*2.2. Experimental Design*

Two feeding experiments in two successive winters were run with 74 and 62 young stallions. Experiment 1 lasted from 10 January 2018 to 10 May 2018, Experiment 2 from 12 November 2018 to 14 May 2019. In Experiment 1, there were 15 horses born in 2015, 24 horses born in 2016 and 35 horses born in 2017. From those 24 horses born in 2016 and

35 horses born in 2017, 11 and 27 horses, respectively, were also included in Experiment 2, according to the rule that the knotweed-fed horses from Experiment 1 were also knotweedfed in Experiment 2 to avoid affecting the control horses with recent treatment. In addition, 24 new horses born in 2018 were included in Experiment 2. Each group of 1–3 years old stallions (foals) was divided in both seasons into two subgroups based on age, body weight and blood test values. The resulting experimental groups were not significantly different before the experiments in any of the baseline parameters, including weight, blood test results, age, breed and/or parentage. The health status of the stallions was monitored by testing blood samples.

#### *2.3. Feed and Feeding Regimen*

All stallions were fed individually in boxes, receiving hay ad libitum, a regular feed supplement and the experimental mixture, which were always consumed without leftovers. As they were kept on a deep straw bed, straw also became an occasional component of their diet.

Each horse was fed daily with:



**Table 1.** Amounts, composition and nutrient contents of the feed for 3-year-old warmblood horses.

The amounts of the feed supplement and composition of the experimental mixture for individual horses respecting their breed, age, weight and condition were determined according to the nutrient requirements recommended by the NRC (National Research Council, 2007), based on experience with the development of similar feeds for horses enriched with bioactive compounds of plant origin, such as Fitmin herbs Regeneration, Fitmin herbs Calmer, or Fitmin Bronchial, see https://www.fitmin.cz (accessed on 30 October 2021).

The control group of horses (not fed knotweed) received 500 g/day/individual barley pellets with soya oil and beet sugar; horses from the treatment group (fed knotweed) received a mixture of dry aboveground knotweed biomass and barley with soya oil and beet sugar (500 g/day/individual). Each horse from the treatment group received daily 150 g of dry knotweed biomass containing 0.087 g of emodin, 0.028 g of resveratrol, 0.554 g of oxyresveratrol and 0.139 g of piceid. Oxyresveratrol, which is resveratrol with an additional -OH group, and piceid, which is resveratrol glucoside, split into glucose and resveratrol in the gut, thus contributing to the overall amount of available resveratrol administered daily to each individual horse, which ultimately amounted to 0.63 g. As some of these horses had been involved in both experiments, the stallions that had already received knotweed in Experiment 1 continued receiving it in Experiment 2.

There was a warm and dry season between the two experiments, from May to November 2018, during which the horses only fed on grass from pastures.
