*4.1. General Information*

All experimental procedures involving animals were carried out in compliance with Polish regulations setting forth the terms and conditions of animal experimentation (Opinions No. 12/2016 and 45/2016/DLZ of the Local Ethics Committee for Animal Experimentation the University of Warmia and Mazury in Olsztyn, Poland in of 27 April 2016 and 30 November 2016).

### *4.2. Experimental Animals and Feed*

The in vivo experiment was performed at the Department of Veterinary Prevention and Feed Hygiene of the Faculty of Veterinary Medicine at the University of Warmia and Mazury in Olsztyn on 60 clinically healthy pre-pubertal gilts with initial BW of 14.5 ± 2 kg [9]. The animals were housed in pens with free access to water. All groups of gilts received the same feed throughout the experiment. They were randomly assigned to three experimental groups (group ZEN5, group ZEN10 and group ZEN15; *n* = 15) and a control group (group C; *n* = 15 — control group) [48,49]. Group ZEN5 gilts were orally administered ZEN (Sigma-Aldrich Z2125-26MG, St. Louis, MO, USA) at 5 μg ZEN/kg BW, group ZEN10 pigs — at 10 μg ZEN/kg BW, and group ZEN15 pigs — at 15 μg ZEN/kg BW. Analytical samples of ZEN were dissolved in 96 μL of 96% ethanol (SWW 2442-90, Polskie Odczynniki SA, Poland) in weight-appropriate doses. Feed containing different doses of ZEN in an alcohol solution was placed in gel capsules. The capsules were stored at room temperature before administration to evaporate the alcohol. In the experimental groups, ZEN was administered daily in gel capsules before morning feeding. The animals were weighed at weekly intervals, and the results were used to adjust individual mycotoxin doses. Feed was the carrier, and group C pre-pubertal gilts were administered the same gel capsules, but without mycotoxins [7–9].

The feed administered to all experimental animals was supplied by the same producer. Friable feed was provided ad libitum twice daily, at 8:00 a.m. and 5:00 p.m., throughout the experiment. The composition of the complete diet, as declared by the manufacturer, is presented in Table 4.


**Table 4.** Declared composition of the complete diet.

1Composition of the vitamin-mineral premix per kg: vitamin A — 500,000 IU; iron — 5000 mg; vitamin D3 — 100,000 IU; zinc — 5000 mg; vitamin E (alpha-tocopherol) — 2000 mg; manganese — 3000 mg; vitamin K — 150 mg; copper (CuSO4·5H2O) — 500 mg; vitamin B1 — 100 mg; cobalt — 20 mg; vitamin B2 — 300 mg; iodine — 40 mg; vitamin B6 — 150 mg; selenium — 15 mg; vitamin B12 — 1500 μg; L-lysine — 9.4 g; niacin — 1200 mg; DL-methionine + cystine — 3.7 g; pantothenic acid — 600 mg; L-threonine — 2.3 g; folic acid — 50 mg; tryptophan — 1.1 g; biotin — 7500 μg; phytase + choline — 10 g; ToyoCerin probiotic + calcium — 250 g; antioxidant + mineral phosphorus and released phosphorus — 60 g; magnesium — 5 g; sodium; calcium — 51 g.

The proximate chemical composition of diets fed to pigs in groups C, ZEN5, ZEN10, and ZEN15 was determined using the NIRS™ DS2500 F feed analyzer (FOSS, Hillerød, Denmark), a monochromator-based NIR reflectance and transflectance analyzer with a scanning range of 850–2500 nm.

### *4.3. Determination of Mycotoxins in Feed*

Feed was analyzed for the presence of mycotoxins and their metabolites: ZEN, α-ZEL and DON. Mycotoxin concentrations in feed were determined by separation in immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA; DON-TestTM DON Testing System, VICAM, Watertown, MA, USA) and high-performance liquid chromatography (HPLC system, Hewlett Packard type 1050 and 1100) — mass spectrometry (MS) and chromatographic column (Atlantis T3 3 μm 3.0 × 150 mm Column No. 186003723, Waters, AN Etten-Leur, Ireland). The mobile phase was a water and acetonitrile mixture with an 80:10 solvent ratio and 2 ml of CH3 COOH. The flow rate was 0.4 mL/min. The obtained values did not exceed the limits of quantitation (LoQ) of 2 ng/g for ZEN and 5 ng/g for DON. The analyzed compounds were quantified at the Department [50].

### *4.4. Blood Sampling*

Blood was sampled from 5 gilts from every group on three analytical dates: exposure day 7 (D1), exposure day 21 (D2) and exposure day 42 (D3). Directly before slaughter, blood samples of 20 mL each were collected from all gilts (blood was sampled within 20 s after immobilization [51]) by jugular venipuncture into syringe containing 0.5 mL of heparin solution. Blood was centrifuged at 3000 rpm for 20 min at 4 ◦C. The obtained plasma samples were stored at – 18 ◦C until the analyses of ZEN, α-ZEL, β-ZEL, estradiol (E2), progesterone (P4) and testosterone (T) concentrations.

### *4.5. Determination of Hormone Concentrations*

### 4.5.1. Estradiol

Estradiol concentration was determined at the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn, Poland. Blood plasma concentrations of E2 were analyzed by the radioimmunoassay (RIA) method with a commercially available kit (ESTR-US-CT, CIS BIO ASSAYS), as described previously [52,53]. All measurements were performed in duplicate for every cultured probe. The extraction yield for E2 was 90.67% <sup>±</sup> 0.73%. The radioactivity of samples with J125 was measured with the Wallac 1470 WIZARDÆ automatic gamma scintillation counter (Perkin Elmer, Waltham, MA, USA). Radioactivity was determined within 1 min with a Geiger counter (counting

efficiency, 75%). The sensitivity of the E2 assay was 1.36 pg/mL. The standard curve range was from 2.72 to 550 pg/mL. The intra- and inter-assay coefficients of variation were 5% and 5%, respectively.

### 4.5.2. Progesterone and Testosterone

Progesterone and testosterone were quantified at the Analytical Laboratory of the Municipal Hospital with Polyclinic in Olsztyn, Poland, by the ECLIA electrochemiluminescence assay with the use of Elecsys Progesterone II and Elecsys Testosterone II assays and the Cobas c6000 analyzer (Hitachi, Tokyo, Japan). In the first stage, the samples were incubated with biotinylated monoclonal antibodies specific for P4 and T and for P4 and T derivatives labeled with a ruthenium complex. The extent to which the hormones were bound to antibodies was determined by their concentrations. Streptavidin-coated microspheres were added in the second stage, and the complex was bound to the solid phase during the interactions between biotin and streptavidin. The quantity of labeled P4 bound to the solid phase was inversely proportional to the concentration of P4 in the sample. The reaction mix was sucked into the measuring cell where microspheres were magnetically captured on the surface of the electrode. Unbound compounds were removed with the ProCell. Voltage was applied to the electrode, and the resulting chemiluminescence was measured with a photomultiplier. The results were read from a two-point calibration curve and a standard curve developed with a barcode verifier. The analytical range of the method was determined by the lower limit of detection and the highest point on the calibration curve at 0.03–60 ng/mL for P4 and 0.025–15 ng/mL for T. All determinations were performed in accordance with the manufacturer's instructions.

### 4.5.3. Statistical Analysis

Hormone concentrations were measured in three experimental groups and the control group on three analytical dates. The results were expressed as mean values (*x*) and standard deviation (SD) for each sample. The following assays were performed for every hormone: (i) the differences between means were analyzed for the experimental groups and the control groups on fixed dates and (ii) the differences between means were analyzed in a fixed group on each analytical date. In both cases, the determinations were made by one-way ANOVA. If the differences between group means were statistically significant, the differences between pairs of means were determined by Tukey's multiple comparison test. The equality of variances in the compared groups was evaluated with Levene's test and the Brown–Forsythe test. If the equal variance hypothesis was rejected in both tests, the significance of differences was evaluated with the Kruskal–Wallis non-parametric test. In each analysis, the tested values were regarded as highly significant at *P* < 0.01 (\*\*) and as significant at 0.01 < *P* < 0.05 (\*). Linear correlations between the concentrations of steroid hormones in fixed groups were determined based on the values of the Pearson's correlation coefficient [22]. Data were processed in Statistica v. 13 (TIBCO Software Inc., Silicon Valley, CA, USA, 2017).

### *4.6. Extraction Procedure*

The presence of zearalenone, α-ZEL and β-ZEL in the blood plasma were determined with the use of immuno-affinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA) and different protocols for each compound. All extraction procedures were performed according to the recommendations of column manufacturers. After extraction, the eluates were placed in a water bath at 50 ◦C, and the solvent was evaporated in a stream of nitrogen. Next, 0.5 mL of 99.8% methanol was added to dry residues to dissolve the mycotoxin.

### 4.6.1. Quantification of ZEN and Its Metabolites

The presence of ZEN, α-ZEL and β-ZEL in the blood plasma was determined by various separation methods with the use of immuno-affinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA) and the Agilent 1100 series liquid chromatography (LC)/mass spectrometry (MS) system. The prepared sample was estimated with the use of a

chromatographic column (Atlantis T3 3 μm 3.0 × 150 mm Column No. 186003723, Waters, AN Etten-Leur, Ireland). The mobile phase consisted of 70% acetonitrile (LiChrosolvTM, No. 984 730 109, Merck-Hitachi, Mannheim, Germany), 20% methanol (LiChrosolvTM, No. 1.06 007, Merck-Hitachi, Mannheim, Germany) and 10% deionized water (Milipore Water Purification System, Millipore S.A. Molsheim-France, 2 mL of CH3 COOH). The immunoaffinity bed in the column was washed with demineralized water (Millipore Water Purification System, Millipore S.A., Molsheim, France). The flow rate was 0.4 mL/min., and the temperature of the oven column was 40 ◦C. The chromatographic analysis was completed in 4 min. The column was eluted with 99.8% methanol (LIChrosolvTM, No. 1.06 007, Merck-Hitachi, Mannheim, Germany) to remove the bound mycotoxin. The eluates were placed in a water bath at 50 ◦C, and the solvent was evaporated in a stream of nitrogen. In the next step, 0.5 mL of 99.8% methanol was added to dry residues to dissolve the mycotoxin. Mycotoxin concentrations were determined according to the external standard and were expressed in ppb (ng/mL).

Matrix-matched calibration standards were used for quantification to avoid matrix effects which can reduce sensitivity. The calibration standards were dissolved in the sample matrix prepared according to the same procedure as the remaining samples. The material used for the preparation of calibration standards was mycotoxin-free. The limits of detection (LODs) for individual mycotoxins were determined as the concentrations at which the signal-to-noise ratio decreased to 3. Alpha-ZEL and beta-ZEL were also determined. Derivative concentrations were below the LODs, and they were separated from their respective parent compounds during purification.

### 4.6.2. Statistical Analysis

The concentrations of ZEN and its metabolites in the blood plasma of prepubertal gilts were analyzed in the control group and in three experimental groups on three analytical dates. The results were expressed as mean values (*x*) and standard deviation (SD) for each sample. The following assays were performed for every hormone: (i) the differences between means were analyzed for the three ZEN doses (experimental groups) and the control groups on fixed dates, and (ii) the differences between means were analyzed for a fixed ZEN dose (group) on each analytical date. In both cases, the differences between means were determined by one-way ANOVA. If the differences between group means were statistically significant, the differences between pairs of means were determined with Tukey's multiple comparison test. If all values were below the limit of detection (mean and variance equal to zero) in any group, one-way ANOVA was performed for the remaining groups (if the number of the remaining groups was higher than two), and the means of these groups were compared against zero with the use of Student's *t*-test. The differences between groups were determined with Student's *t*-test. In each analysis, the tested values were regarded as highly significant at *P* < 0.01 (\*\*) and as significant at 0.01 < *P* < 0.05 (\*). Data were processed in Statistica v. 13 (TIBCO Software Inc., Silicon Valley, CA, USA, 2017).

**Author Contributions:** The experiments were conceived and designed by M.G. and M.T.G. The experiments were performed by K.C., A.R., E.O. and M.D. Data were analyzed and interpreted by K.C., M.D., M.B., S.L.-Z. and M.G. The manuscript was drafted by K.C. and M.G. and critically edited by A.B., Ł.Z. and M.T.G.

**Funding:** The study was supported by the "Healthy Animal - Safe Food" Scientific Consortium of the Leading National Research Centre (KNOW) pursuant to a decision of the Ministry of Science and Higher Education No. 05-1/KNOW2/2015.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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