Application of a biological metabolism marker in detection of pregnancy of sows

Abstract

The application relates to the fields of biological detection and livestock breeding technology, and particularly relates to application of a biological metabolic marker in sow pregnancy detection. The biological metabolic marker is glutathione, a chemical molecular formula of which is C 10 H 17 N3O6S, wherein the expression level of glutathione in the excrement of a pregnant sow is higher than that in the excrement of a non-pregnant sow. The application provides a new and effective detection method for early sow pregnancy detection, and provides a new direction for product development of early sow pregnancy diagnosis.

Description

Technical Field

[0001] This invention relates to the fields of biological detection and animal husbandry technology, and in particular to the application of a biological metabolic marker in the detection of pregnancy in sows. Background Technology

[0002] In modern intensive pig farming, reproductive efficiency is the core determinant of economic benefits. Early and accurate pregnancy diagnosis is crucial for shortening non-productive days in sows, optimizing herd structure, and increasing annual parity. However, the diagnostic methods currently widely used in the industry have significant limitations and cannot fully meet the industry's demand for "earlier, more accurate, and more convenient" methods.

[0003] Common methods for diagnosing sow pregnancy include: (1) estrus observation method: relies on behavioral observation 18-24 days after mating, is highly subjective, has a high rate of missed detection, and the timing of diagnosis is significantly delayed; (2) ultrasound diagnosis method (B-scan): usually performed 21-28 days after mating, the accuracy is limited by the operator's experience, and the equipment is expensive, restraining animals causes stress, making it difficult to achieve large-scale screening; (3) blood hormone detection method: judges by measuring hormone levels such as progesterone and estrone, which has high accuracy, but blood collection itself is an invasive operation, which can easily cause animal stress, and the process is complicated and costly, making it unsuitable for daily rapid screening in farms.

[0004] Given the limitations of the aforementioned methods, the industry has begun actively seeking non-invasive diagnostic solutions based on bodily fluids (such as saliva and urine). Several potential early pregnancy-related metabolic biomarkers have been identified in sow saliva, bovine serum, and urine, demonstrating the feasibility of using metabolomics to identify pregnancy-specific biomarkers. However, saliva sample collection carries the risk of contamination, and urine collection remains inconvenient in large-scale pig farms. In contrast, fecal samples offer significant advantages, including extremely simple collection, complete non-invasiveness, and zero stress, making them an ideal source of samples for large-scale screening. However, currently, no early pregnancy diagnostic method based on sow fecal-specific metabolic biomarkers has been established. Summary of the Invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an application of biological metabolic markers in the detection of pregnancy in sows.

[0006] Another object of the present invention is to provide the application of a reagent for detecting the above-mentioned biochemical markers in the preparation of sow pregnancy detection products.

[0007] Another object of the present invention is to provide a method for detecting early pregnancy in sows.

[0008] The objective of this invention is achieved through the following technical solution: The application of a biomarker in sow pregnancy detection, wherein the biomarker is glutathione (GSH), whose chemical formula is C0. 10 H 17 N3O6S, whose structural formula is shown in Formula I, shows that the expression level of glutathione in the feces of pregnant sows is higher than that in the feces of non-pregnant sows.

[0009]

[0010] Formula I; The application of a reagent for detecting a biochemical metabolic marker in the preparation of a sow pregnancy detection product, wherein the biochemical metabolic marker is glutathione (GSH), and its chemical formula is C0. 10 H 17 N3O6S, among which, the expression level of glutathione in the feces of pregnant sows was higher than that in the feces of non-pregnant sows.

[0011] The reagent is used to detect the content of the biochemical metabolic marker in the sample to be tested.

[0012] The reagents include biomolecules that specifically hybridize with glutathione or other detection reagents that use glutathione as the target analyte.

[0013] Preferably, the biomolecule includes at least one of a probe and an antibody.

[0014] The object of the test was a sample of sow feces.

[0015] The detection methods include, but are not limited to, spectroscopic methods, immunological methods, and precision instrument analysis methods. Spectroscopic methods include spectrophotometry, immunological methods include enzyme-linked immunosorbent assay (ELISA), Western spectroscopy, colloidal gold assay, etc., and precision instrument analysis methods include mass spectrometry (such as high performance liquid chromatography, liquid chromatography-mass spectrometry, etc.), electrochemical methods, etc. Those skilled in the art should understand that other conventional methods known in the art can be used to determine the expression level of the biological metabolite glutathione.

[0016] Preferably, the sow pregnancy detection product can be a chip, reagent, test strip, drug, preparation, reagent kit or high-throughput screening system, etc.

[0017] The test strip can be colloidal gold test strip.

[0018] The kit described can be an enzyme-linked immunosorbent assay (ELISA) kit.

[0019] The sow pregnancy test mentioned refers to the early pregnancy test in pigs.

[0020] Preferably, the pregnancy test is performed on the 15th day after the sow is bred.

[0021] Preferably, the pig is a Large White sow.

[0022] More preferably, the pig is a French Large White sow.

[0023] A method for detecting early pregnancy in sows includes the following steps: The expression level of glutathione, a biomarker of biological metabolism, in sow feces was detected and used to determine the pregnancy status of sows.

[0024] The detection methods include, but are not limited to, spectroscopic methods, immunological methods, and precision instrument analysis methods. Spectroscopic methods include spectrophotometry, immunological methods include enzyme-linked immunosorbent assay (ELISA), Western spectroscopy, colloidal gold assay, etc., and precision instrument analysis methods include mass spectrometry (such as high performance liquid chromatography, liquid chromatography-mass spectrometry, etc.), electrochemical methods, etc. Those skilled in the art should understand that other conventional methods known in the art can be used to determine the expression level of biological metabolites.

[0025] Preferably, the detection is performed on the 15th day after the sow is bred.

[0026] Preferably, the detection method is enzyme-linked immunosorbent assay (ELISA).

[0027] The method for determining pregnancy is as follows: when the glutathione concentration is greater than 32.07 ng / mL, the patient is considered pregnant; when the glutathione concentration is less than 32.07 ng / mL, the patient is considered non-pregnant.

[0028] Preferably, the pig is a Large White sow.

[0029] More preferably, the pig is a French Large White sow.

[0030] In this invention, the applications or methods described do not include the purpose of treating or diagnosing diseases.

[0031] The present invention has the following advantages and effects compared with the prior art: (1) This invention confirms that glutathione can be used as a metabolic marker for early pregnancy detection in sows by non-targeted metabolomics detection and analysis of sow feces. In particular, the level of glutathione metabolite in the feces of sows in early pregnancy is significantly higher than that in non-pregnant sows.

[0032] (2) This invention uses glutathione, a metabolic marker in sow feces, as the target for early pregnancy detection in sows. It has early detection timing, high sensitivity and specificity, simple detection process, low cost, and no damage to sows (completely non-invasive, zero stress), and is suitable for large-scale screening.

[0033] (3) This invention provides new and effective detection targets and detection methods for early pregnancy detection in sows, and also provides a new direction for the development of products for early pregnancy diagnosis in sows. Attached Figure Description

[0034] Figure 1 This is a graph showing the expression levels of glutathione in the feces of pregnant and non-pregnant sows in Example 1 of the present invention, based on non-targeted metabolomics technology. * indicates P < 0.05.

[0035] Figure 2 This is a graph showing the expression level of the metabolite glutathione in the feces of pregnant and non-pregnant sows in Example 1 of the present invention, using enzyme-linked immunosorbent assay (ELISA). *** indicates P < 0.001.

[0036] Figure 3 This is a graph showing the expression level of the metabolite glutathione in the feces of pregnant and non-pregnant sows in Example 2 of the present invention, using enzyme-linked immunosorbent assay (ELISA). *** indicates P < 0.001.

[0037] Figure 4 This is an ROC curve showing the expression of glutathione, a metabolite of Example 3 of the present invention, in the feces of sows on the 15th day after mating. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0040] Example 1: Screening for differential metabolites in feces of pregnant and non-pregnant sows on day 15 post-mating In this embodiment, feces from pregnant and non-pregnant sows were collected on day 15 post-mating. Then, non-targeted metabolomics technology and bioinformatics analysis were used to screen for differentially expressed metabolites in early pregnancy. The specific steps are as follows: 1. Screening of differentially expressed metabolites based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology (1) Collection of fecal samples The experimental samples used in this embodiment were non-pregnant French Large White sows of the same parity from the Wenshi Dongcheng farm, with similar genetic background, growth status, feeding conditions, and parity. The day of artificial insemination was recorded as day 1. The sows were then fed normally until day 15, at which point fecal samples were collected from sows on day 15 of gestation (P15) and day 15 of non-gestation (C15). An ultrasound examination was performed on day 30 of artificial insemination to confirm pregnancy. A total of 7 fecal samples were collected from each of the P15 and C15 days. All collected samples were flash-frozen in liquid nitrogen and then stored at -80°C for later use.

[0041] (2) Pretreatment of fecal samples Take 100 mg of fecal sample ground with liquid nitrogen, place it in an EP tube, then add 500 μL of 80% methanol aqueous solution, vortex, and let stand in an ice bath for 5 min; after centrifugation at 15000×g and 4℃ for 20 min, take a certain amount of supernatant and dilute it with mass spectrometry grade water to a methanol content of 53% (v / v), centrifuge again at 15000×g and 4℃ for 20 min, collect the supernatant for non-targeted metabolomics analysis.

[0042] (3) Preparation of quality control samples An equal volume of sample was taken from each test sample and vortexed to serve as a quality control sample (QC). This QC sample was used to balance the chromatography-mass spectrometry system and determine the instrument's condition, and to evaluate the system's stability throughout the experiment. A 53% (v / v) methanol-water solution was used as a blank sample in place of the test samples, primarily to remove background ions.

[0043] (4) Non-targeted metabolomics detection based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology The test samples prepared in step (2) were analyzed by LC-MS / MS using a Vanquish UHPLC system and a Q Exactive™ HF series mass spectrometer. The specific experimental conditions are as follows: Chromatographic conditions: The sample to be tested was injected into a Hypersil Goldcolumn (C18) column at a flow rate of 0.2 mL / min. The eluent in positive mode was mobile phase A (0.1% formic acid solution) and mobile phase B (methanol). The eluent in negative mode was mobile phase A (5 mM ammonium acetate, pH = 9.0) and mobile phase B (methanol). The chromatographic gradient elution program is shown in Table 1.

[0044] Table 1 Chromatographic gradient elution program Time / min Mobile phase A / % Mobile phase B / % 0 98 2 1.5 98 2 3 15 85 10 0 100 10.1 98 2 11 98 2 12 98 2 Mass spectrometry conditions were as follows: scan range m / z 100-1500; ESI source settings were as follows: spray voltage 3.5 kV, sheath gas flow rate 35 psi, auxiliary gas flow rate 10 L / min, ion transfer tube temperature 320 ℃, ion introduction RF level 60, auxiliary gas heater temperature 350 ℃, polarity positive (positive ion mode) and negative (negative ion mode), and MS / MS secondary scan was a data-dependent scan.

[0045] (5) Metabolite identification The raw data were imported into Compound Discoverer 3.3 (CD3.3) for processing. Simple screening of parameters such as retention time and mass-to-charge ratio was performed on each metabolite. Peak area correction was then performed using QC samples to improve identification accuracy. Subsequently, peak extraction was performed with parameters including a mass deviation of 5 ppm, a signal intensity deviation of 30%, minimum signal intensity, and adduct ions. Simultaneously, peak area quantification was performed. Target ions were then integrated, and molecular formula prediction was performed using molecular ion peaks and fragment ions, compared with the mzCloud, mzVault, and Masslist databases. Background ions were removed using blank samples. The raw quantitative results were standardized using the formula: Raw quantitative value of sample / (Total quantitative values ​​of metabolites in sample / Total quantitative values ​​of metabolites in QC sample) to obtain the relative peak area. Compounds with a relative peak area CV greater than 30% in the QC samples were deleted. Finally, the identification and relative quantification results of the metabolites were obtained.

[0046] (6) Screening of differential metabolites Partial least squares discriminant analysis (PLS-DA) was used to establish relational models for the identified metabolites. The screening of differentially expressed metabolites mainly referenced VIP, FC, and... P The three parameters are: VIP (Variance Value), FC (Fold Value), and t-test (Statistical Significance of Metabolites Between Two Groups). VIP refers to the projected importance of the variable in the first principal component of the PLS-DA model, and the VIP value represents the contribution of the metabolite to the grouping. FC is the fold difference, which is the ratio of the mean of all biological replicate quantitative values ​​of each metabolite in the comparison group. P Value). Select VIP > 1.0, FC > 1.2 or FC < 0.833, and P Metabolites with a value < 0.05 were used as the screening criteria for differential metabolites.

[0047] This embodiment collects fecal samples from pregnant and non-pregnant sows on day 15 post-mating, analyzes differentially expressed metabolites in the fecal samples, and identifies glutathione (GSH) as a differentially expressed metabolite with the chemical formula C0. 10 H17 N3O6S.

[0048] The expression levels of glutathione in the feces of pregnant and non-pregnant sows are shown in the figure. Figure 1 As can be seen, the expression level of glutathione in the pregnant group (P15) was significantly higher than that in the non-pregnant group (C15) (VIP = 1.53, FC = 7.11). P The value was 0.0139 < 0.05, indicating that glutathione is an upregulated metabolite. The expression level of this metabolite will increase significantly after the sow becomes pregnant, suggesting that glutathione can be used as a diagnostic marker for pregnancy.

[0049] 2. ELISA was used to verify the expression levels of the metabolite glutathione in different groups. To further validate the expression of glutathione in feces using non-targeted metabolomics, enzyme-linked immunosorbent assay (ELISA) was used to detect and validate the expression of glutathione in feces from pregnant and non-pregnant sows. The specific steps are as follows: (1) Add fecal samples to PBS buffer at a mass ratio of 1:10, centrifuge at 5000×g for 8 min to remove precipitates and impurities, and take the supernatant; repeat the above steps, then aliquot the supernatant and store it in a -80 ℃ freezer to avoid repeated freeze-thaw cycles.

[0050] (2) The ELISA enzyme-linked immunosorbent assay was performed according to the instructions of the glutathione detection kit (Shanghai Keabob Biotechnology Co., Ltd., catalog number CB10768-Pg). The specific operation procedure is as follows: ① Prepare the working solutions for each component of the reagent kit.

[0051] ② Arrange the standard wells, blank wells, and sample wells as follows: add 50 μL of graded concentration standards to the standard wells, add 50 μL of the sample to be tested to the sample wells, and leave the blank wells empty.

[0052] ③ Except for the blank wells, 100 μL of enzyme-labeled reagent was added to both the standard wells and the sample wells for antigen detection.

[0053] ④ After sealing, incubate at 37 ℃ for 60 min.

[0054] ⑤ Fill with washing solution and let stand for 20 seconds. Repeat 5 times.

[0055] ⑥ Mix colorimetric reagent A and colorimetric reagent B at a volume ratio of 1:1 to obtain a mixed solution. Add 100 μL to each well, seal the plate, and react in the dark at 37 °C for 15 min.

[0056] ⑦ Add 50 μL of stop solution to each well, then run the instrument for detection and analyze the OD value of each well at a wavelength of 450 nm.

[0057] ⑧ Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis. Calculate the corresponding concentration of the sample from the standard curve based on the OD value of the test sample, and then multiply by the dilution factor to calculate the sample concentration.

[0058] Figure 2 The expression levels of the metabolite glutathione in the feces of pregnant and non-pregnant sows, as detected by ELISA, were shown. The results were consistent with those obtained from non-targeted metabolomics analysis, indicating that the expression level of glutathione in the pregnant group (P15) was significantly higher than that in the non-pregnant group (C15). P A value < 0.001 suggests that glutathione has potential use as a diagnostic marker for pregnancy.

[0059] Example 2: Population validation of glutathione expression in feces of different groups of sows To further verify the expression of glutathione, a fecal metabolite identified in Example 1, this example selected a new sow population (validation population) and used ELISA technology to detect and verify the expression of glutathione in the feces of pregnant and non-pregnant sows. Following step 1 of Example 1, fecal samples were collected from 7 sows each from P15 and C15; following step 2 of Example 1, ELISA enzyme-linked immunosorbent assay was performed.

[0060] Figure 3 The expression levels of the metabolite glutathione in the feces of pregnant and non-pregnant sows, as detected by ELISA, were shown. These results were consistent with the non-targeted metabolomics and ELISA results in Example 1, indicating that the expression level of glutathione in the pregnant group (P15) was significantly higher than that in the non-pregnant group (C15). P (value < 0.001).

[0061] Example 3: Analysis of Glutathione ROC Curve and Area Under the Curve (AUC) Data The receiver operating characteristic (ROC) curve is a line graph constructed with 1 - specificity (false positive rate) as the X-axis and sensitivity (true positive rate) as the Y-axis. It is a comprehensive indicator of the continuous variables of response sensitivity and specificity. In this field, the area under the ROC curve represents the prediction accuracy, called the AUC value (Area under curve). The AUC value is between 0 and 1; a larger value indicates higher prediction accuracy, and vice versa. Based on the ELISA experimental results of the experimental population in Example 1 and the validation population in Example 2, ROC curves were constructed using the Wilson / Brown method with 95% confidence intervals for the pregnancy status corresponding to the fecal metabolite glutathione. The AUC area and confidence level were calculated to determine the diagnostic value of glutathione for pregnancy outcomes in sows.

[0062] The results are shown in Table 2 and Figure 4 In the table, the Youden index = sensitivity + specificity - 1, and the optimal cut-off value is the cut-off value when the specificity is 1.000.

[0063] The results showed that on day 15 after mating, the AUC value of glutathione, a metabolite in sow feces, was 1. P A significant value indicates that, on day 15 of pregnancy, detecting the expression level of the fecal metabolite glutathione has very high diagnostic value for pregnancy. Glutathione is a reliable diagnostic biomarker for early pregnancy.

[0064] Table 2 Summary of ROC curve results According to the ROC curve results, the optimal threshold concentration of the metabolite glutathione is 32.07 ng / mL. It is an upregulated metabolite. When the glutathione concentration detected by ELISA is greater than 32.07 ng / mL, it is judged as a pregnancy state, and when it is lower than this concentration, it is judged as a non-pregnant state.

[0065] In summary, the metabolite glutathione in sow feces is closely related to early pregnancy status and can effectively distinguish between pregnant and non-pregnant states. This confirms that the glutathione provided by this invention is suitable as a biomarker for early pregnancy diagnosis in sows, exhibiting excellent sensitivity and specificity.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a biological metabolic marker in the detection of pregnancy in sows, characterized in that... The aforementioned biomarker is glutathione, whose chemical formula is C0. 10 H 17 N3O6S, among which, the expression level of glutathione in the feces of pregnant sows was higher than that in the feces of non-pregnant sows.

2. The application of a reagent for detecting biological metabolic markers in the preparation of sow pregnancy detection products, characterized in that... The aforementioned biomarker is glutathione, whose chemical formula is C0. 10 H 17 N3O6S, among which, the expression level of glutathione in the feces of pregnant sows was higher than that in the feces of non-pregnant sows.

3. The application according to claim 2, characterized in that: The reagent is used to detect the content of the biochemical metabolic marker in the sample to be tested.

4. The application according to claim 3, characterized in that: The reagents include biomolecules that specifically hybridize with glutathione or other detection reagents that use glutathione as the target analyte.

5. The application according to claim 4, characterized in that: The biomolecules include at least one of probes and antibodies.

6. The application according to claim 2, characterized in that: The object of the test was a sample of sow feces.

7. The application according to claim 2, characterized in that: The detection method is a spectroscopic method, an immunological method, or a precision instrument analysis method.

8. The application according to claim 2, characterized in that: The sow pregnancy test mentioned above refers to the early pregnancy test in pigs. The pregnancy test was conducted on the 15th day after the sow was bred.

9. A method for detecting early pregnancy in sows, characterized in that... It includes the following steps: The expression level of glutathione, a biomarker of biological metabolism, in sow feces was detected and used to determine the pregnancy status of sows.

10. The method for detecting early pregnancy in sows according to claim 9, characterized in that: The detection method is enzyme-linked immunosorbent assay (ELISA). The method for determining pregnancy is as follows: when the glutathione concentration is greater than 32.07 ng / mL, the patient is considered pregnant; when the glutathione concentration is less than 32.07 ng / mL, the patient is considered non-pregnant.

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