confidence interval (CI) is 95%.

[0067] 2.3 Experimental results and analysis As Figure 5 shown, there are significant differences in the levels of the biomarker metabolite Adenosine between the healthy control group and the heat - stress group individuals. Compared with the healthy control group, the level of Adenosine in the heat - stress group individuals is significantly reduced; in addition, Figure 6 the AUC curve in

[0068] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "embodiment" or "specific embodiment" etc. means that the specific features, structures, materials or characteristics described in connection with that embodiment are included in at least one embodiment of the present invention. In this specification, the schematic representations of the above - mentioned terms do not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments and the features of different embodiments described in this specification.

[0069] Although the embodiments of the present invention have been shown and described above, it can be understood that the above - mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above - mentioned embodiments within the scope of the present invention.

Claims

1. A metabolic marker for screening heat stress-resistant broilers, characterized in that, The biomarker includes adenosine.

2. Use of a reagent for detecting the metabolic biomarker according to claim 1 in the preparation of a kit for screening heat stress-resistant broilers.

3. A kit, characterized in that The kit contains a reagent suitable for detecting the metabolic biomarker according to claim 1.

4. Use of the metabolic biomarker according to claim 1 in screening heat stress-resistant broilers.

5. The use according to claim 4, characterized in that, That the abundance of the metabolic biomarker of the broiler reaches or is higher than a predetermined threshold is an indication that the broiler is a heat stress-resistant broiler.

6. A method for screening heat stress-resistant broilers, characterized in that, Comprising: (1) Determining the abundance of the metabolic biomarker according to claim 1 in a serum sample of a broiler to be tested; (2) Comparing the abundance obtained in step (1) with a predetermined threshold to determine whether the broiler to be tested is a heat stress-resistant broiler; Optionally, step (1) is achieved by an ultra-high performance liquid chromatography-mass spectrometry analysis method; Optionally, that the abundance obtained in step (1) reaches or is higher than the abundance value of the metabolic biomarker according to claim 1 in a known heat stress-resistant broiler is an indication that the broiler is a heat stress-resistant broiler; Optionally, the threshold includes the abundance values of the metabolic biomarker according to claim 1 in known heat stress-resistant broilers or non-heat stress-resistant broilers.

7. A device for determining whether a broiler to be tested is a heat stress-resistant broiler, characterized in that, Comprising: A metabolic biomarker abundance determination unit for determining the abundance of the metabolic biomarker according to claim 1 in a serum sample of the broiler to be tested; A comparison unit for comparing the obtained abundance with a predetermined threshold to determine whether the broiler to be tested is a heat stress-resistant broiler.

8. A device for screening heat-stress-resistant broilers, characterized in that, Comprising: A computer-readable storage medium having stored thereon a computer program for performing the method according to claim 6; And one or more processors for executing the program in the computer-readable storage medium.

9. Use of a preparation for increasing the concentration of the metabolic biomarker according to claim 1 in the preparation of a drug for preventing, alleviating and / or treating heat stress in broilers.

10. A drug, characterized in that, A preparation containing an agent for increasing the abundance of the metabolic biomarker according to claim 1.

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