A method for obtaining a biomarker of estrus period of buffalo and application of the biomarker to identifying estrus period of buffalo

Abstract

The application relates to the field of biotechnology, and particularly relates to a method for obtaining a biological marker of a buffalo estrus period and application of the biological marker to identifying the buffalo estrus period, S1, collecting milk samples of a female buffalo, and observing follicle development of the female buffalo by using B ultrasonic waves; S2, after the milk samples are extracted, analysis is conducted by using a quadrupole-electric field orbitrap high-resolution mass spectrometry system and an ultrafast high-performance liquid chromatography system; S3, after original data are converted into an mzXML format, extraction data are obtained by using XCMS software processing, the extraction data are processed and analyzed, and significant difference metabolites of cow milk in an estrus period are screened out; S4, expression amounts of the significant difference metabolites of the cow milk in the estrus period and a non-estrus period are detected, the expression amount changes are compared with B ultrasonic wave results, and a biological marker of the buffalo estrus period is obtained. Compared with the prior art, pyridoxamine and homovanillic acid are obtained as the biological marker of the estrus period of the female buffalo, and are used for identifying whether the female buffalo is in the estrus period.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for obtaining estrus biomarkers in buffalo and the application of these biomarkers in identifying estrus in buffalo. Background Technology

[0002] Water buffalo is one of the important economic livestock in southern my country, possessing advantages such as strong adaptability, disease resistance, tolerance to roughage, and rich dairy products, and is recognized as a specialty livestock with development value. Water buffalo milk is rich in components, containing various trace elements and nutrients, and the market share of water buffalo milk products is increasing year by year, indicating huge product development potential and a broad market prospect. However, water buffalo are seasonally estrous animals, especially in summer when estrus signs are not obvious. The high incidence of silent estrus in water buffalo, coupled with the lack of accurate biomarkers for detecting the estrus period, makes it difficult to formulate appropriate breeding plans, severely reducing the reproductive efficiency of the herd and hindering the progress of water buffalo breed improvement, thus imposing a significant economic burden on the water buffalo industry. Therefore, obtaining accurate and efficient methods for estrus detection biomarkers is of great significance for improving water buffalo reproductive efficiency and promoting water buffalo breed improvement.

[0003] The estrous cycle of a buffalo is about 21 days, which is usually divided into four stages: proestrus, estrus, metestrus and estrus. Based on the physiological changes of the estrus cycle, a series of estrus detection methods have been developed, including external observation, estrus testing, rectal examination, and automated monitoring. External observation involves observing the cow's estrus symptoms to determine if she is in estrus; however, this method has low accuracy due to a lack of standardized quantitative criteria and the subjective influence of the observer's experience. The estrus testing method uses a vasectomized bull to test the cow's estrus by observing her acceptance of mounting. However, this method is not ideal due to difficulties in observing mounting at night and in densely populated areas. Rectal examination involves palpating the ovaries through the rectal wall and assessing follicle size, texture, and undulation to determine if the cow is in estrus. However, this is an invasive procedure that can easily cause stress in the cow and increases the risk of gynecological infections, affecting the cow's health. Automated monitoring involves equipping the cow's legs or neck with a pedometer, using different algorithms and thresholds to define activity levels and intensity, tracking the cow's activity level in real time and uploading the data to identify estrus status. However, this method, which relies solely on activity intensity to determine the estrus status of cows, is insufficient to accurately and effectively identify the estrus state of buffaloes and cannot achieve the desired identification effect.

[0004] In summary, existing methods for detecting estrus in buffalo have significant shortcomings. Efficient and accurate pregnancy diagnosis technology can help managers identify non-pregnant and pregnant cows, reducing the number of days of non-pregnancy in the herd and the economic losses in calving, milk production, labor, and feed caused by non-pregnancy. Therefore, based on the dynamic changes in the content of various substances in milk during different estrus cycles, the inventors screened biomarkers from numerous substances such as total solids, fat, protein, and metabolites to determine the estrus period in buffalo. These biomarkers serve as auxiliary evidence for determining the estrus period in buffalo and provide a new direction for the development of estrus detection kits. Summary of the Invention

[0005] This invention provides a method for obtaining estrus biomarkers in buffalo and the application of these biomarkers in identifying estrus in buffalo, aiming to solve the problems of unsatisfactory detection results and lack of biomarkers and screening methods in existing buffalo estrus identification methods.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for obtaining estrus biomarkers in buffalo, the method comprising: S1. Collect milk samples from the female buffalo and observe the follicle development of the female buffalo using ultrasound. S2, take the milk sample and methanol solution, vortex and mix them, place the mixed milk sample on ice for 4-8 min, centrifuge for 18-22 min and take the supernatant, dilute the supernatant with ultrapure water, centrifuge for 20 min, and take the supernatant for analysis. Take equal volumes of all milk samples and mix them as quality control samples. After extraction, analyze them using a quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system and an ultra-fast high-performance liquid chromatography system. S3. After the raw data is converted into mzXML format, XCMS software is used to perform peak alignment, retention time correction and peak area extraction to obtain extracted data. The extracted data is then subjected to metabolite structure identification, data preprocessing, experimental data quality evaluation and data analysis in sequence to screen out significantly different metabolites in milk during the estrous cycle. S4, detect the expression levels of the significantly differentially expressed metabolites in the milk during estrus and non-estrus periods, compare the changes in expression levels with ultrasound results, and obtain buffalo estrus biomarkers.

[0007] Preferably, in the above technical solution, the milk sample and the methanol solution are mixed evenly at a volume ratio of 1:4, and the volume fraction of the methanol solution is 80%.

[0008] Preferably, in the above technical solution, in step S2, diluting the supernatant with ultrapure water specifically means diluting the methanol volume fraction in the supernatant with ultrapure water to a methanol volume fraction of 53%.

[0009] According to the above technical solution, this application first mixes and shakes the milk sample with an 80% methanol solution by volume. The high concentration of methanol is used to fully denature and precipitate the protein components in the milk, reducing the matrix effect. Then, the methanol volume fraction is diluted from 80% to 53% before analysis, which allows the impurities in the milk sample to fully precipitate and the metabolites to fully dissolve, reducing the matrix effect, protecting the chromatographic column and improving the stability and accuracy of detection. However, if the extraction is not diluted, the high concentration of methanol will cause problems such as column damage, abnormal peak shape, enhanced matrix interference, and poor data repeatability.

[0010] Preferably, in the above technical solution, the milk sample of the female buffalo specifically refers to the milk sample of Murrah female buffalo from 2 to 5 parities.

[0011] According to the above technical solution, this application uses 2-5 parity Murrah buffalo as experimental subjects. Compared with primiparous or high-parity Murrah buffalo, 2-5 parity Murrah buffalo have mature and stable reproductive functions, small individual differences, and reliable experimental results. This solves the problems of hormonal disorders in primiparous buffalo and the potentially unstable expression of metabolites in milk samples during the estrous cycle. It also avoids the problems of decreased reproductive performance and irregular fluctuations in follicle development in high-parity buffalo.

[0012] Preferably, in the above technical solution, the chromatographic conditions of the ultra-fast high-performance liquid chromatography system include: ACQUITYUPLCBEHC18 chromatographic column, the column having dimensions of 50 mm × 2.1 mm and 1.7 μm; The column temperature is 30℃; the autosampler temperature is 10℃. In both positive and negative ion modes, mobile phase A is 0.1% formic acid in water, and mobile phase B is 99.9% methanol. The sample gradient elution program for the chromatographic system is as follows: 0-2 min, 95%A-95%A; 2-13 min, 95%A-0%A; 13-16 min, 0%A-0%A; 16-16.1 min, 0%A-95%A; 16.1-21 min, 95%A-95%A. The flow rate was 0.3 mL / min; The injection volume was 2 μL.

[0013] The accuracy of the peak area of ​​a biomarker determines the accuracy of its relative concentration calculation. However, existing chromatographic conditions suffer from problems such as sparse peak numbers, insufficient information coverage, poor information abundance, and weak main peak signal intensity, leading to inaccurate relative concentration calculations using the peak area of ​​biomarkers and consequently, low accuracy in identifying the estrus period of female buffalo. To solve these technical problems, this invention employs the aforementioned chromatographic conditions, resulting in highly uniform peak distribution and complete information in both positive and negative ion maps. Furthermore, the biomarker expression peaks are clear and accurate, significantly increasing the accuracy of relative concentration calculations using the biomarker expression peaks obtained under these chromatographic conditions.

[0014] Preferably, in the above technical solution, the mass spectrometry conditions of the quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system include: Detection was performed using electrospray ionization in both positive and negative ion modes. The electrospray ionization source and mass spectrometry settings were as follows: the ion source was a heated electrospray ionizer at 350°C; the spray voltage in negative ion mode was 3.0 kV; the transmission capillary temperature was 320°C; the sheath gas was 35 psi; and the auxiliary gas flow rate was 10 psi. The scanning mode was FullMS / dd-MS2 with a mass range of 200-2000 m / z, and the resolutions for the first and second scans were 70,000 and 17,500, respectively. The collision gas was high-purity nitrogen.

[0015] Preferably, in the above technical solution, the biomarker is one or both of pyridoxine and homovanillic acid.

[0016] Based on extensive experimental data analysis, the inventors obtained the relative concentrations of pyridoxine calculated from the peak area during estrus, proestrus, and mesestrus as follows: The relative concentration of pyridoxine during proestrus was 0.0800±0.0362, during estrus it was 0.0240±0.0114, and during mesestrus it was 0.0413±0.0125. Thus, the relative concentrations of pyridoxine calculated from the peak area during estrus, proestrus, and mesestrus are significantly different. Furthermore, based on this, the inventors determined that a relative concentration of pyridoxine in the milk sample of 0.03 can be used as the estrus threshold for Mora female buffalo. This threshold is used to determine whether a Mora female buffalo has entered the estrus period. Specifically, when the relative concentration calculated from the expression peak area of ​​pyridoxine in the milk sample to be tested is lower than 0.03, the Mora buffalo to be identified corresponding to the milk sample to be tested is in the estrus period. When the relative concentration calculated from the expression peak area of ​​pyridoxine in the milk sample to be tested is higher than or equal to 0.03, the Mora buffalo to be identified corresponding to the milk sample to be tested is in the non-estrus period.

[0017] Based on extensive experimental data analysis, the inventors obtained the relative concentrations of homovanillic acid calculated from the peak area during estrus, proestrus, and mesestrus as follows: The relative concentration of homovanillic acid during proestrus was 0.1257±0.1273, during estrus it was 0.0047±0.0031, and during mesestrus it was 0.1122±0.0908. Thus, the relative concentrations of homovanillic acid calculated from the peak area during estrus, proestrus, and mesestrus are significantly different. Furthermore, based on this, the inventors determined that a relative concentration of homovanillic acid in the milk sample of 0.05 can be used as the estrus threshold for Mora female buffalo. This threshold is used to determine whether a Mora female buffalo has entered the estrus period. Specifically, when the relative concentration calculated from the expression peak area of ​​homovanillic acid in the milk sample to be tested is lower than 0.05, the Mora buffalo to be identified corresponding to the milk sample to be tested is in the estrus period. When the relative concentration calculated from the expression peak area of ​​pyridoxine in the milk sample to be tested is higher than or equal to 0.05, the Mora buffalo to be identified corresponding to the milk sample to be tested is in the non-estrus period.

[0018] Preferably, in the above technical solution, the relative concentration of pyridoxine in the proestrus stage is 0.0800±0.0362, the relative concentration in the estrus stage is 0.0240±0.0114, and the relative concentration in the postestrus stage is 0.0413±0.0125. The relative concentration of homovanillic acid was 0.1257±0.1273 in the proestrus stage, 0.0047±0.0031 in the estrus stage, and 0.1122±0.0908 in the postestrus stage.

[0019] To achieve the above objectives, the present invention also provides an application of buffalo estrus biomarkers for identifying buffalo estrus, wherein a buffalo estrus identification kit is prepared using pyridoxine and / or homovanillic acid as biomarkers.

[0020] Preferably, in the above technical solution, when the biomarker is pyridoxine, the relative concentration of pyridoxine in milk of 0.03 is used as the judgment threshold. If the concentration is lower than the threshold, the buffalo to be identified is determined to be in estrus.

[0021] Preferably, in the above technical solution, when the biomarker is homovanillic acid, the relative concentration of homovanillic acid in milk of 0.05 is used as the judgment threshold. If the concentration is lower than the threshold, the buffalo to be identified is determined to be in estrus.

[0022] A method for identifying the estrus period of buffalo using a buffalo estrus biomarker, the method comprising the following steps: S1, Collect milk samples from the female buffalo to be tested; S2, after thawing the milk sample on ice, 100 μL of the milk sample was vortexed and mixed with 400 μL of 80% methanol solution. The mixed milk sample was placed on ice for 5 min, then centrifuged at 4℃ and 15000×g for 20 min. The supernatant was collected, and the methanol solution in the supernatant was diluted to 53% by volume using ultrapure water. Finally, it was centrifuged for another 20 min, and the supernatant was analyzed. Equal volumes of all milk samples were mixed as quality control samples. After extraction, the samples were analyzed using a quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system (ThermoScientific, Q-Exactive) and an ultra-fast high-performance liquid chromatography system (ThermoScientific, UltiMate3000). The ultra-fast high-performance liquid chromatography... The chromatographic conditions of the system are as follows: ACQUITYUPLCBEHC18 column (50 mm × 2.1 mm, 1.7 μm); column temperature: 30℃; autosampler temperature: 10℃; mobile phase A in positive and negative ion modes is 0.1% formic acid water, and mobile phase B is 99.9% methanol; the sample gradient elution program of the chromatographic system is: 0-2 min, 95%A-95%A; 2-13 min, 95%A-0%A; 13-16 min, 0%A-0%A; 16-16.1 min, 0%A-95%A; 16.1-21 min, 95%A-95%A; flow rate: 0.3 mL / min; injection volume: 2 μL; the relative concentration of the biomarker is obtained by detecting the expression peak of the biomarker according to the method. S3, compare the relative concentration of the biomarker with the determination threshold of the biomarker. If the concentration is lower than the determination threshold, the female buffalo to be tested is in estrus; otherwise, the female buffalo to be tested is in non-estrus.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a marker for detecting estrus in buffalo from buffalo milk, and screens out two new estrus markers, pyridoxine and homovanillic acid. By monitoring the expression levels of pyridoxine or homovanillic acid in the milk of female buffalo, it is possible to determine whether the female buffalo has entered estrus. This solves the problems of stress or uterine infection in female buffalo caused by invasive detection, and also solves the problem of low detection accuracy in existing technologies, thus meeting the needs of estrus identification in large-scale buffalo farming. Attached Figure Description

[0024] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings.

[0025] Figure 1 These are peak diagrams of different gradients in positive ion mode; Figure 2 These are peak diagrams of different gradients in negative ion mode; Figure 3 This is a volcano plot of differential metabolite analysis in buffalo milk during proestrus and estrus under positive ion mode; Figure 4 This is a volcano plot showing the differential metabolite analysis of buffalo milk during proestrus and estrus under negative ion mode; Figure 5 This is a volcano plot of differential metabolite analysis in buffalo milk during estrus and postestrus under positive ion mode; Figure 6 This is a volcano plot showing the differential metabolites in buffalo milk during estrus and postestrus under negative ion mode. Figure 7 This is an OPLS-DA score graph of differential metabolite analysis in buffalo milk during estrus and proestrus under positive ion mode; Figure 8 This is an OPLS-DA score graph of differential metabolite analysis of buffalo milk during estrus and proestrus under negative ion mode; Figure 9 This is an OPLS-DA score graph of differential metabolite analysis in buffalo milk during estrus and mesrus under positive ion mode; Figure 10 This is an OPLS-DA score graph showing the differential metabolites analysis of buffalo milk during estrus and postestrus under negative ion mode. Figure 11 This is a bar chart showing the changes in pyridoxine expression levels during different estrous cycles; Figure 12 This is a bar chart showing the changes in the expression level of homovanillic acid during different estrous cycles; Figure 13 This is an ultrasound image of a cow in estrus during an experiment. Detailed Implementation

[0026] The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] The experimental cattle used in this application were Murrah female buffaloes from the Guangxi Buffalo Research Institute, with similar body condition, normal estrus, and good health, and 2-5 calvings.

[0028] The experimental instruments and reagents used in this application include: Quadrupole-electrostatic track trap high-resolution mass spectrometry system (ThermoScientific, Q-Exactive), ultra-fast high-performance liquid chromatography system (ThermoScientific, UltiMate3000), low-temperature high-speed centrifuge (Eppendorf, 5424R), methanol (Fisher). Example 1

[0029] A method for obtaining a biomarker of buffalo estrus, wherein the biomarker is homovanillic acid, the method comprising: S1. Under the same management conditions, samples were collected at three estrus stages. At 4:00 AM on the first, third, and eighth days of proestrus (day 1), estrus (day 3), and mesestrus (day 8), the dairy buffaloes were driven to the restraint area and milked using milking equipment. Milk samples were collected from 12 dairy buffaloes. After collection, the milk samples were transported to the laboratory, aliquoted into 5mL centrifuge tubes, and stored at -80℃.

[0030] Under the same management conditions, ultrasound was used to simultaneously monitor the follicular development of buffalo ovaries. The buffalo were herded into a restraint cage, and after clearing any impacted feces from their rectum, the position of the uterus and both ovaries was preliminarily determined using a rectal examination. A small amount of paraffin oil was applied to the ultrasound probe for lubrication, and the probe was inserted into the rectum. The probe was slid back and forth or swung left and right to change the angle for examination. The ovaries were appropriately fixed, and the probe was placed on them to observe real-time follicular development images, obtaining direct evidence of estrus and ovulation in the buffalo.

[0031] S2, Sample Preparation: After thawing the milk sample on ice, 100 μL of the milk sample was vortexed and mixed with 400 μL of 80% methanol solution. The mixed milk sample was placed on ice for 5 min, then centrifuged at 15000×g for 20 min at 4℃. The supernatant was collected, and the methanol solution in the supernatant was diluted to 53% by volume with ultrapure water. Finally, it was centrifuged for another 20 min, and the supernatant was collected for analysis. All milk samples were mixed in equal volumes as quality control samples. After extraction, the samples were analyzed using a quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system (ThermoScientific, Q-Exactive) and an ultra-fast high-performance liquid chromatography system (ThermoScientific, UltiMate3000). The chromatographic conditions of the ultra-fast high-performance liquid chromatography system are as follows: ACQUITYUPLCBEHC18 column (50mm×2.1mm, 1.7μm); column temperature 30℃; autosampler temperature 10℃; mobile phase A in positive and negative ion modes is 0.1% formic acid water, and mobile phase B is 99.9% methanol; the sample gradient elution program of the chromatographic system is: 0-2min, 95%A-95%A; 2-13min, 95%A-0%A; 13-16min, 0%A-0%A; 16-16.1min, 0%A-95%A; 16.1-21min, 95%A-95%A; flow rate 0.3mL / min; injection volume 2μL.

[0032] Studies have found that the mobile phase gradient elution program for this sample suffers from several problems: "Setting a fast gradient results in a significant loss of early elution information in the positive ion spectrum and an overly dense mid-term peak cluster in the negative ion spectrum; setting a slow gradient results in sparse peaks in the positive ion spectrum and sparse peak clusters in the later stages of the negative ion spectrum with insufficient information coverage; setting a conventional slope gradient results in low main peak signal intensity in the positive ion spectrum and a small number of intermediate peaks in the negative ion spectrum." To address these technical issues, this application employs a precise segmented gradient elution program: 0-2 min, 95%A-95%A; 2-13 min, 95%A-0%A; 13-16 min, 0%A-0%A; 16-16.1 min, 0%A-95%A; 16.1-21 min, 95%A-95%A. Through the coordinated and synergistic effect of the different gradient segments, the program ultimately achieves uniform peak distribution and complete information coverage throughout the entire time period.

[0033] Positive and negative ion peak diagrams for different mobile phase gradient elution programs: Mobile phase gradient 1: 0-2 min, 95% A-95% A; 2-13 min, 95% A-40% A; 13-16 min, 40% A-40% A; 16-16.1 min, 40% A-95% A; 16.1-21 min, 95% A-95% A; The obtained positive ion peak diagram is shown below. Figure 1 As shown in Figure A, the negative ion peak diagram is as follows: Figure 2 As shown in Figure A.

[0034] Mobile phase gradient 2: -2 min, 95% A - 95% A; 2 - 13 min, 95% A - 30% A; 13 - 16 min, 30% A - 30% A; 16 - 16.1 min, 30% A - 95% A; 16.1 - 21 min, 95% A - 95% A; The obtained positive ion peak diagram is shown below. Figure 1 As shown in B, the negative ion peak diagram is as follows: Figure 2 As shown in B.

[0035] Mobile phase gradient 3: 0-2 min, 95% A-95% A; 2-13 min, 95% A-20% A; 13-16 min, 20% A-20% A; 16-16.1 min, 20% A-95% A; 16.1-21 min, 95% A-95% A; The obtained positive ion peak diagram is shown below. Figure 1 As shown in C, the negative ion peak diagram is as follows: Figure 2 As shown in C.

[0036] Mobile phase gradient 4: 0-2 min, 95% A-95% A; 2-13 min, 95% A-10% A; 13-16 min, 10% A-10% A; 16-16.1 min, 10% A-95% A; 16.1-21 min, 95% A-95% A; The obtained positive ion peak diagram is shown below. Figure 1 As shown in D, the negative ion peak diagram is as follows: Figure 2 As shown in D.

[0037] Mobile phase gradient 5: 0-2 min, 95% A-95% A; 2-13 min, 95% A-0% A; 13-16 min, 0% A-0% A; 16-16.1 min, 0% A-95% A; 16.1-21 min, 95% A-95% A; The obtained positive ion peak diagram is shown below. Figure 1 As shown in E, the negative ion peak diagram is as follows: Figure 2 As shown in E.

[0038] The above Figure 1 This is an LC-MS total ion chromatogram (TIC), with acquisition times ranging from 0 to 21 min. From Figure 1 A- Figure 1 E shows that positive ions Figure 1E has the most uniform peak distribution and the highest information integrity: a high-abundance early elution peak is visible within 0-2 min (0.89 min), the baseline is stable from 2-10 min, a dense and continuous peak cluster forms from 11-21 min, especially in the 14-15 min region where the main peak cluster signal is strong and clear, and identifiable continuous peak signals are still retained from 16-21 min, which can comprehensively reflect the sample component information. Figure 1 A and Figure 1 The peak plot of B has problems such as sparse peaks, insufficient information coverage, and poor information abundance. Figure 1 The peaks in the peak diagram of C are richer in the later stages, but there is a problem of poor signal strength of the main peak. Figure 1 The peak of D is prominent in the latter half of its peak plot, but it suffers from problems such as the lowest overall signal strength and significant information loss in the 0-12 min region. Therefore... Figure 1 E performs best in terms of peak integrity, signal richness, and time coverage, making it suitable as a representative spectrum for subsequent data analysis.

[0039] The above Figure 2 This is an LC-MS total ion chromatogram (TIC), with acquisition times ranging from 0 to 21 min. From Figure 2 A- Figure 2 E shows that negative ions Figure 2 E has the most uniform peak distribution and the best information completeness: a high-abundance early elution peak exists from 0-2 min (0.90 min), the line is stable from 2-12 min with continuous low-abundance characteristic peaks, and a coherent peak cluster forms from 13-21 min, especially in the 18-19 min region where the main peak cluster signal is prominent, which can comprehensively reflect the sample component information. Figure 2 A and Figure 2 The peak diagram of B has the problem of sparse peak clusters and insufficient information coverage in the second half. Figure 2 The peak diagram of C has the problem of a small number of peaks in the middle section; Figure 2 The peak clusters in the peak diagram of D are relatively dense from 14 to 20 minutes, and there is a problem of uneven peak distribution throughout the entire time period. Figure 2 E performs best in terms of peak integrity, information coverage, and baseline stability, making it suitable as a representative spectrum for subsequent data analysis.

[0040] Mass spectrometry conditions of the quadrupole-electrostatic track trap high-resolution mass spectrometry system: After separation using an Ultimate3000 ultra-fast high-performance liquid chromatography system, the sample was analyzed by mass spectrometry using a Q-Exactive quadrupole-electrostatic track trap high-resolution mass spectrometry system, with detection performed in both electrospray ionization (ESI) positive and negative ion modes. The ESI source and mass spectrometry settings were as follows: ion source: heated electrospray ionization (HESI), temperature: 350℃; spray voltage: 3.0 kV in negative ion mode; transport capillary temperature: 320℃; sheath gas: 35 psi; auxiliary gas flow rate: 10 psi; scan mode: Full MS / dd-MS2, mass range: 200~2000 m / z; primary and secondary scan resolutions: 70000 and 17500, respectively. Collision gas: high-purity nitrogen.

[0041] The collected samples were subjected to mass spectrometry detection and data acquisition. Non-targeted metabolomics methods were then used to analyze the mass spectrometry data. Specifically, this step involves detecting the collected samples using liquid chromatography-mass spectrometry (LC-MS) to obtain relevant data, which are then analyzed using non-targeted metabolomics methods. The LC-MS data analysis primarily utilizes both positive and negative ion scanning modes. Figure 3 This is a volcano plot showing the differential metabolite analysis of buffalo milk during proestrus and estrus in positive ion mode. Figure 4 This is a volcano plot showing the differential metabolite analysis of buffalo milk during proestrus and estrus under negative ion mode. Figure 5 This is a volcano plot showing the differential metabolite analysis of buffalo milk during estrus and postestrus in positive ion mode. Figure 6 This is a volcano plot analyzing differential metabolites in buffalo milk during estrus and mesrus under negative ion mode. Red represents significantly upregulated metabolites (LOG2(FoldChange) > 1, -LOG10(P) > 1.3), green represents significantly downregulated metabolites (LOG2(FoldChange) < -1, -LOG10(P) > 1.3), and gray represents insignificant metabolites. The results indicate that there are differences in metabolites in buffalo milk between proestrus and estrus, and between estrus and mesrus. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used for between-group differential analysis. On the OPLS-DA score plot, the horizontal axis represents predicted principal components, reflecting the maximization of between-group differences, and the vertical axis represents orthogonal principal components, reflecting within-group variation. In the figure, points of the same color represent biological replicates within a group, and the distribution of points reflects the degree of difference between and within groups.

[0042] S3. The raw data was converted to mzXML format using ProteoWizard, and then peak alignment, retention time correction, and peak area extraction were performed using XCMS software. The data extracted by XCMS were first subjected to metabolite structure identification and data preprocessing, followed by experimental data quality evaluation, and finally data analysis. Data analysis included univariate statistical analysis, multidimensional statistical analysis, and differential metabolite screening. Significantly differential metabolites in buffalo milk from female buffalo during proestrus, estrus, and metestrus are shown in Tables 1-4.

[0043] Table 1. Significantly Upregulated Metabolites in Milk Between Proestrus and Estrus Groups Arachidonic acid [M+H]+1 305.25 3.65 1.04 2.68 0.0122 lipids and lipid molecules Tyramine [M+H]+1 138.09 6.85 1.38 1.71 3.00E-06 benzene compounds Glutaryl-CoA [M+DMSO+H]+1 960.17 12.42 1.30 2.11 0.0002 lipids and lipid molecules Succinylacetone [M+H]+1 159.07 2.06 1.03 1.08 0.0095 Organic acids and their derivatives 7-Oxycholesterol [M+Na]+1 423.32 15.13 1.44 2.67 3.00E-08 lipids and lipid molecules 5α-Dihydrotestosterone [M+H]+1 291.23 12.91 1.33 1.04 5.00E-05 lipids and lipid molecules androstanolone [M+H]+1 291.23 0.52 1.34 2.53 2.00E-05 lipids and lipid molecules Coenzyme A-20-carboxyl-LTE4 [M+H]+1 1215.29 12.64 1.33 2.93 0.0001 lipids and lipid molecules 18E-20-oxo-20-coenzyme A-LTE4 [M+H]+1 1213.28 17.74 1.12 2.79 0.0079 lipids and lipid molecules 4-Hydroxycoumarin [M+H]+1 163.04 10.74 1.25 1.31 3.90E-04 Phenylacetic compounds and polyketides Eicosapentaenoic acid [M+H-H2O]+1 285.22 2.33 1.43 1.41 2.00E-07 lipids and lipid molecules Methyl palmitate [M+H]+1 271.26 0.71 1.41 1.41 4.88E-07 lipids and lipid molecules Ureidine diphosphate glucose [M+H-H2O]+1 549.05 10.80 1.30 1.38 0.0001 Nucleosides, nucleotides and their analogues Cyclamen aldehyde [M+H]+1 191.14 18.41 1.46 1.35 4.20E-09 lipids and lipid molecules Jasmonic acid [M+H]+1 211.13 20.63 1.33 1.32 6.98E-05 lipids and lipid molecules High-gamma-linolenic acid [M+H]+1 307.26 2.58 1.42 1.32 3.07E-07 lipids and lipid molecules Carbolic acid [M+H]+1 138.97 18.80 1.23 1.15 0.0008 Organometallic compounds Table 2. Significantly downregulated metabolites in milk between the proestrus and estrus groups. N-acetyl-L-aspartic acid [M+H]+1 176.06 1.92 1.03 -2.43 0.0111 Organic acids and their derivatives Leukotriene B4 [M+H]+1 337.24 16.80 1.22 -1.27 0.0026 lipids and lipid molecules High vanillic acid [M+H]+1 183.07 4.72 1.27 -4.38 0.0004 benzene compounds Pyridoxine [M+H]+1 169.10 3.76 1.13 -1.73 0.0032 Organic heterocyclic compounds Table 3. Table of significantly upregulated metabolites in milk between the estrus and postestrus groups. 5'-Pyridoxal phosphate [M+H]+1 248.03 1.55 1.29 2.30 0.0013 Organic heterocyclic compounds Pyridoxal [M+H]+1 168.07 1.22 1.14 1.11 0.0126 Organic heterocyclic compounds High vanillic acid [M+H]+1 183.07 4.72 1.30 4.16 0.0020 benzene compounds L-Tryptophan [M+H]+1 205.10 5.64 1.19 1.37 0.0071 Organic heterocyclic compounds Glutaryl-CoA [M+DMSO+H]+1 960.17 12.42 1.23 1.91 0.0055 lipids and lipid molecules xanthine [M+H]+1 153.04 4.27 1.32 2.28 0.0012 Organic heterocyclic compounds hypoxanthine [M+H]+1 137.05 1.68 1.16 3.52 0.0089 Organic heterocyclic compounds adenine [M+H]+1 136.06 1.99 1.34 1.76 0.0007 Organic heterocyclic compounds 2,8-Dihydroxyadenine [M+H]+1 168.05 1.45 1.18 1.76 0.0078 Organic heterocyclic compounds L-threonine [M+K]+1 159.03 0.93 1.46 1.25 1.04E-05 Organic acids and their derivatives L-Isoleucine [M+H]+1 132.10 1.87 1.15 1.08 0.0105 Organic acids and their derivatives Citric acid [M+Na]+1 215.02 1.35 1.25 1.12 0.0031 Organic acids and their derivatives Succinic acid [M+H]+1 119.03 1.34 1.33 1.72 0.0008 Organic acids and their derivatives D-glucosamine 6-phosphate [M+H]+1 260.05 0.94 1.24 1.453 0.0033 Organic oxygen compounds (R)-3-hydroxybutyrylcarnitine [M+H]+1 248.15 1.23 1.48 7.79 1.68E-06 lipids and lipid molecules Pyridoxine [M+H]+1 169.10 3.76 1.13 1.38 0.0045 Organic heterocyclic compounds Table 4. Comparison of significantly downregulated metabolites in milk from the estrus and postestrus groups. 5α-androstan-3β-ol [M+H+MeOH]+1 309.28 0.23 1.20 -1.06 0.0080 lipids and lipid molecules methyl arachidonic acid [M+H]+1 319.26 14.72 1.43 -1.43 6.57E-05 lipids and lipid molecules Palmitamide [M+H]+1 256.26 10.80 1.29 -1.44 0.0016 lipids and lipid molecules As shown in Tables 1-4, by comparing the metabolites in the milk of female buffalo at different stages, 39 significantly different metabolites, including arachidonic acid, leukotrienes B4, and pyridoxine, were identified. Among them, only pyridoxine and homovanillic acid showed significant changes, decreasing significantly in the proestrus and estrus comparison group and increasing significantly in the estrus and mesrus comparison group. This suggests that the content of pyridoxine and homovanillic acid in the milk of female buffalo may decrease during estrus and increase during non-estrus.

[0044] The relative concentrations of pyridoxine and homovanillic acid were further calculated based on the peak areas of their metabolites. Table 5 shows the results of the relative concentration analysis of pyridoxine and homovanillic acid at different estrus stages.

[0045] Table 5. Changes in relative concentrations of pyridoxine and homovanillic acid in buffalo milk at different estrus stages. Pyridoxine <![CDATA[0.0800±0.0362 a ]]> <![CDATA[0.0240±0.0114 c ]]> <![CDATA[0.0413±0.0125 b ]]> High vanillic acid <![CDATA[0.1257±0.1273 a ]]> <![CDATA[0.0047±0.0031 b ]]> <![CDATA[0.1122±0.0908 a ]]> The superscripts in the same row for a, b, and c indicate significant differences (P < 0.05). Table 5 shows that the relative concentrations of pyridoxine in proestrus were 0.0800±0.0362, in estrus 0.0240±0.0114, and in metestrus 0.0413±0.0125; the relative concentrations of homovanillic acid in proestrus were 0.1257±0.1273, in estrus 0.0047±0.0031, and in metestrus 0.1122±0.0908. This further demonstrates that the relative concentrations of pyridoxine and homovanillic acid in the milk of female buffalo decrease during estrus, while increasing during non-estrus periods.

[0046] Figure 11 This is a graph showing the changing trend of pyridoxine expression peak area at different stages of the estrous cycle, where Proestrus represents proestrus, Estrus represents estrus, and Metestrus represents metestrus. From... Figure 11 It can be seen that the expression level of pyridoxine is greater than 2×10 during proestrus and metestrus. 6 During estrus, the expression level of pyridoxine is much lower than 2×10. 6 Therefore, the expression level of pyridoxine is significantly increased during the non-estrus period and significantly decreased during the estrus period, indicating that the expression level of pyridoxine in cow's milk is significantly different between the estrus and non-estrus periods. By detecting the expression level of pyridoxine in cow's milk, it is possible to detect whether buffalo are in estrus. Pyridoxine can be used as a biomarker of buffalo estrus.

[0047] Figure 12 This is a graph showing the changing trend of the expression peak area of ​​homovanillic acid at different stages of the estrous cycle, where Proestrus represents proestrus, Estrus represents estrus, and Metestrus represents metestrus. From... Figure 12 It can be seen that the expression level of homovanillic acid in proestrus and mesestrus is greater than 2×10. 5 During estrus, the expression level of homovanillic acid is much lower than 1×10⁻⁶. 5 Therefore, the expression level of homovanillic acid is significantly increased during the non-estrus period and significantly decreased during the estrus period, indicating that the expression level of homovanillic acid in milk is significantly different between the estrus and non-estrus periods. By detecting the expression level of homovanillic acid in milk, buffalo can be detected to determine whether they are in estrus. Homovanillic acid can be used as a biomarker for buffalo estrus.

[0048] The method for determining whether a Mora female buffalo has entered estrus using pyridoxine and homovanillic acid involves calculating the relative concentration of pyridoxine or homovanillic acid by measuring the peak area of ​​the expression in the milk sample of the buffalo to be identified. The relative concentration is then used to determine whether the buffalo has entered estrus.

[0049] Twelve Mora female buffaloes from the Guangxi Buffalo Research Institute, with similar body condition, normal estrus, and good health, and belonging to the 2nd to 5th parity, were selected as the buffaloes to be identified. The estrus status of the buffaloes was determined by detecting whether the relative concentration of pyridoxine expression peak area in the milk samples of the Mora female buffaloes was lower than 0.03 and by observing follicular development images by ultrasound. The results are shown in Table 6.

[0050] Table 6. Results of pyridoxine application in buffalo estrus diagnosis. 1 A value below 0.03 indicates the animal is in estrus. estrus 100% 2 A value below 0.03 indicates the animal is in estrus. estrus 100% 3 A value below 0.03 indicates the animal is in estrus. estrus 100% 4 A value above 0.03 indicates the person is not in estrus. Late estrus 100% 5 A value above 0.03 indicates the person is not in estrus. Proestrus 100% 6 A value below 0.03 indicates the animal is in estrus. estrus 100% 7 A value above 0.03 indicates the person is not in estrus. Proestrus 100% 8 A value below 0.03 indicates the animal is in estrus. estrus 100% 9 A value below 0.03 indicates the animal is in estrus. estrus 100% 10 A value above 0.03 indicates the person is not in estrus. Proestrus 100% 11 A value above 0.03 indicates the person is not in estrus. Late estrus 100% 12 A value above 0.03 indicates the person is not in estrus. Postestrus 100% Twelve Mora female buffaloes from the Guangxi Buffalo Research Institute, with similar body condition, normal estrus, and good health, and belonging to the 2nd to 5th parity, were selected as the buffaloes to be identified. The estrus status of the buffaloes was determined by detecting whether the relative concentration of homovanillic acid expression peak area in the milk samples of the buffaloes was lower than 0.05 and by observing follicular development images by ultrasound. The results are shown in Table 7.

[0051] Table 7 Results of using homovanillic acid for estrus diagnosis in buffalo. 1 A value below 0.05 indicates the animal is in estrus. estrus 100% 2 A value below 0.05 indicates the animal is in estrus. estrus 100% 3 A value above 0.05 indicates the person is not in estrus. Late estrus 100% 4 A value below 0.05 indicates the animal is in estrus. estrus 100% 5 A value above 0.05 indicates the person is not in estrus. Proestrus 100% 6 A value above 0.05 indicates the person is not in estrus. Postestrus 100% 7 A value below 0.05 indicates the animal is in estrus. estrus 100% 8 A value below 0.05 indicates the animal is in estrus. estrus 100% 9 A value above 0.05 indicates the person is not in estrus. Proestrus 100% 10 A value above 0.05 indicates the person is not in estrus. Proestrus 100% 11 A value above 0.05 indicates the person is not in estrus. Postestrus 100% 12 A value above 0.05 indicates the person is not in estrus. Proestrus 100% From Table 6, Table 7 and Figure 13 It can be seen that the decrease in the expression levels of pyridoxine and homovanillic acid in cow's milk indicates that the buffalo is in estrus, while the expression levels are higher than the set thresholds for homovanillic acid and pyridoxine, indicating that the buffalo is not in estrus. The results are completely consistent with the ultrasound results used to determine estrus. Therefore, it can be concluded that the expression levels of pyridoxine and homovanillic acid in buffalo milk can be used to determine whether dairy buffalo are in estrus.

[0052] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for obtaining estrus biomarkers in buffalo, characterized in that, The method includes: S1. Collect milk samples from the female buffalo and observe the follicle development of the female buffalo using ultrasound. S2, take the milk sample and methanol solution, vortex and mix them, place the mixed milk sample on ice for 4-8 min, centrifuge for 18-22 min and take the supernatant, dilute the supernatant with ultrapure water, centrifuge for 20 min, and take the supernatant for analysis. Take equal volumes of all milk samples and mix them as quality control samples. After extraction, analyze them using a quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system and an ultra-fast high-performance liquid chromatography system. S3. After the raw data is converted into mzXML format, XCMS software is used to perform peak alignment, retention time correction and peak area extraction to obtain extracted data. The extracted data is then subjected to metabolite structure identification, data preprocessing, experimental data quality evaluation and data analysis in sequence to screen out significantly different metabolites in milk during the estrous cycle. S4, detect the expression levels of the significantly differentially expressed metabolites in the milk during estrus and non-estrus periods, compare the changes in expression levels with ultrasound results, and obtain buffalo estrus biomarkers.

2. The method for obtaining estrus biomarkers in buffalo as described in claim 1, characterized in that, The milk sample and the methanol solution were mixed evenly at a volume ratio of 1:4, and the methanol solution had a volume fraction of 80%.

3. The method for obtaining estrus biomarkers in buffalo as described in claim 1, characterized in that, In step S2, diluting the supernatant with ultrapure water specifically involves diluting the methanol volume fraction in the supernatant with ultrapure water to a methanol volume fraction of 53%.

4. The method for obtaining estrus biomarkers in buffalo as described in claim 1, characterized in that, The chromatographic conditions of the ultrafast high-performance liquid chromatography system include: ACQUITYUPLCBEHC18 chromatographic column, the column having dimensions of 50 mm × 2.1 mm and 1.7 μm; The column temperature is 30℃; the autosampler temperature is 10℃. In negative ion mode, mobile phase A is 0.1% formic acid in water, and mobile phase B is 99.9% methanol; The sample gradient elution program for the chromatographic system is as follows: 0-2 min, 95%A-95%A; 2-13 min, 95%A-0%A; 13-16 min, 0%A-0%A; 16-16.1 min, 0%A-95%A; 16.1-21 min, 95%A-95%A. The flow rate was 0.3 mL / min; The injection volume was 2 μL.

5. The method for obtaining estrus biomarkers in buffalo as described in claim 1, characterized in that, The mass spectrometry conditions of the quadrupole-electrostatic track trap high-resolution mass spectrometry system include: Detection was performed using electrospray ionization in both positive and negative ion modes. The electrospray ionization source and mass spectrometry settings were as follows: the ion source was a heated electrospray ionizer at 350°C; the spray voltage was 3.0 kV in both positive and negative ion modes; the transport capillary temperature was 320°C; the sheath gas was 35 psi; and the auxiliary gas flow rate was 10 psi. The scanning mode was FullMS / dd-MS2 with a mass range of 200-2000 m / z, and the resolutions for the first and second scans were 70,000 and 17,500, respectively. The collision gas was high-purity nitrogen.

6. The method for obtaining estrus biomarkers in buffalo as described in claim 1, characterized in that, The biomarker is one or both of pyridoxine and homovanillic acid.

7. The method for obtaining estrus biomarkers in buffalo as described in claim 1, characterized in that, The relative concentration of pyridoxine during proestrus was 0.0800±0.0362, during estrus it was 0.0240±0.0114, and during metestrus it was 0.0413±0.0125. The relative concentration of homovanillic acid was 0.1257±0.1273 in the proestrus stage, 0.0047±0.0031 in the estrus stage, and 0.1122±0.0908 in the postestrus stage.

8. An application of a buffalo estrus biomarker for identifying the estrus period in buffaloes, characterized in that, A kit for identifying the estrus cycle of buffalo was prepared using pyridoxine and / or homovanillic acid as biomarkers.

9. The application of the buffalo estrus biomarker as described in claim 8 for identifying the estrus period in buffaloes, characterized in that, When the biomarker is pyridoxine, the relative concentration of pyridoxine in milk of 0.03 is used as the threshold. If the concentration is lower than this threshold, the buffalo to be identified is determined to be in estrus.

10. The application of the buffalo estrus biomarker as described in claim 8 for identifying the estrus period in buffaloes, characterized in that, When the biomarker is homovanillic acid, the relative concentration of homovanillic acid in milk of 0.05 is used as the judgment threshold. If the concentration is lower than this threshold, the buffalo to be identified is determined to be in estrus.

Images