RU2565412C1 - Method of diagnostics of ruminal digestion disorder in ruminants - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: in the rumen contents the amount of substances having a "medium" (500-5000 D) molecular weight range is determined. The clinical health state of animals is evidenced by the content level of middle molecules (CMM) in the rumen fluid, determined on the wave of 237 nm - less than 2.0 c.u., on the wave of 254 nm - less than 1.0 c.u., on the wave of 280 nm - less than 1.0 c.u. The presence of pathology of rumen digestion is evidenced by the increase of CMM on the wave of 237 nm as over 2.0 c.u., on 254 nm - over 1.0 c.u., and on 280 nm - over 1.0 c.u.

EFFECT: method enables to detect pathology at early stages of development, and in patients - to assess the degree of impairment of rumen digestion and effectiveness of the treatment.

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Description

The invention relates to veterinary medicine and animal husbandry, relates to a method for assessing cicatricial digestion and diagnosing its disorders in ruminants in order to take measures to optimize feeding, increase the safety and productivity of animals.

A feature of the digestion of ruminants is the ability to digest fiber of vegetable feed with relatively high efficiency and in large volumes. This ability is inherent in ruminants due to the presence of a multi-chamber stomach and ruminant process. Along with the transformation of the components of the feed into compounds accessible for animals to assimilate, vitamins of groups B and K are formed in the rumen, in an amount that fully meets the body's need for them. The breakdown products of plant proteins, non-protein nitrogen-containing substances of the diet and urea coming from the blood are absorbed in the rumen by bacteria and ciliates. As a result, a microbial protein is formed, providing 20-30% of the animal’s protein needs [Aliev A.A. Metabolism in ruminants. - M: SIC “Engineer”. - 1997. - 564 p .; Georgievsky V.I. Physiology of farm animals. M .: Agropromizdat, 1990 .-- 511 p .; Kurilov N.V., Krotkova A.P. Physiology and biochemistry of digestion of ruminants. M .: Kolos. - 1971. - 432 p.]. Violation of cicatricial digestion is accompanied by a decrease in the activity of the processes of destructuring the components of the diet and increasing the microbiological mass, which negatively affects the feeding efficiency and the level of animal productivity.

Animal health depends to a large extent on the usefulness of the functions of the pancreas. Scar dysfunctions are relatively widespread among ruminants. They are the earliest symptoms of damage to the digestive system, the incidence of which is within 40% of all diseases [Inozemtsev V.P. Prevention of non-communicable diseases basis of animal preservation / Veterinary medicine. - 2000. - No. 11. - S. 9-13]. In this case, primary (atony and hypotension of the scar, acidosis and alkalosis of the scar, etc.) and a secondary lesion of the pancreas are observed. Secondary dystonia of the pancreas occurs in 19.5-31.6% of cows. In the etiological structure of secondary dystonia, complex hypomicroelementosis (33.8%) and hypovitaminosis A, B, Ε (29.9%), hepatodystrophy (11.7%), rumen acidosis (10.8%), and ketosis (8.2%) and to a lesser degree osteodystrophy (5.6%) [Khmelkov Y.T. Secondary hypotension of the scar and its determining factors / Ya.T. Khmelkov, E.G. Yakovleva // Bull. scientific works / Belgorod State Agricultural Academy. - Issue 3. - Belgorod, 2005. - S. 42-46].

To identify scar dysfunctions, physical research methods are most often used. Inspection determines the shape of the abdomen and the degree of filling of hungry pits. The method of palpation assesses the sensitivity and tension of the scar wall, strength, rhythm and the frequency of its contractions. Auscultation allows you to determine the frequency and phase of contraction of the pancreas. In order to unify and increase the informational content of clinical research methods, a tonometry of the scar and a graphical recording of its abbreviations are carried out using various designs of ruminographs and tonometers [Kumsky, Sh.A. Digestive apparatus diseases. - M .: Kolos 2006. - 563 p .; Kalyuzhny I.I. Acidosis of the scar. - Saratov, Volga Book Publishing House. - 1996. - 238 p.].

However, changes in ruminography, auscultation, palpation and tonometry of the scar are already symptoms of diseases of the pancreas, indicating the presence of a clinical stage of pathology. They have a relatively low diagnostic value at the subclinical stage of the pathology and limited informational content in assessing the mechanisms of the development of the disease.

In clinical practice, studies of scar contents are relatively often performed. In this case, the diagnostic pathophysiological significance of studies depends on the spectrum of the determined parameters. The most commonly determined color, smell, texture, pH of the contents and the rate of formation of sediment in it. The most informative is the pH of the cicatricial content.

A known method of remote control of the pH in the rumen of cows with a special device, which includes a pH sensor inserted into the cavity of the rumen, external equipment providing an radiopaque image of the rumen [US 6694161 B2; US 10 / 125.319; published on February 17th. 2004]. This device includes sophisticated equipment that limits its use in clinical practice.

A known method of monitoring the pH of cicatricial content using a bolus of dielectric material, which is introduced into the cavity of the scar. A sensor located in the bolus detects and transmits to the receiver electromagnetic parameters of the cicatricial contents [US 8694091 B2; PCT / SE 2011/051154; public date 8.04. 2014].

This research method is safe and allows for a long time to control the acidity of the scar contents. However, this device is acceptable mainly for scientific research, as the need for special equipment limits its use in clinical practice.

The portable equipment for obtaining and analyzing cicatricial fluid has wider capabilities, which allows, based on generally accepted parameters, to identify subclinical stages of the disease in cattle and small cattle in the field. The equipment includes a yawner, a set of gastric tubes, a pH meter and reagents for setting it [US 6241688 B1; US09 / 142.064; public date 5.06. 2001]. This equipment simplifies the process of sampling cicatricial content and determining pH, but its diagnostic capabilities are limited to detecting only acidosis or alkalosis of the scar.

A method for assessing the degradation of feed fibers makes it possible to evaluate the functionality of cicatricial digestion. This method includes the steps of sampling cicatricial content, introducing into it a substrate of carbohydrates and incubating in an airtight container [US 20090272889 A1; US 12 / 405,650; publ. November 5, 2009]. This method significantly expands the information content of the results, allowing to identify the early stages of digestive disorders in the rumen and the mechanisms of their development. However, this method is applicable only when conducting scientific research, because requires special equipment and is acceptable only when examining individual animals.

In order to study the state of digestion in the rumen, the number of bacteria and ciliates, the concentration of ammonia, the total amount of volatile fatty acids, acetic, propionic and butyric acids are also determined in its content [Prevention of metabolic disorders in farm animals / Ed. A.A. Aliyev. - M: Agropromizdat, 1986. - 384 p .; Shevelev N.S., Grushkin A.G., Tarakanov B.V. The physiological role of microbiota in cicatricial digestion / Agricultural Biology, 2005, No. 6, S. 9-13]. The indicators determined at the same time expand the possibility of identifying the early stages of pathology, mechanisms of dysfunction of the pancreas and secondary lesions of other organs. However, the need to increase the informative value of diagnostic tests and deepen knowledge of the pathogenesis of cicatricial digestion disorders determine the need for new research methods. The main direction of the search for new tests is the reflection of processes preceding changes in pH, enzyme activity and the number of microflora.

A known method for determining the cicatricial content of leukocytes and / or cytokines using specific antibodies, which allows to evaluate the activity of inflammation, the permeability of blood vessels of the rumen wall and the risk of secondary lesions, as well as to identify allergic properties in feed and feed additives [WO 2011154924 A1; PCT / IB 2011/052540; public date 12/15/2011]. However, the indicators of the number of leukocytes and the activity of cytokines do not reflect the main mechanisms of impaired cicatricial digestion, in particular the mechanism of defaunization of the cavities of the pancreas. The main reason for the death of bacteria, yeast and ciliates is the adverse living conditions or the presence of substances with a cytotoxic effect. Moreover, if the change in the environment in the rumen can be judged by the pH level, then there are no tests to assess the toxicity of cicatricial content in clinical practice.

Thus, the theoretical justification of the proposed method is the assumption that the cause of the decrease in the number of bacteria, yeast and ciliates in the rumen may be the presence of toxic substances in the rumen contents. Given that scar defaunization is a constant component of the pathogenesis of most diseases of the pancreas, an assessment of the toxicity of cicatricial content will not only clarify the pathogenesis, but also determine the direction of medical manipulations.

As a prototype, a method for determining the blood content of substances with a molecular weight of from 500 to 5000 D, conventionally called "average", was chosen, because the molecular weight of most toxins of endogenous origin is in this weight range. The principle of the method is the precipitation of high molecular weight (> 5000 D) compounds with an acid solution, followed by determination of optical density, the parameters of which in certain spectral regions are proportional to the content of fractions of “medium” molecules [Alekhin, Yu.N. Endogenous intoxication in animals and their diagnosis (guidelines) / Yu.N. Alekhine - Voronezh, 2000. - 12 p.].

However, the prototype is aimed at determining the content of "average" molecules in the blood and does not reflect their number in the content of the scar, which excludes the possibility of evaluating the toxicity of the contents of the scar and identifying the pathology of cicatricial digestion. In addition, the method indicated in the prototype is adapted for blood analysis and it is not known whether it is acceptable for the study of cicatricial contents.

The invention solves the problem of developing a method for diagnosing disorders of cicatricial digestion in ruminants with the achievement of a technical result - an assessment of the content of toxic substances in cicatricial content.

To solve the problem and achieve a technical result in a method for diagnosing cicatricial digestion disorders in ruminants by photometric determination of the level of “average” molecules (CCM) in the animal’s body at 237, 254 and 280 nm according to the invention, CCM is determined in the cicatricial content, toxic content is evaluated substances and in the presence of SSM at a wave of 237 nm more than 2.0 conv. units, at a wavelength of 254 nm - more than 1.0 srvc. units and at a wavelength of 280 nm - more than 1.0 srvc. units diagnose the occurrence of pathology of cicatricial digestion, given that in clinically healthy animals this indicator is when determining it at a wavelength of 237 nm <2.0 conv. units, at a wavelength of 254 nm <1.0 srvc. units, at a wavelength of 280 nm <1.0 srvc. units

The method allows to evaluate the content of toxic substances in cicatricial content. An increase in CCM is used for early diagnosis of cicatricial digestive disorders and is observed before the onset of traditional symptoms (pH, microflora, etc.), and the severity of CCM changes in patients reflects the severity of cicatricial disturbance.

The essence of the method is illustrated by examples.

Example 1. Determination of the optimal variant of the studied material and the concentration of trichloroacetic acid in the study of the content of "medium" molecules in the cicatricial content.

The object of the study was 30 clinically healthy calves aged 5-6 months, which were kept in a typical room with optimal microclimate parameters. The animals received a diet developed in accordance with the norms of feeding, which included mixed grass hay 2.0 kg, grain barley 8.0 kg, compound feed (P63-1) 2.3 kg, beet molasses 0.4 kg.

Sampling of cicatricial contents was carried out using a mouth-esophageal probe 3 hours after the next feeding. Samples of cicatricial content obtained were examined within 2 hours after collection. A sample from each animal was filtered through 4 layers of gauze and divided into 9 equal parts. No. 1, 4 and 7 were diluted 2 times with distilled water before the study, No. 2, 5 and 8 were diluted 4 times, No. 3, 6 and 9 were not diluted (native material was examined). In the study of samples No. 1 and 2, for the precipitation of proteins used a 9% solution of trichloroacetic acid (TCA), for samples No. 3 and a 4-10% solution of TCA, and for No. 5 and 6-11% TCA solution. When determining the content of “medium” molecules (CCM), 0.5 ml of trichloroacetic acid solution was added to 1 ml of cicatricial content. After 2 minutes, the contents were centrifuged for 15 minutes at 3000 rpm. 4.5 ml of distilled water was added to 0.5 ml of the supernatant, and the solution was photographed at a wavelength of 254 nm against a comparison solution (1 ml of 10% trichloroacetic acid and 29 ml of distilled water).

The results are presented in table No. 1, from the data of which it is seen that the dilution of the scar content is accompanied by a decrease in CCM when using a 9% solution of TCA by 44.8%, a 10% solution of TCA by 50.0% and in the sample with 11% TCA solution - 43.4%. A 4-fold dilution resulted in a decrease in SSM of 2.28; 3.58 and 2.52 times, respectively.

Thus, dilution of cicatricial content leads to a decrease in CCM, but the severity of this decrease does not correspond to the degree of dilution. Therefore, more objective data is obtained using undiluted cicatricial content.

A change in the concentration of TCA also affects the determined indicator (see Table 1). Thus, in the study of undiluted samples, the coefficient of variation was minimal when using a 10% solution of TCA, while it increased by 1.7 and 1.3% when using a 9 and 11% solution, respectively.

Thus, a change in the concentration of the working solution of TCA has an unreliable change in the coefficient of variation of CCM, however, the lowest variability was noted when using a 10% solution of TCA.

The results showed that the most stable and objective indicators of CCM are obtained in the study of undiluted cicatricial content using 10% TCA solution for protein precipitation.

Example 2. Unification of the method for determining SSM and the study of its diagnostic information content.

Using the modeling method with the help of special diets, we reproduced the most common variants of cicatricial indigestion in cows - acidosis and alkalosis.

The object of the study was clinically healthy cows of the red-motley and Holstein-Friesian breed, of which three groups were formed on the 40-50th day of lactation. All animals involved in the experiment were kept on a leash in individual machines. For each group, diets were developed that are identical in content of nutrients, vitamins and minerals, but differ in the level of protein and carbohydrates. Animals from group No. 1 (control, n = 100) received a basic diet corresponding to the recommended norms. In addition to the basic diet, cows of group No. 2 (n = 30) were given 4 kg of beet molasses, and group No. 3 (n = 30) were given 250 g of brand A urea. They were accustomed to a high dose of urea for 15 days.

On the first day of the experiment, as well as on days 7 and 25, a detailed examination of the cows was carried out with the sampling of blood and cicatricial contents. Samples were taken 3 hours after the first (morning) feeding. Most of the cicatricial samples were examined in their native form within 2 hours after collection, but 10 samples from each group were preserved with 10% formalin solution (4 drops per 10 ml of contents).

The results of monitoring cows from the control group showed that their clinical condition did not change significantly during the experiment. In animals from group No. 2, changes in clinical status were observed only on the 25th day of the experiment, when the majority (93.3%) of them showed a decrease in productivity by 8.7% (group No. 1 - 19.4 kg, No. 2 - 17 , 7 kg) and the fat content in milk by 19.0% (35.9 and 29.1 g / l). In addition, these cows showed a decrease in appetite, gnashing of teeth and lacrimation. In group No. 3, in individual animals (10%), diarrhea and a temporary decrease in appetite were observed during the first 7 days of the experiment. On the 25th day of the experiment, a decrease in productivity by 5.1% was noted, and muscle tremble was observed in 16.7% (5 animals) of animals. Most cows experienced increased heart rate (110-160 / min) and respiration (28-34 / min).

From the data in Tables Nos. 2 and 3, it can be seen that at the beginning of the experiment, all the animals involved in the experiment had cicatricial digestion within the normal range.

Animals from group No. 1 (Tables 2 and 3) remained healthy throughout the entire experiment, and the observed changes in the indices of cicatricial content and blood were caused by the functional load on the body of cows during milking.

In cows from group No. 2, in comparison with the initial level, there were no significant changes in the rumination and composition of the scar content during the first 7 days of the experiment. On the 25th day, the pH decreased by 1.7%, the shades of gray (milk) intensified in the color of the contents, and the smell became acidic. In comparison with the initial level, the sedimentation rate increased by 90.0%, the number of protozoa decreased by 62.1%, and the number of scar reductions by 4 times.

In animals of group No. 3, significant changes in the indices of cicatricial digestion were noted only on the 25th day of the experiment, when, in comparison with the initial level, the pH increased by 0.9 units, the sedimentation time decreased by 2.8 times, and the number of infusoria by 39 , 0%. The color of the contents became darker, and the smell was initially ammonia, and then putrefactive shades appeared. The number of ruminant movements during chewing one gum decreased by 27.6%, and rumination weakened by 2 times.

Thus, using the modeling method, rumen acidosis was formed in cows from group No. 2 and rumen alkalosis in group No. 3. Changes in the motility of the scar and the properties of its contents, characteristic of each metabolic profile, appeared during the period from the 7th to the 25th day of the action of adverse feeding factors.

Using the basic version of the method for determining SSM (see Example 1) on the 25th day of the experiment, we evaluated the dynamics of SSM in the range from 200 to 300 nm. An analysis of the results showed that in the range from 200 to 233.0 ± 0.28 nm, a high variability of indicators is observed. So the coefficient of variability (C) in healthy animals was 37.2%, in cows with rumen acidosis - 29.5%, and in alkalosis - 26.8%. Then, in healthy animals, the diagram forms relatively stable intervals from 233.7 ± 0.32 to 245.2 ± 0.5 nm (C = 10.14%) and from 250.2 ± 0.5 to 285.0 ± 0.025 nm (C = 8.07%). In patients with acidosis and alkalosis, there is only one relatively stable interval (C = 6.8%) in the range from 234.9 ± 0.45 to 266.0 ± 0.6 nm.

Thus, in the study of cicatricial content, the most acceptable measurements are SSM in the ranges of 235-245 and 250-285 nm. Given this, and also to preserve the possibility of comparing blood indices with cicatricial content, it is necessary to determine CCM at wavelengths of 237; 254 and 280 nm.

The results obtained made it possible to form an optimal variant of the method for determining SSM in cicatricial content.

Reagents: 1) working solution: 10% solution of trichloroacetic acid; 2) comparison solution: mix 1 ml of working solution and 29 ml of distilled water.

Materials for the study: scar content, which before the study is filtered through 4 layers of gauze. If it is not possible to investigate within 2 hours after collection, the samples are preserved with a 10% formalin solution, adding 4 drops per 10 ml of filtered contents.

Equipment: centrifuge, spectrophotometer.

The course of determination. Into a centrifuge tube make 1 ml of scar contents and 0.5 ml of working solution, mix with a glass rod. After 2 minutes, centrifuged for 15 minutes at 3000 rpm. Add 0.5 ml of the supernatant and 4.5 ml of distilled water to a clean tube, mix and photometer. Measure the extinction at long wavelengths of 237, 254 and 280 nm in a cuvette with a layer thickness of 1 cm against the comparison solution.

The results of the study of CCM in serum and cicatricial content are presented in table No. 3, from the data of which it can be seen that in the control animals during the experiment there were no significant deviations of the CCM in blood and cicatricial content.

In cows from group No. 2, the SMS in the cicatricial content at a wavelength of 237 nm increased 2.7 times on day 7 of the experiment, and 3.0 times on day 25. At the wavelength of 254 nm on the indicated days of the experiment, the growth of indicators was 46.8 and 82.4%, and at 280 nm - 21.0 and 34.8%. In group No. 3, when analyzed at a wavelength of 237 nm, the CCM in animals increased 3.9 times after 7 days, and 4.1 times after 25 days. When determining the indicator at a wavelength of 254 nm, it increased by 43.6% and 1.6 times, and at 280 nm - by 43.5 and 64.6%.

In serum, a significant increase in CCM on the 7th day of the experiment was observed only at a wavelength of 254 nm. On day 25, in cows from group No. 2, the CCM increased at a wavelength of 237 nm by 2.2 times, 254 nm by 43.4% and 280 nm by 23.7%. In animals from group No. 3, similar indicators increased 2.3 times, by 40.5 and 30.9%, respectively.

Comparative studies of native and canned cicatricial contents showed that the introduction of a formalin solution does not have a significant effect on the determined parameters. The exception is SSM, which in a canned sample increases on average by 0.08 ± 0.011 (-0.05 ÷ 0.22) srvc. units In this case, the most pronounced effect of the preservative was noted when using wavelengths of 237 and 280 nm. However, the observed effect of formalin is close to the range of individual variation of CCM in animals (5-12% or 0.08-0.150 conv. Units), which gives reason to ignore the effect of preservative on this indicator.

Calculation of the CCM reference interval in cicatricial content in healthy animals was performed using the Reference Value Advisor program based on Microsoft Excel [Geffre A., Concordet D., Braun JP, Trumel C. Reference Value Advisor: a new freeware set of macro instructions to calculate reference intervals with Microsoft Excel, Vet Clin Pathol. 2011 40 (1) 107-112]. The results showed that the reference interval of healthy animals (n = 100) SSM when determined at a wavelength of 237 nm is 0.5-2.0 srvc. units, 254 nm 0.3-1.0 conv. units and 280 nm 0.25-1.0 conv. units

Thus, the normal values of CCM in the cicatricial content should be considered: at a wavelength of 237 nm - <2.0; at 254 and 280 nm - <1.0 srvc. units (conventional units of optical density).

The degree of connection of the CCM indicator in the cicatricial content with metabolic profile disorder in the rumen was evaluated on the basis of the chi-square (χ ² ) connection reliability indicator [Khamitov GP, Vedernikova TI Probabilities and statistics, Irkutsk: BSUEP, 2006. - 272 p.]. The information content of CCM indicators as an early diagnosis test was evaluated by comparing its level on the 7th day of the experiment and the frequency of cicatricial digestion disorders at the end of the experiment (Table 4). The results obtained from this showed that in the early stages of acidosis and alkalosis (day 7 of the experiment), when there are no significant deviations of traditional indices of cicatricial digestion, there are already significant deviations of CCM. So, the χ ² value at a wavelength of 237 nm was 124.8, and the determination coefficient (r _d ) was 0.77 (Ρ0.001). When determining the SSM at 254 nm, these parameters were 90.0 and 0.49, and at 280 nm - 65.7 and 0.4 (P0.001).

This indicates that there is a reliable correlation between the level of SMM in cicatricial content in ruminants and metabolic disturbances in the rumen when using a strong wavelength of 237 nm, and a moderate degree at 254 and 280 nm. The share of the effect of increasing CCM at a wavelength of 237 nm (above 2.0 srvc), 254 nm (above 1.0 srvc) and 280 nm (above 1.0 srvc) on cicatricial digestion respectively 77.0; 49.0 and 40.0%, which allows us to regard it as a reliable indicator of early diagnosis with a high level of prognostic value.

SSM, determined at a wavelength of 237 nm, exceeds the norm in 94.0% of animals with severe symptoms of acidosis and alkalosis (day 25 of the experiment), and the correlation coefficient (r) with changes in pH was 0.88. At a wavelength of 254 nm, these indicators were at 254 nm - 85.0% and 0.5, and at 280 nm - 72.5% and 0.46.

Thus, a new method for the diagnosis of cicatricial digestion disorders in ruminants is characterized by a relatively high prognostic and diagnostic information content. Its use allows you to identify violations of cicatricial digestion in the early stages of their development, which is the basis for adjusting the diet or preventive therapy, which is more effective and less costly. The use of this test also allows you to assess the severity of metabolic disorders in the rumen, which is the basis for determining treatment tactics and evaluating its effectiveness.

Claims (1)

A method for diagnosing cicatricial digestion disorders in ruminants by photometric determination of the level of “average” molecules (CCM) in the animal’s body at 237, 254 and 280 nm, characterized in that the CCM is determined in the scar content, proteins are pre-precipitated with trichloroacetic acid solution, the content is evaluated toxic substances and in the presence of SSM at a wave of 237 nm more than 2.0 conv. units, at a wavelength of 254 nm - more than 1.0 srvc. units and at a wavelength of 280 nm - more than 1.0 srvc. units diagnose the occurrence of pathology of cicatricial digestion, given that in clinically healthy animals this indicator is when determining it at a wavelength of 237 nm <2.0 conv. units, at a wavelength of 254 nm <1.0 srvc. units, at a wavelength of 280 nm <1.0 srvc. units