4.1.2. Thrombocytes

Thrombocytes are non-nuclear blood elements that are crucial in the onset of blood clotting. Their number in the blood depends on their production in the bone marrow, consumption and losses. They account for the smallest proportion of blood cells. As their

average lifespan in horse blood is 4–6 days [39], their population size sensitively reflects both their production and their destruction. They contain many substances involved in inflammatory reactions, blood coagulation and other specific reactions. It is impossible to assess immune function based on the thrombocyte count, as their absolute numbers do not correlate with thrombocyte function. Therefore, although stallions fed knotweed produced more thrombocytes than the control stallions in Experiment 2, the informative value of such a finding is limited with regard to the effect of knotweed supplementation on immunity. On the contrary, the number of thrombocytes decreased in the blood of knotweed-fed pigs in our previous study [40]. The different types of knotweed biomass in the feed supplements, i.e., stems and leaves in the supplement for horses, and rhizomes and roots in the supplement for the pigs (varying in their contents of bioactive substances, namely carotenoids, resveratrol, piceid and emodin), might help explain this difference and serve as a hint for further research. The decrease in thrombocyte numbers in pigs could be explained by high resveratrol content in knotweed roots [41,42] while the increase in thrombocyte numbers in horses could be due to other bioactive compounds in knotweed leaves and stems, such as carotenoids. It was reported that tomato puree rich in carotenoids improved mice health and increased their thrombocyte numbers in mice exposed to toxic fluorides [43]. The cellular membranes of thrombocytes in the carotene-deficient blood of people infected with the dengue virus suffered from dryness, which led to the death of these cells [44]. Carotenoids were not targeted in this study, however; their considerable content in knotweed leaves has been reported [7] and they deserve further study.

PCT, plateletcrit, is a number describing ratio between the volume of thrombocytes and plasma. A higher thrombocytes number means a higher PCT [42]. In this study, PCT was higher in the blood of knotweed-fed horses than in the blood of control ones.

#### 4.1.3. Activity of Alkaline Phosphatase (ALP)

ALP is a non-specific metallo-enzyme (Mg, Zn), a hydrolase removing phosphate group from various proteins and nucleotides and hydrolyzing inorganic phosphate, produced in liver, bones, intestines, kidneys and placenta. Only activities of ALP isoenzymes from liver and bones, both coded by the same gene, could be detected here because the other ALP isoenzymes survive in the blood only for a short amount of time. ALP from bones is typical for young individuals whose bones are growing. When the growth slows down the activity of ALP physiologically decreases, being associated with mineralisation of bones [45]. The ALP, which was lower in the blood of knotweed-fed horses than in the blood of control horses in this study, was probably not produced in the liver but in the bones as there are no statistically significant changes in any other parameters connected with the liver (AST, GMT, bilirubin). It means that the decrease in ALP activity was associated with the physiological process of adolescence. In this study, the ALP decrease was enhanced by knotweed and correlated positively with urea (r = 0.411, *p* = 0.01) in knotweed-fed horses in Experiment 1, indicating a positive effect of knotweed on nitrogen use from the feed, as discussed below in Section 4.1.6. The ALP values from all the horses were within physiological limits [46].
