Toxicological Analysis of Feed

Feed was analyzed for the presence of mycotoxins and their metabolites: ZEN, α-ZEL, and deoxynivalenol (DON). The level of the analyzed mycotoxins in feed samples were determined in accordance with the study protocol described previously [54]. Chromatographic methods were validated at the Department of Veterinary Prevention and Feed Hygiene, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland [55].

#### *5.3. Experimental Animals*

An in vivo experiment was performed at the Department of Veterinary Prevention and Feed Hygiene of the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn, Poland. The experiment involved 60 clinically healthy pre-pubertal gilts with an initial BW of 14.5 ± 2 kg [10,54]. During the experiment, the animals were housed in pens, fed identical diets, and had ad libitum access to water. The gilts were randomly divided into a control group (group C; *n* = 15) and 3 experimental groups (ZEN5, ZEN10, and ZEN15; *n* = 15 each). Groups ZEN5, ZEN10, and ZEN15 were administered ZEN (Sigma-Aldrich Z2125-26MG, St. Louis, MO, USA) per os at 5 µg/kg BW (MABEL dose), 10 µg/kg BW (NOAEL dose), and 15 µg/kg BW (LOAEL dose), respectively. Each experimental group was maintained in a separate pen in the same building. Each pen had an area of 25 m<sup>2</sup> , which complies with the applicable cross-compliance regulations (Regulation (EU) No 1306/2013 of the European Parliament and of the Council Brussels, Belgium of 17 December 2013).
