Protein biomarkers for early pregnancy diagnosis in sows and their applications

Abstract

This invention belongs to the field of biotechnology diagnostics and discloses protein biomarkers for early pregnancy diagnosis in sows and their applications. From the perspective of early pregnancy diagnosis strategies in sows, this invention, through bioinformatics analysis and screening of differentially expressed proteins, identifies porcine complement component 5a receptor 1 (C5aR1), porcine lysosomal acid phosphatase 2 (ACP2), and porcine tetrahexine 7 (TSPAN7) as biomarkers involved in the development and progression of sow pregnancy, which can be used to indicate whether a sow is pregnant. This invention can improve the risk prediction ability of sows after artificial insemination based on known risk factors. In production, it can be used for precise stratification after artificial insemination, improving sow parity and reproductive efficiency, and reducing non-productive days in sows.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology diagnostics, specifically relating to protein biomarkers for early pregnancy diagnosis in sows and their applications. Background Technology

[0002] Parity is a key factor affecting the number of weaned piglets (PSY) produced by a sow per year. However, current pregnancy diagnosis techniques can only be performed 25 days after artificial insemination. For sows that have failed to conceive, timely artificial insemination during the first estrus cycle after artificial insemination is not possible, leading to increased non-productive days, reduced parity, and technical limitations on improving sow reproductive performance. Therefore, improving existing pregnancy diagnosis methods is of great significance to pig farming, and the development of accurate, simple, and rapid early pregnancy diagnosis techniques is urgently needed in sow production.

[0003] Existing methods for diagnosing pregnancy each have their limitations. For example, ultrasound diagnosis is influenced by the user's experience; external characteristic examinations such as abdominal and mammary enlargement lead to diagnostic delays; hormone testing may result in false estrus in a few sows, leading to misidentification; and using boars to identify sows returning to estrus significantly increases the risk of malignant disease transmission. Currently, ultrasound is widely used domestically and internationally for pregnancy diagnosis in sows. However, this technology fails to diagnose pregnancy within the first estrous cycle (21 days) after artificial insemination, which is the most valuable period, thus increasing non-productive days. Small and medium-sized pig farms, lacking equipment and testing technologies such as ultrasound machines, can only determine pregnancy by observing the sow's behavior, resulting in even lower diagnostic accuracy and hindering effective improvement in sow reproductive efficiency. Especially given the current lack of effective control over African swine fever, applying early pregnancy diagnosis technology is a powerful way to reduce costs and increase efficiency.

[0004] Based on this, this application proposes a protein biomarker for early pregnancy diagnosis in sows and its application method. Summary of the Invention

[0005] The technical problem to be solved by this invention is to enable early pregnancy diagnosis and its application in sows by discovering biomarkers on the 15th day after artificial insemination.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical measures:

[0007] The scope of protection of this invention includes:

[0008] The application of a system for detecting the protein levels of C5aR1 (porcine complement component 5a receptor 1), ACP2 (porcine lysosomal acid phosphatase 2), and TSPAN7 (porcine tetrahexameric protein 7) in sows in the preparation of diagnostic products for early pregnancy in sows; the early pregnancy period is 12-25 days after artificial insemination, and the system is a reagent, kit, or instrument.

[0009] The application of a system for detecting the levels of C5aR1, ACP2, and TSPAN7 proteins in sow saliva in the preparation of diagnostic products for early pregnancy in sows; the early pregnancy period is 12-25 days after artificial insemination, and the system is a reagent, kit, or instrument.

[0010] An apparatus for diagnosing early pregnancy in sows, characterized in that: the apparatus includes a data receiving block and a data processing module; the data receiving module is configured to receive the C5aR1, ACP2, and TSPAN7 protein contents in the sow to be diagnosed; the data processing module is used to convert the C5aR1, ACP2, and TSPAN7 protein contents from the receiving module into a determination result for diagnosing early pregnancy in the sow to be tested.

[0011] A computer-readable storage medium for diagnosing early pregnancy in sows, characterized in that: the computer-readable storage medium causes a computer to perform the following steps: detecting the protein content of C5aR1, ACP2 and TSPAN7 in the sow to be tested, and converting the content into a determination result for diagnosing early pregnancy in the sow to be tested.

[0012] In the applications described above, the reagents for detecting protein content include: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, Western blotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin assay for detecting the content of C5aR1, ACP2, and TSPAN7 in sows.

[0013] In the applications described above, the kit mentioned is an ELISA kit.

[0014] In the above applications, when the subject of the test is a crossbred sow, the sow is considered pregnant if the concentration of C5aR1 in the sow's saliva is less than or equal to 676.2 pg / ml, the concentration of ACP2 is greater than or equal to 41 pg / ml, and the concentration of TSPAN7 is greater than or equal to 253.4 pg / ml, and at least two of these conditions are met; otherwise, the sow is considered not pregnant.

[0015] In the above-described applications, preferably, the early pregnancy occurs 12-18 days after artificial insemination.

[0016] The above applications or methods may be for non-disease diagnosis purposes. The above applications or methods may not be for the direct purpose of obtaining disease diagnosis results or health status of living human or animal subjects.

[0017] The above applications or methods may be for non-disease treatment purposes. The above applications or methods may not have the direct purpose of restoring or restoring health or reducing suffering in a living human or animal body.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] This invention, from the perspective of early pregnancy diagnosis strategies for sows, uses bioinformatics analysis and screening of differentially expressed proteins to discover that porcine complement component 5a receptor 1 (C5aR1), porcine lysosomal acid phosphatase 2 (ACP2), and porcine tetrahexine 7 (TSPAN7) can be used as markers involved in the occurrence and development of sow pregnancy to indicate whether a sow is pregnant.

[0020] Furthermore, this invention utilizes ELISA technology to quantify the levels of soluble porcine complement component 5a receptor 1 (C5aR1), porcine lysosomal acid phosphatase 2 (ACP2), and porcine tetrahexine 7 (TSPAN7) in the saliva of sows after artificial insemination. It was found that these soluble levels can serve as predictive indicators for whether sows are pregnant after artificial insemination, and can be used to prepare products that predict or assist in predicting the prognosis of sow pregnancy. By detecting the levels of these soluble porcine complement component 5a receptor 1 (C5aR1), porcine lysosomal acid phosphatase 2 (ACP2), and porcine tetrahexine 7 (TSPAN7) in saliva, the pregnancy status of sows can be predicted. This invention can improve the risk prediction ability of whether sows will become pregnant after artificial insemination based on known risk factors. In production, it can be used to accurately stratify sows after artificial insemination, improve the annual parity and reproductive efficiency of sows, and reduce the number of non-productive days of sows. Attached Figure Description

[0021] Figure 1 Standard curve for protein C5aR1 determination;

[0022] The X-axis represents the concentration of the standard, and the Y-axis represents the absorbance value of the standard.

[0023] Figure 2 Standard curve for ACP2 protein assay;

[0024] The X-axis represents the concentration of the standard, and the Y-axis represents the absorbance value of the standard.

[0025] Figure 3 Standard curve for TSPAN7 protein assay;

[0026] The X-axis represents the concentration of the standard, and the Y-axis represents the absorbance value of the standard.

[0027] Figure 4To construct the expression profiles of C5aR1, ACP2, and TSPAN7 protein biomarkers in early pregnancy in sows.

[0028] Figure 5 ROC curve analysis of population expression of C5aR1 in saliva 15 days after artificial insemination;

[0029] The area under the curve is 0.7771.

[0030] Figure 6 ROC curve analysis of the population expression of ACP2 and TSPAN7 in saliva 15 days after artificial insemination;

[0031] The areas under the curves are 0.8857 and 0.8743.

[0032] Figure 7 ROC curve analysis of the population expression of C5aR1-ACP2, C5aR1-TSPAN7, and ACP2-TSPAN7 in saliva 12 days after artificial insemination in pairs;

[0033] The areas under the curves are 0.8393, 0.8766, and 0.9026.

[0034] Figure 8 ROC curve analysis of the population expression of C5aR1-ACP2, C5aR1-TSPAN7, and ACP2-TSPAN7 in saliva 15 days after artificial insemination in pairs;

[0035] The areas under the curves are 0.94, 0.9143, and 0.9600.

[0036] Figure 9 ROC curve analysis of the population expression of C5aR1-ACP2, C5aR1-TSPAN7, and ACP2-TSPAN7 in saliva 18 days after artificial insemination in pairs;

[0037] The areas under the curves are 0.8866, 0.9664, and 0.8571.

[0038] Figure 10 ROC curve analysis of the population expression of C5aR1-ACP2-TSPAN7 in saliva at 12, 15 and 18 days after artificial insemination;

[0039] The areas under the curves are 0.9242, 0.9886, and 0.9706. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] The preferred embodiments of the present invention are described in detail below.

[0043] In the embodiments of this invention, all pigs used are two-breed sows, and the early pregnancy time is ≤25 days after artificial insemination.

[0044] Example 1: Screening for differentially expressed proteins in the saliva of pregnant and non-pregnant sows on day 15 post-mating using 4D-DIA technology.

[0045] In this embodiment, blood samples were collected from pregnant and non-pregnant sows on day 15 post-mating. Proteins were isolated, and differentially expressed proteins in early pregnancy were analyzed and screened using 4D-DIA technology and bioinformatics methods. The specific steps are as follows:

[0046] 1 test sample

[0047] This experiment selected eight crossbred sows with similar growth age, parity, body condition, and farrowing history. All sows were from a national-level pig production capacity control base in Manas, Xinjiang, and were free of reproductive system diseases, healthy, and without genetic diseases. The eight sows were randomly and equally divided into an experimental group and a control group. Following strict production procedures, including boar induction, estrus detection and artificial insemination were performed on the sows. The experimental group sows received semen via standard procedures, while the control group sows received semen containing inactive sperm (microscopic observation showed complete sperm inactivation).

[0048] 2. Saliva protein extraction

[0049] Remove the frozen sample and freeze-dry it. Add 800 μL of extraction buffer and mix well. Add an equal volume of saturated phenol-Tris-HCl (7.8) solution and mix at 4°C for 30 min, shaking frequently during mixing. Centrifuge at 7100 rpm for 10 min at 4°C and collect the phenol supernatant. Add 5 volumes of pre-cooled 0.1 M ammonium acetate-methanol solution and incubate overnight at -20°C to precipitate. Centrifuge at 12000 rpm for 10 min at 4°C and collect the precipitate. Add 5 volumes of pre-cooled methanol to wash and mix gently. Centrifuge at 12000 rpm for 10 min at 4°C and collect the precipitate, repeating once. Repeat steps 7 and 8 twice with acetone instead of methanol to thoroughly remove methanol. Centrifuge at 12000 rpm for 10 min at 4°C and collect the precipitate. After drying at room temperature (generally about 5 min), dissolve in the sample lysis buffer and dissolve at room temperature for 3-5 min. Centrifuge the solution at 12000 rpm for 10 min at room temperature, collect the supernatant, and centrifuge again to collect the supernatant. The supernatant is the total protein solution of the sample. After protein concentration determination, it is aliquoted and stored at -80℃ for later use.

[0050] 3. Trypsin digestion

[0051] Based on the determined protein concentration, 50 μg of protein was taken from each sample, and the samples from different groups were diluted with lysis buffer to adjust to the same concentration and volume. DTT was added to the protein solution to a final concentration of 5 mM, mixed thoroughly, and incubated at 55°C for 30 min. The solution was then cooled on ice until room temperature was reached. (Note: The solution should not be too cold or too hot to the touch.) An appropriate volume of iodoacetamide was added to a final concentration of 10 mM, mixed thoroughly, and incubated at room temperature in the dark for 15 min. Six volumes of acetone were added to the solution to precipitate the protein, and the solution was incubated at -20°C for at least 4 hours or overnight. The precipitate was collected by centrifugation at 8000 × g for 10 min at 4°C, and the acetone was evaporated for 2-3 min. The precipitate was reconstituted with 100 μL of 50 mM NH4HCO3, and 1 mg / ml Trypsin-TPCK (1 / 50 sample weight) was added. The solution was digested overnight at 37°C. The enzymatic digestion was terminated by adjusting the pH to approximately 3 with phosphate.

[0052] 4-peptide desalting

[0053] The enzymatically hydrolyzed peptides were treated with SOLA. TMSPE 96-well plate desalting. Activation: Activate the column with 200 μL methanol, repeat twice. Equilibration: Equilibrate the column with 200 μL of pure water (containing 0.1% formic acid), repeat twice. Load sample: 50-500 μL of sample, adjust vacuum, maintain a droplet rate of 1 mL / min (approximately 1 drop / s). Repeat loading once. Wash: Wash with 200 μL of pure water (containing 0.1% formic acid), repeat twice. Elute: Elute peptides with 150 μL of 50% acetonitrile-water (containing 0.1% formic acid), repeat twice, for a total of 3 times, to obtain 450 μL of eluent, evaporate to dryness under vacuum.

[0054] 5. Protein LC-MS / MS High-Resolution Mass Spectrometry Detection

[0055] The LC-MS / MS conditions were set as follows: flow rate 300 nL / min, C18 analytical column (25 cm × 75 μm ID, 1.6 μm C18, ionopticks), buffer A was 0.1% FA aqueous solution, and buffer B was 0.1% FA / 80% ACN / 20% water.

[0056] 6 Protein Data Analysis

[0057] Analysis of the raw data from 4-DIA determination was performed using Spectronaut Pulsar 16.2.220903.53000 (Biognosys) software.

[0058] 7 Data Analysis

[0059] The differential protein screening criteria were Foldchange ≥ 1.2 or Foldchange ≤ 1 / 1.2 and p-value < 0.05. FC = 0 and FC = inf both indicate 'presence / absence' differences. Pearson correlation analysis was used for correlation analysis. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0060] 8 Test Results

[0061] In this mass spectrometry analysis, a total of 1904 proteins were identified, of which 1891 were reliable proteins. Based on the screening criteria, 491 differentially expressed proteins were identified, with 172 proteins upregulated and 319 proteins downregulated in the pregnancy group compared to the control group.

[0062] 9 Small Group Validation

[0063] Of the collected samples, samples of culled sows and substandard samples were removed. The remaining sow samples underwent pregnancy checks by ultrasound on days 25 and 35 after AI (autoimmune disease) and were followed up later. All sows gave birth within the normal cycle, including 15 pregnant sows and 6 non-pregnant sows, for a total of 21 sows.

[0064] Based on the results of bioinformatics analysis and the current research progress in screening differentially expressed proteins, nine proteins—MESD, AGAP2, PGRMC1, C5aR1, TINAGL1, PRCP, ACP2, PRSS22, and TSPAN7—were ultimately selected for preliminary validation analysis in 21 sows.

[0065] The results showed that for six potential biomarkers—AGAP2, MESD, PGRMC1, TINAGL1, PRCP, and PRSS22—the p-values ​​were greater than 0.05 on day 15 after artificial insemination in sows, indicating no significant differences, and the AUC values ​​were all less than 0.7. For three potential biomarkers—C5aR1 (porcine complement component 5a receptor 1, A0A4X1W0G9), ACP2 (porcine lysosomal acid phosphatase 2, A0A287A023), and TSPAN7 (porcine tetrahexin 7, A0A4X1VGT2)—the p-values ​​were less than 0.05, indicating significant differences, and the AUC values ​​were 0.8208, 0.8765, and 0.8481, respectively, suggesting further research is needed to determine their feasibility as biomarkers.

[0066] Analysis of the Diagnostic Value of Potential Biomarkers

[0067]

[0068]

[0069] Example 2:

[0070] Pregnancy determination when C5aR1, ACP2, and TSPAN7 proteins are used alone as biomarkers:

[0071] The expression profiles of C5aR1, ACP2, and TSPAN7 in early pregnancy of sows were constructed using ELISA technology. Population analysis was performed on the differential expression of C5aR1, ACP2, and TSPAN7 proteins in saliva from pregnant and non-pregnant sows at 12, 15, and 18 days post-mating. ELISA assays were performed according to the kit instructions (Shanghai Hengyuan Biotechnology Co., Ltd., Porcine C5aR1 ELISA KIT; Porcine ACP2 ELISA KIT; Porcine TSPAN7 ELISA KIT).

[0072] Construction of protein biomarker expression profiles

[0073] Saliva samples were collected from six pregnant sows after artificial insemination on days 0, 10, 12, 15, 18, 20, 25, 30, and 40, and from six non-pregnant sows on days 12, 15, and 18.

[0074] The concentrations of C5aR1, ACP2, and TSPAN7 were determined using ELISA on days 0, 10, 12, 15, 18, 20, 25, 30, and 40 post-gestation in sows, and on days 12, 15, and 18 in non-pregnant sows. Expression profiles were constructed after standardization of the results. Standard curves for the three proteins are shown below. Figure 1-3 As shown.

[0075] The results showed that C5aR1 expression was stable in early pregnancy (P10-P30) (i.e., days 10 to 30), and upregulated in the non-pregnant group on days 12, 15, and 18. ACP2 and TSPAN7 expression was downregulated after day 18 of pregnancy, and upregulated in the non-pregnant group on days 12, 15, and 18. All three proteins showed stable expression during embryo implantation, meeting the requirements for biomarker stability. Figure 4 As shown.

[0076] Analysis of the Diagnostic Effect of Biomarkers in Production

[0077] Of the collected samples, samples from culled sows and those that failed quality control were removed. The remaining sow samples underwent pregnancy checks via ultrasound on days 25 and 35 after artificial insemination, and subsequent follow-up showed that all sows gave birth within their normal gestational periods. The final usable sample size was 158 sows: 46 sows 12 days after artificial insemination (38 pregnant, 8 non-pregnant), 64 sows 15 days after artificial insemination (55 pregnant, 9 non-pregnant), and 48 sows 18 days after artificial insemination (41 pregnant, 7 non-pregnant).

[0078] Saliva sample processing: Centrifuge the sample for approximately 20 minutes (2000-3000 rpm) after collection. Carefully collect the supernatant. If precipitation forms during storage, centrifuge again.

[0079] The ROC curve results showed that the diagnostic AUCs for C5aR1, ACP2, and TSPAN7 at 15 days post-insemination were 0.7771 (specificity: 0.46, sensitivity: 1). Figure 5 The specificity values ​​were 0.8857 (specificity: 0.72, sensitivity: 1) and 0.8743 (specificity: 0.6, sensitivity: 0.8), with ACP2 showing the best diagnostic performance. Figure 6 ).

[0080] The cutoff values ​​were 676.2 pg / ml, 41 pg / ml, and 253.4 pg / ml, respectively, which can be used as biomarkers for early pregnancy diagnosis in sows.

[0081] In production applications, we determined that when the C5aR1 concentration in sow saliva measured by ELISA is less than or equal to 676.2 pg / ml, the sow is considered pregnant; when it is higher than 676.2 pg / ml, the sow is considered not pregnant. Similarly, when the ACP2 concentration in sow saliva measured by ELISA is greater than or equal to 41 pg / ml, the sow is considered pregnant; when it is lower than 41 pg / ml, the sow is considered not pregnant. Likewise, when the TSPAN7 concentration in sow saliva measured by ELISA is greater than or equal to 253.4 pg / ml, the sow is considered pregnant; when it is lower than 253.4 pg / ml, the sow is considered not pregnant.

[0082] Summary of Diagnostic Value Results of Biomarkers

[0083]

[0084] Example 3:

[0085] C5aR1, ACP2, and TSPAN7 proteins are used as biomarkers; the combination of two or all three can be used to determine pregnancy.

[0086] Of the collected samples, samples from culled sows and those that failed quality control were removed. The remaining sow samples underwent pregnancy checks via ultrasound on days 25 and 35 after artificial insemination, and subsequent follow-up showed that all sows gave birth within their normal gestational periods. The final usable sample size was 158 sows: 46 sows 12 days after artificial insemination (38 pregnant, 8 non-pregnant), 64 sows 15 days after artificial insemination (55 pregnant, 9 non-pregnant), and 48 sows 18 days after artificial insemination (41 pregnant, 7 non-pregnant).

[0087] The ELISA (Enzyme-Linked Immunosorbent Assay) assay should be performed according to the kit instructions. After collecting the saliva sample, centrifuge for approximately 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation occurs during storage, centrifuge again.

[0088] The efficacy of C5aR1-ACP2, C5aR1-TSPAN7, ACP2-TSPAN7, and C5aR1-ACP2-TSPAN7 combined in diagnosing pregnancy in sows on days 12, 15, and 18 after artificial insemination was analyzed using a binary logistic regression model combined with ROC curves.

[0089] The results are as follows Figure 7 , Figure 8 and Figure 9As shown: Analysis revealed that the combination of ACP2-TSPAN7 showed the best diagnostic results on days 12 and 15 after artificial insemination, with AUCs of 0.9026 (specificity: 0.72, sensitivity: 1) and 0.96 (specificity: 0.89, sensitivity: 1), respectively; while the combination of C5aR1-TSPAN7 showed the best diagnostic effect on day 18 after artificial insemination, with an AUC of 0.9664 (specificity: 0.94, sensitivity: 1).

[0090] The results are as follows Figure 10 As shown, the diagnostic AUC of C5aR1-ACP2-TSPAN7 combination on days 12, 15 and 18 after artificial insemination were 0.9424 (specificity: 0.91, sensitivity: 1), 0.9886 (specificity: 0.94, sensitivity: 1) and 0.9706 (specificity: 0.97, sensitivity: 1), respectively.

[0091] Based on the above analysis, the combined diagnosis of C5aR1-ACP2-TSPAN7 on day 15 after artificial insemination yielded the best diagnostic AUC value of 0.99, enabling early pregnancy diagnosis in sows. The criteria for diagnosis are: a C5aR1 concentration in the sow's saliva less than or equal to 676.2 pg / ml, and ACP2 and TSPAN7 concentrations greater than or equal to 41 pg / ml and 253.4 pg / ml, respectively, indicating pregnancy; when the C5aR1 concentration measured by ELISA is higher than 676.2 pg / ml, and the ACP2 and TSPAN7 concentrations are lower than 41 pg / ml and 253.4 pg / ml, respectively, the sow is considered not pregnant.

[0092] We can also choose a more comprehensive determination method, namely, if the concentration of C5aR1 in the sow's saliva is less than or equal to 676.2 pg / ml, the concentration of ACP2 is greater than or equal to 41 pg / ml, and the concentration of TSPAN7 is greater than or equal to 253.4 pg / ml, and at least two of these conditions are met, the sow is determined to be pregnant; otherwise, the sow is determined to be not pregnant.

[0093] Summary of the Diagnostic Value of Biomarkers

[0094]

[0095] Example 4:

[0096] Application of C5aR1, ACP2, and TSPAN7 proteins as biomarkers in the diagnosis of early pregnancy in sows:

[0097] Saliva samples were collected from 40 sows 15 days after artificial insemination. Early pregnancy prediction was performed using the method provided in this invention. Pregnancy was assessed using ultrasound on days 25 and 35 after artificial insemination. The ultrasound results served as a control analysis. Subsequent follow-up showed that all pregnant sows delivered within normal gestational periods.

[0098] Saliva sample processing: Centrifuge the sample for approximately 20 minutes (2000-3000 rpm) after collection. Carefully collect the supernatant. If precipitation forms during storage, centrifuge again.

[0099] The concentrations of C5aR1, ACP2, and TSPAN7 proteins in the saliva of this group of sows were determined using an ELISA assay kit, and the results are shown in the table below.

[0100]

[0101] We determined that a sow is pregnant if at least two of the following conditions are met: C5aR1 concentration ≤ 676.2 pg / ml, ACP2 concentration ≥ 41 pg / ml, and TSPAN7 concentration ≥ 253.4 pg / ml. Otherwise, the sow is considered not pregnant. Analysis showed that, based on these indicators, the true negative rate for early pregnancy diagnosis in sows was 100%, the true positive rate was 97%, and the diagnostic accuracy for pregnant sows was 97.14%, which meets current production needs. The diagnostic results and analysis are shown in the table below:

[0102] Combined prediction of C5aR1, ACP2, and TSPAN7 proteins and ultrasound diagnostic results

[0103]

[0104] Analysis of joint prediction results of C5aR1, ACP2 and TSPAN7 proteins

[0105]

[0106] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a system for detecting the content of C5aR1, ACP2 and TSPAN7 proteins in sows in the preparation of a product for diagnosing early pregnancy in sows; the time of early pregnancy is 12-25 days after artificial insemination, and the system is a reagent, a kit or an instrument.

2. The use of a system for detecting the content of C5aR1, ACP2 and TSPAN7 proteins in sows' saliva in the preparation of a product for diagnosing early pregnancy in sows; the time of early pregnancy is 12-25 days after artificial insemination, and the system is a reagent, a kit or an instrument.

3. A device for diagnosing early pregnancy in sows, characterized in that: The device comprises a data receiving module and a data processing module; the data receiving module is configured to receive the content of C5aR1, ACP2 and TSPAN7 proteins in sows to be diagnosed; the data processing module is used to convert the content of C5aR1, ACP2 and TSPAN7 proteins from the receiving module into a judgment result for diagnosing early pregnancy in sows to be tested.

4. A computer readable storage medium for diagnosing early pregnancy in sows, characterized in that: the computer readable storage medium enables a computer to perform the following steps: detecting the content of C5aR1, ACP2 and TSPAN7 proteins in sows to be tested, and converting the content into a judgment result for diagnosing early pregnancy in sows to be tested.

5. The use according to any one of claims 1-2, the device of claim 3 or the computer readable storage medium of claim 4, wherein the sows are binary sows.

6. The use according to any one of claims 1-2, the device of claim 3 or the computer readable storage medium of claim 4, wherein the detection reagent for the content of proteins is a reagent for detecting the content of C5aR1, ACP2 and TSPAN7 in sows by enzyme-linked immunosorbent assay, immunofluorescence method, radioimmunoassay method, immunoprecipitation method, immunoblotting method, high performance liquid chromatography method, capillary gel electrophoresis method, near infrared spectroscopy method, mass spectrometry method, immunochemiluminescence method, colloidal gold immunological technology, fluorescence immunoassay technology, surface plasmon resonance technology, immuno-PCR technology or biotin-avidin technology.

7. The use according to claim 2, wherein the kit is an ELISA kit.

8. The use according to claim 7, wherein the judgment result of the application process is that the concentration of C5aR1 in sows' saliva detected by the ELISA kit is less than or equal to 676.2 pg / ml, the concentration of ACP2 is greater than or equal to 41 pg / ml, and the concentration of TSPAN7 is greater than or equal to 253.4 pg / ml, and at least two of the above conditions are met, then the sow is determined to be pregnant; otherwise, the sow is determined to be not pregnant.

9. The use according to any one of claims 1-2, the device of claim 3 or the computer readable storage medium of claim 4, wherein the time of early pregnancy is 12-18 days after artificial insemination.

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