*4.2. Experimental Feed*

The feed administered to animals (Table 2) was analysed for the presence of ZEN and DON. Mycotoxin content was determined by standard separation techniques using 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) (Hewlett Packard, type 1050 and 1100) [67] with fluorescence and/or ultraviolet detection techniques. The detection limit was 3.0 ng/g for ZEN [19] and 1.0 ng/g for DON [36].


**Table 2.** Mixture of diets for pre-pubertal gilts (first stage of rearing).

Abbreviation: Composition 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; niacin—1200 mg; 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; magnesium—5 g.

#### *4.3. Experimental Design*

The animals were allocated to an experimental group (E = ZEN; n = 18) and a control group (C, n = 18) [68,69]. The animals in group E were orally administered ZEN at a dose of 40 µg/kg BW (Table 3). The pigs in group C were given a placebo. At the time when this test was designed, the above value complied with the recommendations of the European Food Safety Authority (CR 2006/576/EC—2006 [70]) and No-Observed-Adverse-Effect Level (NOAEL) dose. The mycotoxin was administered every morning before feeding, in gel capsules that dissolved in the stomach. In group C, pigs received identical gel capsules, but without the mycotoxin.

**Table 3.** Diurnal feed intake in a restricted feeding regime (kg/day) and the average zearalenone concentration per kg feed (µg ZEN/kg feed).


Zearalenone was biosynthesised at the Faculty of Chemistry at the University of Life Sciences in Pozna´n. The trial lasted 42 days. Zearalenone doses were adapted to the BW of gilts. Zearalenone was served in capsules to avoid potential problems resulting from unequal feed intake. Zearalenone samples were dissolved in 500 µL 96% C2H5OH (96% ethyl SWW 2442-90, Polskie Odczynniki Chemiczne SA, Poland) to obtain the required dose (converted to BW). The solutions were kept at 20 ◦C for twelve hours. The gilts were weighed at weekly intervals to adjust the ZEN dose of each animal. Three gilts from each group (six animals in total) were euthanised on days 7 (date I), 14 (date II), 21 (date III), 28 (date IV), 35 (date V), and 42 (date VI) by intravenous administration of sodium pentobarbital (Fatro, Ozzano Emilia BO, Italy). Directly after cardiac arrest, part of the intestinal tissue were taken and prepared for analysis.

#### *4.4. Reagents*

ZEN was obtained from the Faculty of Chemistry, University of Life Sciences in Pozna ´n based on an earlier developed methodology [71,72] presented in other studies [73].

#### *4.5. Chemicals and Equipment*

The chromatographic analysis of ZEN was conducted at the Faculty of Chemistry, University of Biosciences in Pozna ´n based on an earlier developed methodology [73].

### *4.6. Tissue Samples*

On each experimental day, intestinal tissue samples (approx. 1 × 1.5 cm) were collected from the succeeding segments of the GI tract of gilts: the duodenum—the first part and the third section; the jejunum and ileum—the middle part; the large intestine—the middle parts of the ascending colon, transverse colon and descending colon; and the caecum—1 cm from the ileocecal valve. The samples were rinsed with phosphate buffer.

#### *4.7. Immunohistochemistry*

#### 4.7.1. Localisation of ERα and ERβ

Tissue samples were fixed in four percent paraformaldehyde and embedded in paraffin. Two samples from each test section were stained to determine the ERα and ERβ expression. In the negative control, the primary antibody was omitted. To unmask the antigens, the sections were placed in citrate buffer (Sigma-Aldrich, Saint Louis, MO, USA) and cooked for 20 min in a microwave oven at 800 W. The sections were coated with ready-to-use DAKO REALTM Peroxidase Blocking Solution (DAKO, Glostrup, Denmark) and reacted for 15 min. Non-specific antigen binding areas were blocked with 2.5% normal goat serum solution. The sections were reacted overnight at a temperature of 6 ◦C with the following primary

antibodies: Mouse Anti-Human Oestrogen Receptor α (Clone: 1D5, DAKO Santa Clara, CA, USA) and Mouse Anti-Oestrogen Receptor β (Clone: 14C8, Abcam, Cambridge, UK), diluted to 1:60 and 1:20, respectively. After the reaction, the specimens were rinsed three times with PBS (Sigma-Aldrich, Saint Louis, MO, USA) at five-minute intervals. Secondary antibodies conjugated with horseradish peroxidase-labelled micropolymer (ImmPRESS™ HRP Universal Antibody, Vector Laboratories, Burlingame, CA, USA) were applied to the specimens. The sections were coloured by incubation with DAB (DAKO, Glostrup, Denmark) for 3 min, and H2O<sup>2</sup> was added to visualise the activity of the bound enzyme (brown colour). The sections were washed with water and contrast stained with Mayer's haematoxylin solution (Sigma-Aldrich, Saint Louis, MO, USA). The primary antibody was ignored in the negative control. Negative controls (solvent-coated slides only, no primary antibody) and positive controls were converted together with the slides [74]. The pig's ovary was used as a positive control for ERβ [75].

#### 4.7.2. Scanning of the Coloured Slides

The expressions of ERα and ERβ were analysed on the scanned slides (Pannoramic MIDI scanner, 3DHISTECH, Budapest, H) using the NuclearQuant programme (3DHIS-TECH, H). The slides were converted into digital images (Figures 1 and 2). The profile of nuclear detection and staining intensity were as previously described [59].

#### *4.8. Statistical Analysis*

The activity of ERα and ERβ in the GI tract of pigs was presented on the basis of ± and SD for each sample. The results were compiled using the Statistica programme (StatSoft Inc., USA). Based on the applied ZEN dose and the duration of its application, the arithmetic means for systems with repeatable measurements were compared using one-way analysis of variance. The homogeneity of variance in the compared groups was checked with the Brown–Forsythe test. Differences between groups were analysed using Tukey's honestly significant difference test (*p* < 0.05 or *p* < 0.01).

**Author Contributions:** The experiments were designed and planned by M.G. and M.T.G. The experiments were conducted by I.O.-D., P.B., S.L.-Z. and M.G. The data were analysed and interpreted ˙ by M.D. and M.G. The manuscript was written by M.G. and critically revised by Ł.Z. and M.T.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Polish Ministry of Science and Higher Education as part of Project No. 12-0080-10/2010. The project was financially supported by the Minister of Education and Science under the program entitled "Regional Initiative of Excellence" for the years 2019–2023, Project No. 010/RID/2018/19, amount of funding PLN 12,000,000.

**Institutional Review Board Statement:** All experimental procedures involving animals were carried out in compliance with Polish regulations setting forth the terms and conditions of animal experimentation for 2010–2013 (opinion no. 88/2009 of the local Ethics Committee for Animal Experimentation at the University of Warmia and Mazury in Olsztyn, Poland of 16 Dec 2009). All of the investigators are authorised to perform experiments on animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


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