Analysis of the season-dependent component in the evaluation of morphological and biochemical blood parameters in Shetland ponies of both sexes during exercise

Determination of haematological and biochemical indices of horses’ blood facilitates assessment of their state of health and welfare, nutrient demand, the effects of training already undertaken, and the horses’ suitability for a specific type of exercise (5, 17). Blood parameters in the horse are influenced by a number of factors, including the animal’s age, sex, breed, physiological state, reproductive status, regularity, intensity and recentness of physical exercise, and a set of factors that make up the concept of the season. The literature increasingly often reports variations in haematological and biochemical parameters as well as blood chemical elements in horses which depend on such environmental factors as atmospheric pressure, temperature, light, and other characteristics of seasons (13).

The locomotor activity of horses in classical equestrian sports and recreation imposes physical and mental stress. The species-specific and physiological characteristics of horses suit them to use in riding sports, but participation requires sensitivity to the horse’s welfare from riders and owners (31). Training, testing, monitoring and using horses in the conditions of equestrian competition needs to follow a modern scientifically based system. Assessing the improvement of the horse’s body gained through training needs to be guided by an objective method (36). It is possible to meet these needs only through an in-depth study of the metabolic processes underlying the adaptive reactions in the horse. Therefore, studies of the processes of adaptation of horses of different breeds to systematic physical activity and research to ensure timely correction of functional disorders caused by overtraining of horses remain very relevant.

The blood system is one of the most important integral systems of the body, and its elements are sensitive to various external influences. Therefore, analysis of the composition of peripheral blood is an important component of the veterinary examination of horses (33). Previous studies have shown that haematological parameters and markers of oxidative stress in the blood of horses of different breeds differed significantly (1, 5). This may indicate the adaptive reactions in order to maintain homeostasis coming under greater or lesser strain depending on the breed; however, such factors as season and type of exercise may also affect the efficacy of adaptation.

In our earlier studies, we investigated photoperiod-induced changes and effects of physical exercise on oxidative stress biomarkers, total antioxidant activity, and antioxidant defence activities together with biomarkers of metabolic changes, such as glucose, urea, uric acid, and lactate dehydrogenase activity in the blood of Shetland pony mares and stallions ridden recreationally (15). This research is now pursued further by identifying the impact of the photoperiod component and the effects of sex and exercise on red and white blood cell parameters and basic biochemical indices that are often used in the evaluation of horse welfare during exercise.

It should be noted that there are scant literature data from this line of research and they are mostly single clinical examples from veterinary studies. Besides addressing this knowledge gap, an equally important reason for the research was that this horse breed is increasingly being used in hippotherapy for children in many countries, especially in Poland (3). Therefore, the aim of our study was to determine the effect of environmental conditions related to changes in the season on haematological and biochemical blood parameters in both sexes of Shetland ponies ridden recreationally before and after exercise.

This study assesses the functional status of morphological blood parameters in Shetland ponies during different periods of the year, taking into account such factors as the sex of the animals and whether they had been recently exercised physically. Although studies of this type exist which are focused on physiological (9, 10) and pathological conditions associated with various diseases (25), our research contains new elements of analysis, which have not been previously presented in the literature. Four main directions of analysis are salient.

Firstly, the four MANOVA tests used helped to estimate differences between the three main factors of photoperiod, sex and exercise as well as the role of each parameter in our statistical model. It was revealed that photoperiod and exercise outweighed sex in triggering the changes in the parameters studied. It is important that factors in combination (season × exercise, sex × exercise and season × sex × exercise) were effective in mediating adaptive reactions to maintain or restore blood chemistry homeostasis in the ponies during exercise. Thus, it is clear from our results that both seasonality itself and its combination with the exercise factor have an important influence on the mechanisms of stable homeostasis in the blood of horses.

Previously, this dependence was presented in the literature for different species of animals and especially horses of different breeds (8). It is considered that conditioning to the season is a critical ability of most organisms. At temperate latitudes, the photoperiod is the main synchroniser of seasonal functions. Seasonal changes in physiology and behaviour typically are innately timed long-term processes, requiring weeks or months to wax and wane. Therefore, in addition to photoperiodic readout mechanisms, living creatures have evolved endogenous long-term timing devices, which allow them to anticipate forthcoming season changes. In the most extreme cases, cycles of about 365 days recur for years in animals kept under constant photoperiods; such so-called circannual rhythms exist in a variety of birds and longer-lived mammals (16).

Characteristics of the stable ultradian structure of behaviour were compared by other authors between ruminants and a group of Przewalski’s horses (30). A less stable adaptive temporal structure was established in Przewalski’s horses. The authors singled out the activity parameter as a multipurpose behaviour. In this case, the activity was variable, and feeding had a clear rhythmic and harmonic structure. The percentage of the circadian component and harmonic ultradian components relative to all rhythmic components of the spectrum was high in well-adapted, healthy, and undisturbed individuals. However, these values decreased during periods of adaptation, illness, or social interactions (30).

Secondly, the statistical analysis revealed no statistically significant season-related differences in blood morphology parameters during exercise in either sex of Shetland pony. The absence of any seasonal effect may be explained by the level of exercise in horses having been adequate, not excessively exhausting and causing no severe stress that could be associated with significant changes in the blood morphology. The evolutionarily established pattern in the control of blood morphology and homeostasis is one of the powerful mechanisms of functional stability under physiological stress.

Exercise remains a key aspect of improving a horse’s endurance and performance (14). These traits in horses result from a number of physiological adaptations. In some cases, these adaptative mechanisms are independent of training, such as the lung size, while others change in response to training, such as the blood volume. The athletic performance of horses is attributed to their high aerobic capacity, large intramuscular stores of energy substrates and especially glycogen, a large volume of mitochondria in the muscles, their ability to increase the oxygen capacity of the blood at the beginning of training by contracting the spleen, gait efficiency, and effective thermoregulation (14).

Several studies have shown that acute and chronic exercise is associated with changes in haematological, biochemical, immunological and hormonal parameters (26). Exercise has different effects on haematological parameters depending on its duration and intensity (short-term high-intensity or maximal exercise or alternatively long-term low or submaximal exercise), the animal’s fitness and extent of training already undertaken, and environmental conditions (26). However, many physiological and biochemical changes in the blood caused by exercise are those that can ultimately affect its reduction-oxidation status. Erythrocytes (mainly because of their number) and leukocytes (mainly because of their acute activation during exercise) can be the main generators of reactive particles in the blood during exercise (20).

Systematic and adequate physical activity of horses increases the capacity of the organism to adapt to oxidative stress with changes in metabolism, in particular. Horses adapt to oxidative stress with a gradual increase in mitochondrial biogenesis, which increases the functional capacity of the cardiovascular and muscular systems (18). This has been reported in a previous study (21). All metabolic reactions are carried out by enzymes; therefore, the regulation of metabolism during exercise leads to the regulation of their activity and de novo synthesis. Measuring biochemical and haematological blood parameters in animals during exercise facilitates determination of the functional state of the organism and changes in the process of adaptation to systematic physical activity (7, 22). Also beyond gaining understanding of the equine response to exertion, haematological studies of adaptive capabilities and monitoring of welfare and performance remain important in modern equine physiology management (29).

It has been suggested that the dynamics of humoral and cellular immunity and the intensity of free radical oxidation processes during physical activity indicate a dose-dependent effect of such loads on the immune system, i.e. physical activity with maximum intensity suppresses the immune system, while exercise with intermediate intensity normalises and stimulates this system (4). At the same time, immune system dysfunction may be one of the leading factors limiting physical performance. The main mechanisms for improving the immunological status comprise the complement system and the formation of immunoglobulins in the blood, mobilisation of the reticular-endothelial system, and increased free radical–induced oxidation. It has been established that muscular activity accelerates the migration of some lymphocytes to the bone marrow, thereby stimulating its haematopoietic function and releasing excess red blood cells and lymphocytes into the bloodstream (37).

During moderate physical activity, when stress is not a significant damaging factor, there is an initiation of adrenergic regulation adequate to the intensity of and the animal’s capacity for physical activity, with corresponding energy expenditure and the development of physiological hypoxia. To optimise tissue oxygenation, the structural and functional state of red blood cells is of great importance, as it determines their shape, size, ability to deform and degree of aggregation. Tissue hypoxia that accompanies intense physical activity causes the release of erythropoietin and stimulation of the erythroid part of haematopoiesis (19).

Thirdly, the results of the MANOVA statistical analysis using the β-coefficient presented a series of relationships: of RDW and GRA with the season; of RBC, HCT, MCV and MPV with sex; and of RBC, RDW, WBC and GRA with exercise. It should be noted that the movement of horses around pasture which is only possible in the warm seasons also affects the animals’ haematological parameters. This movement ensures the intake of fodder with a sufficient nutrient composition and content of mineral compounds. The intake and digestion of different types of feed have a significant effect on changes in some biochemical and haematological blood parameters. For example, increased concentrations of glucose, Ca, Mg, Fe and inorganic phosphorus have been observed in the serum (11). Conversely, prolonged starvation may result in a decrease in such haematological blood parameters as RBC and WBC and a decrease in the HGB concentration (35). Similar changes in the peripheral blood of horses related to the nutritional process may also result from a shortage of water or overwatering due to hot weather or/and intense exercise in these conditions, which is associated with weather peculiarities of the season (34). Monitoring of morphological and biochemical blood parameters can be used as a tool to assess the overall health and welfare of horses. By having analysed blood parameters during exercise, the study can provide breeders with insights into how different seasons affect the physiological responses of horses to exercise. This information can guide breeders in optimising exercise programmes for their ponies throughout the year. This understanding may help breeders to anticipate and manage seasonal changes in equine health and performance.

Seasonal changes in the environment influence the physiological responses of animals. Changes in such haematological parameters as RBC are of value in determining the adaptation of animals to the environment (28). In spring, water intake with grazing may limit the effect of haemoconcentration induced by physical activity and/or thermolysis (27). The greater ALT activity observed in mares and stallions after exercise in spring and lesser activity in this phase in autumn and winter can also be explained by the period of transition to additional grazing following winter. Satué and et al. (28) observed an increase in erythrocytes, HCT and PLT counts in Carthusian broodmares in summer. The higher temperatures prevailing in summer months may contribute to the development of an adaptive response to heat stress (27). These high environmental temperatures activate thermoregulatory mechanisms, with loss of water through sweating and evaporation through respiratory mucosa. This in turn may lead to a decrease in plasma volume and an increase in the HCT level (27, 28).

It has been noted that intense muscle activity is accompanied by significant changes in metabolic and haematological parameters (23). As a physical training regimen intensifies, the attendant biochemical changes indicate how the main energy substrates in the horse are mobilised and utilised and what destructive changes are taking place (22). Markers of such metabolic changes include the activity of energy metabolism enzymes such as LDH and ALT and AST, which are involved in the transfer of amino groups from amino acids to ketoacids, and also include blood lactate and pyruvate levels. Changes in the activity and level of these biomarkers in the dynamics of physical activity reflect specific transformations in the metabolism of horses. In trained horses, the content of lactic acid after physical exertion in response to exhausting but systematic exercise is significantly lower than in untrained animals. In response to intensive systematic training loads, the dynamics of changes in the levels of lactate and pyruvate in the blood of sports horses remain almost the same, and their ratio determines the intensity of anaerobic processes (6).

The physiological increase in the activities of ALT and AST has been shown to occur without any tissue destruction. The degree of this increase depends on the type of exercise. As a muscle-derived enzyme, AST is commonly used as an indicator of muscle damage in athletes or sports horses. Levels of AST in horses have been reported to increase immediately after a 160-km endurance race (24). After four and six months of training, there were no differences in pre-training performance. Increased muscle mass and increased activity of certain metabolic enzymes, combined with changes in the expression of structural proteins, are known to promote significant adaptations in muscles and metabolism, leading to improvements in muscle strength, endurance, and overall metabolic efficiency (7). These adaptations are often observed in response to regular physical activity, exercise training, and other physiological stimuli. They play a fundamental role in enhancing athletic performance, maintaining muscle health, and supporting metabolic homeostasis.

Fourthly, the role of biomarkers demonstrating the relationship between aerobic and anaerobic levels of energy metabolism in the blood should be mentioned, as they clarify the relationships in the regression analysis in our statistical model for LDH activity, levels of lactate and pyruvate, lactate-to-pyruvate ratios, and ALT and AST activity. Importantly, none of these parameters in our regression analysis showed any dependence on sex, which may indicate the universality of these parameters in such types of dependencies. In this case, statistically significant β-coefficients were obtained for photoperiod and exercise, the determining factors for ALT, which confirms their important role.

Defence against the lack of oxygen in hypoxia-tolerant animals implicates a reduction in energy turnover and/or an improvement of energetic efficiency in other metabolic processes. An increase in LDH, creatine kinase or AST activity has been observed in exercised race horses, after endurance rides and after show-jumping competitions (2). Lactate is widely used to examine the effects of training, diagnose positive performance and assess the level of fitness in sports horses (22).

Identification of the season-dependent components and the effects of sex and exercise on the changes in haematological and biochemical parameters that are often used in equine physiological assessment was the main goal of the current study. The analysis of these effects revealed the leading role of the photoperiod and exercise factors and of their combination on the formation of effective adaptive reactions for maintenance of morphological blood indices in the ponies during exercise. There were no statistically significant differences in the blood morphology parameters in either Shetland pony mares or stallions depending on the season or the undertaking of recent exercise. Maximum ALT activity was found in the spring period in both mares and stallions after exercise. This activity was significantly weaker in the autumn and especially in the winter photoperiod, compared not only to the result in spring but also to that in summer. Lactate dehydrogenase activity exhibited seasonal changes in the stallions before exercise in summer and after exercise in autumn. The lactate-to-pyruvate ratio was statistically significantly higher in the winter period in both mares and stallions after exercise.