Serum metabolism marker glycine of heat-resistant mutton sheep as well as screening method and application of serum metabolism marker glycine
By combining metabolomics technology to screen out the serum metabolic marker glycine, the problem of difficult early identification of heat resistance in meat sheep breeding was solved, and the effect of early accurate identification and breed improvement was achieved.
Patent Information
- Application Number
- CN202510845559.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-23
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technologies lack effective breeding methods in meat sheep breeding, making it difficult to accurately identify and evaluate the heat resistance of meat sheep in the early stages, resulting in low breeding efficiency.
A combination of non-targeted and targeted metabolomics was used to screen out the serum metabolic marker glycine. Ultra-high performance liquid chromatography-mass spectrometry and orthogonal-partial least squares discriminant analysis were used to screen out serum metabolic markers that can be used to early identify the heat tolerance of meat sheep.
It has achieved early and accurate identification of the heat resistance of meat sheep, improved breeding efficiency, shortened the breeding cycle, and provided a theoretical basis for the improvement of the Hu sheep breed.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of screening livestock heat stress markers, and in particular relates to glycine, a serum metabolic marker of heat-resistant mutton sheep, and a screening method and application thereof. Background Art
[0002] Evaluating heat tolerance in meat sheep is a crucial foundation for breeding heat-stress-resistant breeds, primarily through a comprehensive assessment using multidimensional indicators. Current heat tolerance assessment systems for meat sheep primarily include correlation analysis with production performance (e.g., feed intake change rate, daily weight gain, reproductive parameters), evaluation of physiological indicators (e.g., rectal temperature, respiratory rate, heart rate, skin conductivity), and blood biochemical analysis (e.g., adrenocorticotropic hormone, cortisol). However, these traditional evaluation indices have significant limitations: They are often measured at a single time point, overlooking dynamic processes such as the individual's ability to recover from sustained stress (e.g., thermoregulation rate) and metabolic adaptation (e.g., oxidative stress repair rate). They also change only when heat stress is significant, making them difficult to predict early and are susceptible to interference from circadian rhythms, nutritional status, disease, measurement stress, and environmental changes. Consequently, they fail to fully and accurately reflect physiological adaptation mechanisms and production performance under heat stress. Therefore, there is an urgent need to establish an objective, stable, and scalable molecular evaluation system to enable early and accurate identification and genetic improvement of heat tolerance traits in meat sheep.
[0003] Metabolomics, a core area of systems biology research, focuses on the dynamics of small-molecule metabolites within organisms. These small molecules (such as sugars, amino acids, organic acids, lipids, and vitamins), as direct products of metabolic activity within organisms, not only reflect the end-products of gene expression and protein function but also sensitively respond to complex factors such as environmental stimuli, nutritional status, and pathological stress, reflecting both the physiological state and pathological changes of an organism. Leveraging high-precision analytical techniques such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR), metabolomics can systematically separate, detect, and identify a variety of biological samples, including blood, saliva, and intestinal contents. Metabolomics analyzes the dynamics of small-molecule metabolites within organisms under heat stress conditions. Its advantages include high sensitivity, non-invasive or minimally invasive testing (e.g., in blood, urine, and saliva samples), and strong dynamic tracking capabilities. Its high degree of coupling with production traits makes it a valuable tool for understanding the mechanisms of heat stress in animals.
[0004] In recent years, with the advancement of metabolomics technology, multiple studies have confirmed that the dynamic changes in serum metabolites can sensitively characterize the physiological changes in individual animals under heat stress. Therefore, the development of molecular markers for heat tolerance in mutton sheep based on serum metabolomics has become a key breakthrough in animal stress tolerance breeding, providing a new technical path for achieving precise and efficient heat-tolerant mutton sheep breeding. Summary of the Invention
[0005] This invention provides a serum metabolic marker for heat-tolerant mutton sheep, glycine, and its screening method and application, aiming to address the lack of effective breeding methods to increase the number of heat-tolerant mutton sheep remaining in the mutton sheep breeding process. This invention combines non-targeted metabolomics and targeted metabolomics, leveraging the advantages of both technologies to systematically screen for serum metabolic markers for heat-tolerant mutton sheep.
[0006] The technical solution of the present invention is achieved as follows:
[0007] A serum metabolic marker for heat-resistant mutton sheep, wherein the serum metabolic marker is glycine.
[0008] A method for screening serum metabolic markers of heat-resistant mutton sheep comprises the following steps:
[0009] (1) Collect serum samples from heat-tolerant and heat-sensitive sheep under heat stress conditions;
[0010] (2) Serum samples were pretreated by protein precipitation, dilution, sample correction with internal standard method, and filtration;
[0011] (3) using ultra-high performance liquid chromatography-mass spectrometry to determine the metabolite concentrations in each blood sample;
[0012] (4) Orthogonal-partial least squares discriminant analysis (OPLS-DA) method was used to analyze the samples and screen serum metabolic markers of heat-resistant meat sheep.
[0013] In step (1), the serum sample is collected, specifically, by collecting blood from mutton under heat stress conditions and placing it in a blood collection tube, centrifuging it and taking the supernatant to obtain a serum sample.
[0014] In step (1), the blood collection tube does not contain anticoagulant substances such as EDTA.
[0015] In step (1), the centrifugation is performed at 1500 r / min for 10-15 min.
[0016] In step (1), a serum sample is obtained. If no subsequent determination is performed in time, the serum sample is stored in a -80°C refrigerator for future use.
[0017] In step (2), the protein precipitation specifically includes the following steps: taking a serum sample into a 2 mL centrifuge tube, adding 400 uL of 10% formic acid methanol solution-water (1:1, v / v), vortexing for 30 seconds, and then centrifuging at 12000 rpm at 4°C for 5 minutes.
[0018] In step (2), the dilution is specifically as follows: the supernatant is aspirated into a centrifuge tube, and 10% formic acid methanol solution-water (1:1, v / v) is added to dilute 10 times, and vortexed for 30 seconds.
[0019] In step (2), the internal standard method is used to correct the sample, specifically: use a pipette to draw 100uL of the diluted supernatant, add it to a centrifuge tube, and add 100uL of an internal standard solution containing 20ng / mL Trp-d3, and vortex for 30s.
[0020] In step (2), the filtration is specifically as follows: filtering the solution containing the internal standard using a 0.22 μm filter membrane, transferring the filtered solution into a detection bottle, and preparing for ultra high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) analysis.
[0021] In step (3), the chromatographic conditions in the ultra-high performance liquid chromatography-mass spectrometry are: using ACQUITY BEH C18 column, injection volume 5 μL, column temperature 40°C, mobile phase A: 50% methanol in water, mobile phase B: 10% methanol in water; gradient elution conditions: 0-6.5 min, 90-70% B; 6.5-7 min, 70-0% B; 7-14 min, 0% B; 14-14.5 min, 0-90% B; 14.5-17.5 min, 90% B; flow rate: 0-8.0 min, 0.3 mL / min; 8.0-17.5 min, 0.4 mL / min.
[0022] The methanol water is methanol water containing 0.1% formic acid.
[0023] In step (3), the mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry are as follows: mass spectrometry detection is performed by a SCIEX mass spectrometer after chromatographic separation; electrospray ionization source, positive ion ionization mode; ion source temperature 500°C, ion source voltage 5500V, collision gas 6psi, curtain gas 30psi, nebulizing gas and auxiliary gas are both 50psi; scanning is performed using multiple reaction monitoring.
[0024] In step (4), the orthogonal partial least squares discriminant analysis (OPLS-DA) method was used for analysis. This method can effectively reduce the complexity of the model and enhance the explanatory power of the model without reducing the predictive power of the model, thereby maximizing the differences between the groups.
[0025] The orthogonal-partial least squares discriminant analysis model will obtain a variable weight value (Variable Importance for the Projection, VIP value). Generally, the VIP value is used to illustrate the importance of a variable (characteristic peak) in explaining the X data set and the associated Y data set; the sum of the squares of all VIP values is equal to the total number of variables in the model; therefore, their average value is 1; when the VIP of a variable is greater than 1, it indicates that the variable is important, and the conditions of VIP value > 1 and P value < 0.05 are used as a screening method for serum metabolic markers of heat-resistant meat sheep.
[0026] The mutton sheep is preferably Hu sheep.
[0027] Glycine, a serum metabolic marker for heat-tolerant mutton sheep, is used in the breeding and selection of heat-tolerant mutton sheep. The critical glycine level for distinguishing heat-tolerant Hu sheep from heat-stressed Hu sheep is 15.56.
[0028] Compared with other existing technologies, the innovations of this technical solution lie in the following aspects:
[0029] 1. The serum metabolic marker glycine obtained by the present invention enables accurate early identification of heat-resistant mutton sheep. The metabolic marker glycine screened by the present invention can be used as an indicator for early and accurate identification. By detecting the marker content in serum during the young mutton sheep or in the early stages of heat stress, the heat tolerance of mutton sheep can be accurately determined, thereby enabling targeted breeding, improving breeding efficiency, and shortening the breeding cycle.
[0030] 2. The glycine serum metabolic marker obtained by this invention for heat-resistant mutton sheep is used for breed improvement. This analysis of serum metabolic markers in Hu sheep will facilitate in-depth research into the molecular mechanisms of Hu sheep heat tolerance and provide a theoretical basis for further improving the Hu sheep breed and enhancing its heat tolerance. During the breeding process, the glycine metabolic marker can be used as an important breeding indicator and combined with traditional breeding methods to accelerate the improvement of Hu sheep breeds.
[0031] In summary, compared with the existing technology, the present invention uses a dual-omics technology system to screen serum metabolic markers of Hu sheep. The data is scientific and reliable, the results are accurate, and it can perform early monitoring of heat-resistant meat sheep. It can also screen and obtain the serum metabolic marker glycine of heat-resistant meat sheep. It is suitable for promotion and use in actual production, and helps provide a basis for further improvement of the Hu sheep breed. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 This is a statistical result diagram of the difference in glycine concentration between the two groups in Example 1.
[0033] Figure 2 Graph showing the normal distribution of glycine concentrations in the HR group and the HS group in Example 1. DETAILED DESCRIPTION
[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.
[0035] In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in this field can implement them. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.
[0036] Currently, there is a lack of effective breeding methods to increase the number of heat-resistant mutton sheep in the existing mutton sheep breeding process. In order to solve the above technical problems, the present invention provides a serum metabolic marker of heat-resistant mutton sheep, glycine, and its screening method and application.
[0037] Example 1
[0038] A method for screening serum metabolic markers of heat-resistant mutton sheep comprises the following steps:
[0039] 1. Sample Collection and Processing
[0040] (1) Blood collection: Blood was collected from 26 heat-resistant (HR) Hu sheep and 26 heat-sensitive (HS) Hu sheep under heat stress conditions. The blood was collected in blood collection tubes that did not contain anticoagulants such as ethylenediaminetetraacetic acid (EDTA). The sheep number, collection date, collection time, and other information were marked on the blood collection tubes.
[0041] (2) Serum preparation: After the blood has naturally coagulated, centrifuge at 1500 rpm for 10-15 minutes and collect the supernatant (serum) in a collection tube;
[0042] (3) Sample storage: If the sample needs to be tested within a short period of time, it can be placed in a 4°C refrigerator for future use. If long-term storage is required, the collected serum sample should be stored in a -80°C refrigerator for future use. Avoid repeated freezing and thawing during storage to avoid affecting the sample quality.
[0043] 2. Amino acid pretreatment
[0044] (1) Protein precipitation: Take an appropriate amount of serum sample into a 2 mL centrifuge tube, add 400 μL of 10% formic acid methanol solution-water (1:1, v / v), vortex for 30 seconds, and centrifuge at 12000 rpm at 4°C for 5 minutes.
[0045] (2) Dilution: Pipette an appropriate amount of supernatant into a new centrifuge tube and add an appropriate amount of 10% formic acid methanol solution-water (1:1, v / v) to dilute 10 times, and vortex for 30 seconds.
[0046] (3) Calibration of samples by internal standard method: Use a pipette to draw 100 μL of the diluted supernatant, add it to a new centrifuge tube, and add 100 μL of 20 ng / mL Trp-d3 internal standard solution, and vortex for 30 seconds.
[0047] (4) Filtration: Filter the solution containing the internal standard using a 0.22 μm filter membrane. Transfer the filtered solution to a test bottle and prepare for UPLC-MS / MS analysis.
[0048] 3. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) analysis
[0049] (1) Chromatographic conditions (UPLC):
[0050] Using ACQUITY A BEH C18 column (2.1×100 mm, 1.7 μm, Waters, USA) was used with an injection volume of 5 μL. The column temperature was 40°C, and the mobile phases A and B consisted of 50% methanol in water (containing 0.1% formic acid) and 10% methanol in water (containing 0.1% formic acid). The gradient elution conditions were: 90% to 70% B (0–6.5 min); 70% to 0% B (6.5–7 min); 0% B (7–14 min); 0% B (14–14.5 min); 90% B (14.5–17.5 min). The flow rate was 0.3 mL / min (0–8.0 min); and 0.4 mL / min (8.0–17.5 min).
[0051] (2) Mass spectrometry conditions (MS):
[0052] After chromatographic separation, mass spectrometry was performed on a SCIEX mass spectrometer (QTRAP 6500+). Electrospray ionization (ESI) was used in positive ionization mode. The source temperature was 500°C, the source voltage was 5500 V, the collision gas was 6 psi, the curtain gas was 30 psi, and the nebulizer and auxiliary gases were both 50 psi. Multiple reaction monitoring (MRM) was used for scanning.
[0053] (3) Data statistics
[0054] Multivariate statistical analysis was performed using the R language ropls package, and then the data were analyzed using SPSS23.0, and graphs were drawn using GraphPadPrism10.
[0055] 4. Data Processing
[0056] The samples were analyzed using Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). This method effectively reduces model complexity and enhances its explanatory power without compromising its predictive power, thereby maximizing the ability to identify differences between groups. The OPLS-DA model generates variable importance for the projection (VIP). VIP values are typically used to indicate the importance of a variable (characteristic peak) in explaining the X dataset and its associated Y dataset. The sum of the squares of all VIP values equals the total number of variables in the model, resulting in a mean of 1. When a variable's VIP is greater than 1, it indicates that the variable is important—a common screening criterion for biomarkers.
[0057] By using the targeted metabolomics method, the concentration of the standard was used as the ordinate and the corresponding absorbance (OD value) as the abscissa. Computer software was used to fit the four-parameter logistic curve (4-pl) to create a standard curve equation. The concentration of the sample was calculated using the equation according to the absorbance (OD value) of the sample to obtain Table 2. SPSS23 was then used for one-way analysis of variance and Origin was used for normal distribution curve plotting. Figure 2 , to find the critical value of the difference in relative glycine concentration between the HS group and the HR group.
[0058] turn out:
[0059] Through the OPLS-DA model, glycine (Gly) was screened as a differential metabolite between the two groups based on the conditions of VIP>1 and P<0.05, and was upregulated in the HR group ( Table 1 , Figure 1 ), glycine can be used as a biomarker of heat resistance in meat sheep.
[0060] Table 1 VIP values of differential metabolites glycine
[0061]
[0062] The heat-resistant group (HR) and the heat-sensitive group (HS) were clearly distinguishable (Table 2). The glycine content in the serum metabolites of the heat-resistant group was significantly higher than that of the heat-sensitive group. Figure 2 It can be seen that the glycine concentration corresponding to the intersection of the two normal distribution curves of the HR group and the HS group is 15.56. It is predicted that glycine is a serum metabolite related to the heat stress resistance of meat sheep. The glycine critical value for distinguishing heat-resistant Hu sheep from heat-stressed Hu sheep is 15.56. In this way, glycine can be used as a serum metabolic marker for screening heat-resistant Hu sheep to achieve the screening of heat-resistant Hu sheep.
[0063] Table 2 Glycine concentration in each group of samples
[0064]
[0065]
[0066] Glycine, a serum metabolic marker for heat-tolerant mutton sheep, is used in the breeding and selection of heat-tolerant mutton sheep. The critical glycine level for distinguishing heat-tolerant Hu sheep from heat-stressed Hu sheep is 15.56.
[0067] Compared with other existing technologies, the innovations of this technical solution lie in the following aspects:
[0068] 1. The serum metabolic marker glycine obtained by the present invention enables accurate early identification of heat-resistant mutton sheep. The metabolic marker glycine screened by the present invention can be used as an indicator for early and accurate identification. By detecting the marker content in serum during the young mutton sheep or in the early stages of heat stress, the heat tolerance of mutton sheep can be accurately determined, thereby enabling targeted breeding, improving breeding efficiency, and shortening the breeding cycle.
[0069] 2. The glycine serum metabolic marker obtained by this invention for heat-resistant mutton sheep is used for breed improvement. This analysis of serum metabolic markers in Hu sheep will facilitate in-depth research into the molecular mechanisms of Hu sheep heat tolerance and provide a theoretical basis for further improving the Hu sheep breed and enhancing its heat tolerance. During the breeding process, the glycine metabolic marker can be used as an important breeding indicator and combined with traditional breeding methods to accelerate the improvement of Hu sheep breeds.
[0070] In summary, compared with the existing technology, the present invention uses a dual-omics technology system to screen serum metabolic markers of Hu sheep. The data is scientific and reliable, the results are accurate, and it can perform early monitoring of heat-resistant meat sheep. It can also screen and obtain the serum metabolic marker glycine of heat-resistant meat sheep. It is suitable for promotion and use in actual production, and helps provide a basis for further improvement of the Hu sheep breed.
[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A serum metabolic marker for heat-resistant mutton sheep, characterized by: The serum metabolic marker is glycine.
2. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 1, characterized in that: The following steps are involved: (1) Collect serum samples from heat-tolerant and heat-sensitive sheep under heat stress conditions; (2) Serum samples were pretreated by protein precipitation, dilution, sample correction with internal standard method, and filtration; (3) using ultra-high performance liquid chromatography-mass spectrometry to determine the metabolite concentrations in each blood sample; (4) Orthogonal-partial least squares discriminant analysis was used to analyze the samples and screen serum metabolic markers of heat-resistant meat sheep.
3. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, characterized in that: In step (1), the serum sample collection is specifically as follows: blood of a sheep under heat stress conditions is collected and placed in a blood collection tube, and the supernatant is collected by centrifugation to obtain a serum sample; In step (1), the blood collection tube is a blood collection tube that does not contain EDTA anticoagulant; In step (1), the centrifugation is performed at 1500 r / min for 10-15 min.
4. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, wherein: In step (2), the protein precipitation specifically comprises the following steps: taking a serum sample into a 2 mL centrifuge tube, adding 400 μL of 10% formic acid methanol solution-water, vortexing for 30 seconds, and then centrifuging at 12000 rpm at 4°C for 5 minutes; In step (2), the dilution is specifically as follows: the supernatant is aspirated into a centrifuge tube, and 10% formic acid methanol solution-water (1:1, v / v) is added to dilute 10 times, and vortexed for 30 seconds.
5. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, characterized in that: In step (2), the internal standard method is used to correct the sample, specifically: use a pipette to draw 100uL of the diluted supernatant, add it to a centrifuge tube, and add 100uL of 20ng / mL Trp-d3 internal standard solution, and vortex oscillation for 30s; In step (2), the filtration is specifically as follows: filtering the solution containing the internal standard using a 0.22 μm filter membrane, transferring the filtered solution into a detection bottle, and preparing for ultra-high performance liquid chromatography-mass spectrometry analysis.
6. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, characterized in that: In step (3), the chromatographic conditions in the ultra-high performance liquid chromatography-mass spectrometry are: using ACQUITY C18 column, injection volume 5 μL, column temperature 40°C, mobile phase A: 50% methanol in water, mobile phase B: 10% methanol in water; gradient elution conditions: 0-6.5 min, 90-70% B; 6.5-7 min, 70-0% B; 7-14 min, 0% B; 14-14.5 min, 0-90% B; 14.5-17.5 min, 90% B; flow rate 0-8.0 min, 0.3 mL / min; 8.0-17.5 min, 0.4 mL / min; The methanol water is methanol water containing 0.1% formic acid.
7. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, characterized in that: In step (3), the mass spectrometry conditions in the ultra-high performance liquid chromatography-mass spectrometry are as follows: mass spectrometry detection is performed by a SCIEX mass spectrometer after chromatographic separation; electrospray ionization source, positive ion ionization mode; ion source temperature 500°C, ion source voltage 5500V, collision gas 6psi, curtain gas 30psi, nebulizing gas and auxiliary gas are both 50psi; scanning is performed using multiple reaction monitoring.
8. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, characterized in that: In step (4), the orthogonal-partial least squares discriminant analysis model will obtain the variable weight VIP value. When the VIP of the variable is greater than 1, it means that the variable is important. The conditions of VIP value > 1 and P value < 0.05 are used as a screening method for serum metabolic markers of heat-resistant meat sheep.
9. The method for screening serum metabolic markers of heat-resistant mutton sheep according to claim 2, characterized in that: The mutton sheep is preferably Hu sheep.
10. Use of the serum metabolic marker glycine of heat-resistant mutton sheep according to claim 1 in the breeding and selection process of heat-resistant mutton sheep.