Serum metabolism marker histidine for screening heat-resistant mutton sheep as well as screening method and application of serum metabolism marker histidine

Abstract

The invention discloses a serum metabolism marker histidine for screening heat-resistant mutton sheep as well as a screening method and application of the serum metabolism marker histidine, and belongs to the technical field of animal molecular breeding and serum biomarker screening. The serum metabolism marker is histidine; the critical value of histidine for distinguishing heat-resistant mutton sheep and heat-stress mutton sheep in a mutton sheep serum sample is 3.18 ug / ml; under the heat stress condition, if the histidine content in the mutton sheep serum sample is higher than 3.18 ug / ml, the mutton sheep is heat-resistant mutton sheep; if the content of histidine in the mutton sheep serum sample is lower than 3.18 ug / ml, the mutton sheep is heat-sensitive mutton sheep. According to the screening method, a liquid chromatography-mass spectrometry combined mode and an orthogonal-partial least square discriminant analysis method are adopted for analyzing a sample, and the serum metabolism marker histidine for screening the heat-resistant mutton sheep is screened out. The serum metabolism marker histidine disclosed by the invention has higher accuracy and stability, and can be used for accurately evaluating the heat resistance of the Hu sheep in the early stage.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal molecular breeding and serum biomarker screening, and in particular relates to a serum metabolic marker histidine for screening heat-resistant mutton sheep, and a screening method and application thereof. Background Art

[0002] Heat stress has become a significant environmental factor restricting the development of the mutton industry, particularly in the context of global warming. Sustained high temperatures significantly impact the growth, development, reproductive performance, and immune function of sheep, leading to reduced production efficiency and even mortality, posing a serious threat to the sustainable development of animal husbandry. Especially in southern regions or during the hot summer months, mutton sheep with poor heat tolerance are more susceptible to a range of heat stress reactions, including elevated body temperatures, reduced feed intake, and reproductive failure. Therefore, selecting and breeding mutton breeds with superior heat tolerance is a key strategy for improving production efficiency and adapting to future climate change.

[0003] Currently, the evaluation of heat tolerance in meat sheep mainly relies on the measurement of physiological indicators (such as respiratory rate, rectal temperature, heart rate, etc.) or stress-related hormone levels (such as cortisol and HSP70, etc.). However, these indicators are affected by multiple factors such as age, breed, and environment, and are highly volatile and time-sensitive, making it difficult to achieve high-throughput and accurate individual screening. Therefore, there is an urgent need to develop a more stable, specific, and scalable molecular marker for the precise screening and early identification of heat tolerance in meat sheep.

[0004] Heat tolerance is a crucial economic trait in meat sheep farming, particularly in high-temperature environments. Sheep with poor heat tolerance are prone to slow growth and reduced reproductive rates. Therefore, screening and breeding heat-tolerant meat sheep is crucial for improving farming profitability. Currently, methods for selecting heat-tolerant meat sheep mainly include the following: 1. Phenotypic selection: This involves observing sheep's performance in high-temperature environments, such as body temperature, respiratory rate, and feed intake, to select individuals with better heat tolerance. While this method is simple and intuitive, it suffers from high subjectivity, low accuracy, and an inability to identify individuals with excellent heat tolerance at an early stage. 2. Marker-assisted selection: This utilizes genetic markers associated with heat tolerance for assisted selection. While this method has some scientific basis, heat tolerance is a complex trait influenced by multiple genes and environmental factors. The effectiveness of screening with a single genetic marker is limited, and it can be unstable across different environments and genetic backgrounds.

[0005] Although the above methods can be used to screen heat-resistant meat sheep to a certain extent, they each have obvious shortcomings: 1. Limitations of phenotypic selection: Phenotypic selection mainly depends on the performance of sheep in high temperature environments. This method is not only time-consuming and labor-intensive, but also easily interfered with by environmental factors, resulting in low accuracy of screening results. In addition, phenotypic selection cannot accurately assess the heat tolerance of sheep in the early stages, which is not conducive to early selection and breeding work. 2. Limitations of gene marker-assisted selection: Although gene marker-assisted selection has a certain scientific basis, since heat tolerance is a complex trait controlled by multiple genes, a single gene marker is difficult to fully reflect the heat tolerance of sheep. In addition, the stability of gene markers in different environments and genetic backgrounds is poor, which limits their wide application in actual production.

[0006] Metabolomics is an important part of multi-omics research, which mainly studies the small molecule metabolites (relative molecular mass <1000×10 3 Metabolomics relies on high-throughput platforms (such as nuclear magnetic resonance spectroscopy (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS)) to systematically analyze metabolic signatures in blood, urine, or tissues, and, by combining multi-omics data, reveal the molecular mechanisms underlying changes in physiological states.

[0007] Therefore, the development of serum metabolic markers that can be used to identify the heat tolerance of meat sheep based on metabolomics methods is expected to provide efficient, molecular-level technical means for the screening of meat sheep germplasm resources and the breeding of new varieties, and promote the intelligent and precise process of animal molecular breeding. Summary of the Invention

[0008] The present invention provides a serum metabolic marker histidine for screening heat-tolerant mutton sheep, as well as a screening method and application thereof. These methods aim to address the problem that phenotypic selection cannot accurately assess the heat tolerance of sheep at an early stage, hindering early selection and breeding efforts; as well as the poor stability of marker-assisted selection in different environments and genetic backgrounds. The present invention combines non-targeted metabolomics and targeted metabolism, leveraging the advantages of both technologies to screen serum metabolic markers for heat-tolerant mutton sheep.

[0009] The technical solution of the present invention is achieved as follows:

[0010] A serum metabolic marker for screening heat-resistant mutton sheep, wherein the serum metabolic marker is histidine.

[0011] Preferably, the critical value of histidine in serum samples of meat sheep for screening heat-resistant meat sheep is 3.18 ug / ml, which distinguishes heat-resistant meat sheep from heat-stressed meat sheep. Under heat stress conditions, the content of histidine in serum samples of meat sheep is higher than 3.18 ug / ml, which is heat-resistant meat sheep; the content of histidine in serum samples of meat sheep is lower than 3.18 ug / ml, which is heat-sensitive meat sheep.

[0012] A screening method for screening serum metabolic markers of heat-resistant meat sheep, comprising the following steps:

[0013] (1) Collecting serum samples of meat sheep under heat stress conditions;

[0014] (2) Determining the concentration of metabolites in each blood sample by liquid chromatography-mass spectrometry;

[0015] (3) Analyzing the samples by orthogonal partial least squares discriminant analysis (OPLS-DA), and screening the serum metabolic marker histidine for screening heat-resistant meat sheep according to the screening conditions of variable weight value VIP (Variable Importance for the Projection) obtained by the orthogonal partial least squares discriminant analysis model being greater than 1 and statistical significance P<0.05.

[0016] In step (1), the serum sample collection is specifically: collecting blood of meat sheep under heat stress conditions into a blood collection tube without EDTA and other anticoagulants, centrifuging to obtain the supernatant, and obtaining the serum sample;

[0017] In step (1), the centrifugation is at 1500 r / min for 10-15 min.

[0018] The serum sample obtained in step (1) is stored in a-80℃ refrigerator for standby use.

[0019] Before determining by liquid chromatography-mass spectrometry in step (2), the serum sample is treated as follows: taking the serum sample into a centrifuge tube, adding 400 μL of 10% formic acid methanol solution-water, vortexing for 30 s; centrifuging at 12000 rpm and 4℃ for 5 min; taking the supernatant, adding 10% formic acid methanol solution-water to dilute 10 times, vortexing for 30 s; taking 100 μL of the supernatant, adding 100 μL of 20 ng / mL Trp-d3 internal standard, vortexing for 30 s, passing the supernatant through a 0.22 μm filter membrane, and adding the filtrate to a detection bottle;

[0020] The volume ratio (v / v) of the 10% formic acid methanol solution-water is 1:1.

[0021] In step (2), the chromatographic conditions in the liquid chromatography-mass spectrometry are: using ACQUITY BEHC18 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;

[0022] 7~14min, 0%B; 14~14.5min, 0~90%B; 14.5~17.5min, 90%B; flow rate 0~8.0min, 0.3mL / min; 8.0~17.5min, 0.4mL / min;

[0023] The methanol water is methanol water containing 0.1% formic acid.

[0024] In step (2), the mass spectrometry conditions in the 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.

[0025] In step (3), the orthogonal-partial least squares discriminant analysis 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.

[0026] In the orthogonal-partial least squares discriminant analysis model, the VIP value is used to illustrate that the characteristic peak of the variable can explain the importance of 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, its average value is 1; when the VIP of a variable is greater than 1, it means that the variable is important, and the conditions of VIP value greater than 1 and P value less than 0.05 are used as conditions for screening serum metabolic markers for heat-resistant meat sheep, thereby screening and obtaining the serum metabolic marker histidine for screening heat-resistant meat sheep

[0027] The mutton sheep is preferably Hu sheep.

[0028] The serum metabolic marker histidine used to screen heat-resistant mutton sheep is obtained by the above screening method. Further preferably, the cutoff value of histidine for distinguishing heat-resistant mutton sheep from heat-stressed mutton sheep is preferably 3.18ug / ml.

[0029] The above-mentioned application of serum metabolic markers for screening heat-resistant meat sheep is specifically used in the early selection and breeding of heat-resistant meat sheep, especially in the precise screening and early identification of the heat resistance of meat sheep. The serum metabolic marker used for screening heat-resistant meat sheep is histidine.

[0030] The present invention systematically screens out heat-resistant Hu sheep serum metabolic markers by combining non-targeted metabolomics and targeted metabolomics technologies.

[0031] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0032] 1. This study uses targeted metabolomics to screen for metabolic markers in the serum of heat-resistant Hu sheep. Targeted metabolomics accurately quantifies and verifies these potential markers, ensuring their accuracy and reliability.

[0033] 2. The present invention uses a systematic metabolomics analysis screening method to screen out the highly specific heat-resistant Hu sheep serum metabolite marker - histidine. The serum metabolite marker histidine can accurately reflect the metabolic response of Hu sheep in high temperature environments, providing a scientific basis for the screening of heat-resistant Hu sheep. Compared with existing phenotypic selection and gene marker-assisted selection methods, the serum metabolite marker histidine of the present invention has higher accuracy and stability, and can accurately assess the heat tolerance of Hu sheep at an early stage.

[0034] 3. The metabolite markers of the present invention can be used to assess heat tolerance in the early stages of Huyang sheep breeding, providing a basis for early seed selection and breeding. The screening method of the present invention not only improves screening efficiency but also reduces misjudgments due to environmental factors and individual differences, thus having important practical application value. Compared with existing metabolomics studies, the markers of the present invention have higher specificity and practicality, making them suitable for promotion and application in actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the difference in histidine concentration between the two groups in Example 1.

[0036] Figure 2 This is the normal distribution diagram of the histidine concentration in the HR group and the HS group in Example 1. DETAILED DESCRIPTION

[0037] 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.

[0038] In addition, the technical solutions among various embodiments can be combined with each other, but it must be based on that a person skilled in the art can realize, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is not within the protection scope required by the present application.

[0039] At present, the existing screening of heat-resistant mutton sheep has the defects that phenotype selection cannot accurately evaluate the heat resistance of sheep at an early stage, which is not conducive to early selection and breeding work, and the stability of gene marker-assisted selection in different environments and genetic backgrounds is poor. In order to solve the above technical problems, the present application proposes a serum metabolic marker histidine for screening heat-resistant mutton sheep and a screening method and application thereof.

[0040] Example 1

[0041] A screening method of a serum metabolic marker for screening heat-resistant mutton sheep, comprising the following steps:

[0042] I. Collection of serum samples

[0043] (1) Collect the blood of 18 heat-resistant mutton sheep (HR, heat-resistant Hu sheep) with good performance and 18 heat-sensitive mutton sheep (HS, heat-sensitive Hu sheep) under heat stress conditions into a negative pressure tube without EDTA and other anticoagulants. After centrifugation at 1500g for 10-15min, the supernatant is obtained, which is the required 36 serum samples. The serum samples are stored in a 80℃ refrigerator for standby.

[0044] II. Sample liquid chromatography-mass spectrometry analysis

[0045] (1) The concentration of metabolites in each blood sample is determined by liquid chromatography-mass spectrometry.

[0046] The 36 serum samples are sequentially subjected to ultra-high performance liquid chromatography-mass spectrometry analysis, and the experimental conditions are as follows:

[0047] An appropriate amount of sample is taken into a 2mL centrifuge tube, 400μL of 10%formic acid methanol solution-water (1:1, v / v) is accurately added, vortexed for 30s, centrifuged at 12000rpm and 4℃ for 5min, an appropriate amount of supernatant is taken and diluted 10 times with 10%formic acid methanol solution-water (1:1, v / v), vortexed for 30s, 100μL of supernatant is taken, 100μL of 20ng / mL Trp-d3 internal standard is added, vortexed for 30s, the supernatant is filtered through a 0.22μm filter membrane, and the filtered liquid is added to a detection bottle.

[0048] Chromatographic conditions (UPLC):

[0049] 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).

[0050] Mass spectrometry conditions (MS):

[0051] 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.

[0052] (3) Multivariate statistical analysis and biomarker screening:

[0053] 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 potential biomarkers.

[0054] 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 histidine concentration between the HS group and the HR group.

[0055] turn out:

[0056] Through the OPLS-DA model, histidine (His) 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 , P<0.05). Histidine is a serum metabolic marker for screening heat-resistant mutton sheep.

[0057] Table 1 VIP values ​​of differential metabolites histidine

[0058]

[0059] The heat-resistant group (HR) and the heat-sensitive group (HS) were clearly distinguishable (Table 2). The content of histidine in serum metabolites in the heat-resistant group was significantly higher than that in the heat-sensitive group. Figure 2 It can be seen that the histidine concentration corresponding to the intersection of the two normal distribution curves of the HR group and the HS group is 3.18ug / ml. It is predicted that histidine is a serum metabolite related to the heat stress resistance of meat sheep. The critical value of histidine for distinguishing heat-resistant Hu sheep from heat-stressed Hu sheep is 3.18ug / ml. In this way, histidine is used as a serum metabolic marker for screening heat-resistant meat sheep to achieve the screening of heat-resistant Hu sheep.

[0060] Table 2 Histidine concentration in each group of samples

[0061]

[0062] Therefore, histidine, a serum metabolic marker for screening heat-resistant mutton sheep, is used in the breeding of heat-resistant mutton sheep. The critical value of glycine for distinguishing heat-resistant Hu sheep from heat-stressed Hu sheep is 3.18ug / ml.

[0063] Compared with other existing technologies, the innovations of this technical solution lie in the following aspects:

[0064] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0065] 1. This study uses targeted metabolomics to screen for metabolic markers in the serum of heat-resistant Hu sheep. Targeted metabolomics accurately quantifies and verifies these potential markers, ensuring their accuracy and reliability.

[0066] 2. The present invention uses a systematic metabolomics analysis screening method to screen out the highly specific heat-resistant Hu sheep serum metabolite marker - histidine. The serum metabolite marker histidine can accurately reflect the metabolic response of Hu sheep in high temperature environments, providing a scientific basis for the screening of heat-resistant Hu sheep. Compared with existing phenotypic selection and gene marker-assisted selection methods, the serum metabolite marker histidine of the present invention has higher accuracy and stability, and can accurately assess the heat tolerance of Hu sheep at an early stage.

[0067] 3. The metabolite markers of the present invention can be used to assess heat tolerance in the early stages of Huyang sheep breeding, providing a basis for early seed selection and breeding. The screening method of the present invention not only improves screening efficiency but also reduces misjudgments due to environmental factors and individual differences, thus having important practical application value. Compared with existing metabolomics studies, the markers of the present invention have higher specificity and practicality, making them suitable for promotion and application in actual production.

[0068] 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 screening heat-resistant mutton sheep, characterized by: The serum metabolic marker is histidine.

2. The serum metabolic marker for screening heat-resistant mutton sheep according to claim 1, characterized in that: The critical value of histidine in sheep serum samples for distinguishing heat-resistant sheep from heat-stressed sheep is 3.18ug / ml; under heat stress conditions, sheep with a higher histidine content in serum samples than 3.18ug / ml are heat-resistant sheep; sheep with a lower histidine content in serum samples than 3.18ug / ml are heat-sensitive sheep.

3. A method for screening serum metabolic markers for heat-resistant mutton sheep, characterized in that: The following steps are involved: (1) Collect serum samples from sheep under heat stress conditions; (2) determination of metabolite concentrations in each blood sample using liquid chromatography-mass spectrometry; (3) The samples were analyzed by orthogonal partial least squares discriminant analysis. The variable weight value VIP obtained by the orthogonal partial least squares discriminant analysis model was used to screen the serum metabolic marker histidine for screening heat-resistant meat sheep according to the screening conditions of variable weight value VIP>1 and statistical significance P<0.

05.

4. The method for screening serum metabolic markers for heat-resistant mutton sheep according to claim 3, characterized in that: In step (1), the serum sample is collected by collecting blood from sheep under heat stress conditions and placing it in a blood collection tube that does not contain EDTA anticoagulant, centrifuging and taking the supernatant to obtain a serum sample; In step (1), the centrifugation is performed at 1500 r / min for 10-15 min.

5. The method for screening serum metabolic markers for heat-resistant mutton sheep according to claim 3, characterized in that: In step (2), before the liquid chromatography-mass spectrometry determination, the serum sample was treated as follows: the serum sample was placed in a centrifuge tube, 400 μL of 10% formic acid methanol solution-water was added, and the sample was vortexed for 30 seconds; the sample was centrifuged at 12000 rpm and 4°C for 5 minutes; the supernatant was diluted 10 times by adding 10% formic acid methanol solution-water, and the sample was vortexed for 30 seconds; 100 μL of the supernatant was taken, 100 μL of 20 ng / mL Trp-d3 internal standard was added, the sample was vortexed for 30 seconds, the supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was added to the detection bottle; The volume ratio (v / v) of the 10% formic acid methanol solution to water is 1:

1.

6. The method for screening serum metabolic markers for heat-resistant mutton sheep according to claim 3, characterized in that: In step (2), the chromatographic conditions in the 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 for heat-resistant mutton sheep according to claim 3, characterized in that: In step (2), the mass spectrometry conditions in the 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 for heat-resistant mutton sheep according to claim 3, characterized in that: In step (3), in the orthogonal-partial least squares discriminant analysis model, the conditions of VIP value>1 and P value<0.05 are used as conditions for screening serum metabolic markers of heat-resistant meat sheep, thereby screening and obtaining the serum metabolic marker histidine for screening heat-resistant meat sheep.

9. The method for screening serum metabolic markers for heat-resistant mutton sheep according to claim 3, characterized in that: The meat sheep are Hu sheep.

10. The use of the method according to any one of claims 1 or 2 for screening serum metabolic markers of heat-resistant mutton sheep, characterized in that: The application in the early selection and breeding of heat-resistant meat sheep is that the serum metabolic marker used for screening heat-resistant meat sheep is histidine.

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