Feces metabolism marker for screening heat-stress-resistant dairy cows and application of faeces metabolism marker

Abstract

The invention belongs to the field of animal husbandry production, and particularly relates to an excrement metabolism marker for screening heat-stress-resistant dairy cows and application of the excrement metabolism marker. According to the invention, through metabonomics analysis with high flux, high sensitivity and high precision, metabolite prostaglandin E2 with significant difference in heat stress tolerant cattle manure and heat stress sensitive cattle manure is screened out. The marker can be used for screening heat stress resistant dairy cows. The prostaglandin E2 is used as the marker for screening the heat-stress-resistant dairy cows, has the characteristics of accuracy, simplicity, convenience and noninvasive property, and can provide reference for breeding of heat-stress-resistant dairy cow varieties.

Description

Technical Field

[0001] The invention belongs to the field of animal husbandry production, and particularly relates to a feces metabolic marker for screening heat stress-resistant dairy cows and an application thereof. Background Art

[0002] Heat stress has become one of the major challenges facing the global animal husbandry industry, especially in the southern region. The high temperature and high humidity climate characteristics in summer cause dairy cows to be exposed to a heat stress environment for a long time, causing disorders in the cows' body temperature regulation, which in turn has a significant negative impact on metabolism, endocrine, immune function and production performance. Specifically, heat stress can lead to increased levels of oxidative stress in dairy cows, inhibit the activity of rumen microorganisms, and affect the metabolism and utilization efficiency of nutrients. At the same time, heat stress can also cause increased secretion of stress hormones, disrupt normal endocrine balance, inhibit the function of immune cells, and reduce the body's disease resistance. The damage to the above series of physiological functions ultimately leads to a significant decrease in milk production and quality of dairy cows, and a significant decrease in reproductive performance, which is manifested in reduced conception rates and abnormal embryonic development.

[0003] For a long time, the selection of heat stress-resistant dairy cows has mainly relied on physiological indicators such as rectal temperature, respiratory rate, and red blood cell potassium concentration. Cows with lower rectal temperatures tend to have stronger resistance to heat stress, and there is a negative correlation between rectal temperature and productivity. The above methods require close contact with dairy cows or blood sampling, which is not suitable for large-scale and real-time monitoring, and the labor cost is high. In recent years, with the development of molecular marker-assisted selection technology and genome-wide association analysis technology (GWAS), more and more gene markers have been used in the selection and breeding of heat stress-resistant dairy cows. For example, genes such as HSP70, S441, S463, S2011, S275, HSF1, ATP1A1 and ATP1B2 can be used as candidate genes for heat resistance traits in dairy cows. However, genetic testing requires high costs and a long time, and the screening accuracy of a single gene is limited.

[0004] Metabolomics, as an emerging omics technology developed after genomics and proteomics, can qualitatively and quantitatively study the changes in metabolites of animals under external stimulation, pathological or physiological conditions. Metabolomics captures the comprehensive changes of metabolites in cells, tissues or organisms through high-throughput, high-sensitivity and high-precision detection technology, and combines bioinformatics analysis to screen out metabolites with significant differences, further analyze metabolic pathways, and reveal their potential mechanisms at the molecular level. It has the advantages of rapidity, accuracy and high sensitivity. Although there have been reports on the application of metabolomics in the study of heat stress in dairy cows, most of these studies are comparative analyses of the changes in dairy cow metabolites in different seasons. In comparison, there are few reports on the screening of heat stress-resistant dairy cows by changes in specific metabolites in dairy cow feces under high temperature conditions in summer. Therefore, screening heat stress-resistant dairy cows based on fecal metabolic markers is of great significance for the selection and breeding of heat stress-resistant dairy cows. In addition, feces is a non-invasive and non-invasive test, and it is easy to operate and suitable for large-scale screening. However, to date, there has been no report on the use of fecal metabolites as biomarkers for screening heat stress-resistant dairy cows. Summary of the invention

[0005] The present invention aims to provide a fecal metabolic marker for screening heat stress-resistant animals and application thereof.

[0006] The present invention is based on high-throughput metabolomics, and systematically analyzes the metabolites with significant differences in the feces of known heat stress tolerant cows (HR) and heat stress sensitive cows (HS), and screens out biomarkers with high specificity and strong sensitivity. Based on this, the technical solution of the present invention is as follows:

[0007] Firstly, the present invention provides a feces metabolic marker for screening heat stress resistant dairy cows, which is prostaglandin E2.

[0008] Secondly, the present invention provides the use of the above-mentioned prostaglandin E2 as a marker in screening mammals or birds with resistance to heat stress. Specifically, the prostaglandin E2 is used as a marker for detecting heat stress resistance in feces.

[0009] The present invention also provides the use of prostaglandin E2 in the preparation of a heat stress resistance detection reagent or kit. Those skilled in the art should understand that prostaglandin E2 can be used as a heat stress resistance fecal marker, and the concentration or content of prostaglandin E2 can be quickly detected by developing a suitable reagent or kit for detection.

[0010] Thirdly, the present invention also provides a method for screening heat stress resistant animals, by detecting the concentration of prostaglandin E2 markers in animal feces to judge the heat stress resistance of animals. The higher the concentration of prostaglandin E2 in feces, the stronger the heat stress resistance of the animal; conversely, the weaker the heat stress resistance.

[0011] Specifically, the method may include the following steps: (1) using fresh feces of an individual or group of animals known to be heat stress tolerant as a standard, and using fresh feces of the animal to be tested as a test sample; (2) using a high performance liquid chromatography-mass spectrometry analysis method to detect the concentration of prostaglandin E2 in the fecal sample; (3) calculating the average value of prostaglandin E2 in the standard of heat stress tolerant animals as a reference value. If the average value of prostaglandin E2 in the sample to be tested is higher than the reference value, the animal to be tested has a higher ability to resist heat stress than the known heat stress tolerant animal. Conversely, if the average value of prostaglandin E2 in the sample to be tested is lower than the reference value, the animal to be tested has a lower ability to resist heat stress than the known heat stress tolerant animal.

[0012] The present invention also provides the use of the marker or reagent, the kit and the method in livestock and poultry stress resistance biological breeding. The stress resistance here includes heat stress resistance biological characteristics, and may also include other stress resistance characteristics. Other stress resistance characteristics can be detected by corresponding methods, and the present invention will not be expanded.

[0013] The present invention also provides the use of the marker in the preparation of anti-heat stress drugs for livestock and poultry. For example, those skilled in the art can screen drugs that can be used for anti-heat stress by detecting the marker in livestock and poultry feces.

[0014] The present invention screens out the metabolite prostaglandin E2 with significant differences in the feces of heat stress tolerant cows and sensitive cows through high-throughput, high-sensitivity and high-precision metabolomics analysis. The marker can be used to screen heat stress resistant dairy cows, and can also be used for heat stress resistance screening of other animals. The use of prostaglandin E2 as a marker to screen heat stress resistant dairy cows is accurate, simple and non-invasive, and can provide a reference for the breeding of heat stress resistant dairy cows. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is the change in temperature and humidity index (THI) of the cowshed during the experimental period.

[0016] Figure 2 These are comparison charts of physiological and biochemical indicators of the tested heat-resistant cattle and non-heat-resistant cattle. Figure A is a comparison chart of rectal temperature, Figure B is a comparison chart of respiratory rate, Figure C is a comparison chart of heat shock protein HSP-70, and Figure D is a comparison chart of heat shock protein HSP-90.

[0017] Figure 3 Shown are PCA analysis, PLS-DA, and OPLS-DA analyses of the tested fecal samples.

[0018] Figure 4 Shown are the correlations of the differential metabolites with rectal temperature, HSP-70, and HSP-90.

[0019] Figure 5 Shown is the relative abundance of prostaglandin E2 in different subjects. DETAILED DESCRIPTION

[0020] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0022] Example 1: Screening of heat stress tolerant dairy cows (HR) and heat stress sensitive dairy cows (HS)

[0023] The dairy cows used in this experiment were all from a local dairy farm in Wuhan. In mid-July, the temperature and humidity of the dairy farm were continuously monitored, and the temperature and humidity index (THI) was calculated to ensure that the tested dairy cows were in a moderate heat stress state ( Figure 1 Subsequently, the rectal temperature (RTN) and respiratory rate of the tested cows were dynamically monitored for seven consecutive days ( Figure 2 ), and blood was collected from the tail vein on the 8th day. After the blood sample was allowed to stand for 2 hours, it was centrifuged at 3000r / min for 15 minutes, and the serum was collected and stored at -80℃ for later use. The ELISA method was used to determine the content of heat stress-related proteins HSP-70 and HSP-90 in the serum of the tested dairy cows ( Figure 2 ).

[0024] The rectal temperature, respiratory rate, HSP-70 and HSP-90 of dairy cows were subjected to principal component analysis, and 10 cows were selected for heat stress tolerance (HR) and heat stress sensitivity (HS). This method provides a scientific basis for the accurate screening of stress tolerance and heat stress individuals by comprehensively considering multiple physiological and biochemical indicators, and also provides high-quality sample support for subsequent research.

[0025] Example 2: Collection and processing of fresh feces samples from tested dairy cows

[0026] The rectal fecal sampling method is used for fecal sample collection to minimize cross-contamination between samples and ensure the integrity and representativeness of fecal samples. The specific operation steps are as follows: wear disposable long-arm gloves before sampling, prepare an appropriate amount of medical lubricant and sterile sampling container, and clean the tail and anus of the cow to reduce the impact of external contamination sources on the sample. During the operation, gently insert the finger into the rectum, and use a rotating motion to obtain about 10-20g of fresh fecal sample, quickly place it in a 50mL sterile centrifuge tube, seal it and minimize the exposure of the sample to the outside world, and label it to indicate the sampling time and cow number. The sample is then stored in an ultra-low temperature refrigerator at -80℃ to maintain the stability of the metabolites and ensure the accuracy and reliability of the sample in metabolomics analysis.

[0027] Example 3: Screening of differential metabolites in fecal samples of heat stress tolerant and sensitive cattle

[0028] This experiment uses Thermo's high-resolution LC-MS platform, combined with non-targeted metabolomics technology, to conduct comprehensive qualitative and quantitative analysis of samples to reveal the full picture of metabolites and their changes under heat stress in dairy cows. Using the new generation of commercial software Compound Discoverer 3.3, combined with various types of MS / MS spectrum databases, metabolites were accurately identified and relatively quantified. The specific experimental steps are as follows:

[0029] 1. Sample Preparation for Mass Spectrometry Analysis

[0030] Take an appropriate amount of the above fecal sample, add pre-cooled methanol, vortex and grind thoroughly to ensure the homogenization of the sample. Then, use ultrasonic treatment for 10 minutes to enhance the solubility and extraction efficiency of metabolites. Then, after the sample is allowed to stand at -20°C for 30 minutes, it is centrifuged at 12,000 rpm and 4°C for 10 minutes, and the supernatant is taken for subsequent analysis.

[0031] 2. Chromatographic conditions

[0032] An ACQUITY UPLC HSS T3 column (100A, 1.8 μm, 2.1 mm × 100 mm) was used, the column temperature was set at 40°C, the flow rate was 0.4 mL / min, the autosampler temperature was maintained at 8°C, and the injection volume was 2 μL each time. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. This optimized chromatographic condition ensured good separation and detection sensitivity of metabolites.

[0033] 3. Mass spectrometry conditions

[0034] Thermo Orbitrap Exploris 120 high-resolution mass spectrometer was used in combination with Xcalibur software (version: 4.7, Thermo Fisher) to collect DDA (data-dependent acquisition) mass spectrometry data in positive and negative ion modes. The key parameters of the HESI source include spray voltage 3.5kV / -3.0kV, sheath gas flow rate 40arb, auxiliary gas flow rate 15arb, capillary temperature 325℃ and auxiliary air temperature 300℃. The resolution of the primary mass spectrometry was 60,000, the scan range was 100-1000m / z, the AGC target was standard mode, and the maximum ion injection time (Max IT) was 100ms. The first four parent ions with the highest response in the primary mass spectrum were fragmented in the secondary mode, with a dynamic exclusion time of 8sec, a secondary resolution of 15,000, a collision energy of 30%, an AGC target of standard mode, and a maximum ion injection time of automatic mode. All formal samples and QC samples were analyzed strictly in accordance with the above chromatographic and mass spectrometric methods. Before the formal injection, balance the system by inserting 2-4 QC samples. During the formal injection process, insert 1 QC sample every 5-10 samples to facilitate subsequent data quality evaluation and control.

[0035] 4. Data Analysis and Biomarker Screening

[0036] The R software package Ropls was used to perform a variety of dimensionality reduction analyses on the collected sample data, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA). By drawing score graphs, loading graphs and S-plot graphs, the composition differences of sample metabolites between different groups are clearly displayed. In addition, KEGG enrichment analysis of differential metabolites was performed in combination with clusterProfiler (V4.6.0) to mine significantly enriched metabolic pathway information. The abundance scores of differential metabolites in specific metabolic pathways were further calculated to reveal the overall change trend of all metabolites in the metabolic pathway, thereby accurately identifying key metabolic pathways and providing an important reference for screening key biomarkers.

[0037] The systematicity and precision of this research method provide technical support for in-depth analysis of the metabolic mechanism of heat stress in dairy cows and lay a solid foundation for building a metabolomics-based screening model.

[0038] (2) Results Analysis

[0039] This study conducted a comprehensive metabolomics analysis of fecal samples from heat stress tolerant (HR) and heat stress sensitive (HS) cows, and evaluated the relative importance of metabolites in screening heat stress resistant cows based on mass spectrometry combined with multivariate statistical methods. The results of multivariate statistical PCA, PLS-DA, and OPLS-DA showed that the sample groups of heat tolerant and heat non-tolerant cows were well separated and had obvious discrimination ( Figure 3 ), and the variation within the sample group was small. The relative importance (VIP value) of each metabolite in classification and prediction was evaluated. On this basis, combined with the availability of metabolites and their statistical significance (P value), the top 40 differential metabolites with VIP values ​​greater than 1 and P values ​​less than 0.05 were screened. These metabolites are considered to be potential biomarkers for screening heat stress-resistant dairy cows, providing important candidate indicators for further evaluating the heat stress resistance of dairy cows.

[0040] Spearman correlation analysis was performed on the screened differential metabolites and the rectal temperature, respiratory rate, HSP-70 and HSP-90 of dairy cows. It was found that prostaglandin E2 was significantly negatively correlated with rectal temperature, and significantly positively correlated with HSP-70 and HSP-90 ( Figure 4 ).

[0041] Furthermore, the changes of the above markers in heat stress resistant dairy cows and the corresponding predicted lower area (AUC) are shown in Table 1 below. The AUC value of the metabolite is 0.97, indicating that the diagnostic value of the metabolite as heat stress resistance is very high.

[0042] Table 1 Changes of prostaglandin E2 in heat stress resistant dairy cows

[0043] Metabolites Molecular formula Molecular weight Heat stress resistance concentration direction CAS AUC Prostaglandin E2 <![CDATA[C 20 H 32 O5]]> 352.465 High expression 363-24-6 0.97

[0044] The changes of the above markers in the feces of heat-tolerant and non-heat-tolerant cows are shown in Figure 5 , the content of prostaglandin E2 in non-heat stress resistant cows was significantly decreased.

[0045] Example 4: Screening method for heat stress resistant dairy cows using prostaglandin E2 as a marker

[0046] A group of fresh feces from cows known to be resistant to heat stress (10 heads) were taken as standard test products, and feces from the cows to be tested (30 heads) were taken as test samples and stored at -80°C.

[0047] Detection of the concentration of prostaglandin E2 in stool samples: This example uses a quantitative analysis method using high performance liquid chromatography-mass spectrometry to obtain the concentration of prostaglandin E2. The detailed steps are shown in Examples 3.1, 3.2, and 3.3.

[0048] The average value of the prostaglandin E2 concentration of the standard test sample is used as the reference level, and the prostaglandin E2 concentration of the tested cow is compared with the average value. If it is higher than the average value of the HR standard, it indicates that the tested cow has a higher ability or probability to resist heat stress than the standard test sample. Otherwise, it indicates that the tested cow has a lower probability of resisting heat stress than the standard test sample.

[0049] In this embodiment, the relative concentration average value of prostaglandin E2 in the feces of 10 heat stress resistant cows is 2988420.48. If the concentration of prostaglandin E2 in the feces of the tested cows is higher than 2988420.48, it means that the tested cows have a higher heat stress resistance than the known cows, and vice versa. After testing, the relative concentrations of prostaglandin E2 of 13 of them are higher than 2988420.48, and the others are lower than this value. The temperature and humidity of the dairy farm are continuously monitored, and the temperature and humidity index (THI) is calculated to ensure that the tested cows are in a moderate heat stress state. The method of Example 1 is used for detection, and the results show that the cows with a relative concentration higher than the average value of prostaglandin E2 have good heat stress resistance, while those with a relative concentration lower than the average value have poor heat stress resistance or are sensitive to heat stress, and the screening accuracy is as high as 100%.

Claims

1. A fecal metabolic marker for screening heat stress-resistant dairy cows, characterized in that: The fecal metabolism marker is prostaglandin E2.

2. Application of prostaglandin E2 as a marker in screening mammals or birds resistant to heat stress.

3. The use according to claim 2, characterized in that Prostaglandin E2 is used as a marker in feces to detect resistance to heat stress.

4. The use according to claim 2, characterized in that The animals are Holstein cows.

5. Application of prostaglandin E2 in the preparation of anti-heat stress detection reagents or kits.

6. A method for screening animals resistant to heat stress, characterized in that: The animal's ability to resist heat stress can be determined by detecting the concentration of prostaglandin E2 markers in animal feces.

7. The method according to claim 6, characterized in that The higher the concentration of prostaglandin E2 in feces, the stronger the animal's ability to resist heat stress; conversely, the weaker its ability to resist heat stress.

8. The method according to claim 6, characterized in that The method comprises the following steps: (1) using an individual or group of animals known to be heat stress tolerant and fresh feces of the animal to be tested as a test sample; (2) using a high performance liquid chromatography-mass spectrometry analysis method to detect the concentration of prostaglandin E2 in the fecal sample; (3) calculating the average value of prostaglandin E2 in a standard product of heat stress tolerant animals as a reference value. If the average value of prostaglandin E2 in the sample to be tested is higher than the reference value, the animal to be tested has a higher ability to resist heat stress than the known heat stress tolerant animal. Conversely, if the average value of prostaglandin E2 in the sample to be tested is lower than the reference value, the animal to be tested has a lower ability to resist heat stress than the known heat stress tolerant animal.

9. Use of the marker according to claim 1 or the method according to any one of claims 6 to 8 in livestock and poultry stress-resistant biological breeding.

10. Use of the marker according to claim 1 in the preparation of anti-heat stress drugs for livestock and poultry.

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