RU2837378C1 - Method for determining copper level in muscular tissue of holstein cattle - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary medicine, namely to clinical and laboratory diagnostics. Hair is sampled from cattle. Then, atomic emission analysis is performed to estimate the level of iron in the hair and to calculate the copper content by the original formula.

EFFECT: method enables lifetime and non-invasive determination of copper content in muscle tissue by iron content in hair, which in turn enables to avoid diseases associated with copper imbalance.

1 cl, 2 tbl, 1 ex

Description

The invention relates to animal husbandry, ecology, veterinary science and is intended for use as a test for assessing the level of copper in the muscles of Holstein cattle.

Copper is an essential microelement for the normal functioning of animals and plants. It is a part of more than 30 copper-containing proteins and enzymes that play an important role in accelerating metabolic processes, enhancing tissue respiration, and accelerating the process of glucose oxidation. Together with iron, it plays an important role in hematopoiesis. Copper activates the reaction of blood heme formation. The formation of this complex reduces the activation energy of the hemoglobin synthesis reaction. The main copper-containing enzymes are oxygenases and hydroxylases. Oxidases include such an important respiratory enzyme as cytochrome oxidase, which catalyzes the final stage of tissue respiration. Ceruloplasmin, as a copper-containing protein of blood plasma, catalyzes iron oxidation, participating in hematopoiesis. In addition, it performs a transport function, regulates the balance of copper and ensures the removal of its excess from the body [1].

It is necessary to identify and control the level of copper in the body of animals, since its imbalance can lead to diseases, which in turn will affect the quality of the resulting products.

There is a method for determining copper in cattle meat [2]. However, this method only allows one to determine the concentration of copper in muscle tissue of animals that have already been killed, or it requires a biopsy, which is difficult. A method for determining copper in fish muscles by the concentration of copper in scales [3] and in sheep liver by the content of testosterone and estradiol in the blood serum of animals [4] is also known. However, these methods are only suitable for fish of the Stizostedion lucioperca species and Romanov sheep, respectively.

The evaluation of the prototype [2] did not show any signs of similarity with the proposed method in terms of the research substrate. The presented solution is non-invasive and can obtain results in vivo.

The technical task of the invention is to evaluate the amount of copper contained in the muscles of Holstein cattle after conducting a chemical study of hair obtained from living animals and determining in it such a chemical element as iron, for which a regression equation is used.

The task is accomplished by determining the iron level in cattle hair with subsequent calculation of copper concentration in muscles using the regression equation: x = 1.0 – 0.0022 × y, where x is the copper content “mg/kg” in muscles, y is the iron content “mg/kg” in hair.

Example of implementation

Hair samples were taken from Holstein bulls aged 16-18 months. The animals were kept under technological conditions typical for dairy cattle breeding for up to 6 months, then until the end of rearing the bulls were kept in groups on deep non-replaceable straw bedding. The maintenance corresponded to zoohygienic and veterinary-sanitary requirements for cattle. Feeding was complete and standardized. The feeding and watering front, microclimate indicators were typical for the species and age of the animals. Rations changed depending on the age and live weight of the bulls, the planned average daily gain was 800-1000 g. Watering was carried out from our own sources of domestic and drinking water supply of the second class with the elimination of deviations to the requirements of SanPiN 2.1.4.1074-01. Cattle were vaccinated in accordance with the plan of veterinary measures.

It is necessary to collect samples of long terminal hair from animals. Then their sample weighing at least 2 g is immersed in a 50 ml laboratory beaker with water at a temperature of plus 40 to plus 60 ° C for 2-3 hours. After the specified time, the water is drained, and the laboratory beaker is placed under running water with high pressure, and the hair is washed until visible dirt disappears, breaking it with a glass rod. Then the sample is poured with bidistilled water, and the beaker with it is placed in an ultrasonic bath (operating frequency 35 kHz) for 60 minutes. After the specified time, the water is drained, the sample is filled with 40% ethyl alcohol and again placed in an ultrasonic bath for 60 minutes. Then the alcohol is drained, the sample is filled with bidistilled water and again subjected to cleaning in an ultrasonic bath for 1 hour. The sample is dried in a drying oven at 40°C.

For analysis, a 100 mg sample is weighed and placed in a quartz cup. The cup with the sample is placed in a cold quartz furnace. The furnace temperature is raised to 250°C. The sample is held at this temperature for 15 minutes. Then the temperature is raised to 450°C and held for 15 minutes. The sample is left in the furnace to cool to room temperature. After charring, the sample is ground in quartz cups to a black powder state. Then, a 10 mg sample is taken from the prepared sample and mixed with 50 mg of graphite powder and 40 mg of a spectroscopic buffer (15% sodium chloride and graphite powder). 20 mg of the resulting mixture are taken directly for analysis.

The iron content in hair is determined using atomic emission spectral analysis using a two-jet arc plasma torch "Fakel" and a multi-channel atomic emission spectrometer "Grand" manufactured by VMK-Optoelectronics LLC (Russia).

A Perkin Elmer 360 atomic absorption spectrophotometer (USA) is used for muscle tissue analysis. Sample preparation for atomic absorption analysis occurs in the following sequence:

1. After washing in a soap solution, the dishes are washed with tap water and rinsed with bidistilled water, then dried.

2. Samples weighing 100 g are ground to a homogeneous mass, then dried in an oven at a temperature of 60-70°C for about 12 hours until constant mass.

3. From the resulting dry residue, 3 g are weighed out and ashed in a muffle furnace at a temperature of 500-550°C. After 10-15 hours, mineralization is complete, and the ash becomes gray or white.

4. After this, the samples are cooled at room temperature.

5. The resulting ash residue weighing 3 g is dissolved in 3 ml of 50% hydrochloric acid.

6. The samples are heated on an electric stove to obtain a dry residue, then it is transferred to a flask by diluting in 25 ml of distilled water.

The resulting finished solution is tested for copper content.

The data on the content of copper in muscle tissue and iron in hair are presented in Table 1. The iron level is characterized by high phenotypic variability, and the copper level is relatively low. The ratio of the extreme variants was 1:15 and 1:21, respectively. The established values can be preliminarily used as the normative values for this species and breed of animals in the conditions of Western Siberia.

Table 1 - Content of chemical elements in muscle tissue and hair of Holstein bulls, mg/kg

Chemical element ±S Q ₁ Q ₃ IQR Lim Cu _{muscle tissue} 0.97±0.09 0.55 0.90 0.35 0.1-2.1 Fe _hair 123.80±13.00 66.50 157.90 91.40 19-290

±S -arithmetic mean and error of the mean; Q 1 and Q 3-first and third quartile; IQR-interquartile range; Lim-maximum and minimum value.

Table 2 shows the mean correlation between skeletal muscle copper and hair iron levels in Holstein cattle.

Table 2 - Associations between muscle copper and hair iron in Holstein bulls

Correlating features n r±Sr x= a + b × y Cu _{muscle tissue} - Fe _hair 32 -0.561±0.151 x = 1.0 - 0.0022 × y

r ± Sr is the correlation coefficient and the error of the correlation coefficient, x= a + by is the regression equation.

On this basis, a model for predicting the concentration of copper in muscle tissue is constructed: x = 1.0 - 0.0022 × y, where x is the copper content "mg/kg" in muscles, y is the iron content "mg/kg" in hair. It is estimated using the coefficients of the regression model, all of which are individually statistically significant (P<0.001), as is the model as a whole, since the F-statistics value was 16.9 (P<0.001), the distribution of the residuals was normal.

The use of the presented regression model allows us to predict the copper content in muscle tissue of Holstein bulls during their lifetime by establishing the concentration of iron in the hair of the animals.

This method makes it possible to evaluate such an animal interior parameter as copper content in the muscle tissue of cattle during life and non-invasively, using only hair as the subject of the study.

Sources of information:

1. Aidarova F.R. Biological role of copper and detection of copper in pharmaceutical preparations / F.R. Aidarova, O.V. Neelova // Advances in modern natural science. - 2011. - No. 8. - P. 221-222.

2. GOST 26931-86. Raw materials and food products. Methods for copper determination. - M: Standartinform, 2010. - 10 p.

3. Patent No. RU 2555518 C1 Russian Federation, IPC G01N 33/48, G01N 33/12. Method for determining copper content in fish muscle tissue / Korotkevich O.S., Miller I.S., Konovalova T.V. et al.; applicant and patent holder Novosibirsk State Agrarian University. - No. 2014131162/15; declared 28.07.2014; published 10.07.2015.

4. Patent No. 2765236 C1 Russian Federation, IPC G01N 33/53. Method for assessing copper content in sheep liver / Saurbaeva R.T., Andreeva V.A., Klimanova E.A. et al.; applicant and patent holder Novosibirsk State Agrarian University. - No. 2021106117; declared 09.03.2021; published 26.01.2022.

Claims (1)

A method for determining the copper content in muscle tissue of Holstein cattle, characterized in that hair is collected from an animal of the given species, atomic emission analysis is performed, assessing the iron level in the hair, and the copper content is calculated using the regression equation: x = 1.0 – 0.0022 × y, where x is the copper content “mg/kg” in muscle, y is the iron content “mg/kg” in hair.