Biomarker for related state of early pregnancy of dairy cow and application of biomarker

Abstract

The invention discloses a biomarker for related states of early and early pregnancy of dairy cows and application of the biomarker, biological samples of a pregnancy group, a non-pregnancy group and a return ostrus group of the dairy cows after insemination are analyzed, and the biomarker with significant difference with the related states of the early and early pregnancy is determined by utilizing a metabonomics technology; the method can be used for quickly, simply, economically, effectively and accurately detecting and / or evaluating and / or identifying the related state of the early pregnancy of the dairy cow, and provides a new means for quickly finding non-pregnant dairy cows (including return-estrus dairy cows and non-pregnant dairy cows), shortening the interval time of re-hybridization, optimizing the feeding management of the pregnant dairy cows, improving the breeding efficiency of the dairy cows and the like; and the method has important economic value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to biomarkers of early pregnancy-related states of dairy cows and applications thereof. Background Art

[0002] At present, candidate factors related to the fate of early embryonic development have become a research hotspot. For example, Sakonju et al. detected the content of early pregnancy factor (EPF) in the serum of cows on days 13-16 and 25 of pregnancy, and compared it with the serum EPF concentration of non-pregnant cows at the corresponding stage. The results showed that there was a significant difference in EPF concentration between the two groups. However, there are also studies that detect pregnancy in cows after mating by detecting EPF levels in the blood, but it is found that the probability of correctly detecting empty cows is only about 50%. In addition, when the same cow is tested multiple times within 4 weeks, the test results lack consistency, reflecting the limited reliability of EPF as an indicator of early pregnancy detection. In addition, cows that successfully conceive begin to produce fetal-related protein bPSP-B in their bodies on the 16th day after mating, and as the pregnancy progresses, the concentration of bPSP-B in the mother's blood gradually increases. In the process of distinguishing pregnant from empty cows by detecting bPSP-B, the false positive rate is as high as 56.7%. Since the half-life of bPSP-B is 7-8 days, when used to detect cows with embryonic death in the late embryonic development (16-42 days), the bPSP-B level still cannot effectively distinguish these cows as empty cows. The fifth day after insemination of dairy cows is a critical time node for early pregnancy detection. At this stage, progesterone (P4) secreted by the corpus luteum plays a key role in the implantation of the fertilized egg, uterine function and embryo growth. Studies have shown that the relationship between P4 concentration in dairy cow blood and embryonic development is already apparent on the fifth day of pregnancy. Previous studies have found that a decrease in P4 concentration in milk on the fifth day after artificial insemination will have an adverse effect on the uterus initiating blastocyst development, resulting in a decrease in pregnancy rate and impaired embryonic development. From ovulation to the seventh day after ovulation, the metabolites of the dairy cow uterus change significantly to adapt to the different functions of the uterus in sperm transport, immune defense and early embryonic development. Statistics show that about 70% to 80% of embryo losses occur in the first two weeks after artificial insemination, and the proportion of embryo deaths before the seventh day after insemination is significantly higher than in other periods. However, there are still few indicators for detecting early pregnancy in dairy cows, and they are not reliable and accurate enough.

[0003] The establishment of dairy cow pregnancy is an extremely complex physiological process, involving a series of highly coordinated reactions such as ovulation, fertilization, blastocyst formation, conceptus growth, secretion of pregnancy recognition signals, and development of the embryo and placenta. Failure to identify non-pregnant cows in time may lead to a delay in the next estrus insemination time, which in turn prolongs the calving interval, resulting in a series of problems such as decreased pasture reproductive efficiency and increased breeding costs. Studies have shown that when a dairy cow is empty for more than 18-24 days (i.e., one estrus cycle), its milk production in one lactation cycle will decrease by 168 to 315 kg. Therefore, exploring a quick, simple, economical, effective and accurate early pregnancy detection method and optimizing the feeding and management of pregnant dairy cows will help to promptly start the next reproductive cycle of non-pregnant or embryo-dead dairy cows, which has important economic value. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, the technical solution provided by the present invention is as follows:

[0005] In a first aspect, the present invention provides a biomarker for early pregnancy-related status in dairy cows, wherein the biomarker comprises at least one of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine;

[0006] There are statistically significant differences in the measured values ​​of the biomarkers in the biological samples of the returning estrus group cows, the measured values ​​in the biological samples of the pregnant group cows, and the measured values ​​in the biological samples of the non-pregnant group cows;

[0007] Among them, the measurement values ​​of alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in the biological samples of the returning estrus group cows were significantly higher than the measurement values ​​in the biological samples of the pregnant group cows and the measurement values ​​in the biological samples of the non-pregnant group cows;

[0008] The measurement value of D-lactic acid in the biological samples of the pregnant group of dairy cows was significantly higher than the measurement value in the biological samples of the returning group of dairy cows and the measurement value in the biological samples of the non-pregnant group of dairy cows;

[0009] The measured values ​​of trans-aconitic acid, desipramine and taurodeoxycholic acid in biological samples of non-pregnant cows were significantly higher than those in biological samples of re-estrus cows and those in biological samples of pregnant cows; the measured values ​​of phosphatidylcholine, maleic acid, methyl fumaric acid, succinic semialdehyde and β-octylglucoside in biological samples of pregnant cows and those in biological samples of non-pregnant cows were significantly higher than those in biological samples of re-estrus cows;

[0010] The measurement values ​​of 5-aminovaleric acid and triethanolamine in the biological samples of the returning estrus group and the non-pregnant group were significantly higher than those in the biological samples of the pregnant group.

[0011] The measurement values ​​of valine-methionine in biological samples of cows in the estrus group and in biological samples of cows in the pregnancy group were significantly higher than those in biological samples of cows in the non-pregnancy group.

[0012] In the present invention, the terms "significantly differential metabolites" and "biomarkers" can be used interchangeably.

[0013] In some preferred embodiments, the biomarker comprises at least two of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine.

[0014] In some more preferred embodiments, the biomarkers include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine.

[0015] In some preferred embodiments, the biological sample comprises cow plasma.

[0016] In the present invention, early pregnancy refers to within 7 days after fertilization.

[0017] In the present invention, the early pregnancy-related states include estrus state, early pregnancy state (or pregnancy state), and non-pregnancy state.

[0018] In some embodiments, the time of the early pregnancy-related state is 4.5-5.5 days after insemination. In some preferred embodiments, the time of the early pregnancy-related state is 5 days after insemination.

[0019] In the present invention, the criteria for statistical significance include:

[0020] (1) variable projection importance VIP; and

[0021] (2) t-test; and

[0022] (3) Univariate statistical analysis of the difference in FC.

[0023] In some embodiments, the statistically significant screening criteria include at least one of the following (a1)-(a3):

[0024] (a1) variable projection importance VIP>1; and

[0025] (a2) P < 0.05; and

[0026] (a3) FC>1.3 or FC<0.77.

[0027] It should be understood that when multiple biomarkers are used together, the AUC value is higher than when used alone, and the detection accuracy is higher.

[0028] Furthermore, in some preferred embodiments, the statistically significant screening criteria also include an AUC value greater than 0.7.

[0029] In some preferred embodiments, the biomarkers of the cows in the estrus group and the cows in the pregnancy group are selected from at least one of phosphatidylcholine, succinic semialdehyde, methyl fumaric acid, β-octylglucoside, D-lactic acid, maleic acid, heptadecanoic acid, alanine-leucine, deoxycholic acid and γ-L-glutamyl-L-phenylalanine, and the AUC value of each of the above biomarkers is greater than 0.7.

[0030] In some preferred embodiments, the biomarkers of the pregnant group of cows and the non-pregnant group of cows are selected from at least one of taurine deoxycholic acid, triethanolamine, desipramine, trans-aconitic acid, 5-aminovaleric acid, D-lactic acid and valine-methionine, and the AUC value of each of the above biomarkers is greater than 0.7.

[0031] In some preferred embodiments, the biomarkers of the cows in the estrus group and the cows in the non-pregnant group are selected from at least one of taurodeoxycholic acid, desipramine, trans-aconitic acid, phosphatidylcholine, succinic semialdehyde, methyl fumaric acid, β-octylglucoside, maleic acid, valine-methionine, alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine, and the AUC value of each of the above biomarkers is greater than 0.7.

[0032] In some more preferred embodiments, the biomarkers of the estrus group cows and the pregnancy group cows are selected from at least one of β-octylglucoside, D-lactic acid and heptadecanoic acid, and the AUC value of each of the above biomarkers is greater than 0.8.

[0033] The biomarkers of the pregnant group of cows and the non-pregnant group of cows are selected from at least one of triethanolamine, 5-aminovaleric acid and valine-methionine, and the AUC value of each of the above biomarkers is greater than 0.8.

[0034] The biomarkers of the cows in the estrus group and the cows in the non-pregnant group are selected from at least one of methyl fumaric acid, maleic acid and heptadecanoic acid, and the AUC value of each of the above biomarkers is greater than 0.8.

[0035] In a second aspect, the present invention provides a test paper, a reagent or a kit for detecting the above-mentioned biomarkers of early pregnancy-related states in dairy cows.

[0036] In a third aspect, the present invention provides a method for detecting early pregnancy-related states in dairy cows, the method comprising:

[0037] A) obtaining a biological sample containing biomarkers from a cow to be tested;

[0038] B) detecting a measurement value of a biomarker in the biological sample, wherein the biomarker comprises at least one of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine; and

[0039] C) computer processing of the measured values ​​of the biomarkers, wherein the measured values ​​of the biomarkers in the biological samples of the estrus group of dairy cows, the measured values ​​in the biological samples of the pregnant group of dairy cows, and the measured values ​​in the biological samples of the non-pregnant group of dairy cows are statistically significantly different;

[0040] Among them, the measurement values ​​of alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in the biological samples of the returning estrus group cows were significantly higher than the measurement values ​​in the biological samples of the pregnant group cows and the measurement values ​​in the biological samples of the non-pregnant group cows;

[0041] The measurement value of D-lactic acid in the biological samples of the pregnant group of dairy cows was significantly higher than the measurement value in the biological samples of the returning group of dairy cows and the measurement value in the biological samples of the non-pregnant group of dairy cows;

[0042] The measurement values ​​of trans-aconitic acid, desipramine and taurodeoxycholic acid in the biological samples of the non-pregnant group of dairy cows were significantly higher than those in the biological samples of the estrus group of dairy cows and the biological samples of the pregnant group of dairy cows;

[0043] The measured values ​​of phosphatidylcholine, maleic acid, methylfumaric acid, succinic semialdehyde and β-octylglucoside in biological samples of pregnant cows and those in biological samples of non-pregnant cows were significantly higher than those in biological samples of returning cows.

[0044] The measurement values ​​of 5-aminovaleric acid and triethanolamine in the biological samples of the returning estrus group and the non-pregnant group were significantly higher than those in the biological samples of the pregnant group.

[0045] The measurement values ​​of valine-methionine in biological samples of cows in the estrus group and in biological samples of cows in the pregnancy group were significantly higher than those in biological samples of cows in the non-pregnancy group.

[0046] This indicates whether the tested dairy cow is in estrus, early pregnancy or non-pregnant state.

[0047] In a fourth aspect, the present invention provides a device for detecting early pregnancy-related states in dairy cows, the device comprising:

[0048] 1) A data acquisition device for acquiring information of biological samples containing biomarkers obtained from the tested dairy cows and acquiring measurement values ​​of the biomarkers in the detected biological samples;

[0049] 2) a data processing device for processing the measured value of the biomarker in the biological sample detected, wherein the biomarker comprises at least one of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine;

[0050] There are statistically significant differences in the measured values ​​of the biomarkers in the biological samples of the returning estrus group cows, the measured values ​​in the biological samples of the pregnant group cows, and the measured values ​​in the biological samples of the non-pregnant group cows;

[0051] Among them, the measurement values ​​of alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in the biological samples of the returning estrus group cows were significantly higher than the measurement values ​​in the biological samples of the pregnant group cows and the measurement values ​​in the biological samples of the non-pregnant group cows;

[0052] The measurement value of D-lactic acid in the biological samples of the pregnant group of dairy cows was significantly higher than the measurement value in the biological samples of the returning group of dairy cows and the measurement value in the biological samples of the non-pregnant group of dairy cows;

[0053] The measurement values ​​of trans-aconitic acid, desipramine and taurodeoxycholic acid in the biological samples of the non-pregnant group of dairy cows were significantly higher than those in the biological samples of the estrus group of dairy cows and the biological samples of the pregnant group of dairy cows;

[0054] The measured values ​​of phosphatidylcholine, maleic acid, methylfumaric acid, succinic semialdehyde and β-octylglucoside in biological samples of pregnant cows and those in biological samples of non-pregnant cows were significantly higher than those in biological samples of returning cows.

[0055] The measurement values ​​of 5-aminovaleric acid and triethanolamine in the biological samples of the returning estrus group and the non-pregnant group were significantly higher than those in the biological samples of the pregnant group.

[0056] The measurement values ​​of valine-methionine in the biological samples of the returning estrus group cows and the measurement values ​​in the biological samples of the pregnant group cows are significantly higher than the measurement values ​​in the biological samples of the non-pregnant group cows; and

[0057] 3) An output device for outputting the result of computer processing of the measurement value of the biomarker, thereby indicating whether the tested dairy cow is in a state of returning to estrus, an early pregnancy state, or a non-pregnant state.

[0058] In a fifth aspect, the present invention provides the above-mentioned biomarkers of early pregnancy-related states in dairy cows, the above-mentioned test paper, reagent or kit for detecting the above-mentioned biomarkers of early pregnancy-related states in dairy cows, the above-mentioned method for detecting early pregnancy-related states in dairy cows, and the use of the above-mentioned device for detecting early pregnancy-related states in dairy cows in detecting and / or evaluating and / or identifying early pregnancy-related states in dairy cows.

[0059] The beneficial effects of the present invention include at least the following:

[0060] The present invention aims to develop biomarkers that can be used to detect and / or evaluate and / or identify early pregnancy-related states in dairy cows by analyzing the changing patterns of metabolites in biological samples about 4.5-5.5 days after insemination of dairy cows. Based on UHPLC-QTOF-MS non-targeted metabolomics technology, the plasma metabolic profiles of estrus, pregnant and non-pregnant cows are analyzed in combination with principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). The present invention provides a combination of biomarkers related to the early pregnancy state of dairy cows and applications thereof. The present invention analyzes biological samples of a pregnant group, a non-pregnant group and an estrus group of dairy cows, and uses metabolomics technology to determine significantly different biomarkers related to the early pregnancy state, so as to effectively and accurately detect and / or evaluate and / or identify the early pregnancy state related to dairy cows. The present invention can quickly, simply, economically, effectively and accurately detect and / or evaluate and / or identify the early pregnancy state related to dairy cows, and provides a new means for quickly discovering non-pregnant dairy cows (including estrus dairy cows and non-pregnant dairy cows), shortening the interval between re-breeding, optimizing the feeding and management of pregnant dairy cows, and improving the reproductive efficiency of dairy cows, etc., and has important economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The total ion current diagram of the QC sample detected by UHPLC-QTOF-MS in positive ion mode according to an embodiment of the present invention is shown.

[0062] Figure 2 The positive ion TIC graph of the QC sample of the embodiment of the present invention is shown.

[0063] Figure 3 The PCA-X one-dimensional distribution diagram of the QC sample in the negative ion mode of an embodiment of the present invention is shown.

[0064] Figure 4 A correlation analysis diagram of positive ion QC samples according to an embodiment of the present invention is shown.

[0065] Figure 5 The PCA score graphs of positive and negative ion modes of the dairy cows in Group A, Group B and Group C 5 days after artificial insemination in the embodiment of the present invention are shown.

[0066] Figure 6 The OPLS-DA score graphs of positive and negative ion modes of the dairy cows in Group A, Group B and Group C 5 days after artificial insemination in the embodiment of the present invention are shown.

[0067] Figure 7 The OPLS-DA permutation test graphs of positive and negative ion modes of group A, group B and group C of dairy cows in the embodiment of the present invention are shown 5 days after artificial insemination.

[0068] Figure 8The Venn distribution diagram of differential metabolites among group A, group B and group C of dairy cows in the embodiment of the present invention on the 5th day after artificial insemination is shown.

[0069] Fig. 9 The heat map of the clustering of significantly different metabolites of dairy cows on the 5th day after artificial insemination in the embodiment of the present invention is shown, wherein red indicates up-regulation and blue indicates down-regulation.

[0070] Figure 10-1 The ROC curves showing the significantly different metabolites between the estrus group and the pregnancy group of dairy cows on the 5th day after artificial insemination in the embodiment of the present invention.

[0071] Figure 10-2 The ROC curve of the metabolites with significant differences between the pregnant group and the non-pregnant group of dairy cows on the 5th day after artificial insemination in the embodiment of the present invention is shown.

[0072] Figure 10-3 The ROC curve of the metabolites with significant differences between the estrus group and the non-pregnant group of dairy cows on the 5th day after artificial insemination in the embodiment of the present invention is shown.

[0073] Fig.11 Metabolic pathway analysis showing differential metabolites on the 5th day after artificial insemination, where the horizontal axis is the pathway influence, the vertical axis is -log (P value), the darker the circle, the more significant the metabolite change in the metabolite pathway, and the larger the circle, the greater the pathway impact; where a represents butyrate metabolism, b represents pyruvate metabolism, and c represents alanine, aspartate, and glutamate metabolism. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0075] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values ​​(such as ± 5%). For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. In the description of this application, unless otherwise specified, the meaning of "multiple / multiple" and similar words is two / kinds or more than two / kinds.

[0076] The present invention is further described in detail below in conjunction with the examples. It should be understood that if the experimental methods in the following examples are not specified with specific conditions, they are usually implemented according to conventional conditions or according to the conditions adopted by the partners.

[0077] Example 1

[0078] 1 Materials and methods

[0079] 1.1 Materials

[0080] 1.1.1 Test instruments and equipment

[0081] Table 1 Test instruments

[0082] instrument Manufacturer 1290UHPLC Ultra High Performance Liquid Chromatography Agilent Technologies QTOF6550 High Resolution Mass Spectrometer Agilent Technologies TripleTOF6600 High Resolution Mass Spectrometer ABSciex BSA124S-CW Balance Sartorius JXFSTPRP-24 grinding machine Shanghai Jingxin Technology Co., Ltd. Heraeus Fresco 17 centrifuge ThermoFisherScientific PS-60AL Ultrasonic Instrument Shenzhen Redbond Electronics Co., Ltd. Mingche D24UV water purifier MerckMillipore ACQUITY UPLC BEHAmide 1.7μm 2.1*100mm column Waters

[0083] 1.1.2 Experimental materials and reagents

[0084] Table 2 Test reagents

[0085]

[0086] 1.2 Sampling method

[0087] 1.2.1 Experimental animal grouping and blood collection and storage

[0088] 63 healthy Holstein cows with 2-3 parities and similar age and body condition (body condition score BCS around 2.75) were selected. Blood was collected from the tail vein of the cows at the end of estrus before morning feeding on the 5th day after artificial insemination (recorded as 5d), and the blood was collected with sodium heparin anticoagulant tubes and centrifuged to separate the plasma and stored at -80℃. The cows were divided into the estrus return group (Group A, n=20) according to the return of estrus 18-24 days after mating, and the results of pregnancy detection by B-ultrasound at 32±3 days, and the cows that were pregnant during pregnancy detection were divided into the pregnancy group (Group B, n=24), and the cows that were not pregnant during pregnancy detection were divided into the non-pregnancy group (Group C, n=19).

[0089] 1.2.2 Sample collection and processing

[0090] Twelve samples were randomly selected from each group on the fifth day after artificial insemination. UHPLC-Q / TOF-MS non-targeted metabolomics technology was used to analyze the plasma of dairy cows in groups A, B, and C.

[0091] 1.3 Sample testing method

[0092] 1.3.1 Metabolite extraction

[0093] Take 100 μL of sample, add 400 μL of extract containing internal standard (methanol and acetonitrile volume ratio = 1:1, internal standard concentration 5 μg / mL), vortex mix for 30 seconds; ultrasonicate for 5 minutes (ice water bath); let stand at -20℃ for 1 hour; centrifuge the sample at 4℃, 12000r / min for 15 minutes; carefully take out 425 μL of supernatant and put it in EP tube; dry the extract in a vacuum concentrator; add 100 μL of extract (acetonitrile and water volume ratio: 1:1) to the dried metabolite for re-dissolution; vortex for 30 seconds, ultrasonicate for 10 minutes in ice water bath; centrifuge the sample at 4℃, 12000r / min for 15 minutes; carefully take out 60 μL of supernatant and put it in 2mL injection bottle, take 10 μL of each sample to mix into QC sample, and then take 60 μL for machine detection.

[0094] 1.3.2 On-machine testing

[0095] The target compounds were separated by chromatography on an Agilent 1290 (Agilent Technologies) ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEH Amide (2.1*100mm, 1.7μm) liquid chromatography column. Phase A of the liquid chromatography was an aqueous phase containing 25mmol / L ammonium acetate and 25mmol / L ammonia water, and phase B was acetonitrile. Gradient elution was used: 0-0.5min, 95% B; 0.5-7min, 95%-65% B; 7-8min, 65%-40% B; 8-9min, 40% B; 9-9.1min, 40%-95% B; 9.1-12min, 95% B. Mobile phase flow rate: 0.5mL / min, column temperature: 25℃, sample tray temperature: 4℃, injection volume: 2μL for positive ions; 2μL for negative ions.

[0096] 1.4 Data Processing

[0097] The obtained raw mass spectrometry data were processed by ProteoWizard software and converted into mzXML format. The converted data were processed by XCMS program according to peak alignment, peak area extraction and peak integration. Data integrity and missing values ​​were checked, and data within the group with extreme values ​​or missing values ​​greater than 50% were deleted. After standardization and normalization of the data, parallel comparisons between metabolites and samples were performed.

[0098] 1.5 Statistical analysis

[0099] After the data were normalized to the total peak area, the data were processed by pareto-scaling using SIMCA-P 14.1 software, and then multivariate statistical analysis was performed: including principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA); at the same time, the model data were permutation tested (Permutation Test) and the variable projection importance (VIP) was calculated during the modeling process. On this basis, the data were subjected to univariate statistical analysis including Student's t-test and fold change (FC) analysis, and differential metabolites were screened according to the P value and FC value of each metabolite.

[0100] 1.6 Identification of differential metabolites and bioinformatics analysis

[0101] The significantly different metabolites were screened based on the criteria of multidimensional statistical analysis VIP>1, univariate statistical analysis P<0.05, and FC>1.3 or FC<0.77. After screening, cluster analysis and metabolic pathway analysis were performed using the MetaboAnalys online analysis platform (https: / / www.metaboanalyst.ca / ), and ROC curve analysis of the differential metabolites was further performed using Origin 8.0 software.

[0102] 2 Results

[0103] 2.1 Experimental stability evaluation

[0104] The total ion currents of the QC samples in UHPLC-QTOF-MS positive and negative ion modes were compared to see the spectral overlap effect. Figure 1 As shown, the chromatographic peak, signal intensity and retention time of the QC sample BPC overlap well in the spectrum, indicating that the variation caused by instrument error in the entire experimental process is small and the stability of the instrument used in this experiment is good.

[0105] To test and verify the stability of the instrument, see Figure 2 It can be seen that the TIC retention time and peak area of ​​the QC samples overlap well, indicating that the instrument stability is very good.

[0106] Depend on Figure 3 From the PCA-X one-dimensional distribution diagram of QC samples in positive and negative ion modes, it can be seen that all QC samples are distributed within 3STD, indicating that the data quality is high.

[0107] The closer the QC value is to 1, the higher the correlation of the sample (at least ≥ 0.7), which means the higher the stability during the test and the smaller the correlation error. Figure 4 It can be seen that the QC value is close to 1, indicating that the QC samples are highly correlated, indicating that the data quality of this experiment is very high.

[0108] 2.2 Multivariate statistical analysis

[0109] 2.2.1 Principal Component Analysis (PCA) Analysis

[0110] The principal component analysis of metabolites was performed using the PCA method to observe the overall distribution trend among all samples and find possible discrete points. The PCA model parameter R 2 X represents the model's explanation rate for the X variable, and the PCA model parameter Q 2 It indicates the model's predictive ability for the X variable. 2 X≥0.5 indicates that the model is stable and reliable. The PCA model spectrum obtained after cyclic interaction verification is as follows Figure 5 The results show that the three groups cannot be well distinguished in 5 days, but there is still a certain separation trend.

[0111] 2.2.2 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) Analysis

[0112] OPLS-DA is a supervised discriminant analysis statistical method. At the same time, VIP is calculated to measure the influence and explanatory power of the expression pattern of each metabolite on the classification and discrimination of each group of samples, thereby assisting the screening of marker metabolites (usually VIP score>1.0 is used as the screening criterion). OPLS-DA model is established, such as Figure 6 As shown. The model evaluation parameter R obtained after cyclic interactive verification 2 Y and Q 2 , R 2 Y≥0.5 and Q 2 ≥0.5 The model is stable and reliable. 2 The closer it is to 1, the more stable and reliable the model is, and the higher its predictive ability is. Figure 7 As shown in Table 3, according to the permutation test diagram of the OPLS-DA model for positive and negative ion mode data, Q 2 The intercepts are all less than 0 (Q 2 The intercept <0) indicates that the OPLS-DA model established for the positive and negative ion mode data is not overfitted.

[0113] Table 3 Evaluation parameters of OPLS-DA model and permutation test

[0114]

[0115] 2.3 Screening of differential metabolites

[0116] The VIP values ​​were calculated according to the multivariate statistical OPLS-DA model, and differential metabolites were screened. In this experiment, a total of 16 significant metabolites were screened in group A (return of estrus group), group B (pregnancy group) and group C (non-pregnancy group) in positive and negative ion modes on the 5th day after artificial insemination. Among them, alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in group A were significantly higher than those in group B and group C; D-lactic acid in group B was significantly higher than that in group A and group C; trans-aconitic acid, desipramine and taurodeoxycholic acid in group C were significantly higher than those in group A and group B; phosphatidylcholine, maleic acid, methylfumaric acid, succinic semialdehyde and β-octylglucoside in groups B and C were significantly higher than those in group A; 5-aminovaleric acid and triethanolamine in groups A and C were significantly higher than those in group B; valine-methionine in groups A and B was significantly higher than that in group C. Differential metabolites identified in positive and negative ion modes refer to Figure 8 and Table 4.

[0117] Table 4 Differential metabolites identified in positive and negative ion modes in groups A, B and C on day 5 after artificial insemination

[0118]

[0119]

[0120]

[0121] 2.4 Cluster analysis of differential metabolites

[0122] MetaboAnalyst analysis platform was used to perform cluster analysis on the significantly different metabolites. Fig. 9 As shown, it was found that the samples in the same group could be clustered together well, indicating that the metabolites in the same group had similar expression patterns, indicating that the screened significant difference metabolites were reasonable and accurate.

[0123] 2.5 ROC curve analysis of differential metabolites

[0124] The ROC curve was used to further screen the significantly different metabolites to analyze the classification and recognition ability of these significantly different metabolites. When the area under the ROC curve (AUC) is close to 0.5-1, it indicates that the recognition ability is good. The closer the AUC value is to 1, the better the recognition effect. Ten metabolites were screened out in the return group A and the pregnancy group B, namely phosphatidylcholine, succinic semialdehyde, methyl fumaric acid, β-octylglucoside, D-lactic acid, maleic acid, heptadecanoic acid, alanine-leucine, deoxycholic acid and γ-L-glutamyl-L-phenylalanine (such as Figure 10-1). Seven metabolites were screened out in the pregnant group B and the non-pregnant group C, namely taurodeoxycholic acid, triethanolamine, desipramine, trans-aconitic acid, 5-aminovaleric acid, D-lactic acid and valine-methionine (such as Figure 10-2 ). Thirteen metabolites were screened out in the estrus group A and the non-pregnant group C, namely taurodeoxycholic acid, desipramine, trans-aconitic acid, phosphatidylcholine, succinic semialdehyde, methyl fumaric acid, β-octylglucoside, maleic acid, valine-methionine, alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine (e.g. Figure 10-3 ).

[0125] 2.6 Metabolic pathway analysis

[0126] The MetaboAnalyst analysis platform was used to perform KEGG metabolic pathway analysis on the significantly different metabolites. The main metabolic pathways were screened based on an influence greater than 0.2 (Pathway impact>0.2). Fig.11 As shown, 3 metabolic pathways were screened out from the 16 significantly differential metabolites screened on the 5th day after artificial insemination: butyrate metabolism (a); pyruvate metabolism (b); alanine, aspartate and glutamate metabolism (c).

[0127] 3 Results Analysis

[0128] The embodiments of the present invention reveal the changing patterns of plasma metabolic characteristics of dairy cows under three pregnancy states by differential analysis of plasma metabolites of pregnant, non-pregnant and re-estrus groups of dairy cows on the 5th day after artificial insemination. The inventors of the present invention found that the differential metabolites are mainly concentrated in butyrate metabolism, pyruvate metabolism, and alanine, aspartate and glutamate metabolic pathways. The present invention screened out 16 biomarkers that can be used as potential biomarkers for detecting, evaluating or identifying early pregnancy dairy cows. The above findings provide an important basis for the accurate detection of early pregnancy status of dairy cows after mating, specifically including:

[0129] Compared with the pregnant and non-pregnant groups, the plasma levels of phosphatidylcholine and succinic semialdehyde in the returning estrus group were downregulated, and the plasma lactate concentration in the pregnant group was significantly higher than that in the non-pregnant and returning estrus groups, which has the potential to be used as a biomarker for identifying returning estrus cows on the 5th day after artificial insemination.

[0130] The present invention found that the phosphatidylcholine content in the plasma of the returning estrus group of dairy cows decreased significantly, which may be related to the reduction of lipid transport capacity, thus affecting the success rate of pregnancy.

[0131] The metabolites of dairy cows undergo significant changes in the early stages of the estrus cycle, and these metabolites play a key role in regulating the development of the embryo before implantation. The present invention found that the level of succinic semialdehyde in the plasma of pregnant and non-pregnant dairy cows was significantly higher than that of the estrus group, and was involved in butyrate metabolism and alanine, aspartate and glutamate metabolic pathways. The increase in SSA levels may provide sufficient energy for embryonic development by promoting the increase in succinate content, thereby supporting pregnancy.

[0132] The placenta requires a lot of energy to support its rapid growth, maturation and material transport process, among which fatty acids are used as the main energy substrate. The results of the present invention show that the lactic acid content in the plasma of the pregnant group of dairy cows on the 5th day after artificial insemination is significantly higher than that of the non-pregnant group and the estrus group. This phenomenon may be related to the role of lactic acid in promoting embryonic development, thereby helping to improve the pregnancy success rate of dairy cows.

[0133] 4 Conclusion

[0134] The present invention develops biomarkers that can be used to detect and / or evaluate and / or identify early pregnancy-related states in dairy cows by analyzing the change patterns of metabolites in biological samples about 4.5-5.5 days after insemination of dairy cows. Based on UHPLC-QTOF-MS non-targeted metabolomics technology, combined with principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), the plasma metabolic profiles of estrus, pregnant and non-pregnant dairy cows were analyzed. The results showed that compared with the pregnant and non-pregnant groups of dairy cows, phosphatidylcholine and succinic semialdehyde in the plasma of the estrus group of dairy cows were both down-regulated, and they have the potential to be used as biomarkers for detecting, evaluating or identifying estrus dairy cows on the 5th day after artificial insemination of dairy cows, and these metabolites are mainly involved in butyrate metabolism and alanine, aspartate and glutamate metabolic pathways. The lactate concentration in the plasma of the pregnant group of dairy cows was significantly higher than that of the non-pregnant group and the estrus group, and it is expected to be used as a biomarker for identifying pregnant dairy cows on the 5th day after artificial insemination of dairy cows, and its metabolic process involves pyruvate metabolism. The results of the present invention provide an important reference for early pregnancy detection after breeding of dairy cows.

[0135] The present invention provides a combination of biomarkers related to the early pregnancy state of dairy cows and applications thereof. The present invention analyzes biological samples of a pregnancy group, a non-pregnancy group and a return estrus group of dairy cows, and uses metabolomics technology to determine significantly different biomarkers related to the early pregnancy state, so as to effectively and accurately detect and / or evaluate and / or identify the early pregnancy state related to dairy cows. The present invention can quickly, simply, economically, effectively and accurately detect and / or evaluate and / or identify the early pregnancy state related to dairy cows, and provides a new means for quickly discovering non-pregnant dairy cows (including return estrus dairy cows and non-pregnant dairy cows), shortening the interval between re-breeding, optimizing the feeding and management of pregnant dairy cows, and improving the reproductive efficiency of dairy cows, and has important economic value.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A biomarker for early pregnancy-related status in dairy cows, characterized in that: The biomarker comprises at least one of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine; There are statistically significant differences in the measured values ​​of the biomarkers in the biological samples of the returning estrus group cows, the measured values ​​in the biological samples of the pregnant group cows, and the measured values ​​in the biological samples of the non-pregnant group cows; Among them, the measurement values ​​of alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in the biological samples of the returning estrus group cows were significantly higher than the measurement values ​​in the biological samples of the pregnant group cows and the measurement values ​​in the biological samples of the non-pregnant group cows; The measurement value of D-lactic acid in the biological samples of the pregnant group of dairy cows was significantly higher than the measurement value in the biological samples of the returning group of dairy cows and the measurement value in the biological samples of the non-pregnant group of dairy cows; The measurement values ​​of trans-aconitic acid, desipramine and taurodeoxycholic acid in the biological samples of the non-pregnant group of dairy cows were significantly higher than those in the biological samples of the estrus group of dairy cows and the biological samples of the pregnant group of dairy cows; The measured values ​​of phosphatidylcholine, maleic acid, methylfumaric acid, succinic semialdehyde and β-octylglucoside in biological samples of pregnant cows and those in biological samples of non-pregnant cows were significantly higher than those in biological samples of returning cows. The measurement values ​​of 5-aminovaleric acid and triethanolamine in the biological samples of the returning estrus group and the non-pregnant group were significantly higher than those in the biological samples of the pregnant group. The measurement values ​​of valine-methionine in biological samples of cows in the estrus group and in biological samples of cows in the pregnancy group were significantly higher than those in biological samples of cows in the non-pregnancy group.

2. The biomarker for early pregnancy-related status in dairy cows according to claim 1, characterized in that: The biomarkers include at least two of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine.

3. The biomarker for early pregnancy-related status in dairy cows according to claim 1, characterized in that: The biological sample includes cow plasma.

4. The biomarker for early pregnancy-related status in dairy cows according to claim 1, characterized in that: The time of the early pregnancy-related state of the dairy cow is 4.5-5.5 days after insemination.

5. The biomarker for early pregnancy-related status in dairy cows according to claim 1, characterized in that: The biomarkers of the returning estrus group cows and the pregnant group cows are selected from at least one of phosphatidylcholine, succinic semialdehyde, methyl fumaric acid, β-octylglucoside, D-lactic acid, maleic acid, heptadecanoic acid, alanine-leucine, deoxycholic acid and γ-L-glutamyl-L-phenylalanine; and / or The biomarkers of the pregnant cows and the non-pregnant cows are selected from at least one of taurodeoxycholic acid, triethanolamine, desipramine, trans-aconitic acid, 5-aminovaleric acid, D-lactic acid and valine-methionine; and / or The biomarkers of the cows in the estrus group and the cows in the non-pregnant group are selected from at least one of taurodeoxycholic acid, desipramine, trans-aconitic acid, phosphatidylcholine, succinic semialdehyde, methyl fumaric acid, β-octylglucoside, maleic acid, valine-methionine, alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine.

6. The biomarker for early pregnancy-related status in dairy cows according to claim 1, characterized in that: The biomarkers of the returning estrus group cows and the pregnant group cows are selected from at least one of β-octyl glucoside, D-lactic acid and heptadecanoic acid; and / or The biomarkers of the pregnant cows and the non-pregnant cows are selected from at least one of triethanolamine, 5-aminovaleric acid and valine-methionine; and / or The biomarkers of the returning estrus group cows and the non-pregnant group cows are selected from at least one of methyl fumaric acid, maleic acid and heptadecanoic acid.

7. A test paper, reagent or kit for detecting a biomarker of an early pregnancy-related state in a dairy cow according to any one of claims 1 to 6.

8. A method for detecting early pregnancy-related conditions in dairy cows, characterized in that: The method comprises: A) obtaining a biological sample containing biomarkers from a cow to be tested; B) detecting a measurement value of a biomarker in the biological sample, wherein the biomarker comprises at least one of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine; and C) computer processing of the measurement values ​​of the biomarkers, wherein the measurement values ​​of the biomarkers in biological samples of the estrus group of dairy cows, the measurement values ​​in biological samples of the pregnancy group of dairy cows, and the measurement values ​​in biological samples of the non-pregnancy group of dairy cows are statistically significantly different; Among them, the measurement values ​​of alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in the biological samples of the returning estrus group cows were significantly higher than the measurement values ​​in the biological samples of the pregnant group cows and the measurement values ​​in the biological samples of the non-pregnant group cows; The measurement value of D-lactic acid in the biological samples of the pregnant group of dairy cows was significantly higher than the measurement value in the biological samples of the returning group of dairy cows and the measurement value in the biological samples of the non-pregnant group of dairy cows; The measurement values ​​of trans-aconitic acid, desipramine and taurodeoxycholic acid in the biological samples of the non-pregnant group of dairy cows were significantly higher than those in the biological samples of the estrus group of dairy cows and the biological samples of the pregnant group of dairy cows; The measured values ​​of phosphatidylcholine, maleic acid, methylfumaric acid, succinic semialdehyde and β-octylglucoside in biological samples of pregnant cows and those in biological samples of non-pregnant cows were significantly higher than those in biological samples of returning cows. The measurement values ​​of 5-aminovaleric acid and triethanolamine in the biological samples of the returning estrus group and the non-pregnant group were significantly higher than those in the biological samples of the pregnant group. The measurement values ​​of valine-methionine in biological samples of cows in the estrus group and in biological samples of cows in the pregnancy group were significantly higher than those in biological samples of cows in the non-pregnancy group. This indicates whether the tested dairy cow is in estrus, early pregnancy or non-pregnant state.

9. A device for detecting early pregnancy-related conditions in dairy cows, characterized in that: The device comprises: 1) A data acquisition device for acquiring information of biological samples containing biomarkers obtained from the tested dairy cows and acquiring measurement values ​​of the biomarkers in the detected biological samples; 2) a data processing device for processing the measured value of the biomarker in the biological sample detected, wherein the biomarker comprises at least one of taurodeoxycholic acid, triethanolamine, heptadecanoic acid, γ-L-glutamyl-L-phenylalanine, desipramine, trans-aconitic acid, 5-aminovaleric acid, alanine-leucine, deoxycholic acid, phosphatidylcholine, succinic semialdehyde, methylfumaric acid, β-octylglucoside, D-lactic acid, maleic acid, and valine-methionine; There are statistically significant differences in the measured values ​​of the biomarkers in the biological samples of the returning estrus group cows, the measured values ​​in the biological samples of the pregnant group cows, and the measured values ​​in the biological samples of the non-pregnant group cows; Among them, the measurement values ​​of alanine-leucine, heptadecanoic acid, deoxycholic acid and γ-L-glutamyl-L-phenylalanine in the biological samples of the returning estrus group cows were significantly higher than the measurement values ​​in the biological samples of the pregnant group cows and the measurement values ​​in the biological samples of the non-pregnant group cows; The measurement value of D-lactic acid in the biological samples of the pregnant group of dairy cows was significantly higher than the measurement value in the biological samples of the returning group of dairy cows and the measurement value in the biological samples of the non-pregnant group of dairy cows; The measurement values ​​of trans-aconitic acid, desipramine and taurodeoxycholic acid in the biological samples of the non-pregnant group of dairy cows were significantly higher than those in the biological samples of the estrus group of dairy cows and the biological samples of the pregnant group of dairy cows; The measured values ​​of phosphatidylcholine, maleic acid, methylfumaric acid, succinic semialdehyde and β-octylglucoside in biological samples of pregnant cows and those in biological samples of non-pregnant cows were significantly higher than those in biological samples of returning cows. The measurement values ​​of 5-aminovaleric acid and triethanolamine in the biological samples of the returning estrus group and the non-pregnant group were significantly higher than those in the biological samples of the pregnant group. The measurement values ​​of valine-methionine in the biological samples of the returning estrus group cows and the measurement values ​​in the biological samples of the pregnant group cows are significantly higher than the measurement values ​​in the biological samples of the non-pregnant group cows; and 3) An output device for outputting the result of computer processing of the measurement value of the biomarker, thereby indicating whether the tested dairy cow is in a state of returning to estrus, an early pregnancy state, or a non-pregnant state.

10. The biomarker of early pregnancy-related status in dairy cows according to any one of claims 1-6, the test paper, reagent or kit for detecting the biomarker of early pregnancy-related status in dairy cows according to any one of claims 1-6 according to claim 7, the method for detecting early pregnancy-related status in dairy cows according to claim 8, and the use of the device for detecting early pregnancy-related status in dairy cows according to claim 9 in detecting and / or evaluating and / or identifying early pregnancy-related status in dairy cows.

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