4.1. Biochemical Parameters of Blood Related to Nutritional Status
The values of the total protein and urea are related, among other things, to the intake of nitrogenous substances in the feed ration and are indicative of the adequacy of the daily protein dose [
18]. The albumin concentration reflects proteosynthetic ability of the liver [
19], so the proteosynthesis rate increased in the horses of the experimental group. Silymarin proves the ability to regulate proteosytesis and can increase the albumin levels [
20] in the disruption of proteosynthetic liver function (reduced albumin). No horse showed health disorders in our experimental observation. The horses were fed with milk thistle seed cakes and received more crude protein (by 35 g) compared to the previous month when all horses received the same granular mixture. The albumin values and urea did not change in the control group (
Table 4;
Figure 1). Possibly, the protein contained in milk thistle seed cakes is better used for the horses, and the silymarin effect may also have stimulated the proteosynthetic processes [
21]. The FD effect with low (up to 1.5%) and high fat content (up to 11.5%) was compared with plasma concentrations of TAG, NEFA, HDL, and LDL [
20]. Ponies with FD of higher fat content had higher HDL concentrations and lower TAG. This trend was confirmed by our results (
Table 4;
Figure 2), but the differences in values were not significant in the total values. ALP value responds first to an increase in cholestasis [
22]. This ALP value was not changed, indicating a total increase in cholesterol levels in the experimental horses, but no pathological effect was seen in cholesterol metabolism. Silymarin has been reported to have the ability to regulate higher cholesterol, triglycerides, and lipoprotein levels [
23]. An individual assessment showed a statistically significant difference (
Figure 2) after 56 days of milk thistle seed cakes feeding in the experimental group. An increase in HDL values was also observed. Silymarin has the ability to reduce intestinal cholesterol absorption and increase HDL levels [
24]. The monitored parameters were detected in the reference range of the values for the horses.
A slight decrease in GGT (
Figure 3) could theoretically be related to the antioxidant effects of silymarin because GGT protects the cells against oxidative stress [
19]. Many authors report that silymarin feeding reduces higher GGT levels. A significant difference was detected in AST values in the second sample between the experimental and control groups. The assessment of the AST parameter also confirmed this difference (
Table 4,
Table 5 and
Table 6;
Figure 3) in the individual horses. A significant reduction in AST values has been observed after milk thistle seed cakes feeding in many studies [
25]. The silymarin active substance demonstrably reduced AST values [
26]. The feeding of milk thistle seed cakes significantly reduced the AST level in the blood of the horses. A stabilizing effect has also been observed on GGT values using silymarin in liver damage [
26,
27,
28].
4.2. Biochemical Parameters of Blood Related to Physical Exercise and Stress
Arfuso et al. [
29] found out a positive correlation with an increase in albumin levels in the horses after exercise. The experimental monitoring of the training lasted for almost two hours in summer; therefore, partial dehydration of the organism was the cause of the albumin level increase in the blood of the monitored horses (
Figure 4). The intensive training led to significant changes in acid-base balance in the horses, with a significant increase in lactate levels after exercise (
Figure 5). A statistically significant difference was found between the values before and after exercise. No difference was observed between the groups of horses. These changes were caused by the release of muscle energy during anaerobic metabolism. A high positive correlation was detected between the plasma lactate value and the intensity of physical exercise in the horses [
30]. As can be seen in
Figure 6, the physical exercise did not affect the AST level. A significant decrease in the AST value was detected in the experimental group. Apparently, the significant AST decrease was not related to the physical exercise in the experimental group, probably due to the feeding of milk thistle seed cakes in the feed dose.
Cortisol is a major representative of glucocorticoids, commonly referred to as stress hormones. Cortisol released in horses is associated with the length and intensity of exercise [
31].
Table 6 shows a significant difference between the groups of the horses in the fourth sample. Significantly lower values were recorded in the experimental group compared to the control one. Previous studies suggested that maximum cortisol concentration was reached earlier in trained horses and these horses had a faster recovery time compared to resting values. On average, maximum cortisol levels were observed approximately 30 min after exercise. A rapid transition is desirable to achieve from catabolic to anabolic state after the training [
31], which was confirmed in the experimental group. Thakare et al. [
32] described in their study that silymarin reduced corticosterone levels in mice exposed to acute stress. Possibly, the silymarin effect may have increased the resistance to stress in the experimental group and achieved the desired acceleration of the body transition into an anabolic state after physical exercise in sport horses.
Glucose homeostasis is closely related to the endocrine system. Gluconeogenesis is accelerated, and glucose absorption in the tissue is reduced, and insulin sensitivity is lower in response to the leaching of glucocorticoids into the blood. An effect from the sampling order on plasma glucose concentration was not found during statistical data processing. A statistically significant difference was detected between the groups in the fourth sample (
Table 6). We suppose that the significant difference in glucose levels between the groups in the fourth sample is related to the significant difference in cortisol levels in the fourth sample taking into account the gluconeogenic effect of cortisol [
33]. Neither hypoglycaemia nor hyperglycaemia was observed in the horses during the experimental monitoring.
NEFA are highly energy-rich molecules competing with glucose, especially in aerobic exercise [
29], and reflect the lipolysis level. NEFA and TAG are a source of energy in plasma, especially at long-term exercise with low and medium intensity. NEFA mobilization from fat stores begins relatively early to start physical exercising. The oxidative processes begin fully within minutes in horses [
31]. The lipolysis level is controlled by catecholamines (adrenaline, noradrenaline, and dopamine) and a decrease in insulin activity [
34]. Glucocorticoids (cortisol) also stimulate lipolysis, thus saving glycogen in trained horses [
35]. As can be seen in
Figure 5, exercise significantly affected the NEFA levels in the blood, which is logical with taking into account the endurance (aerobic) type of exercise.
The effect of physical exercise on the NEFA level in horses has been addressed by other authors [
36,
37]. Cortisol stimulates the mobilization of energy substrates by accelerating gluconeogenesis, mobilizing NEFA through lipolysis, and increasing the availability of amino acids by proteolysis at the start of physical exercising (or stress). These processes could theoretically delay the beginning of the total fatigue during physical exercise [
31]. Higher NEFA values could appear as beneficial energy sources. Higher NEFA utilization as an energy source leads to a decrease in plasma lactate levels [
34]. This process did not occur in the control group. No difference in lactate levels was found out between the groups (the average lactate values were even lower in the experimental group), which means that the higher NEFA level was not used in the control group of the horses. Higher NEFA values may be due to the low ability to use NEFA as an energy source after physical exercise in horses [
29]. The increased lipolysis, serving as a major energy source in endurance exercise, does not prevent hepatic and muscle glycogen depletion associated with a steady decrease in blood glucose levels despite the increased NEFA concentrations [
35]. In addition, NEFA can slow glycolysis [
33], and thus, the ATP recovery from glycolysis can occur. Higher NEFA levels tend to be in the blood at doses with a higher fat proportion in FD, contrary to the experimental group. NEFA utilization was determined to be at a higher level in the experimental group, and NEFA levels were detected lower after exercise, thus, saving the energy. The utilization of the energy sources was found out more efficient in the experimental group discussed below for Pi concentrations. In plasma, NEFA levels could also be related to the higher PUFA (polyunsaturated fatty acid) content within the FD in the experimental group of the horses. Piccione et al. [
38] observed lower NEFA values treated with FD with a higher PUFA proportion after exercise in the experimental groups of the horses. The opinion of Assenza et al. [
37] is that the lower NEFA levels may be due to either decreased fatty acid mobilization or increased NEFA utilization (as an energy source) after the PUFA supplementation diet in the blood in the experimental group compared to the control one.
Phosphorus is essential in energy metabolism, indispensable in the ATP synthesis and 2,3-diphosphoglycerate (oxygen dissociation from hemoglobin), and can also modulate the activity of several metabolic pathways [
39].
Figure 6 shows the P
i values (PO
43−). The sampling order had no effect on P
i levels. Statistically significant differences were observed in the values between the groups after exercise. The values were not changed in the experimental group. A significant decrease was detected in the values of phosphate anion in the control group. Doubek et al. [
19] concluded that hypophosphatemia could be occurring according to a reference range. But Kraft and Dür [
22] stated that P
i values were detected at the lower limit of the reference value range. The physiological decrease in P
i values is usually due to an increase in insulin levels or P
i consumption during glycolysis [
19]. Vervuert et al. [
40] monitored, among other things, the P
i values in the blood of horses before and after physical exercise and observed a significant decrease in the value of all horses after exercise (in the range of 30–120 min). If the lower phosphorus values bordering on hypophosphatemia due to higher insulin levels were detected, no increase would be recorded in glucose concentration in the fourth sample in the control group. Lower P
i values were observed probably due to the need for ATP recovery as an energy source, suggesting that the experimental group proved a better use/recovery of the energy sources. No normal decrease was observed in phosphorus values after exercise. Lipolysis was used more efficiently as an energy source (lower NEFA values after exercise in the experimental group), and thus, energy sources were saved from glycolysis.
Muscular work is necessary for the performance of a sport horse, but even small deviations in muscle function can have an effect on performance, coordination, endurance, and work determination. Skeletal muscle damage (e.g., acute rhabdomyolysis) is readily diagnosed by determining AST and CK levels [
41]. No pathological conditions occurred in any of the horses after the evaluation of these two biochemical indicators. Slightly increased AST values could be a sign of the training process in the first sample. An increase in the lactate values was observed in all horses after exercise. No difference was found in the CK values catalyzing phosphorylation.
Silymarin may reduce the impact of acute stress [
32,
42]. Longer-term effects of glucocorticoids suppress the immune responses and contribute to the development of negative side effects [
33], including insulin resistance [
43]. The relationship between cortisol and insulin may have been reflected in glucose results in the fourth sample (demonstrably higher glucose levels in the control group than in the experimental group). Silymarin can have the ability to increase insulin sensitivity and reduce elevated insulin levels in addition to its antioxidant properties and may protect the pancreas from toxic effects, among other things [
42]. A rapid decrease should occur in plasma cortisol concentrations in healthy, trained horses. Negative health effects can reduce the immunity of sport horses if higher cortisol concentrations persist for a long time after exercise [
33]. A more significant decrease was observed in cortisol values one hour after physical exercise in the experimental group, showing again a positive phenomenon in the group of the horses fed with milk thistle seed cakes. Oxidative stress is also associated with physical exercise and stress. A long-term training can increase the markers of the antioxidant system according to the results [
34] and thus to increase the resistance to oxidative stress. TAS and GSH-Px analyses served as the markers of the antioxidant capacity. No difference was detected between the groups or the effect of the blood sampling order on these indicators. The changes may not be detected in the antioxidant values immediately after exercise but may appear until 16–24 h after exercise. In addition, the onset of fatigue activates the sympathetic nerve (catecholamine secretion), promoting the NEFA lipomobilization, thereby optimizing the metabolic response to exercise and stress. Among other things, exercise improves energy metabolism at the muscle level, reflected in higher NEFA utilization [
36]. If sport horses were fed by milk thistle seed cakes, the NEFA would be more efficient. Also, the adaptation of the horses to training in energy metabolism, stress and regeneration processes would be accelerated and is desirable