A system and application for predicting ketoacidosis fatty liver syndrome in dairy cows using blood biochemical indicators

Abstract

The present invention relates to the field of biological detection technology, and provides a system and application for predicting ketoacidosis fatty liver syndrome in dairy cows using blood biochemical indicators. The blood biochemical indicators include bile acid, glycocholic acid, taurocholic acid or deoxycholic acid. When bile acid>3.54μg / mL, glycocholic acid>6.802μg / mL, taurocholic acid>4.403μg / mL or deoxycholic acid>0.392μg / mL in the blood of dairy cows, it is predicted that the dairy cow suffers from ketoacidosis fatty liver syndrome. The system of the present invention can be used to non-invasively monitor the incidence of ketoacidosis fatty liver syndrome in dairy cows. Compared with the existing detection methods, the detection indicators are simpler and the clinical operation is easier, which provides effective technical support for the prevention and control of the disease and effectively reduces the economic losses of the ranch.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a system and application thereof for predicting dairy cow ketoacidosis fatty liver syndrome by utilizing blood biochemical indicators. Background Art

[0002] With the development of society, dairy farming occupies a very important position in the livestock farming industry around the world. Dairy cows are not only the main source of dairy products, but also play an important role in promoting social and economic development. Ketosis fatty liver syndrome, as a nutritional metabolic disease that causes a high mortality rate in dairy cows, has received widespread attention. The peripartum period is the high incidence period of ketoacidosis fatty liver syndrome in dairy cows. When the disease occurs, it not only leads to decreased liver function, but also reduces the resistance and immune function of dairy cows, causing great losses to reproduction, lactation and labor. In severe cases, dairy cows will have symptoms such as retained placenta, endometritis and production paralysis. Due to the lack of obvious symptoms of the disease, it is often difficult to diagnose and control, thus endangering the health of dairy cows. The increased mortality rate of dairy cows can account for about 20% of the annual mortality rate, causing huge economic losses to the dairy industry. Ketosis-induced fatty liver syndrome in dairy cows can lead to increased triglyceride content in the liver, as well as increased concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA) in the blood and urine. However, the disease is currently mainly detected through comprehensive indicators such as blood NEFA, BHBA, and aspartate aminotransferase (AST), which have the problems of complex detection indicators and difficult clinical operations. Summary of the invention

[0003] The purpose of the present invention is to provide a system and application for predicting ketoacidosis and fatty liver syndrome in dairy cows by using blood biochemical indicators. The system of the present invention can be used to non-invasively monitor the incidence of ketoacidosis and fatty liver syndrome in dairy cows. Compared with existing detection methods, the detection indicators are simpler and the clinical operation is easier.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides application of a reagent for measuring blood biochemical indexes in preparing a reagent for predicting dairy cow ketosis fatty liver syndrome. The blood biochemical indexes include bile acid, glycocholic acid, taurocholic acid or deoxycholic acid.

[0006] Furthermore, when bile acid>3.54μg / mL, glycocholic acid>6.802μg / mL, taurocholic acid>4.403μg / mL or deoxycholic acid>0.392μg / mL in the blood of dairy cows, it is predicted that the dairy cows suffer from ketotic fatty liver syndrome.

[0007] The present invention also provides a system for predicting dairy cow ketosis fatty liver syndrome, the system comprising:

[0008] Data acquisition module: used to obtain the concentration data of biochemical indicators in the blood of dairy cows, wherein the biochemical indicators include bile acid, glycocholic acid, taurocholic acid or deoxycholic acid;

[0009] Result determination module: used to receive the concentration data acquired by the data acquisition module, and predict the dairy cow ketoacidosis fatty liver syndrome according to the concentration data.

[0010] Furthermore, the method for predicting ketoacidosis fatty liver syndrome in dairy cows based on concentration data is as follows: when bile acid>3.54μg / mL, glycocholic acid>6.802μg / mL, taurocholic acid>4.403μg / mL or deoxycholic acid>0.392μg / mL in the blood of dairy cows, it can be predicted that the dairy cows suffer from ketoacidosis fatty liver syndrome.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention collects blood from dairy cows in the early stage of lactation and detects bile acid levels through LC-MS technology. Pearson correlation analysis and ROC curve analysis are performed based on the content of bile acid (CA), glycocholic acid (GCA), taurocholic acid (TCA), and deoxycholic acid (DCA) in the blood of dairy cows with ketoacidic fatty liver syndrome and healthy dairy cows. It is determined that when CA>3.540μg / mL, GCA>6.802μg / mL, TCA>4.403μg / mL or DCA>0.392μg / mL, it can be predicted that the dairy cow suffers from ketoacidic fatty liver syndrome. This method can non-invasively monitor the incidence of ketoacidic fatty liver syndrome in dairy cows, provide effective technical support for the prevention and control of the disease, and effectively reduce the economic losses of pastures. DETAILED DESCRIPTION

[0013] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0014] Example 1

[0015] This embodiment provides blood biochemical indicators that can predict ketoacidosis and fatty liver syndrome in dairy cows. The specific steps are as follows:

[0016] 1. Selection of experimental animals

[0017] The experiment was conducted in a large intensive dairy farm in Heilongjiang Province. Thirty Holstein cows with similar parity (2-3) and body condition (2.75-3.75) at 3-14 days postpartum were randomly selected and divided into a ketosis fatty liver syndrome group (BHBA>1.4mmol / L, Y<1) and a healthy group (BHBA<1.2mmol / L, Y>1, without any other symptoms) according to the concentration of blood β-hydroxybutyric acid (BHBA) and Y value (Y=-0.51-0.003NEFA+2.84GLU-0.0528AST). There were 10 cows in the healthy group and 11 cows in the ketosis fatty liver syndrome group.

[0018] 2. Blood Sample Collection

[0019] 10 mL of blood samples were collected from the tail vein of the experimental cattle before feeding in the early morning. The blood samples were left to stand at room temperature for 30 minutes, centrifuged at 3000 r / min for 10 minutes after natural coagulation, and stored at -20°C for testing. The plasma bile acid level was detected by LC-MS. The reagents and instruments used in the detection process are shown in Tables 1, 2, 3, and 4.

[0020] Table 1 Metabolite extraction reagents

[0021]

[0022] Table 2 Metabolite extraction instruments and equipment

[0023]

[0024] Table 3 Metabolomics detection instruments

[0025]

[0026] Table 4 Material information of bile acid standards

[0027]

[0028]

[0029] 3. Detection Methods

[0030] Take 5 μL of each of the above four bile acid standards, take an appropriate amount of each mother solution to prepare a mixed standard, dilute one by one with methanol to prepare a working standard solution. The concentration points of bile acids and the specific calculation method are shown in Table 5.

[0031] Accurately pipette 100 μL of sample into a 2 mL EP tube, add 600 μL of methanol (-20°C), vortex for 60 seconds, centrifuge at 12000 rpm, 4°C for 10 minutes, take 400 μL of supernatant, concentrate and dry, take 200 μL of 30% methanol to re-dissolve the sample, vortex for 30 seconds, take 50 μL of supernatant, add 200 μL of 30% methanol, vortex for 30 seconds, and add to the detection bottle for detection. Using ACQUITY BEH C18 chromatographic column (2.1×100 mm, 1.7 μm, Waters, USA), injection volume 5 μL, column temperature 40°C, mobile phase A-0.01% formic acid water, B-acetonitrile. Gradient elution conditions: 0-4 min, 25% B; 4-9 min, 25-30% B; 9-14 min, 30-36% B; 14-18 min, 36-38% B; 18-24 min, 38-50% B; 24-32 min, 50-75% B; 32-33 min, 75-90% B; 33-35.5 min, 90-25% B. Flow rate 0.25 mL / min. Electrospray ionization (ESI) source, negative ionization mode. The ion source temperature was 500°C, the ion source voltage was -4500V, the collision gas was 6psi, the curtain gas was 30psi, and the nebulizer gas and auxiliary gas were both 50psi. Multiple reaction monitoring (MRM) was used for scanning.

[0032] Table 5 Bile acid standard curve concentration (ng / mL)

[0033]

[0034] Calculation formula: content = C*0.7*5 / 2 / sampling volume (content unit: μg / mL; C unit: ng / mL; sampling volume unit: μL)

[0035] Each working standard solution was tested by LC-MS, and the concentration of the working standard solution was used as the abscissa and the peak area was used as the ordinate to examine the linear range and draw a standard curve. The linear regression equations of each bile acid obtained are shown in Table 6, and the correlation coefficient r>0.99.

[0036] Table 6 Linear regression equation of bile acid in dairy cow blood

[0037]

[0038] 4. Experimental Results

[0039] Independent sample T test was used to analyze the differences between groups. The results of blood bile acid components are shown in Table 7. The GCA and DCA in the blood of cows with ketoacidic fatty liver syndrome were significantly higher than those in the healthy group (P<0.05), and the CA and TCA were extremely significantly higher than those in the healthy group (P<0.01).

[0040] Table 7 Comparison of blood bile acid indexes between healthy dairy cows and dairy cows with ketoacidosis and fatty liver syndrome

[0041]

[0042] Note: *(p<0.05) indicates significant difference, **(p<0.01) indicates extremely significant difference.

[0043] The blood Y values ​​and BHBA results of healthy cows and cows with ketotic fatty liver syndrome are shown in Table 8.

[0044] Table 8 Y values ​​and BHBA of healthy cows and cows with ketoacidosis and fatty liver syndrome

[0045]

[0046] Pearson linear correlation analysis was used to analyze the correlation between each index and ketoacidosis fatty liver syndrome. The results are shown in Table 9. Blood CA, GCA, TCA, and DCA were all significantly negatively correlated with the diagnostic criteria for ketoacidosis fatty liver syndrome (Y value) (P<0.01), and blood CA, GCA, TCA, and DCA were all significantly positively correlated with the diagnostic criteria for ketoacidosis fatty liver syndrome (BHBA) (P<0.01).

[0047] Table 9 Significant correlation indicators of dairy cows with ketoacidosis and fatty liver syndrome

[0048]

[0049] Note: *(p<0.05) indicates significant difference, **(p<0.01) indicates extremely significant difference.

[0050] The ROC curve was used to analyze the early warning effect of blood bile acid CA, GCA, TCA or DCA on ketoacidosis and fatty liver syndrome in dairy cows. The results are shown in Table 10. The areas under the curve of blood CA, GCA, TCA and DCA were 0.889, 0.917, 0.875 and 0.933, respectively. This shows that blood CA, GCA, TCA or DCA has a good early warning effect on ketoacidosis and fatty liver syndrome in dairy cows. When blood CA>3.540μg / mL, the sensitivity of predicting ketoacidosis and fatty liver syndrome in dairy cows was 100%, and the specificity was 66.7%; when GCA>6.802μg / mL, the sensitivity of predicting ketoacidosis and fatty liver syndrome in dairy cows was 100%, and the specificity was 75%; when TCA>4.403μg / mL, the sensitivity of predicting ketoacidosis and fatty liver syndrome in dairy cows was 100%, and the specificity was 75%; when DCA>0.392μg / mL, the sensitivity of predicting ketoacidosis and fatty liver syndrome in dairy cows was 100%, and the specificity was 80%.

[0051] Table 10 Prediction parameters of blood CA, GCA, TCA, DCA

[0052]

[0053] Experimental example

[0054] Ten Holstein cows from 3-14 days postpartum were randomly selected from Jiusan Farmland Dairy Cooperative in Heilongjiang Province, and blood was collected from the tail vein to detect bile acid levels using LC-MS technology. Ketosis fatty liver syndrome in dairy cows was predicted based on the standards of CA>3.540μg / mL, GCA>6.802μg / mL, TCA>4.403μg / mL or DCA>0.392μg / mL, and the prediction effect was evaluated based on the gold standard of ketoacidosis fatty liver syndrome in dairy cows (BHBA>1.4mmol / L, Y<1).

[0055] The results are shown in Table 11. There were 5 cows with ketosis fatty liver syndrome, 5 of which were detected based on the CA>3.540μg / mL index, 5 were detected based on the blood GCA>6.802μg / mL index, 5 were detected based on the TCA>4.403μg / mL index, and 4 were detected based on the DCA>0.392μg / mL index.

[0056] Table 11 Case study of blood bile acid testing for ketosis and fatty liver syndrome

[0057]

[0058] Note: + indicates that the cow tested positive for ketotic fatty liver syndrome, - indicates that the cow tested positive for healthy liver syndrome.

[0059] In order to make the identification result more accurate, the biochemical detection kit of Sinopharm Biotechnology Co., Ltd. was used to detect the blood GLU (healthy group GLU>2.8; diseased group GLU<2.8), AST (healthy group AST<100; diseased group AST>100), NEFA (healthy group NEFA<0.8; diseased group NEFA>0.8) and BHBA content of dairy cows on Mindray BS-350S fully automatic biochemical analyzer and calculate the Y value, as shown in Table 12. By comparing Table 11 and Table 12, it can be seen that the blood biochemical indicators of the present invention can well predict whether dairy cows suffer from ketoacidosis fatty liver syndrome.

[0060] Table 12 Blood GLU, AST, NEFA and BHBA content and Y value detection ketosis fatty liver syndrome implementation case

[0061]

[0062]

[0063] It can be seen from the above embodiments that the present invention provides a system and application for predicting ketoacidosis and fatty liver syndrome in dairy cows by using blood biochemical indicators. The system of the present invention can be used to non-invasively monitor the incidence of ketoacidosis and fatty liver syndrome in dairy cows. Compared with existing detection methods, the detection indicators are simpler and the clinical operation is easier.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of a reagent for measuring blood biochemical indicators in the preparation of a reagent for predicting ketoacidosis and fatty liver syndrome in dairy cows, characterized in that: The blood biochemical indexes include taurocholic acid or deoxycholic acid; When taurocholic acid>4.403μg / mL or deoxycholic acid>0.392μg / mL in the blood of dairy cows, it is predicted that the cows suffer from ketosis fatty liver syndrome.

2. A system for predicting ketoacidosis fatty liver syndrome in dairy cows, characterized in that: The system includes: Data acquisition module: used to obtain the concentration data of biochemical indicators in the blood of dairy cows, wherein the biochemical indicators include: taurocholic acid or deoxycholic acid; Result determination module: used to receive the concentration data acquired by the data acquisition module, and predict the dairy cow ketoacidosis fatty liver syndrome according to the concentration data.

3. The system according to claim 2, characterized in that The method for predicting ketoacidosis fatty liver syndrome in dairy cows based on concentration data is: when taurocholic acid>4.403μg / mL or deoxycholic acid>0.392ug / mL in the blood of dairy cows, it can be predicted that the dairy cows suffer from ketoacidosis fatty liver syndrome.

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