Kit for detecting beef cattle nutrition metabolite level and application thereof

Abstract

The invention discloses a kit for detecting the beef cattle nutrition metabolite level and application thereof, and belongs to the technical field of animal husbandry detection.The kit for detecting the beef cattle nutrition metabolite level comprises a urease-indophenol method kit or a Beckmann Coulter method kit used for detecting urea nitrogen in beef cattle blood. The invention also discloses a non-esterified fatty acid detection kit for detecting energy factors in beef cattle blood. According to the invention, beef cattle metabolites such as beef cattle blood urea nitrogen and blood energy factors can be rapidly detected, and the nutritional status of beef cattle can be evaluated by effectively detecting symbolic metabolites such as urea nitrogen and energy factors existing in blood related to the health and nutrition level of beef cattle; feed raw material and formula adjustment can be performed for feeding management, body condition scoring and the like of beef cattle in different periods, so that the feed can be widely applied to animal husbandry and beef cattle industry.

Description

Technical Field

[0001] The invention relates to the technical field of animal husbandry detection, in particular to a kit for detecting the level of nutritional metabolites of beef cattle and an application thereof. Background Art

[0002] The purpose of raising beef cattle breeding cows is to produce high-quality calves and provide a high-quality source of seeds and cattle for the healthy and sustainable development of the beef cattle industry. In recent years, my country's beef cattle farms have been continuously put into production, and many large beef cattle farms with a single unit of 10,000 heads have emerged, and even beef cattle farms with a level of 10,000 heads or quasi-10,000 heads have appeared in China. At present, the correlation between the nutritional status of beef cows and their production efficiency, as well as the importance of high-quality and healthy beef production, are increasingly receiving high attention for the healthy development of the beef cattle industry. Especially for grassroots farmers, the output and quality of newborn calves are closely related to the economic benefits of the industry.

[0003] Through the analysis of the current status of research on factors affecting the efficient production performance of cows, the nutritional status of cows has always been a neglected issue. The production performance of cows is often improved by using reproductive biotechnology and other means to improve the reproduction rate and conception rate of cows and regulate the birth time of calves, so as to improve the seasonal survival rate of calves and reduce the labor intensity of farmers. However, with the continuous increase in the scale and number of cow farming, the health status and production performance of cows are not in a virtuous cycle. The metabolic diseases and body condition problems caused by insufficient or excessive nutrition in different physiological periods are becoming more and more prominent, resulting in the extension of the calf birth interval and the continuous increase in economic losses of farmers. On the other hand, although artificial insemination of cows is carried out in a timely manner, due to the differences in the nutritional status of cows, problems such as embryo abortion, birth of deformed calves, and insufficient milk production to maintain the maximum growth rate of calves often occur. Therefore, an evaluation basis and method for evaluating the nutritional status of cows is needed.

[0004] In summary, in order to solve these key issues that are directly related to the healthy development of the beef cattle industry and the direct benefits of farmers, it is necessary to develop a more efficient, accurate and rapid diagnostic technology that can monitor and evaluate the nutritional level of breeding cows in the beef cattle industry.

[0005] However, in the field of beef production, existing technologies for monitoring the health level and nutritional status of beef breeding cows face different challenges, mainly the following problems: 1. Lack of accuracy: Traditional monitoring methods rely too much on empirical observations rather than quantitative biochemical indicators, making it difficult to accurately assess the nutritional health and production performance of cows. 2. Data delay: The current technology lacks real-time or accurate real-time data feedback, resulting in insufficient response speed for adjustment measures of cow nutrition and reproductive status. 3. Low specificity: Existing technology is not sufficient to effectively distinguish between pregnant and non-pregnant cows using specific biomarkers, and it is difficult to accurately assess ovulation efficiency and nutritional needs after conception. 4. Low cost efficiency: Traditional detection methods often involve high-cost detection, long waiting time for results, and rely on expensive equipment and professionals, resulting in low resource utilization efficiency. Summary of the invention

[0006] The purpose of the present invention is to provide a kit for detecting the level of nutritional metabolites of beef cattle to solve the problems raised in the above background technology.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions: A kit for detecting the level of nutritional metabolites in beef cattle, comprising a urease-indophenol method kit or a Beckman Coulter method kit for detecting urea nitrogen in the blood of beef cattle, and a non-esterified fatty acid detection kit for detecting energy factors in the blood of beef cattle; The urease-indophenol method kit comprises potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, urease, phenol, sodium hypochlorite and sodium hydroxide; The Beckman Coulter method kit includes urease, 2-oxoglutarate, NADH and glutamate dehydrogenase; The non-esterified fatty acid detection kit comprises acyl-CoA synthetase, coenzyme A, adenosine triphosphate, 4-aminoantipyrine, ascorbic acid oxidase, sodium azide, buffer, acyl-CoA oxidase, peroxidase and 3-methyl-N-(β-hydroxyethyl)aniline.

[0008] Preferably, the urease is urease derived from Jack Bean.

[0009] Another object of the present invention is to provide a use of the above-mentioned kit for detecting the level of nutritional metabolites in beef cattle for non-diagnostic purposes.

[0010] Preferably, the method for detecting urea nitrogen in the blood of beef cattle using a urease-indophenol method kit comprises the following steps: Adding distilled water, potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, and urease to the blood of the tested beef cattle to react and obtain a product containing ammonia and carbon dioxide; Phenol, sodium hypochlorite and sodium hydroxide are added to a product containing ammonia and carbon dioxide to obtain a product containing blue indigophenol, and the urea nitrogen concentration in the blood of beef cattle is detected by the product.

[0011] Preferably, the final concentration of potassium dihydrogen phosphate is 12-16mmol / L; the final concentration of disodium hydrogen phosphate monohydrate is 40-45mmol / L; the final concentration of ethylenediaminetetraacetic acid is 34-36mmol / L; the final concentration of sodium nitroprusside is 1.5-2mmol / L; the final concentration of urease is 0.5-1.5g / L; the final concentration of phenol is 100-140mmol / L; the final concentration of sodium hypochlorite is 25-30mmol / L; and the final concentration of sodium hydroxide is 120-160mmol / L.

[0012] Preferably, the method for detecting urea nitrogen in the blood of beef cattle using the Beckman Coulter method kit comprises the following steps: After adding distilled water and urease to the blood of the tested beef cattle for reaction, 2-oxoglutaric acid, NADH and glutamate dehydrogenase are added for reaction to obtain a product containing blue L-glutamic acid, and the urea nitrogen concentration in the blood of the beef cattle is detected by the product.

[0013] Preferably, the method for detecting energy factors in beef cattle blood using a non-esterified fatty acid detection kit comprises the following steps: Using non-esterified fatty acids as markers of energy factors, acyl-CoA synthetase, coenzyme A, and adenosine triphosphate are added to the blood of tested beef cattle to react and obtain products containing acyl-CoA. Adding acyl-CoA oxidase, ascorbic acid oxidase, sodium azide and buffer to the product containing acyl-CoA to carry out oxidation reaction, thereby obtaining a product containing 2,3-trans-enoyl-CoA, hydrogen peroxide and hydroperoxide; 4-aminoantipyrine, peroxidase and 3-methyl-N-(β-hydroxyethyl)aniline are added to a product containing 2,3-trans-enoyl-CoA, hydrogen peroxide and hydroperoxide to react to obtain a final product, and the energy factor in the blood of beef cattle is detected by the product.

[0014] Preferably, the final concentration of acyl-CoA synthetase is 0.5-0.6U / mL; the final concentration of coenzyme A is 0.2-0.4mmol / L; the final concentration of adenosine triphosphate is 4-5mmol / L; the final concentration of 4-aminoantipyrine is 1-2mmol / L; the final concentration of ascorbic acid oxidase is 2-3U / mL; the final concentration of acyl-CoA oxidase is 10-15U / mL; and the final concentration of 3-methyl-N-(β-hydroxyethyl)aniline is 2-3mmol / L.

[0015] Another object of the present invention is to provide an application of the above-mentioned kit for detecting the level of nutritional metabolites of beef cattle in regulating the nutrition of beef cattle feed.

[0016] The kit provided by the present invention for detecting the level of nutritional metabolites of beef cattle can quickly detect metabolic substances of beef cattle such as blood urea nitrogen and blood energy factors, and can evaluate the nutritional status of beef cattle by effectively detecting the marker metabolites urea nitrogen and energy factors in the blood related to the healthy nutritional level of beef cattle, and can adjust feed raw materials and formulas for feeding management and body condition scoring of beef cattle at different stages, so it can be widely used in animal husbandry and beef cattle industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the chemical reaction route of the urease-indophenol method.

[0018] Figure 2 This is the chemical reaction route of the Beckman Coot process.

[0019] Figure 3 This is the result of detecting standard samples using the urease-indophenol method.

[0020] Figure 4 This is a graph showing the test results of the reaction time and color change of the urease-indophenol method.

[0021] Figure 5 This is a graph showing the results of repeated experiments using the urease-indophenol method.

[0022] Figure 6 This is the chemical reaction route for the non-esterified fatty acid detection method.

[0023] Figure 7 This is the result of testing the standard sample using the non-esterified fatty acid detection method.

[0024] Figure 8 This is a graph showing the test results of the reaction time versus color change of the non-esterified fatty acid detection method.

[0025] Fig. 9 This is a graph showing the results of repeated experiments using the non-esterified fatty acid detection method. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] In the process of efficient beef cattle breeding, the nutrition of breeding cows plays a vital role in the entire beef cattle industry. In view of the current problem that the nutritional level of beef cows is not taken seriously and the average production performance cannot be evaluated, the embodiment of the present invention will clarify whether the feed nutritional level of cows in different physiological periods is balanced through a method of quickly detecting the concentration level of characteristic nutritional metabolites in the blood of cows, and provide an evaluation and decision-making basis for the healthy breeding of cows and the regulation of nutritional levels.

[0028] In view of the above problems, the purpose of the embodiments of the present invention is mainly: 1. Improve accuracy: Develop a new method based on blood metabolite analysis to more accurately evaluate the health and nutritional status of beef cows. 2. Realize timely monitoring of the nutritional level of cows: Provide a technology that can timely track the nutritional and reproductive status of cows to ensure that feeding and management measures can be adjusted in time. 3. Enhance specificity: Innovative specific biomarker detection technology can accurately distinguish the difference in nutritional levels of cows during pregnancy and non-pregnancy. 4. Improve cost efficiency: Design an economical, efficient and easy-to-operate detection method to reduce dependence on expensive equipment and professional operators and improve work efficiency.

[0029] Specifically, in one embodiment of the present invention, a kit for detecting the level of nutritional metabolites in beef cattle is provided, which includes a urease-indophenol kit or a Beckman Coulter kit for detecting urea nitrogen (BUN) in the blood of beef cattle, and a non-esterified fatty acid detection kit for detecting energy factors (NEFA) in the blood of beef cattle; The urease-indophenol method kit includes potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid (EDTA), sodium nitroprusside dihydrate, urease, phenol, sodium hypochlorite solution and sodium hydroxide. In addition, the kit may also include urea as a standard.

[0030] The Beckman Coulter method kit includes urease, 2-oxoglutarate, NADH and glutamate dehydrogenase (GLDH); wherein NADH refers to reduced coenzyme I, which is the reduced state of nicotinamide adenine dinucleotide. In addition, the kit may also include urea as a standard.

[0031] The non-esterified fatty acid detection kit includes Acyl-coenzyme A synthetase (ACS), Coenzyme A (Coenzyme A), Adenosine triphosphate (ATP), 4-aminoantipyrine (4-aminoantipyrine), Ascorbate oxidase (AOD), Sodium azide (Sodium azide), buffer, Acyl-coenzyme A oxidase (ACOD), Peroxidase (POD) and 3-methyl-N-(beta-hydroxyethyl)-aniline (3-methyl-N-(beta-hydroxyethyl)-aniline, MEHA. The buffer can be a phosphate buffer with a pH of 7. In addition, the kit can also include oleic acid as a standard.

[0032] In a preferred embodiment of the present invention, the urease is urease derived from Jack Bean (Canavalia ensiformis).

[0033] In a preferred embodiment of the present invention, the method for detecting urea nitrogen in the blood of beef cattle using a urease-indophenol method kit comprises the following steps: S1. Add distilled water, potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, and urease to the blood of the tested beef cattle to react and obtain a product containing ammonia and carbon dioxide; S2. Add phenol, sodium hypochlorite and sodium hydroxide to the product containing ammonia and carbon dioxide to obtain a product containing blue indigophenol, and use the product to detect the urea nitrogen concentration in the blood of beef cattle.

[0034] In a preferred embodiment of the present invention, the final concentration of potassium dihydrogen phosphate is 12-16 mmol / L; the final concentration of disodium hydrogen phosphate monohydrate is 40-45 mmol / L; the final concentration of ethylenediaminetetraacetic acid is 34-36 mmol / L; the final concentration of sodium nitroprusside is 1.5-2 mmol / L; the final concentration of urease is 0.5-1.5 g / L; the final concentration of phenol is 100-140 mmol / L; the final concentration of sodium hypochlorite is 25-30 mmol / L; and the final concentration of sodium hydroxide is 120-160 mmol / L.

[0035] In a preferred embodiment of the present invention, the method for detecting urea nitrogen in the blood of beef cattle using the Beckman Coulter method kit comprises the following steps: After adding distilled water and urease to the blood of the tested beef cattle for reaction, 2-oxoglutaric acid, NADH and glutamate dehydrogenase are added for reaction to obtain a product containing blue L-glutamic acid, and the urea nitrogen concentration in the blood of the beef cattle is detected by the product.

[0036] In a preferred embodiment of the present invention, the method for detecting energy factors in beef cattle blood using a non-esterified fatty acid detection kit comprises the following steps: S1. Using non-esterified fatty acids as markers of energy factors, adding acyl-CoA synthetase, coenzyme A, and adenosine triphosphate to the blood of tested beef cattle for reaction to obtain a product containing acyl-CoA; S2, adding acyl-CoA oxidase, ascorbic acid oxidase, sodium azide, and buffer to the product containing acyl-CoA to carry out an oxidation reaction to obtain a product containing 2,3-trans-enoyl-CoA, hydrogen peroxide, and hydroperoxide; S3. Add 4-aminoantipyrine, peroxidase and 3-methyl-N-(β-hydroxyethyl)aniline to the product containing 2,3-trans-enoyl-CoA, hydrogen peroxide and hydroperoxide to react and obtain a final product, and use the product to detect the energy factor in the blood of beef cattle.

[0037] In a preferred embodiment of the present invention, the final concentration of acyl-CoA synthetase is 0.5-0.6U / mL; the final concentration of coenzyme A is 0.2-0.4mmol / L; the final concentration of adenosine triphosphate is 4-5mmol / L; the final concentration of 4-aminoantipyrine is 1-2mmol / L; the final concentration of ascorbic acid oxidase is 2-3U / mL; the final concentration of acyl-CoA oxidase is 10-15U / mL; and the final concentration of 3-methyl-N-(β-hydroxyethyl)aniline is 2-3mmol / L.

[0038] Example 1: This example provides a urease-indophenol method kit and method for detecting urea nitrogen in the blood of beef cattle, wherein the urease-indophenol method kit includes potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, urease from sword bean, phenol, sodium hypochlorite, sodium hydroxide and a certain amount of standard urea.

[0039] The production performance and reproductive efficiency of beef cows are significantly affected by their nutritional level, among which the concentration of urea nitrogen in the blood is one of the important influencing factors and can be used as a detection marker. Generally, when the concentration of urea nitrogen in the blood is 8-14 mg / dL, the production performance of the cow is good. In the embodiment of the present invention, the urease-indophenol method is used for detection. Specifically, the method for detecting urea nitrogen in the blood of beef cattle comprises the following steps: S1. Add distilled water, potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, and urease to the blood of the tested beef cattle to react and obtain a product containing ammonia and carbon dioxide; S2. Add phenol, sodium hypochlorite and sodium hydroxide to the product containing ammonia and carbon dioxide to obtain a product containing blue indigophenol, and use the product to detect the urea nitrogen concentration in the blood of beef cattle.

[0040] Among them, the final concentration of potassium dihydrogen phosphate is 14mmol / L; the final concentration of disodium hydrogen phosphate monohydrate is 42.9mmol / L; the final concentration of ethylenediaminetetraacetic acid is 34.8mmol / L; the final concentration of sodium nitroprusside is 1.8mmol / L; the final concentration of urease is 1g / L; the final concentration of phenol is 120mmol / L; the final concentration of sodium hypochlorite is 27mmol / L; and the final concentration of sodium hydroxide is 140mmol / L.

[0041] like Figure 1 As shown, in the above method, when distilled water and urease are added to urea, a primary reaction of ammonia and carbon dioxide can be carried out. At this time, hypochlorite and phenol are added to the generated ammonia to further react to form blue indophenol.

[0042] In addition, if Figure 2 As shown, the embodiment of the present invention can also use the Beckman Coulter method to detect urea nitrogen in the blood. This method is to add 2-ketoglutaric acid and NADH under the catalysis of glutamate dehydrogenase after the primary reaction of the urease-indophenol method to form blue L-glutamate.

[0043] The above two methods are both methods for detecting the urea concentration in the blood, and both are methods for detecting free protein in the blood through the blood concentration.

[0044] BUN method standard product analysis: Using the urease-indophenol method kit provided in Example 1 above, the effect of urea concentration changes on colorimetry was confirmed by testing different concentrations of standard products. After using the reagents contained in the kit to react with the standard products, the naked eye can distinguish high, medium, and low levels (specifically, Figure 3 As shown in the figure, the correlation with the analytical instrument results is as high as 99%. At the same time, compared with the commercially available reagent (GLDH method), the sensitivity of the concentration response is more linear. In addition, the effect of the reaction time of the urease-indophenol method kit on the color change was tested, and it was found that the reaction time of the kit did not change much before and after 7 minutes, indicating that the test time error is small (as shown in the figure). Figure 4In order to prove the repeatability of the urease-indophenol method kit provided in the embodiment of the present invention, the same experiment was repeated 8 times using a standard product, and the coefficient of variation (CV)% was 1.08% based on the OD value (630 nm), indicating that the urease-indophenol method kit provided in the embodiment of the present invention has high repeatability between tests (specifically as shown in FIG. Figure 5 as shown).

[0045] Example 2: This example provides a non-esterified fatty acid detection kit and method for detecting energy factors in beef cattle blood, wherein the non-esterified fatty acid detection kit includes acyl-CoA synthetase, coenzyme A, adenosine triphosphate, 4-aminoantipyrine, ascorbic acid oxidase, sodium azide, buffer, acyl-CoA oxidase, peroxidase, 3-methyl-N-(β-hydroxyethyl)aniline and a certain amount of standard oleic acid. The buffer can be a phosphate buffer with a pH of 7.

[0046] In order to detect the energy factor released in the blood of cows, the embodiment of the present invention uses non-esterified fatty acids (NEFA) as a marker. Specifically, the method for detecting the energy factor in the blood of beef cattle using a non-esterified fatty acid detection kit includes the following steps: S1. Using non-esterified fatty acids as markers of energy factors, adding acyl-CoA synthetase, coenzyme A, and adenosine triphosphate to the blood of tested beef cattle for reaction to obtain a product containing acyl-CoA; S2, adding acyl-CoA oxidase, ascorbic acid oxidase, sodium azide, and buffer to the product containing acyl-CoA to carry out an oxidation reaction to obtain a product containing 2,3-trans-enoyl-CoA, hydrogen peroxide, and hydroperoxide; S3. Add 4-aminoantipyrine, peroxidase and 3-methyl-N-(β-hydroxyethyl)aniline to the product containing 2,3-trans-enoyl-CoA, hydrogen peroxide and hydroperoxide to react and obtain a final product, and use the product to detect the energy factor in the blood of beef cattle.

[0047] Among them, the final concentration of acyl-CoA synthetase is 0.53U / mL; the final concentration of coenzyme A is 0.31mmol / L; the final concentration of adenosine triphosphate is 4.3mmol / L; the final concentration of 4-aminoantipyrine is 1.5mmol / L; the final concentration of ascorbic acid oxidase is 2.6U / mL; the final concentration of acyl-CoA oxidase is 12U / mL; the final concentration of 3-methyl-N-(β-hydroxyethyl)aniline is 2.4mmol / L; the final concentration of buffer is 50mmol / L; sodium azide can be added twice: once alone, with a final concentration of 0.062%; the other time it is added together with the buffer, with a final concentration of 0.05%.

[0048] like Figure 6 As shown, in the above method, a substance containing a carboxyl group (RCOOH) (such as standard oleic acid), ATP and coenzyme A (CoA) are reacted with acyl-CoA synthetase (ACS) to convert into acyl-CoA (acyl-CoA), and the acyl-CoA formed in this process is oxidized by acyl-CoA oxidase (acyl-CoA oxidase, ACOD) to generate 2,3-trans-enoyl-CoA, hydrogen peroxide and hydrogen peroxide; then, peroxidase (peroxidase) and 4-aminoantipyrine (4-aminoantipyrine), 3-methyl-N-ethyl-(β-hydroxyethyl)-aniline (MEHA) are added to react to form a blue-purple reaction product, and the energy factor in the blood can be detected by this method. Therefore, the kit provided in the embodiment of the present invention can quickly detect the energy factor in the blood of beef cattle.

[0049] NEFA standard sample analysis: The non-esterified fatty acid detection kit provided in the embodiment of the present invention is used to analyze the standard oleic acid of different concentrations, and the three levels of high, medium and low can be distinguished by naked eyes (specifically as follows Figure 7 As shown in the figure, the correlation of the results obtained using the analytical instrument also reached 99%. In addition, by extending the reaction time of the non-esterified fatty acid detection kit and observing the changes over time, it was found that the reaction did not change much around 7 minutes of the kit's reaction time, thus confirming that the test time error was small (as shown in the figure). Figure 8 as shown).

[0050] In order to verify the repeatability of the non-esterified fatty acid detection kit provided in the embodiment of the present invention, the same standard was analyzed 8 times. The results showed that the coefficient of variation (CV)% based on the OD value (530 nm) was 2.27%, indicating that the non-esterified fatty acid detection kit provided in the embodiment of the present invention has a high degree of repeatability (specifically, Fig. 9 as shown).

[0051] Example 3: This example is an experiment on the effect of the nutritional components of beef cattle TMR feed on the metabolic substances in cattle blood, as follows: In order to analyze the relationship between changes in blood metabolites of beef cattle and the nutritional level of cows according to the nutritional adjustment of feed, the composition of five total mixed feeds (TMR) was analyzed. The general nutritional composition analysis results of each TMR feed (A, B, C, D, E) are shown in Table 1 below.

[0052]

[0053] According to the above five feed types, energy factors such as free NEFA, glucose (GLU), protein (BUN), cholesterol (CHOL) and liver protein factors (AST and GGT) in the blood of cows were analyzed. The analysis results are shown in Table 2:

[0054] As a result of the blood metabolite analysis in the embodiment of the present invention, the GLU concentration in the blood of cows on farms fed with TMR-C feed was the highest, while the GLU concentration in the blood of cows on farms fed with TMR-E feed was the lowest. NEFA is an important indicator for the mobilization of body fat utilization in beef cattle. The NEFA concentration in the blood of cows on farms fed with TMR-E feed was the highest, while the GLU concentration was the lowest; followed by farms fed with TMR-D, TMR-A, TMR-C and TMR-B feeds. It is reported that when the body fat of cows decreases, the concentration of NEFA in the blood will temporarily increase. Therefore, the reason why the NEFA in the blood of cows on farms fed with TMR-E feed is high can be judged to be insufficient feed intake caused by the low energy concentration of TMR-E feed.

[0055] Under normal conditions, the increase in NEFA concentration in the blood may be mainly due to the mobilization of body fat caused by insufficient feed intake. When cows have pathological problems, such as liver dysfunction, ketosis, and reproductive disorders caused by hypertonia of the sympathetic nervous system due to parturition stress, the NEFA concentration in the blood will also increase. In addition, cows fed TMR-E feed not only showed an increase in NEFA levels, but also showed very high levels of liver function indicators AST and GGT concentrations. Therefore, it can be suspected that individual beef cattle have decreased liver function or reproductive disorders. In addition, cows fed TMR-E feed had the highest blood BUN concentration, and TMR-C had the lowest blood BUN concentration. Studies have shown that when the intake of protein and nitrogen compounds increases or liver function decreases, the level of BUN in the blood will increase. According to the results of nutrient intake analysis (as shown in Table 3), cows fed TMR-E feed had the highest protein intake (0.92kg). Although the total nutrient intake was lower than that of other feeds, the increase in BUN concentration in the blood was mainly due to the high protein (CP) intake. Therefore, the protein level of TMR-E feed needs to be slightly reduced.

[0056]

[0057] Example 4: This example is an experiment on the effect of feed intake on blood metabolites of cows, as follows: The nutritional components of the basic diet of the embodiment of the present invention are shown in Table 4:

[0058] Since the experimental group was designed based on mixed feed, the nutrient concentrations of the embodiments of the present invention were 80%, 100% and 120% of the basic diet, respectively. After the above feed was fed to the cows, the concentration changes of metabolites in the blood were shown in Table 5:

[0059] Example 5: This example provides a feed nutrition regulation strategy for beef cows, as follows: In the above embodiment of the present invention, the feed nutrients and the concentration of metabolites in the blood are analyzed. After the concentration of blood metabolites is confirmed according to the feeding type, the feed is adjusted and improvement measures are sought. The specific strategy for controlling the concentration of blood metabolites and the body condition (BSC) score of the cow is as follows: For feed TMR-A, the analysis of metabolites in the blood and body condition of cows was as follows: the concentration of metabolites in the blood was normal, and the average BCS body condition score of the cows was 5.0, which was high; the control strategy was: it was recommended to reduce the supply of concentrate feed.

[0060] For the feed TMR-B, the analysis of metabolites in the blood and body condition of cows was: the concentration level of metabolites in the blood was normal, and the BCS body condition score of the cows was good; the control strategy was: none.

[0061] For feed TMR-C, the analysis of metabolites and body condition in the cow's blood was: the cow's BSC body condition score was 4.18, which was high; the regulation strategy was: when the cow's BCS was maintained between 3.0-4.0, the cow's production performance was excellent; studies have shown that when BCS=4 or higher, the cow's production performance decreased by 30%, so it is necessary to reduce the amount of feed to lower the BCS score.

[0062] For the feed TMR-D, the analysis of metabolites in the blood and body condition of cows showed: the concentration of metabolites in the blood was normal; the control strategy was: none.

[0063] For the feed TMR-E, the analysis of metabolites in the blood and body condition of cows is: the concentrations of NEFA and BUN in the blood are slightly higher, and the intake of BCS and TDN of cows is slightly lower; the control strategy is: when BUN is 8-14mg / dL, the cow's performance is good. The higher the BUN concentration, the lower the reproductive performance of the cow. The BUN concentration in the blood of cows fed TMR-E feed is slightly higher than that of other types of feed, so the CP ratio needs to be adjusted; the reduction of BCS may lead to a decrease in feed intake, so the energy concentration of the feed needs to be slightly increased.

[0064] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A kit for detecting the level of nutritional metabolites of beef cattle, characterized in that: It includes a urease-indophenol method kit or a Beckman Coulter method kit for detecting urea nitrogen in the blood of beef cattle, and a non-esterified fatty acid detection kit for detecting energy factors in the blood of beef cattle; The urease-indophenol method kit comprises potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, urease, phenol, sodium hypochlorite and sodium hydroxide; The Beckman Coulter method kit includes urease, 2-oxoglutarate, NADH and glutamate dehydrogenase; The non-esterified fatty acid detection kit comprises acyl-CoA synthetase, coenzyme A, adenosine triphosphate, 4-aminoantipyrine, ascorbic acid oxidase, sodium azide, buffer, acyl-CoA oxidase, peroxidase and 3-methyl-N-(β-hydroxyethyl)aniline.

2. The kit for detecting the level of nutritional metabolites of beef cattle according to claim 1, characterized in that: The urease is urease derived from sword bean.

3. Use of the kit for detecting the level of nutritional metabolites in beef cattle as claimed in claim 1 or 2 in detecting the level of nutritional metabolites in beef cattle for non-diagnostic purposes.

4. The use according to claim 3, characterized in that: The method for detecting urea nitrogen in the blood of beef cattle using a urease-indophenol method kit comprises the following steps: Adding distilled water, potassium dihydrogen phosphate, disodium hydrogen phosphate monohydrate, ethylenediaminetetraacetic acid, sodium nitroprusside, and urease to the blood of the tested beef cattle to react and obtain a product containing ammonia and carbon dioxide; Phenol, sodium hypochlorite and sodium hydroxide are added to a product containing ammonia and carbon dioxide to obtain a product containing blue indigophenol, and the urea nitrogen concentration in the blood of beef cattle is detected by the product.

5. The use according to claim 4, characterized in that: The final concentration of potassium dihydrogen phosphate is 12-16mmol / L; the final concentration of disodium hydrogen phosphate monohydrate is 40-45mmol / L; the final concentration of ethylenediaminetetraacetic acid is 34-36mmol / L; the final concentration of sodium nitroprusside is 1.5-2mmol / L; the final concentration of urease is 0.5-1.5g / L; the final concentration of phenol is 100-140mmol / L; the final concentration of sodium hypochlorite is 25-30mmol / L; and the final concentration of sodium hydroxide is 120-160mmol / L.

6. The use according to claim 3, characterized in that: The method for detecting urea nitrogen in the blood of beef cattle using the Beckman Coulter method kit comprises the following steps: After adding distilled water and urease to the blood of the tested beef cattle for reaction, 2-oxoglutaric acid, NADH and glutamate dehydrogenase are added for reaction to obtain a product containing blue L-glutamic acid, and the urea nitrogen concentration in the blood of the beef cattle is detected by the product.

7. The use according to claim 3, characterized in that: The method for detecting energy factors in beef cattle blood using a non-esterified fatty acid detection kit comprises the following steps: Using non-esterified fatty acids as markers of energy factors, acyl-CoA synthetase, coenzyme A, and adenosine triphosphate are added to the blood of tested beef cattle to react and obtain products containing acyl-CoA. Adding acyl-CoA oxidase, ascorbic acid oxidase, sodium azide and buffer to the product containing acyl-CoA to carry out oxidation reaction, thereby obtaining a product containing 2,3-trans-enoyl-CoA, hydrogen peroxide and hydroperoxide; 4-aminoantipyrine, peroxidase and 3-methyl-N-(β-hydroxyethyl)aniline are added to a product containing 2,3-trans-enoyl-CoA, hydrogen peroxide and hydroperoxide to react to obtain a final product, and the energy factor in the blood of beef cattle is detected by the product.

8. The use according to claim 7, characterized in that: The final concentration of acyl-CoA synthetase is 0.5-0.6U / mL; the final concentration of coenzyme A is 0.2-0.4mmol / L; the final concentration of adenosine triphosphate is 4-5mmol / L; the final concentration of 4-aminoantipyrine is 1-2mmol / L; the final concentration of ascorbic acid oxidase is 2-3U / mL; the final concentration of acyl-CoA oxidase is 10-15U / mL; and the final concentration of 3-methyl-N-(β-hydroxyethyl)aniline is 2-3mmol / L.

9. Use of a kit for detecting the level of nutritional metabolites of beef cattle as claimed in claim 1 or 2 in regulating the nutrition of beef cattle feed.

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