Rickets in young cattle

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Rickets in young cattle

Ligomina I.P., Polissia National University, Sokoluk V.M., Polissia National University, Sokulskiy I.M., Polissia national university, Gutyj B.V., Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv

Introduction

Agriculture is considered as the primary sector of the national economy for a large part of the world's population [1]. Ukraine is justly acknowledged as one of the biggest agricultural nations in the world [2]. Nowadays, raising livestock is considered as one of the vital and significant sectors of agriculture since it provides valuable nutrients and healthy food for the country's population, raw materials for various industries, hormones, medicines, other biologically active substances for medical and veterinary purposes, and useful organic fertilizers necessary for agriculture. Furthermore, livestock production and its high energy and nutritional products have improved the living standards of the population. It also solves many of the socio-economic problems faced by agricultural enterprises [3, 4].

One of the most important areas in agriculture is the increase of livestock production [5]. A properly developed system of measures aimed at the timely prevention and elimination of losses caused by animal diseases, common among them metabolic disorders, is crucial for the effective implementation of this task [6]. ricket young cattle

The animal body is a complex, dynamic, self-regulating system. Continuous functioning of all functional systems ensures its sustainability [7]. Changes in physiological parameters lead to significant changes in physical processes in biological tissues, such as alterations in temperature, electrical conductivity, dielectric constant, haematological parameters, magnetic perception and potentials, among others [8].

The human and animal body persistently consume energy and various substances throughout their lifespan. Their replenishment sources are nutrients, primarily obtained from the environment. Several structures within the body necessitate an uninterrupted flow of energy. This energy enters an animal's body along with the nutrient-rich feed through the digestive system [9, 10]. The primary functions of the digestive system include the physical and chemical processing of feed, nutrient absorption, and elimination of indigestible components into the environment [11]. Most metabolic disorder-related diseases exhibit a latent stage of progression, which is a unique characteristic [12]. Disorders of phosphorus and calcium metabolism are widespread pathologies in animals, for instance, osteodystrophy in adults and rickets in young animals. They affect almost all body structures, causing a general weakening and the development of secondary, more lifethreatening pathologies. Nutritional deficiencies of macro- and microelements cause growth and developmental delays, particularly in young animals. In acute forms of the pathological process, it even leads to irreversible changes in the structure of the body of calves. The disease becomes hazardous for cows during the pregnancy period, when the animal requires more mineral and vitamin nutrition [13].

These complications can lead to disorders in various systems including but not limited to nervous, muscular, skeletal, respiratory, cardiovascular, digestive, and reproductive. They can also cause anaemia, gastrointestinal and respiratory diseases, emaciation in adult animals, and hypotrophy in young animals, ultimately weakening their ability to resist infectious diseases. This pathology is characterised by multiple complex causes and a complicated clinical manifestation. For instance, a nutritional origin disorder of phosphorus-calcium metabolism could be caused by a deficiency of a complex of nutrients, minerals, and vitamins required by the body [14].

Results of the monitoring revealed that one of the factors responsible for diseases in young animals under industrial farming is the decreased immunological response of the body, which could be a result of primary immunodeficiency, insufficient and unbalanced nutrition, non-adherence to feeding rules and regimes, feed toxicosis, as well as stress factors related to industrial farming such as inadequate animal housing and low qualification of service personnel.

Biochemical blood parameters are widely considered to fully reflect protein, fat, carbohydrate, vitamin, macro- and microelement, hormone, as well as water and mineral characteristics of an organism [19, 20, 21]. Furthermore, various factors, including the provision of nutrients, vitamins and minerals to the body of animals, impact the quantitative characteristics of these blood parameters of cattle [22].

Metabolism, referring to the consumption and breakdown of substances and energy, occurs in a complex chemical process within the body, beginning from nutrient intake down to the excretion of final metabolic products [23]. Morphological and functional changes at various stages in organ cells are accompanied by metabolic disorders with the accumulation of intermediate metabolic products within the body [24]. It is widely recognised that every illness is linked with metabolic dysfunctions to some degree [25]. As per classical definitions, metabolism is the exchange of substances and energy between the body and the environment and also comprises a series of processes involving the transformation of matter and energy that take place directly within living organisms [26]. It is widely recognised that the metabolism of substances and energy forms the basis of the vital activity of organisms and is one of the most significant distinct characteristics of organic matter. The metabolic process, governed by multilevel regulatory systems, comprises many enzyme compounds that facilitate a sequence of chemical reactions ordered temporally and spatially. These genetic biochemical reactions take place successively in particular cell regions, which are facilitated by the principle of cell compartmentalisation. Ultimately, the substances that are ingested by the body are transformed into specific tissue substances and end products that are excreted from the body. During any biochemical transformation, energy is released and absorbed [27].

Hyperphosphatemia, decreased total calcium and increased alkaline phosphatase activity were observed in animals showing signs of mineral metabolism disorders, nutritional anaemia and leukocytosis [28].

Cellular metabolism executes four main functions: extraction of energy from the environment and its conversion into high-energy chemical compounds enough to satisfy the energy demands of the cell; formation of intermediate compounds from exogenous substances that are precursors of high molecular weight cellular components; synthesis of proteins, nucleic acids, carbohydrates, lipids and other cellular components from these precursors; synthesis and degradation of specific biomolecules, the formation and decay of which are linked to the performance of specific functions within a cell [29].

Metabolic disorders, which may occur as a result of insufficient or imbalanced nutrition in terms of nutrients and biologically active substances, non-compliance with the feeding regime and the structure of the diet, taking into account the physiological state and the lactation period, feeding of low- quality silage and haylage containing excess oil, leading to a decrease in milk production of cows, but also to the development of diseases caused by metabolic disorders: osteodystrophy, A and D hypovitaminosis, ketosis, postpartum hypocalcemia and hypophosphatemia); heart disease (myocardial dystrophy); liver disease (cirrhosis, hepatodystrophy); digestive system (foregut dystonia, rumen acidosis, rennet shift); and development of polymorbid (multiple) internal pathology [30, 31, 32].

In order to maintain metabolic processes at a normal physiological level, it is necessary to ensure that all nutrients are constantly supplied to the cow's body in optimal amounts (a deficiency or excess of at least one of them causes an imbalance). In most cases, a complex deficiency of nutrients is emphasised, which makes it much more difficult to detect pathological changes in the body [33]. Optimal feeding and housing conditions for pregnant cows ensure the birth of healthy, well-developed calves. [34].

Nutritional value should be understood as the ability of the feed (food) to meet the animal's need for nutrients: proteins, fats, carbohydrates, vitamins and minerals. The higher the nutritional value, the better the health and productivity of the animals and the quality of the products. The nutritional value of a feed is determined by its chemical composition and the digestibility of the nutrients in the digestive tract.

Physiologically, the animal's body cannot function properly unless it is supplied with the optimum amount of macro- and micronutrients in water and feed. This is also reflected in the blood composition, as haematological changes during animal ontogeny are associated with feeding, housing and health factors [35].

Thus, it has been scientifically proven and confirmed in practice that regular analysis of the composition of the feed and constant monitoring of the biochemical parameters of the blood serum of heifers can normalise the metabolism in the animal's body and prevent the development of hepatosis and metabolic disorders at the beginning of lactation. All this makes it possible to better exploit the genetic potential of post-calving milk production in cows, maintain animal health, extend the period of their economic use and ultimately increase the economic efficiency of the farm [36].

One of the most pressing problems of age-related morpho- functionality of dairy breeds is the formation of the physiological and biochemical status of the calf's organism during the periods of early postnatal ontogenesis (newborn, milk feeding and intensive growth), which are among the most critical in the process of individual development of animals, as they are associated with profound morphological and biochemical changes in organs, tissues and systems of the organism as a whole. It should be noted that the periods of early postnatal ontogenesis are characterised by high plasticity of the calf's body, intensive metabolism and increased need for nutrients and biologically active substances. Although the process of individual development is genetically determined, the intensification of production changes the functional activity of the body's physiological systems, which is reflected in the safety of the herd, growth rate and future productivity. Therefore, maintaining and correcting the health of calves during their development is an important issue in modern veterinary medicine. It is known that the physiological maturity of newborn animals depends on the physiological, biochemical and morphological status of the mother cow during the dry period, a change in which initiates the appearance of disorders in the functional system "mother-fetus",

which affect the harmonious development of the fetus in a pregnant cow. Therefore, by correcting the vital processes in the body of dry cows, it is possible to increase the viability of newborn calves [37].

In order to study the processes of growth and development of young animals and the biochemical basis of adult productivity, as well as to determine some genetic characteristics of breeds and individual animals, it is necessary to make extensive use of biochemical methods of blood, tissues and organs, as well as to study the metabolic processes taking place in the animal's body [38]. This is understandable because the state of the blood, as a liquid mobile connective tissue, is an indicator and diagnostic marker of the health of the body [39].

The process of adaptation of the body is associated with a serious forced restructuring of the systems that ensure homeostasis. The immune system maintains the biochemical antigenic individuality of the person. The body's remote effector systems are represented primarily by immune antibodies and complement. Immune antibodies are secreted by special clones of cells of the central and peripheral organs of immunogenesis - B lymphocytes and plasma cells. Antibodies are highly specialised proteins that react specifically with the antigens that cause their formation [40].

One of the main directions for improving breeding methods is the search for reliable markers for predicting early maturity and productive traits in livestock. This is due to the close relationship between biochemical processes in the body and productive traits. Many years of experimental research have shown that the entire metabolic process between the body's cells and the external environment takes place through the blood, which transports nutrients to the cells and removes metabolic products from them. The biochemical parameters of the blood can be considered as the

most important characteristics of the functional state and potential capabilities of the pig organism. Changes in blood composition indicate that metabolic systems can be a link between the genotype of an organism and its phenotype. The processes taking place in the body affect the composition and properties of the blood and can be used to assess the intensity of metabolism, which determines the productive qualities of animals [41].

All the most important biochemical reactions in the body's internal environment take place with the obligatory participation of mineral components: calcium, phosphorus, other macro- and microelements - vitamins D and A, and proteins. Bone tissue contains the majority of calcium in the whole body. Bone tissue consists of an organic matrix - collagen - and a mineral component - calcium phosphate. The presence of collagen in bone tissue gives it strength and elasticity. Biomineralisation of bones with calcium salts is a very complex process involving a large number of vitamins (groups B, A, D, E, K). Bone tissue contains collagen with a specific amino acid composition, which is the organic part of bone. The inorganic matrix consists of hydroxyapatite crystals, a biomineral compound whose main elements are calcium and phosphorus [42]. If there is a deficiency of calcium in the diet and subsequently in the fluid part of the blood, its resorption from the bone tissue is stimulated by the action of the parathyroid hormone and vitamin D. If the calcium level in the blood is sufficient, the thyroid hormone calcitonin prevents its exit from the skeletal bones [43].

A special role is played by vitamin D, whose action is not limited to the regulation of calcium and phosphorus homeostasis, but also extends to lipid and protein metabolism, influences cell proliferation and differentiation, and participates in the regulation of the functional activity of organs [44, 45]. It is known that the development and continued productivity of young animals depend on the active action of vitamin D [46]. It is also known that the absorption and regulation of vitamin D metabolism in the body depends on a number of factors, including the levels of other vitamins [47, 48]. For example, scientists have found that calciferol regulates calcium-phosphorus metabolism, affects the proliferation and differentiation of cells of organs and tissues of embryos, synthesises lipids, proteins, enzymes and hormones, and is actively involved in regulating the functions of many organs and systems of the body, including the cardiovascular, digestive and other systems [49, 50].

Recent studies have greatly expanded our understanding of the spectrum of biological activity of vitamin D in human and animal health. Vitamin D is essential for a wide range of physiological processes and optimal health [51, 52, 53]. Vitamin D is unique in that, unlike other vitamins, it is not only provided by food and feed, but can also be formed in human skin under the influence of ultraviolet radiation, i.e. it is not a vitamin in the classical sense [54]. Vitamin D promotes calcium absorption in the intestine and maintains the necessary levels of calcium and phosphate in the blood to ensure bone mineralisation and prevent hypocalcemic tetany. It is also essential for bone growth and the process of bone remodelling, i.e. the work of osteoblasts and osteoclasts. Without sufficient vitamin D, bones can become thin and fragile [55]. Adequate levels of vitamin D prevent the development of rickets in children and osteomalacia in adults [56, 57]. Vitamin D is also used with calcium to prevent and treat osteoporosis [58].

The key link in the metabolism is provitamin D in the form of ergo- and cholecalciferol, which ensures the absorption of calcium and phosphorus in the small intestine, as well as influencing the regulation of cell mitosis and stimulating the synthesis of a number of hormones. However, some vitamin D is degraded to inactive metabolites in the rumen under the influence of bacteria (59).

The functions of vitamin D are not limited to the control of calcium-phosphorus metabolism; it also affects other physiological processes in the body, including the modulation of cell growth, neuromuscular conduction, immunity and inflammation in animals and humans [60, 61, 62].

Research by many scientific groups has shown that the expression of many genes encoding proteins involved in proliferation, differentiation and apoptosis is regulated by vitamin D. Many cells have receptors for vitamin D [63, 64]. Vitamin D exerts a variety of biological effects on the human body through genomic (gene transcription) and non-genomic mechanisms (rapid extra-genomic responses). For genomic effects, calcitriol interacts with VDRs located in the nucleus, and for extra- genomic effects, it interacts with plasma membranes (rapid response) [65, 66].

Numerous studies have shown that vitamin D receptors are found not only in the small intestine and bones, but also in the kidneys, pancreas, skeletal muscle, vascular smooth muscle, bone marrow cells, bone itself, as well as in lymphocytes, monocytes and macrophages [67].

Many scientists have studied metabolic pathology, particularly D-hypovitaminosis (rickets) in young cattle [68, 69, 70]. This disease is caused by a disorder of vitamin D and phosphorus-calcium metabolism in calves. Rickets is characterised by disruption of bone structure and calcification with subsequent functional changes in the organism [71, 72, 73]. Intrauterine hereditary pathology is characterised by disruption of the mechanisms and enzyme systems involved in calcium and phosphorus metabolism. Congenital rickets has been described in foals, piglets and children. It has been established that low vitamin D status is strongly associated with the risk of developing infectious (acute respiratory viral infections, tuberculosis), cardiovascular (hypertension, heart failure), chronic inflammatory, allergic, autoimmune, etc. diseases [74].

Endogenous factors play an important role in the development of rickets and osteodystrophy. This is mainly due to impaired vitamin D metabolism, liver, kidney, thyroid and parathyroid function. Due to the complexity of the aetiology and pathogenesis, these diseases have been termed endogenous D hypovitaminosis and secondary osteodystrophy. Vitamin D deficiency, disturbances in calcium and phosphorus metabolism and other changes in the body of calves due to vitamin D deficiency require great attention to the prevention and treatment of D hypovitaminosis. The issue of treatment and prevention of rickets is particularly relevant when this disease occurs in calves in the first weeks after birth. Thus, it is recognised that vitamin D has gone beyond the limits of calcium and phosphate metabolism and has become a factor in ensuring the most important physiological functions.

Rickets in calves is associated with vitamin D deficiency [75, 76]. In addition, hypovitaminosis A, B1, C and deficiencies and disorders in the metabolism of calcium, phosphorus, magnesium, cobalt, copper and zinc contribute to the development of this pathology.

The literature shows that the main biological effects of vitamin D include participation in the maintenance of calciumphosphorus homeostasis and bone remodelling. Vitamin D stimulates the expression of a number of protein transporters (TRV5,6 systems, calcium-binding protein calbindin - CaBP-9k, CaBP-28k and others) [77, 78]. The main function of transport proteins is to bind calcium ions, and to a lesser extent magnesium and phosphate ions, with their subsequent transport through ion channels of small intestinal enterocytes to the lymphatic system and then to the blood, as well as calcium reabsorption in the distal nephron [79, 80].

It should be noted that currently the range of diagnostic tests for the above diseases has been significantly expanded by the use of various research methods (haematological, biochemical, morphological, etc.) as well as organ biopsies. The integrated use of different diagnostic methods makes it possible to study metabolic processes at all levels of the structural organisation of living matter (organ, tissue, cellular, subcellular and molecular).

Diagnosis of rickets at the stage of bone deformation is not difficult, as the symptoms of the disease are quite characteristic. Identifying the subclinical course of the disease is more difficult. The diagnosis of rickets in calves should be comprehensive, taking into account the conditions of husbandry, feeding and the results of biochemical blood tests.

Presenting the main material

The fragment of the scientific work (monograph) deals with the problem of vitamin D deficiency in young cattle from a modern point of view. It includes material on the classification, aetiology and pathogenesis of vitamin D deficiency rickets in calves. The role of vitamin D in the prevention of rickets and its importance for overall animal health and welfare is summarised. It is noted that the characteristic feature of this disease is mainly the latent stages of its course. Clinical symptoms of D-hypovitaminosis appear in the late stages of the disease, when it is impossible to restore the health of the animals. The essence of the pathology is a disturbance in the mineralisation of the organic bone matrix (D- hypovitaminosis) or osteolysis of the already formed bone structure. When young animals suffer from rickets, not only morphological changes in skeletal bones are observed, but the systemic pathogenesis of the disease involves the organs of the heart, respiratory system, gastrointestinal tract, kidneys, organs of the endocrine system, endocrine glands, etc. Disturbances in mineral metabolism lead to impaired non-specific resistance and adaptability of animals.

Based on the above, the aim of this study was to investigate the prevalence, aetiology, clinical and biochemical status of calves with D-hypovitaminosis.

The material for the study were clinically healthy and ricket infected calves aged 1-3 months, owned by Mozhary LLC, Zhytomyr region.

The research work was carried out in accordance with the State Initiative Theme: "Biochemical and morphological changes in domestic animals with metabolic and invasive pathologies - State Registration No. 0122U200482", Department of Normal and Pathological Morphology of Hygiene and Expertise of Polissya National University on the topic.

Three groups of animals were formed during the study: clinically healthy calves, calves with subclinical course, and calves with clinically severe course. A total of 83 animals were examined.

It is worth noting that animal research (manipulations) was carried out in accordance with the existing regulations governing the organisation of work with experimental animals and compliance with the principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986), the General Ethical Principles for Animal Experiments approved by the First National Congress on Bioethics (Kyiv, 2001) [81, 82].

General clinical and laboratory research methods were used during the work. To analyse the general condition of calves under production conditions, we performed a clinical examination, measured body temperature and determined respiratory and pulse rates. To analyse haematological parameters in calves under production conditions, blood was collected from the jugular vein before morning feeding using vacuum systems "V-TUBETM" (manufactured by AB Medical, South Korea). The biochemical parameters obtained, biometrically processed, were analysed, compared with the data of other researchers and subsequently accepted by us as the norm (Levchenko et al., 2002).

The state of mineral metabolism in calves was determined by the content of 25-OHN3 in blood serum (immunoenzyme ELISA), total calcium (arsenase III reagent), inorganic phosphorus (by UV detection of phosphomolybdate complex), activity of alkaline phosphatase and its isoenzymes (by the method of Wagner and Putilin) [83].

Statistical processing of the digital data obtained was performed using Microsoft Excel. The arithmetic mean (M), the mean square error (m) and the correlation coefficient (r) were determined. The significance of the differences was assessed using Student's t-test. The results of the obtained digital data of the studied indicators were considered reliable at p<0.05-0.001.

One of the current problems of age physiology of dairy cattle breeds is the formation of physiological and biochemical status of calves in the periods of early postnatal ontogeny (newborn, milk feeding and intensive growth), which are in the process of individual animal development, are important in research and serve as one of the most critical factors, as they are associated with profound morphological, biochemical and physiological changes in organs, tissues and systems of the body as a whole. Protein metabolism is the basis of all vital processes in animals. All changes in the body affect the protein composition of the blood, as it is linked to protein formation processes in other organs and tissues and is responsible for the whole range of metabolic processes.

The diagnosis of vitamin D deficiency is based on a number of factors, including the conditions in which the animals are kept: keeping animals outdoors or indoors can affect their exposure to sunlight, which is an important source of vitamin D for the body. To assess the vitamin and mineral status of animals, rations are analysed, including the levels of vitamin D, calcium and phosphorus (the ratio of calcium, phosphorus and vitamin D). A normal balance between these three components is important to ensure normal calcium and phosphorus metabolism [84]. During the on-farm work, we examined the housing and feeding conditions of the animals and carried out clinical and experimental studies. The diagnosis and prevalence of D-hypovitaminosis in calves aged 1-3 months was investigated using clinical and laboratory methods. It was found that the disease occurred in two forms: subclinical (45.8%) and clinically expressed (24.1%), the latter being much less common. A total of 83 animals were examined (Fig. 1).

It is known from practical experience that rickets is a common disease. The latent form of vitamin D deficiency is detected in 60-100% and clinically expressed in 30-35% of growing cattle [85, 86]. Thus, according to the authors of [87], a latent form of D-hypovitaminosis is detected in 60-90% of calves, which leads to profound disorders of metabolic processes in the animal's body, endochondrial bone formation, caused by a disorder of D-vitamin and phosphorus-calcium metabolism.

Fig. 1. Prevalence of D-hypovitaminosis in calves

The analysis of the care and feeding of young cattle and the results of blood tests suggest that the main aetiological factors of D hypovitaminosis in calves are lack of exercise, insufficient sunlight in the case of non-walking, and low supply of basic nutrients and biologically active substances. Animals in the experimental farm are fed on diets with insufficient vitamin D2 in the feed (hay, haylage, silage, straw), with excess calcium, deficiency (63.2%) or excess (120.05%) of phosphorus and digestible sugar protein, which causes the disease.

When analysing the data obtained, it should be noted that the most commonly used indicators for the diagnosis of rickets in animals in veterinary medicine are: the content of vitamin D, calcium and phosphorus in the diet (phosphorus-calcium metabolism); the results of the analysis of the animal's housing conditions; the biochemical examination of blood serum for macronutrients and the level of alkaline phosphatase activity and synovial fluid [85]. However, it is recognised that a highly informative method for the diagnosis of rickets is the determination of 2,3-diphosphoglyceric acid levels, but due to the complexity of the procedure this technique is not widely used in veterinary laboratory practice [88].

Our studies have shown that the disruption of phosphorus and calcium nutrition is complicated by a pronounced deficiency of vitamin D (25.8%) and a lack of trace elements such as cobalt, copper and zinc, which amounted to 57.6%, 96.2% and 85.6% of the requirement, respectively. Such an imbalance of minerals in the diet leads to the development of specific diseases in animals, including D-hypovitaminosis [89, 90].

The subclinical course of D-hypovitaminosis in calves was not pronounced. It was characterised by non-specific symptoms: decreased appetite, taste distortion (allotriophagia), dry and flaccid skin. The calves showed stiffness of movement, frequent limb kicking and an increase in abdominal volume. Calves would lie down, be difficult to get up from, move with difficulty, and have brief clonicotonic convulsions. Animals were frequently diagnosed with foregut atony. Similar results were obtained in studies [84], which were manifested by characteristic symptoms such as decreased appetite, hair loss and subsequent development of short stature. As a result of research by Stadnyk A.M. (2008) and co-authors, calves have decreased appetite, licking, and stunted growth and development. Movement intensity is impaired and skeletal muscle tone decreases. Animals often step over their limbs, limp, lie down more and have difficulty getting up; some calves develop bone curvature, joint swelling, spinal and thoracic deformities. Analysis of blood and urine parameters of calves showed that haemoglobin content, number of red blood cells, leukocytes, concentration of calcium, inorganic phosphorus and protein were significantly lower in rickets patients compared to clinically healthy ones [91].

Under current conditions, the most characteristic symptoms of the clinical course of rickets in calves are growth and developmental retardation, reduced gain, tendency to lick and allotriophagy, softening and partial resorption of the last ribs. Thickening of the wrist joints, curvature of the limbs (X-shaped posture of the thoracic limbs), osteolysis of the last caudal vertebrae and loosening of the teeth were observed in the affected animals.

Many researchers note that calcium is the most abundant mineral in the animal body. It is the main constituent of teeth and the skeleton. Calcium is involved in the excitability of the nervous system, the normal functioning of the heart and skeletal muscles, regulates the permeability of cell membranes, blood clotting and also affects the availability of phosphorus from the diet [92]. Phosphorus is a component of bone tissue, is found in nucleic acids, is closely related to calcium, and plays an important role in carbohydrate metabolism [93].

The diagnosis of rickets in calves has been carried out comprehensively, taking into account the conditions of housing and feeding, the presence of locomotion, the exposure of the animals to sunlight, and the use of clinical and special research methods. Thus, our studies have shown that in the subclinical course of rickets, hypocalcaemia was diagnosed in 45.8% of experimental calves, with a total calcium content ranging from 1.65 to 2.65 (2.21±0.06 mmol/l), which is significantly less (p<0.01) than in clinically healthy animals and more (p<0.05) than in animals with obvious symptoms of the disease (Table 1).

Table 1

The content of total calcium and inorganic phosphorus in the

Note: p< - animals with subclinical disease compared with clinically healthy animals; p1< - animals with clinical disease compared with clinically healthy animals; p2< - animals with clinically severe disease compared with subclinical disease.

Hypocalcaemia with clinically expressed D-hypovitaminosis was diagnosed in 24.1% of the calves, with a mean value in the group of 2.05±0.05 mmol/l, which is significantly less (p<0.001) than in healthy animals and also less (p<0.05) than in young animals with subclinical disease.

The blood serum inorganic phosphorus content of calves with subclinical disease was 2.05±0.03 mmol/l, which was not significantly different from that of clinically healthy animals.

In the chronic course of the disease, hypophosphatemia was diagnosed in 24.1% of the calves. The inorganic phosphorus content was significantly lower (p<0.001) than in clinically healthy animals and in young animals with subclinical D-hypovitaminosis (p<0.01).

The conclusion from the analysis of literature data [94, 95, 96] suggests that the role of vitamin D3 is not limited to the regulation of calcium and phosphorus metabolism. At the present stage of research, it has been established that vitamin D3 has a regulatory effect on the proliferation of all organs and tissues, as well as on the synthesis of lipids, enzymes, hormones, not only calcium-responsive ones, but also thyrotropin, glucocorticoids, prolactin, gastrin and others. Recent studies also point to the effect of vitamin D3 on carbohydrate metabolism, which has been confirmed experimentally in rats. It has been found that insulin synthesis is dependent on the presence of vitamin D3 in the body and less dependent on calcium levels. It is therefore necessary to take vitamin D3 throughout life in order to maintain the body's vital functions.

Alkaline phosphatase is one of the most important enzymes in the animal body. It is found in many tissues and plays an important role in phosphorus and calcium metabolism, as well as other types of metabolism [97, 98]. Alkaline phosphatase is synthesised by intestinal enterocytes and is involved in the transport of glucose and other monomers across the enterocyte membrane and in phosphorylation reactions [99]. This enzyme has the ability to regulate osteoblast differentiation and serves as a specific marker for osteoblasts. A sharp change in the activity of this enzyme is observed in pathological conditions of the animal body associated with impaired blood supply to organs, bone growth pathologies, etc. Low activity of this enzyme is associated with magnesium and zinc deficiency and protein deficiency. Alkaline phosphatase is involved in maintaining homeostasis, regulating growth and adapting the body to environmental conditions [100, 101]. An increase in serum alkaline phosphatase activity is most commonly seen in liver and bone tissue pathology. Damage to the liver parenchyma causes a slight increase in serum enzyme activity because ALP is tightly bound to cell membranes. In bone pathology, when there is increased osteoblast activity (in the development of rickets, osteodystrophy), the activity of the bone isoenzyme alkaline phosphatase in the blood increases. In these cases, the activity of total alkaline phosphatase in blood serum and synovial fluid increases by 3-10 times.

As a result of our studies, it was found that a significant increase in the activity of alkaline phosphatase in calves with subclinical disease (112.3±7.6 vs. 83.7±5.8 U/l) in clinically healthy young animals (p<0.05) is due to an increase in the activity of bone isozyme.

In calves with clinical course of D-hypovitaminosis, the activity of alkaline phosphatase was on average 129.1±6.5 U/l, which was significantly higher than in clinically healthy young animals (p<0.001) and in animals with subclinical course of the disease (p<0.01; Table 2).

The activity of total alkaline phosphatase and its bone isoenzyme in D-hypovitaminosis increased 1.8-fold, indicating a violation of the process of bone mineralisation (Table 2).

Table 2

Activity of alkaline phosphatase and its isozymes in calves

Note: p < - animals with subclinical disease compared to clinically healthy; pl < - animals with clinical disease compared to clinically healthy; p2 < - animals with clinically expressed D-hypovitaminosis compared to subclinical.

Significant disorders of the functional state of the calves' body in subclinical and clinically expressed course of D-hypovitaminosis are confirmed by the results of biochemical examination of blood serum: a decrease in the content of 25-hydroxycholecalciferol to 2.56 ng/ml (normally - 32.0-90.0 ng/ml), hypocalcaemia, respectively, in 55 and 81% (2.2±0.07 and 2.0±0.04 mmol/l), a significant increase in the activity of alkaline phosphatase and its bone isoenzyme (p<0.001).

Conclusions

D-hypovitaminosis in calves was found to be widespread on the farm. A subclinical course was observed in 45.8% of the animals and a clinical course in 24.1%. Pathology was more frequent in the winter-spring period.

The main aetiological factors of the disease in calves are hypodynamia and insufficient insolation of animals, low supply of cholecalciferol (25.8%), violation of the calcium-phosphorus ratio (2.7-4.2:1 vs. 1.5-2.0:1), deficiency of trace elements - cobalt, zinc, copper, which amounted to 57.6, 85.6 and 96.2% of the requirement, respectively.

The characteristic symptoms of the disease in calves are licking, allotriophagia, thickening of the wrist joints, partial resorption of the last ribs and coccygeal vertebrae, and loose teeth.

The most informative laboratory tests for diagnosing the pathology are the determination of serum cholecalciferol, total calcium, inorganic phosphorus, alkaline phosphatase activity and its bone isoenzyme. The disturbance of mineral homeostasis in calves, especially during the winter period, requires further research into a more comprehensive and in-depth approach to the pathogenesis of D-hypovitaminosis and targeted treatment and prevention measures, especially the use of complex mineral preparations with a prolonged effect.

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