Hu sheep heat stress serum metabolism marker thromboxane B2 as well as screening method and application thereof

Abstract

The invention discloses a Hu sheep heat stress serum metabolism marker thromboxane B2 as well as a screening method and application thereof, and belongs to the technical field of livestock serum markers. The Hu sheep heat stress serum metabolism marker is thromboxane B2; the screening method of the heat-stress-resistant Hu sheep comprises the following steps: (1) preparing a serum sample of to-be-detected Hu sheep; (2) detecting the concentration of thromboxane B2 in the serum sample by using an ELISA (enzyme-linked immuno sorbent assay) method; and (3) analyzing the difference of the concentration of the thromboxane B2 in the heat-sensitive Hu sheep and the heat-resistant Hu sheep, performing omics analysis, judging a critical value, comparing the critical value with the concentration of the thromboxane B2 in the serum sample, distinguishing the heat-sensitive Hu sheep and the heat-resistant Hu sheep, and screening out the heat-resistant stress Hu sheep. Thrombin B2 is used as a marker for detection, has the characteristics of high accuracy and high sensitivity, and can be directly used as an evaluation index for distinguishing heat-sensitive Hu sheep from heat-resistant Hu sheep.

Description

Technical Field

[0001] The present invention belongs to the technical field of livestock serum markers, and in particular relates to a heat stress serum metabolic marker thromboxane B2 of Hu sheep, a screening method thereof, and an application thereof. Background Art

[0002] As the greenhouse effect intensifies and global warming deepens, high temperatures are becoming a major problem for the development of animal husbandry. When raising sheep, high temperatures can cause them to experience symptoms of heat stress.

[0003] Huyang (Huyang) is a rare and valuable sheep breed in China, renowned for its high fertility and high-quality fur. However, in the hot and humid southern region, Huyang farming faces the severe challenge of heat stress. Studies have shown that heat stress can lead to decreased feed intake, significantly increased water intake and frequency, and increased heart and respiratory rates. Prolonged heat stress can lead to emaciation, decreased resistance and immunity, and, in severe cases, death.

[0004] In livestock farming, technicians often struggle to determine whether Hu sheep are experiencing heat stress. Even after obvious heat stress is apparent, diagnosis and treatment are often ineffective. In breeding, more heat-tolerant Hu sheep should be retained, while heat-sensitive ones should be eliminated. Therefore, a simple and efficient method for detecting serum markers of heat stress in Hu sheep is crucial. Summary of the Invention

[0005] The present invention provides a serum metabolic marker for heat stress in Hu sheep, thromboxane B2, and its screening method and application. These methods aim to address the lack of effective detection methods for long-term heat stress in Hu sheep during breeding, as well as the lack of effective breeding methods to increase the number of heat-resistant Hu sheep in Hu sheep breeding. This invention provides an effective auxiliary detection method for detecting heat stress in Hu sheep and for selecting and retaining heat-resistant Hu sheep.

[0006] The technical solution of the present invention is achieved as follows:

[0007] A heat stress serum metabolic marker for Hu sheep, wherein the serum metabolic marker is thromboxane B2.

[0008] A method for screening heat stress-resistant Hu sheep comprises the following steps:

[0009] (1) Using the blood of known heat-sensitive Hu sheep or heat-resistant Hu sheep as the standard test sample and the blood of the Hu sheep to be tested as the test sample, the whole blood specimen collected in the serum separation tube was allowed to stand, and then centrifuged to obtain the supernatant serum sample;

[0010] (2) Detection of thromboxane B2 concentration in serum samples by ELISA;

[0011] (3) Analyze the differences in thromboxane B2 concentrations between heat-sensitive and heat-resistant Hu sheep, conduct omics analysis, determine the critical value, and compare the critical value with the thromboxane B2 concentration in serum samples to distinguish heat-sensitive and heat-resistant Hu sheep, and screen out heat stress-resistant Hu sheep.

[0012] The standing in step (1) is to stand at room temperature for 2 hours or at 4°C overnight.

[0013] The centrifugation in step (1) is performed at 1000-1200×g for 18-25 minutes.

[0014] When the serum sample prepared in step (1) cannot be tested by ELISA in time, the serum should be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided.

[0015] The ELISA method described in step (2) is to detect sheep thromboxane B2 (TXB2) using an ELISA kit for ELISA detection.

[0016] The critical value in step (3) is preferably 70.79 pg / mL.

[0017] The step (3) is described in which the critical value is compared with the thromboxane B2 concentration in the serum sample to distinguish between heat-sensitive sheep and heat-resistant Hu sheep. Specifically, the heat stress resistance of Hu sheep is judged by the concentration of the serum metabolic marker thromboxane B2 in the serum sample of Hu sheep. The lower the thromboxane B2 concentration, the greater the ability of the Hu sheep to resist heat stress, and vice versa. The concentration critical point is determined by omics analysis.

[0018] The application of the above-mentioned heat stress serum metabolic marker thromboxane B2 in the screening of heat stress-resistant Hu sheep is specifically embodied in two aspects: application in detecting whether Hu sheep are in a heat stress state or application in the breeding and screening process of heat-resistant Hu sheep.

[0019] The application of the heat stress serum metabolic marker thromboxane B2 of Hu sheep in screening heat stressed Hu sheep is specifically as follows: the heat stress resistance of Hu sheep is judged by the concentration of the serum metabolic marker thromboxane B2 in the serum sample of Hu sheep. The lower the thromboxane B2 concentration, the greater the ability of the Hu sheep to resist heat stress, and vice versa. The concentration critical point is determined by omics analysis, which provides help for the basic method of screening heat-resistant Hu sheep.

[0020] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0021] Currently, there is no serum metabolic marker for distinguishing between heat-sensitive Hu sheep and heat-resistant Hu sheep. The present invention uses metabolomics data to propose metabolic markers, screening methods and applications for heat-sensitive Hu sheep and heat-resistant Hu sheep from the serum metabolic level. The metabolic marker is thromboxane B2. Detection using thromboxane B2 as a marker has the characteristics of high accuracy and high sensitivity, and can be directly used as an evaluation indicator for distinguishing between heat-sensitive Hu sheep and heat-resistant Hu sheep.

[0022] The present invention provides a serum marker for detecting heat stress in Hu sheep and its application. By measuring the expression of thromboxane B2 in the serum of heat-resistant Hu sheep under high temperatures, it was found that the expression was significantly higher in the serum of heat-resistant Hu sheep than in the serum of heat-sensitive Hu sheep. This serum marker can be used to simply and efficiently distinguish between heat-resistant and heat-sensitive Hu sheep. Using the serum marker thromboxane B2 as an auxiliary test for heat stress in Hu sheep will help explore the physiological mechanisms of heat stress in Hu sheep and identify better therapeutic interventions, thus providing some support for Hu sheep farming in southern China. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a statistical result chart of the average thromboxane B2 concentration detected in the Hu sheep in Example 1.

[0024] Figure 2 This is the normal distribution curve of the average concentration of thromboxane B2 detected in the Hu sheep in Example 1.

[0025] Figure 3 This is the ROC curve of thromboxane B2 in Hu sheep serum in Example 1. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] Currently, there is a lack of effective detection methods for the long-term heat stress experienced by Hu sheep during their breeding process, as well as a lack of effective breeding methods to increase the number of heat-resistant Hu sheep remaining. To address these technical issues, the present invention proposes a Hu sheep heat stress serum marker, thromboxane B2, and its use in screening for heat-stressed Hu sheep.

[0029] Example 1

[0030] The experiment selected 20 heat-sensitive Hu sheep (HS) and 20 heat-resistant Hu sheep (HR) as the Hu sheep to be tested for subsequent testing.

[0031] A method for screening heat stress-resistant Hu sheep, comprising the following steps:

[0032] (1) Using blood from known heat-sensitive or heat-resistant Hu sheep as the standard test sample and the blood of the Hu sheep to be tested as the test sample, place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes, take the supernatant serum, or store the serum at -20°C or -80°C, but avoid repeated freezing and thawing;

[0033] (2) Detection of thromboxane B2 concentration in serum samples by ELISA;

[0034] (3) Analyze the differences in thromboxane B2 concentrations between heat-sensitive and heat-resistant Hu sheep, perform omics analysis, determine a critical value, and compare the critical value with the test serum to distinguish between heat-sensitive and heat-resistant Hu sheep, thereby screening Hu sheep resistant to heat stress. This example provides a basic method for distinguishing between heat-sensitive and heat-resistant Hu sheep by determining the critical value.

[0035] The ELISA method described in step (2) is to detect sheep thromboxane B2 (TXB2) using an ELISA kit for ELISA detection.

[0036] The specific method of the ELISA method detection is as follows:

[0037] 1) Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, seal the remaining strips in a ziplock bag and return them to 4°C;

[0038] 2) Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well;

[0039] 3) Add 50 μL of the sample to be tested to the sample well; do not add any to the blank well;

[0040] 4) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard sample and sample wells, except for the blank wells. Seal the wells with a sealing film and incubate at 37°C in a water bath or incubator for 60 min.

[0041] 5) Discard the liquid, pat dry on absorbent paper, add 350 μL of wash solution to each well, let it stand for 1 minute, discard the wash solution, pat dry on absorbent paper, and repeat this process 5 times (you can also use a plate washer to wash the plate);

[0042] 6) Add 50 μL of substrate A and substrate B to each well and incubate at 37°C in the dark for 15 min;

[0043] 7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0044] Experimental results: With the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis, a standard curve is drawn, and a linear regression equation is obtained. The OD value of the sample is substituted into the equation to calculate the concentration of the sample.

[0045] SPSS was used for one-way analysis of variance and Origin was used for normal distribution curve drawing to find the critical value of the difference in relative concentration of thromboxane B2 between the HS group and the HR group.

[0046] The concentrations of thromboxane B2 in the serum of the 40 tested Hu sheep are shown in Table 1.

[0047] Table 1: Thromboxane B2 concentration (pg / mL)

[0048]

[0049]

[0050] The average concentrations of thromboxane B2 in the serum of the heat-sensitive Hu sheep (HS) and the heat-resistant Hu sheep (HR) are shown in Tables 2 and Figure 1 shown.

[0051] Table 2 Average concentration of thromboxane B2

[0052]

[0053] The normal distribution curve of the average concentration of thromboxane B2 is as follows Figure 2 As shown, the heat-sensitive Hu sheep were grouped as HS, and the heat-resistant Hu sheep were grouped as HR. Figure 2 It can be seen that the thromboxane B2 content of heat-sensitive Hu sheep is significantly greater than that of heat-resistant Hu sheep, and based on the average concentration and normal distribution curve analysis, the critical value is 70.79 pg / mL. This critical value can be used to judge and screen whether Hu sheep are heat-resistant.

[0054] Perform ROC analysis of thromboxane B2:

[0055] The receiver operating characteristic curve (ROC) is a curve drawn according to a series of different binary classification methods (division = decision threshold), with the true positive rate (sensitivity) as the vertical coordinate and the false positive rate (1-specificity) as the horizontal coordinate. The present embodiment draws the ROC curve of thromboxane B2, and calculates the area under the curve (AUC). The area under the ROC curve (AUC) is an indicator of the performance of the ROC curve, and the larger the AUC, the better. The area under the ROC curve is between 1.0 and 0.5. AUC is between 0.5 and 0.7, with low reliability, AUC is between 0.7 and 0.9, with certain reliability, and the closer the AUC is to 1, the better the classification effect. The ROC curve of the above-mentioned differential metabolites is drawn as shown in Figure 3 and Table 3, it can be seen that the AUC value of thromboxane B2 is greater than 0.7, which has high reliability.

[0056] Table 3 Area under the curve of thromboxane B2

[0057]

[0058] There is no serum metabolic marker for distinguishing heat-sensitive Hu sheep and heat-resistant Hu sheep at present, and the present embodiment uses metabolomics data to propose a metabolic marker of heat-sensitive Hu sheep and heat-resistant Hu sheep from the serum metabolic level, and a screening method and application, the metabolic marker is thromboxane B2, and the use of thromboxane B2 as a marker for detection has the characteristics of high accuracy and high sensitivity, and can be directly used as an evaluation index for distinguishing heat-sensitive Hu sheep and heat-resistant Hu sheep.

[0059] The present embodiment determines the heat stress resistance of Hu sheep by the concentration of serum metabolic marker thromboxane B2 in the serum sample of Hu sheep, the lower the concentration of thromboxane B2, the greater the heat stress resistance of the Hu sheep, and vice versa, and the critical point (70.79 pg / mL) of the concentration is determined by omics analysis, which provides help for the basic method of screening heat-resistant Hu sheep.

[0060] The above only describes the preferred embodiments of the present application and does not limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A heat stress serum metabolic marker for Hu sheep, characterized by: The serum metabolic marker is thromboxane B2.

2. A method for screening heat stress-resistant Hu sheep, characterized by: The following steps are involved: (1) Using the blood of known heat-sensitive Hu sheep or heat-resistant Hu sheep as the standard test sample and the blood of the Hu sheep to be tested as the test sample, the whole blood specimen collected in the serum separation tube was allowed to stand, and then centrifuged to obtain the supernatant serum sample; (2) Detection of thromboxane B2 concentration in serum samples by ELISA; (3) Analyze the differences in thromboxane B2 concentrations between heat-sensitive and heat-resistant Hu sheep, conduct omics analysis, determine the critical value, and compare the critical value with the thromboxane B2 concentration in serum samples to distinguish heat-sensitive and heat-resistant Hu sheep, and screen out heat stress-resistant Hu sheep.

3. The method for screening heat stress-resistant Hu sheep according to claim 2, characterized in that: The standing in step (1) is to stand at room temperature for 2 hours or at 4°C overnight.

4. The method for screening heat stress-resistant Hu sheep according to claim 2, characterized in that: The centrifugation in step (1) is performed at 1000-1200×g for 18-25 minutes.

5. The method for screening heat stress-resistant Hu sheep according to claim 2, characterized in that: The ELISA method detection in step (2) uses an ELISA kit for sheep thromboxane B2 for ELISA detection.

6. The method for screening heat stress-resistant Hu sheep according to claim 2, characterized in that: The critical value in step (3) is 70.79 pg / mL.

7. The method for screening heat stress-resistant Hu sheep according to claim 2, characterized in that: The step (3) is described in which the critical value is compared with the thromboxane B2 concentration in the serum sample to distinguish between heat-sensitive sheep and heat-resistant Hu sheep. Specifically, the heat stress resistance of Hu sheep is judged by the concentration of the serum metabolic marker thromboxane B2 in the serum sample of Hu sheep. The lower the thromboxane B2 concentration, the greater the ability of the Hu sheep to resist heat stress, and vice versa. The concentration critical point is determined by omics analysis.

8. Use of the heat stress serum metabolic marker thromboxane B2 of Hu sheep according to claim 1 in screening Hu sheep resistant to heat stress.

9. The use of the heat stress serum metabolic marker thromboxane B2 of Hu sheep according to claim 8 in screening Hu sheep resistant to heat stress, characterized in that: The invention can be used in detecting whether Hu sheep are in a state of heat stress or in the breeding and screening process of heat-resistant Hu sheep.

10. The use of the heat stress serum metabolic marker thromboxane B2 of Hu sheep according to claim 8 in screening Hu sheep resistant to heat stress, characterized in that: The heat stress resistance of Hu sheep is judged by the concentration of serum metabolic marker thromboxane B2 in Hu sheep serum samples. The lower the thromboxane B2 concentration, the greater the ability of the Hu sheep to resist heat stress, and vice versa. The concentration critical point is determined by omics analysis, which provides assistance for the basic method of screening heat-resistant Hu sheep.

Images