RU2801829C1 - Method for assessing endogenous intoxication in cattle - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: saliva is sampled and centrifuged, then the concentration level of the middle molecules is measured by ultraviolet spectrophotometry at a wavelength of 254 nm. A sample of cattle saliva is used, taken in the morning before feeding, in an amount of at least 3 ml, which is centrifuged at 4000 rpm for 20 minutes. In addition, albumin concentration is measured. When the concentration level of middle molecules is more than 0.5 units. op. sq. and albumin is less than 0.1 g/l, endogenous intoxication is detected.

EFFECT: effective non-invasive method for assessing endogenous intoxication in cattle.

1 cl, 1 tbl, 2 ex

Description

The invention relates to veterinary medicine and can be used in the evaluation of endogenous intoxication in cattle.

In recent years, the study of the parameters of endogenous intoxication in the body plays an important role.

Endogenous intoxication (EI) is a complex multicomponent process caused by pathological activity of endogenous products or dysfunction of natural detoxification systems in the body. The substrate responsible for the occurrence of the pathological effects of endogenous intoxication is considered to be protein substances - medium-weight molecules (MSM). It has been established that these compounds have different structures, and their molecular weight is 500-5000 daltons. It has been proven that an excess of MSM has a toxic and mutagenic effect, their individual components suppress cellular immunity, nonspecific reactivity, erythropoiesis, inhibit a number of enzymes, etc. Determining the concentration of MSM in biological media of the body is one of the most informative and accessible methods for assessing the severity of endogenous intoxication.

A known method for determining endogenous intoxication by the content of low and medium molecular weight substances in the blood, when the blood serum is released from high molecular weight proteins by boiling followed by centrifugation, then spectrophotometry of the supernatant aqueous solution is performed in the wavelength range from 200 to 330 nm, after which the estimated indicator is calculated substances of low and medium molecular weight. Based on the spectra obtained, the estimated content of substances of low and medium molecular weight in the blood serum (Δ) is calculated, and at a value of Δ more than 50%, the presence of endogenous intoxication is determined (RF patent No. 2324943).

A known method for diagnosing the syndrome of endogenous intoxication, including blood sampling, determination of medium mass molecules (MSM) in blood serum by direct spectrometry of the deproteinized supernatant obtained after precipitation of proteins with a solution of trichloroacetic acid, at wavelengths (λ) of 280 nm and 254 nm, where additionally determine the MSM at λ 238 nm, calculate the index of medium molecules as the sum of the quotient of the value of the MSM at λ 280 nm to the same value at λ 254 nm and the quotient of the value of the MSM at λ 238 nm to the same value at λ 280 nm and at the value an index of medium molecules of more than 4.5 diagnoses endogenous intoxication (RF patent No. 2475749).

A known method for assessing the functional state of the liver of cattle, including a clinical examination of the animal and a biochemical blood test, in the blood serum determine the concentration of medium-weight molecules (MSM) at a sample irradiation wavelength of 254 nm and ketone bodies. In the case of an MSM concentration above 0.185 units. op. sq. in combination with the concentration of ketone bodies above or equal to 1.033 mmol/l, a pathology of the functional state of the liver in cattle is detected (RF patent No. 2773414).

The disadvantage of these methods is the inability to use them for non-invasive assessment of the level of endogenous intoxication in the body of cattle, since when implementing these methods, blood is used as the studied biological fluid, and to obtain a sample, it is necessary to perform invasive manipulation, which, in addition, may be difficult due to features of the anatomical structure of the vessels or the age of the examined animal.

Therefore, the development and introduction into laboratory practice of new non-invasive methods for studying biological fluids alternative to blood is one of the promising areas of science. Recently, the attention of researchers to the study of the properties of human and animal saliva has increased, as a material with unique properties and diagnostic capabilities. Saliva contains many biological molecules, the change in concentration of which can be used to detect various diseases, as well as to control the body's response to treatment.

A known method for diagnosing endogenous intoxication, when a drop of mixed saliva is dried and the structure of the resulting facies is analyzed under a microscope. The low severity of endogenous intoxication is judged by the presence of dark pigmentation in the very center of the central zone of the facies. The average severity of endogenous intoxication is judged by the presence of dark pigmentation in the form of a ring along the edge of the central zone of the facies. A high degree of endogenous intoxication is judged by the presence of a dark one (RF patent No. 2395087).

The disadvantage of this method is the very high sensitivity of the process of crystallogenesis of biosubstrates to environmental factors, as well as the obligatory condition for obtaining saliva facies by drying for several hours from sampling, which makes the use of crystallography methods inaccessible in animal husbandry. This is especially true for ruminants, which include cattle, since they have a multi-chamber stomach, divided into four sections: scar, net, book and abomasum. Swallowed food feed in cattle will first fall into the vestibule of the scar, and then into the scar, from which after some time it returns to the oral cavity for re-chewing and thorough wetting with saliva. Therefore, saliva of cattle contains a large amount of plant residues and in its native form is unsuitable for assessing endogenous intoxication, including crystallographic methods.

Closest to the claimed technical solution is a method for assessing endogenous intoxication by studying saliva, described in the article "The relationship between the processes of endogenous intoxication, lipid peroxidation and antioxidant protection in kickboxers by studying saliva" authors - Velskaya L.V., Turmanidze A.V. and Turmanidze V.G. (Journal "Sports Medicine: Science and Practice". - 2018. - V. 8. - No. 1. - P. 10-16. https://wvvw.smjoumal.ru/jour/article/view/86?locale= ru_RU). In this method, to study the state of endogenous intoxication processes in athletes, saliva was taken in an amount of 2 ml, followed by centrifugation at 7000 rpm and determination of MSM by ultraviolet spectrophotometry at wavelengths of 254 and 280 nm (prototype).

However, this method is of little use for assessing endogenous intoxication in cattle using ruminant saliva.

The objective of the invention is to develop an effective non-invasive method for assessing endogenous intoxication in cattle.

The technical result is to increase the efficiency of a non-invasive method for assessing endogenous intoxication in cattle.

The technical result is achieved by the fact that the method for assessing endogenous intoxication in cattle, including the selection of saliva, its centrifugation, followed by determining the level of concentration of molecules of average mass by ultraviolet spectrophotometry at a wavelength of 254 nm, characterized in that a sample of cattle saliva is used, selected in the morning before feeding, in an amount of at least 3 ml, which is centrifuged at 4000 rpm for 20 minutes, the concentration of albumin is additionally determined, and if the concentration level of average mass molecules is more than 0.5 units. op. sq. and albumin less than 0.1 g/l is judged on the presence of endogenous intoxication.

The novelty of the proposed method is due to the fact that the possibilities of diagnosing endogenous intoxication in polygastric patients will be expanded.

votnyh non-invasive and accessible methods. The authors selected a method for sample preparation of cattle saliva to be able to determine the level of concentration of the medium mass molecule (MSM) in it. It has been established that the optimal parameters are saliva centrifugation at 4000 rpm for 20 minutes. An additional diagnostic marker in the assessment of endogenous intoxication in cattle is proposed to use the concentration of albumin in saliva, the decrease in the content of which is an important indicator of intoxication of the body.

The features that distinguish the claimed technical solution from the prototype are aimed at achieving a technical result and have not been identified in the study of patent and scientific and technical literature in this and related fields of science and technology and, therefore, meet the criterion of "inventive step".

The proposed method meets the criterion of "industrial applicability", since it can be used in veterinary medicine for the diagnosis of endogenous intoxication in cattle.

The method for assessing endogenous intoxication in cattle is as follows.

In cattle in the morning before feeding, saliva is taken in a volume of at least 3 ml, which is centrifuged at 4000 rpm for 20 minutes, 2 ml of centrifugate is taken, where the concentration of MSM and albumin is determined as follows: MSM - to 1 ml saliva centrifugate add 0.5 ml of trichloroacetic acid, mix thoroughly and after 5 minutes centrifuge at 3000 rpm for 30 minutes, after which 0.5 ml of the supernatant is transferred into a test tube with 4.5 ml of distilled water, mixed and spectrophotometered at a wavelength of 254 nm in a 1 cm cuvette against distilled water (the results are expressed in units of extinction (un. op. pl.)); albumin - determined by the colorimetric method (the results are expressed in g / l).

An example of a specific implementation of the method for assessing endogenous intoxication in cattle.

To prove the effectiveness of the proposed method, scientific and production studies were carried out in livestock farms in the Krasnodar Territory. A survey of the livestock of cattle was carried out, on the basis of which 100 cows were selected - 50 clinically healthy and 50 patients with hepatopathy. All 100 animals were diagnosed with endogenous intoxication by two methods: 1 - control, used the method for diagnosing EI according to the prototype; 2 - experience, where the diagnosis was carried out in accordance with the claimed method.

The results of studies substantiating the effectiveness of the proposed method are presented in the table.

From the data presented in the table, it can be seen that the effectiveness of methods for assessing EI in cattle is hardly comparable.

So, in the experimental group of 50 clinically healthy cows, laboratory studies showed the absence of EI in 46 animals, and EI was established in 4, which was due to the presence of latent inflammatory processes in animals (later, the incidence of these cows was recorded, 3 developed mastitis and 1 - pneumonia). The results obtained confirm the scientific data that the severity of endotoxicosis in the body, determined by the concentration of MSM in biological fluids, is ahead of the manifestation of the clinical picture of the disease. The method used in the control did not reveal EI in these cows.

For sick animals, there is a slight overlap of results between groups: in the control group, EI was registered in 12% of cows; the application of the claimed method made it possible to identify EI in 86% of the livestock with hepatopathology.

Therefore, the use of the proposed method will make it possible to conduct a non-invasive screening study of a large number of livestock as part of a clinical examination, to identify endogenous intoxication, to conduct in-depth diagnostics and start treatment in a timely manner, which will significantly increase the effectiveness of therapeutic measures.

Additional advantages of the effectiveness of the proposed method are less stress on animals (non-invasiveness) and reduced labor costs (vacuum systems are required for taking blood, the duration of the procedure and pain for the animal, and saliva is taken into any test tube without painful manipulations).

The most obvious are the following examples.

Example 1. Cow No. 1689 - in laboratory studies of saliva according to the proposed method, the concentration of MSM at a wavelength of 254 nm was 0.584 units. op. sq., albumin - 0.06 g/l, which indicates the presence of endogenous intoxication in the animal. A clinical examination of the cow revealed general depression, reduced chewing gum and rumination, dullness and ruffled hairline, hepatomegaly, body temperature was normal. Biochemical analysis of blood recorded: total protein - 65 g/l and albumin - 28.3 g/l; ALT - 39 U/l and AsAT - 112 U/l; total bilirubin - 7.8 µmol/l; cholesterol - 6.9 mmol / l. Ultrasound examination showed: the contours of the liver are smooth with roundings, they are visualized indistinctly, the sizes are increased; echogenicity changed, mostly increased; the number of echo-positive signals "light liver" is increased; a few hyperechoic inclusions are located in the parenchyma (most likely foci of lipomatosis); the vascular pattern is mostly unchanged. Conclusion: the cow was diagnosed with fatty hepatosis.

Example 2. Cow No. 1152 - in laboratory studies of saliva according to the proposed method, the concentration of MSM at a wavelength of 254 nm was 0.326 units. op. sq., albumin - 0.15 g/l, which indicates the absence of endogenous intoxication in the animal. During the clinical examination of the cow, all parameters corresponded to the norm. Biochemical analysis of blood recorded: total protein - 81 g/l and albumin - 35.7 g/l; ALT - 21 U/l and AsAT - 84 U/l; total bilirubin - 5.6 µmol/l; cholesterol - 4.7 mmol / l. Ultrasound examination revealed no pathological changes. Conclusion: the cow is healthy.

Thus, the use of the proposed method makes it possible to achieve the desired technical result - to increase the efficiency of assessing endogenous intoxication in cattle using non-invasive and affordable methods.

Claims (1)

A method for assessing endogenous intoxication in cattle, including the selection of saliva, its centrifugation, followed by determining the level of concentration of medium-weight molecules by ultraviolet spectrophotometry at a wavelength of 254 nm, characterized in that a sample of cattle saliva is used, taken in the morning before feeding, in an amount of at least 3 ml, which is centrifuged at 4000 rpm for 20 minutes, the concentration of albumin is additionally determined, and when the concentration level of molecules of average mass is more than 0.5 units. op. sq. and albumin less than 0.1 g/l judge the presence of endogenous intoxication.