Composition of biomarkers for monitoring silent estrus in cows, method of obtaining and use

Abstract

The present application relates to the field of animal husbandry and metabolomics technology, and discloses a biomarker composition for monitoring quiet estrus of cows, and an acquisition method and application thereof.The biomarker composition for monitoring quiet estrus of cows is composed of alpha-tocotrienol, methionine-arginine-lysine and undecanedioic acid.Through screening, alpha-tocotrienol, methionine-arginine-lysine and undecanedioic acid in feces are selected as biomarkers for quiet estrus of cows, the AUC of a single marker is 0.89 or more, and the diagnostic performance is significantly better than that of traditional serum biochemical indicators.The combined application of the three markers can make the AUC reach 1.00, realize non-invasive, accurate and stable monitoring of quiet estrus, and the preparation is simple, the cost is low, and the method is suitable for large-scale breeding.

Description

Technical Field

[0001] This invention relates to the fields of animal husbandry and veterinary medicine and metabolomics, specifically to a biomarker composition for monitoring silent estrus in cows, its acquisition method, and its application. Background Technology

[0002] Estrus in cows is divided into normal estrus and silent estrus. Silent estrus, also known as recessive estrus, is a special physiological phenomenon in cows where only the physiological changes of ovarian follicle development, maturation, and ovulation occur during estrus, without obvious external estrus symptoms such as mounting, vulvar redness and swelling with discharge, or vocalization. Silent estrus has a high incidence in cow populations, especially in high-producing cows, postpartum cows, and peripartum cows, where the incidence can reach 20-40%. Because it cannot be identified for mating in time, it can lead to missed mating opportunities, prolonged estrus, and decreased reproductive rates. The prolonged non-pregnant period for each cow directly increases feed and labor costs, causing huge economic losses to large-scale livestock farming.

[0003] Currently, traditional methods for monitoring estrus in cows mainly rely on external behavioral observation. This method can only identify normal estrus with obvious symptoms and cannot effectively identify silent estrus. Auxiliary monitoring methods include rectal palpation and ultrasound monitoring of ovarian follicle development. Although these methods can accurately determine the estrus status, they are cumbersome, time-consuming, and labor-intensive, requiring on-site operation by professional technicians, which cannot meet the batch monitoring needs of large-scale farming. In addition, existing hormone detection methods (such as serum progesterone and estradiol detection) have problems such as long detection cycles, complex sample processing, insufficient specificity and sensitivity for early silent estrus identification, and high detection costs, making it difficult to popularize them in the field of farming.

[0004] Therefore, finding a non-invasive, highly specific, sensitive, and easy-to-operate method for monitoring silent estrus in cows that meets the needs of livestock farming practices, and screening out biomarkers that can accurately characterize the physiological state of silent estrus in cows, has become an urgent technical bottleneck to overcome in order to solve the difficulty of identifying silent estrus in cows and improve the reproductive efficiency of cows. Summary of the Invention

[0005] The present invention aims to overcome the problems of existing methods for identifying quiet estrus in cows, which rely on invasive operations, are costly, and are difficult to apply on a large scale.

[0006] To achieve the above objectives, the first aspect of the present invention provides a biomarker composition for monitoring silent estrus in cows, the biomarker composition comprising α-tocotrienol, methionine-arginine-lysine (Met-Arg-Lys), and undecanedioic acid.

[0007] A second aspect of the present invention provides a method for obtaining the biomarker composition for monitoring silent estrus in cows as described in the first aspect, comprising the following steps: (1) Select at least 20 cows to form a discovery set. Through estrus monitoring, estrus identification and rectal ovary examination, the cows are divided into a normal estrus group and a quiet estrus group. Collect the cow feces and detect the content of small molecule metabolites in the feces by mass spectrometry. Use multidimensional testing to screen out small molecule metabolites with significant differences in distribution between the normal estrus group and the quiet estrus group. According to VIP value, metabolite abundance and FC value, the top 200 metabolites are screened respectively. Take the intersection of the small molecule metabolites obtained from each group to obtain the candidate differential markers of the discovery set. (2) Select at least 18 cows to form a validation set. Based on the results of traditional clinical serum indicators, estrus identification and veterinary comprehensive diagnosis, they are divided into a suspected normal estrus group and a suspected silent estrus group. Feces are collected and the content of small molecule metabolites in feces is detected by mass spectrometry. Multidimensional tests are used to screen out small molecule metabolites with significant differences in distribution between the normal estrus group and the silent estrus group. The top 200 metabolites are selected according to VIP value, metabolite abundance and FC value. The intersection of the small molecule metabolites obtained from each group is used to obtain the candidate differential markers of the validation set. (3) The candidate differential markers in the discovery set and the candidate differential markers in the validation set are compared and the intersection is taken to obtain potential biomarkers; the potential biomarkers are validated for diagnostic capability to obtain a biomarker composition composed of α-tocotrienol, methionine-arginine-lysine and undecanoic acid.

[0008] A third aspect of the present invention provides the use of the biomarker composition described in the first aspect in the preparation of reagents or kits for monitoring silent estrus in cows.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention, based on metabolomics technology, combines three metabolic markers (covering antioxidant, lipid metabolism, and peptide signaling dimensions) to more comprehensively capture metabolic network changes caused by silent estrus, serving as non-invasive biomarkers for identifying and diagnosing silent estrus cows. Diagnostic capability verification shows that the AUC value of each marker is higher than 0.85, demonstrating outstanding clinical diagnostic value; moreover, the combined diagnostic results of these three biomarkers show even higher diagnostic value. 2. The biomarkers of this invention can be used to identify, characterize and monitor cows in silent estrus. Only fecal samples need to be collected from the cows. There is no need for blood samples or surgical puncture. It is a non-invasive and non-surgical detection method that conforms to the concept of animal welfare and healthy breeding and will not cause stress or side effects to the cows. 3. This invention employs a dual independent sample cohort cross-validation strategy of discovery set and validation set, which ensures the stability and reliability of biomarkers and overcomes the technical challenge of large inter-individual variability in metabolic markers. Attached Figure Description

[0010] Figure 1 In a preferred embodiment of the present invention, a VIP volcano map of fecal samples was discovered. Figure 2 This is a preferred embodiment of the present invention that verifies the VIP volcano map of fecal samples; Figure 3 This is a violin diagram of the differential biomarker Alpha-Tocotrienol in a preferred embodiment of the present invention; Figure 4 This is a violin diagram of the differential biomarker Met-Arg-Lys in a preferred embodiment of the present invention; Figure 5 This is a violin diagram of the differential biomarker Undecanedioic acid in a preferred embodiment of the present invention; Figure 6 This is the ROC curve of the biomarker Alpha-Tocotrienol in a preferred embodiment of the present invention; Figure 7 This is the ROC curve of the biomarker Met-Arg-Lys in a preferred embodiment of the present invention; Figure 8 This is the ROC curve of the biomarker Undecanedioic acid in a preferred embodiment of the present invention. Detailed Implementation

[0011] 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. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0012] As previously stated, the first aspect of the present invention provides a biomarker composition for monitoring silent estrus in cows, the biomarker composition comprising α-tocotrienol, methionine-arginine-lysine and undecanoic acid.

[0013] In a preferred embodiment, the α-tocotrienol, methionine-arginine-lysine, and undecanoic acid are all derived from bovine fecal metabolites.

[0014] As previously described, a second aspect of the present invention provides a method for obtaining the biomarker composition for monitoring silent estrus in cows as described in the first aspect, comprising the following steps: (1) Select at least 20 cows to form a discovery set. Through estrus monitoring, estrus identification and rectal ovary examination, the cows are divided into a normal estrus group and a quiet estrus group. Collect the cow feces and detect the content of small molecule metabolites in the feces by mass spectrometry. Use multidimensional testing to screen out small molecule metabolites with significant differences in distribution between the normal estrus group and the quiet estrus group. According to VIP value, metabolite abundance and FC value, the top 200 metabolites are screened respectively. Take the intersection of the small molecule metabolites obtained from each group to obtain the candidate differential markers of the discovery set. (2) Select at least 18 cows to form a validation set. Based on the results of traditional clinical serum indicators, estrus identification and veterinary comprehensive diagnosis, they are divided into a suspected normal estrus group and a suspected silent estrus group. Feces are collected and the content of small molecule metabolites in feces is detected by mass spectrometry. Multidimensional tests are used to screen out small molecule metabolites with significant differences in distribution between the normal estrus group and the silent estrus group. The top 200 metabolites are selected according to VIP value, metabolite abundance and FC value. The intersection of the small molecule metabolites obtained from each group is used to obtain the candidate differential markers of the validation set. (3) The candidate differential markers in the discovery set and the candidate differential markers in the validation set are compared and the intersection is taken to obtain potential biomarkers; the potential biomarkers are validated for diagnostic capability to obtain a biomarker composition composed of α-tocotrienol, methionine-arginine-lysine and undecanoic acid.

[0015] In the preferred case, in steps (1) and (2), the multidimensional tests are based on VIP>1 and P<0.05.

[0016] In a preferred embodiment, in step (3), the diagnostic capability verification is performed using violin plots and ROC curves.

[0017] Preferably, the mass spectrometry detection is performed using gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.

[0018] As previously stated, a third aspect of the present invention provides the use of the biomarker composition described in the first aspect in the preparation of reagents or kits for monitoring silent estrus in cows.

[0019] According to a preferred embodiment, the monitoring of silent estrus in cows includes: detecting the content of α-tocotrienol, methionine-arginine-lysine and undecanoic acid in cow feces, and determining whether the estrus type of the cow is silent estrus based on the detected content.

[0020] Preferably, the criterion for judgment is: when the relative contents of α-tocotrienol, methionine-arginine-lysine, and undecanoic acid are all significantly reduced compared with those of cows in normal estrus, it indicates silent estrus.

[0021] The present invention will be described in detail below through examples. Unless otherwise specified, the raw materials used are all commercially available products.

[0022] Animal grouping criteria: Normal estrus group: exhibiting mounting behavior, vulvar redness and swelling, ultrasound showing mature follicles, and serum progesterone levels within the reference range for estrus; Quiet estrus group: No external behavioral manifestations, but continuous ultrasound monitoring showed that the follicles matured and ovulated.

[0023] Example 1: This example illustrates the method for obtaining the biomarker composition for monitoring silent estrus in cows provided by the present invention, which includes the following steps: (1) 845 Xinjiang Brown cattle (cows) in their breeding season were selected as the research subjects. All cows were healthy adult individuals. After estrus monitoring, rectal-ovarian examination and serum biochemical index screening, 20 cows were selected to form a discovery set. Through estrus monitoring, estrus identification and rectal-ovarian examination, the cows were divided into a normal estrus group (10 cows) and a quiet estrus group (10 cows). Fresh fecal samples were collected from the cows, freeze-dried, ground, and extracted with organic solvents (such as methanol-water). The supernatant was centrifuged and derivatized (such as methoxypyridine and MSTFA). Gas chromatography-mass spectrometry was used for analysis. The retention time and mass spectrum were compared with the standard and NIST spectral library for qualitative analysis. The relative content of each metabolite was calculated by the internal standard normalization method. Multidimensional tests were used to screen for small molecule metabolites with significantly different distributions between the normal estrus group and the silent estrus group: small molecules with significantly different distributions between the two groups were screened using VIP>1 and P<0.05 as criteria. Figure 1 The image shows the VIP volcano map of the fecal sample collection. Then, based on VIP value, metabolite abundance, and FC value, the top 200 metabolites are selected respectively. The intersection of the obtained small molecule metabolites is taken to obtain the candidate differential markers of the discovery set. (2) Eighteen cows were selected to form a validation set. Based on the results of traditional clinical serum indicators, estrus identification and veterinary comprehensive diagnosis, they were divided into a suspected normal estrus group and a suspected silent estrus group. Feces were collected and the content of small molecule metabolites in the feces was detected by mass spectrometry. Multidimensional tests were used to screen for small molecule metabolites with significantly different distributions between the normal estrus group and the silent estrus group: small molecules with significantly different distributions between the two groups were screened using VIP>1 and P<0.05 as criteria. Figure 2 The VIP volcano plot of the validation set fecal samples is shown; The top 200 metabolites were selected based on VIP value, metabolite abundance, and FC value, and the intersection of the obtained small molecule metabolites was used to obtain the candidate differential markers for the validation set. (3) The candidate differential markers in the discovery set and the candidate differential markers in the validation set are compared and the intersection is taken to obtain potential biomarkers; the potential biomarkers are validated for diagnostic capability to obtain a biomarker composition composed of α-tocotrienol, methionine-arginine-lysine and undecanedioic acid.

[0024] Information on the above three biomarkers is shown in Table 1; Table 1. Basic information on three metabolic biomarkers

[0025] Note: "-" indicates that there is no corresponding annotation information for this compound in the relevant database (HMDB / KEGG).

[0026] Table 1 shows the source, type, HMDB number, KEGG pathway number, and P-value of the t-test for the three markers. It can be seen that the expression of α-tocotrienol, methionine-arginine-lysine, and undecanoic acid differed significantly between the silent estrus group and the normal estrus group (P<0.001), and the FC value (silent estrus group / normal estrus group) showed that all three markers were significantly downregulated in the silent estrus group.

[0027] Figure 3 A diagram of a violin made of Alpha-Tocotrienol is shown. Figure 4 A diagram of a Met-Arg-Lys violin is shown. Figure 5 A violin plot of undecanedioic acid is shown, where the asterisk "*" indicates a significant difference between groups. The statistical criteria are: "*" indicates P < 0.05, indicating a significant difference between groups; "**" indicates P < 0.01, indicating an extremely significant difference between groups; "***" indicates P < 0.001, indicating an extremely highly significant difference between groups; no marker indicates no statistical significance between groups (P ≥ 0.05). See Table 1 for specific P values. The plot shows that compared with cows in the normal estrus group, cows in the quiet estrus group had significantly lower levels of Alpha-Tocotrienol, Met-Arg-Lys, and Undecanedioic acid in their feces. ROC curve analysis shows that... Figures 6-8 ),in, Figure 6The ROC curve of Alpha-Tocotrienol is shown. Figure 7 The ROC curve of Met-Arg-Lys is shown. Figure 8 The ROC curve of undecanedioic acid is shown. The figure shows that the AUC value of each marker is higher than 0.85: Alpha-Tocotrienol has an AUC of 0.94, Met-Arg-Lys has an AUC of 0.90, and undecanedioic acid has an AUC of 0.89. The combined diagnostic result of the three fecal biomarkers can reach an AUC value of 1.00 (Table 2).

[0028] Table 2. Comparison of diagnostic capabilities of different indicators for cows in silent estrus

[0029] Note: AUC > 0.9 indicates excellent discrimination ability, and 0.8 < AUC ≤ 0.9 indicates good discrimination ability.

[0030] The ability of the three biomarkers of this invention to identify silent estrus cows was compared with that of traditional serum biochemical and hormonal indicators, and the results are shown in Table 2. Table 2 shows that the AUC values ​​(0.89-0.94) of each of the three biomarkers of this invention are higher than those of most traditional serum biochemical indicators (0.18-0.92) and some hormonal indicators; the combined diagnostic ability of the three biomarkers is the highest (AUC=1.00). This invention, by combining three fecal biomarkers as a combined diagnostic marker, can significantly improve the diagnostic ability for silent estrus cows.

[0031] The above experimental results demonstrate that this invention, by screening α-tocotrienol, methionine-arginine-lysine, and undecanoic acid from feces as biomarkers for silent estrus in cows, achieves an AUC of over 0.89 for each individual marker, exhibiting significantly superior diagnostic performance compared to traditional serum biochemical indicators. The combined application of these three markers can achieve an AUC of 1.00, enabling non-invasive, accurate, and stable monitoring of silent estrus. Furthermore, the preparation process is simple and inexpensive, making it suitable for large-scale farming.

[0032] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combining the various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A biomarker composition for monitoring silent estrus in cows, characterized in that, The biomarker composition consists of α-tocotrienol, methionine-arginine-lysine, and undecanoic acid.

2. The biomarker composition according to claim 1, characterized in that, The α-tocotrienol, methionine-arginine-lysine, and undecanoic acid mentioned are all derived from bovine fecal metabolites.

3. A method for obtaining the biomarker composition for monitoring silent estrus in cows as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Select at least 20 cows to form a discovery set. Through estrus monitoring, estrus identification and rectal ovary examination, the cows are divided into a normal estrus group and a quiet estrus group. Collect the cow feces and detect the content of small molecule metabolites in the feces by mass spectrometry. Use multidimensional testing to screen out small molecule metabolites with significant differences in distribution between the normal estrus group and the quiet estrus group. According to VIP value, metabolite abundance and FC value, the top 200 metabolites are screened respectively. Take the intersection of the small molecule metabolites obtained from each group to obtain the candidate differential markers of the discovery set. (2) Select at least 18 cows to form a validation set. Based on the results of traditional clinical serum indicators, estrus identification and veterinary comprehensive diagnosis, they are divided into a suspected normal estrus group and a suspected silent estrus group. Feces are collected and the content of small molecule metabolites in feces is detected by mass spectrometry. Multidimensional tests are used to screen out small molecule metabolites with significant differences in distribution between the suspected normal estrus group and the suspected silent estrus group. The top 200 metabolites are selected according to VIP value, metabolite abundance and FC value. The intersection of the small molecule metabolites obtained from each group is used to obtain the candidate differential markers of the validation set. (3) The candidate differential markers in the discovery set and the candidate differential markers in the validation set are compared and the intersection is taken to obtain potential biomarkers; the potential biomarkers are validated for diagnostic capability to obtain a biomarker composition composed of α-tocotrienol, methionine-arginine-lysine and undecanoic acid.

4. The acquisition method according to claim 3, characterized in that, In steps (1) and (2), the multidimensional tests are based on VIP>1 and P<0.

05.

5. The method for obtaining according to claim 3 or 4, characterized in that, In step (3), the diagnostic capability verification is performed using violin plots and ROC curves.

6. The method for obtaining according to claim 3 or 4, characterized in that, The mass spectrometry detection is performed using gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.

7. The use of the biomarker composition of claim 1 or 2 in the preparation of reagents or kits for monitoring silent estrus in cows.

8. The application according to claim 7, characterized in that, The monitoring of silent estrus in cows includes: detecting the content of α-tocotrienol, methionine-arginine-lysine, and undecanoic acid in cow feces, and determining whether the estrus type of the cow is silent estrus based on the detected content.

9. The application according to claim 8, characterized in that, The criteria for judgment are as follows: when the relative contents of α-tocotrienol, methionine-arginine-lysine, and undecanoic acid are all significantly reduced compared with those of cows in normal estrus, it indicates silent estrus.

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