*2.1. Experimental Feed*

The analysed feed did not contain mycotoxins, or its mycotoxin content was below the sensitivity of the method (VBS). The concentrations of masked mycotoxins were not analysed.

### *2.2. Clinical Observations*

The results presented in this paper were acquired during a large-scale experiment where clinical signs of ZEN mycotoxicosis were not observed. However, changes in specific tissues or cells were frequently noted in analyses of selected serum biochemical parameters, heart muscle, coronary artery, genotoxicity of caecal water, selected steroid concentrations, gut microbiota parameters, and the animals' body weight gains. Samples for laboratory analyses were collected from the same pre-pubertal gilts. The results of these analyses were presented in our previous studies [7,16,28,29].

#### *2.3. Concentrations of Zearalenone and Its Metabolites in the Bone Marrow Microenvironment*

Zearalenone concentrations in the bone marrow microenvironment did not differ significantly between the experimental groups, but the lowest values were noted on the first two exposure dates (5.64 ng/g on D1; 4.69 ng/g on D2) in group ZEN5 (5 µg ZEN/kg BW) (see Table 1). In turn, significant differences at *p* ≤ 0.05 were observed between group ZEN5 and group ZEN10 (7.74 ng/g and 7.35 ng/g, respectively) on D3. Significant differences at *p* ≤ 0.01 were noted between group ZEN5 and group ZEN15 (7.74 ng/g and 7.03 ng/g, respectively) on D3, and in group ZEN15 between D2 and D3 vs. D1 (6.84 ng/g and 7.03 ng/g vs. 8.17 ng/g, respectively). Similar values had been reported in other tissues, such as the heart muscle [19]. Zearalenone metabolites, α -ZEL and β-ZEL, were not detected (values below the sensitivity of the method).

#### *2.4. Carry-OVER Factor (CF)*

The carry-over of ZEN from the intestinal lumen to the bone marrow microenvironment collected from the wing of ilium (posterior superior iliac spine), was influenced by the administered dose and time of exposure in each group (see Table 1). The lowest values of the CF were noted in group ZEN5 (in particular on D1 and D3 at 7 <sup>×</sup> <sup>10</sup>−<sup>5</sup> and <sup>6</sup> <sup>×</sup> <sup>10</sup>−<sup>5</sup> , respectively), and the highest CF values were observed in group ZEN15 (in particular on D3 at 98 <sup>×</sup> <sup>10</sup>−<sup>7</sup> ). The values of the CF determined in this study were proportionally higher relative to the values noted in the blood serum [28] and the heart muscle [19] of the same animals.


**Table 1.** The carry-over factor (CF) and mean (*x*) concentrations of ZEN and ZELs (ng/g) in the bone marrow microenvironment of pre-pubertal gilts.

**Abbreviation**: D1, exposure day 7; D2, exposure day 21; D3, exposure day 42. Experimental groups: Group ZEN5, 5 µg ZEN/kg BW; Group ZEN10, 10 µg ZEN/kg BW; Group ZEN15, 15 µg ZEN/kg BW. LOD > values below the limit of detection were expressed as 0. Statistically significant differences were determined at \* *p* ≤ 0.05 and \*\*,•• *<sup>p</sup>* <sup>≤</sup> 0.01; \*, \*\* statistical difference between group ZEN5 vs. group ZEN10 and group ZEN15 on exposure date D3; •• statistical difference in group 3 between exposure date D1 and exposure dates D2 and D3.

#### *2.5. Results of Haematological Analyses*

Only statistically significant differences in all animal groups are presented in the figure drawings. The results of this study and the findings of other authors indicate that even very low concentrations of mycotoxins in feed materials can lead to changes in blood homeostasis in pre-pubertal gilts [10,30–32]. The significant differences in the haematological parameters of pre-pubertal gilts exposed to various doses of ZEN (MABEL, highest NOAEL values, and LOAEL) for 6 weeks did not exceed the reference range [32,33]. In view of the above, the results noted in group C were unambiguous (see Supplementary Materials—Table S1), and they could be used as a reference in a risk assessment analysis, where each result is considered within a range of positive control (increase) or negative control (decrease) values during subchronic exposure to the mycotoxin.

## *2.6. Haematological Analyses*

#### 2.6.1. General Analysis

Significant differences were rarely noted, mostly in erythrocyte parameters in the middle (see Figure 1) and at the end of the experiment (see Figure 2), and in the percentages of white blood cells on the first and last date of exposure (see Figure 2). At the beginning of the experiment, the analysed parameters were generally lower in group C, whereas the reverse was noted towards the end of the study. Significant differences were not observed on date 9.

#### 2.6.2. Accompanying Factors

The results of this study could have been influenced by several accompanying factors, including (i) the manner of ZEN transmission to the body, (ii) ZEN dose, (iii) and/or the kinetic effects of mycotoxin bioassimilation [13]. The latter can be subdivided into several sub-processes, beginning from mycotoxin extraction from the feed matrix to its absorption, distribution, and deposition in tissues, and mycotoxin modification [15]. This is a very important consideration, but it was not studied in the described experiment.

The above can have two effects: (i) higher demand for energy needed for biotransformation and constitutive growth of pre-pubertal gilts [7,34], and (ii) a milder response to the applied mycotoxin doses in the peripheral vascular system (*vena cava cranialis*) from which samples for metabolic analyses were collected. As a result, the exposure to low doses of ZEN with low values of the CF to intestinal tissues (towards the end of the experiment) and the liver probably induced minor but significant changes in the values of selected haematological parameters. Changes in WBC (White Blood Cells), EOS (Eosinophils), BASO (Basophils), MONO (Monocytes), RBC—Red Blood Cells; HGB—Haemoglobin, and

HCT—Haematocrit, values were observed mainly in the first three weeks of exposure (between sampling dates 1 and 6; see Tables 2–4). *Toxins* **2022**, *14*, x FOR PEER REVIEW 5 of 19 *Toxins* **2022**, *14*, x FOR PEER REVIEW 5 of 19

> **Figure 1.** Selected haematological parameters on the fourth analytical date ( *x* , SD) Key: C, control group; Group ZEN5, 5 μg ZEN/kg BW; Group ZEN10, 10 μg ZEN/kg BW; Group ZEN15, 15 μg ZEN/kg BW. Statistically significant differences: \* at *p* ≤ 0.05; \*\* at *p* ≤ 0.01. **Figure 1.** Selected haematological parameters on the fourth analytical date (*x*, SD) Key: C, control group; Group ZEN5, 5 µg ZEN/kg BW; Group ZEN10, 10 µg ZEN/kg BW; Group ZEN15, 15 µg ZEN/kg BW. Statistically significant differences: \* at *p* ≤ 0.05; \*\* at *p* ≤ 0.01. **Figure 1.** Selected haematological parameters on the fourth analytical date ( *x* , SD) Key: C, control group; Group ZEN5, 5 μg ZEN/kg BW; Group ZEN10, 10 μg ZEN/kg BW; Group ZEN15, 15 μg ZEN/kg BW. Statistically significant differences: \* at *p* ≤ 0.05; \*\* at *p* ≤ 0.01.

**Figure 2.** Selected haematological parameters on the tenth analytical date ( *x* , SD). Key: C, control group; Group ZEN5, 5 μg ZEN/kg BW; Group ZEN10, 10 μg ZEN/kg BW; Group ZEN15, 15 μg ZEN/kg BW. Statistically significant differences: \* at *p* ≤ 0.05; \*\* at *p* ≤ 0.01. **Figure 2.** Selected haematological parameters on the tenth analytical date ( *x* , SD). Key: C, control group; Group ZEN5, 5 μg ZEN/kg BW; Group ZEN10, 10 μg ZEN/kg BW; Group ZEN15, 15 μg ZEN/kg BW. Statistically significant differences: \* at *p* ≤ 0.05; \*\* at *p* ≤ 0.01. **Figure 2.** Selected haematological parameters on the tenth analytical date (*x*, SD). Key: C, control group; Group ZEN5, 5 µg ZEN/kg BW; Group ZEN10, 10 µg ZEN/kg BW; Group ZEN15, 15 µg ZEN/kg BW. Statistically significant differences: \* at *p* ≤ 0.05; \*\* at *p* ≤ 0.01.

a very important consideration, but it was not studied in the described experiment.

a very important consideration, but it was not studied in the described experiment.

the applied mycotoxin doses in the peripheral vascular system (*vena cava cranialis*) from which samples for metabolic analyses were collected. As a result, the exposure to low doses of ZEN with low values of the CF to intestinal tissues (towards the end of the experiment) and the liver probably induced minor but significant changes in the values of

the applied mycotoxin doses in the peripheral vascular system (*vena cava cranialis*) from which samples for metabolic analyses were collected. As a result, the exposure to low doses of ZEN with low values of the CF to intestinal tissues (towards the end of the experiment) and the liver probably induced minor but significant changes in the values of

The results of this study could have been influenced by several accompanying factors, including (i) the manner of ZEN transmission to the body, (ii) ZEN dose, (iii) and/or the kinetic effects of mycotoxin bioassimilation [13]. The latter can be subdivided into several sub-processes, beginning from mycotoxin extraction from the feed matrix to its absorption, distribution, and deposition in tissues, and mycotoxin modification [15]. This is

The results of this study could have been influenced by several accompanying factors, including (i) the manner of ZEN transmission to the body, (ii) ZEN dose, (iii) and/or the kinetic effects of mycotoxin bioassimilation [13]. The latter can be subdivided into several sub-processes, beginning from mycotoxin extraction from the feed matrix to its absorption, distribution, and deposition in tissues, and mycotoxin modification [15]. This is

2.6.2. Accompanying Factors

2.6.2. Accompanying Factors


**Table 2.** Selected haematological parameters in group ZEN5 on different analytical dates (*x*, SD).

Key: Group ZEN5, 5 µg ZEN/kg BW; WBC, White Blood Cells; MONO, Monocytes; LUC, Large Unstained Cells; MPV, Mean Platelet Volume. Statistical symbols: <sup>a</sup> , relative to date 1; <sup>b</sup> , relative to date 4; <sup>c</sup> , relative to date 5; d , relative to date 6; <sup>e</sup> , relative to date 9; <sup>f</sup> , relative to date 10. Statistically significant differences: a,b,c,d and <sup>f</sup> at *<sup>p</sup>* <sup>≤</sup> 0.05; bb,cc,ee and ff at *<sup>p</sup>* <sup>≤</sup> 0.01.

Significant differences in PLT (Platelet Count) and PLT clump values were also noted on the same dates (see Table 5). These changes were probably induced by compensatory mechanisms [35].
