*5.4. Toxicological Studies of Reproductive, Hypothalamic, and Pituitary Gland Tissues* 5.4.1. Tissue Samples

Five prepubertal gilts from every group were euthanized on analytical date 1 (D1 exposure day 7), date 2 (D2—exposure day 21), and date 3 (D3—exposure day 42). Initially, general sedation was performed by intravenous administration of pentobarbital sodium (Fatro, Ozzano Emilia BO, Italy) and bleeding. Immediately after cardiac arrest, tissue samples (approximately 1 × 1.5 cm) were collected from the following segments: entire left ovary; left uterine horn (from the ovarian and uterine sections); middle part of the cervix; the entire hypothalamus; and the pituitary gland. The samples were rinsed with phosphate

buffer and prepared for analyses. The collected samples were stored at a temperature of −20 ◦C.

#### 5.4.2. Extraction Procedure

The presence of ZEN, α-ZEL, and β-ZEL in tissue samples was determined in accordance with the with the study protocol described previously [54].

#### 5.4.3. Chromatographic Quantification of Zearalenone and Its Metabolites

Zearalenone and its metabolites were quantified at the Institute of Dairy Industry Innovation in Mr ˛agowo, Poland. The biological activity of ZEN, α-ZEL, and β-ZEL in the tissues was determined in accordance with the study protocol described previously [54].

Mycotoxin concentrations were determined with an external standard and were expressed in ng/mL. Matrix-matched calibration standards were applied in the quantification process to eliminate matrix effects that can reduce sensitivity. Calibration standards were dissolved in matrix samples based on the procedure that was used to prepare the remaining samples. The material for the calibration standards was free of undesirable substances. The limits of detection (LOD) for ZEN, α-ZEL, and β-ZEL were determined as the concentration at which the signal-to-noise proportion decreased to 3. The concentrations of ZEN, alfa-ZEL and beta-ZEL were determined in each group and on 3 analytical dates (see Table 1).

5.4.4. Mass Spectrometry Conditions

The electrospray ionization (ESI) mass spectrometer was operated in the negative ion operation. MS/MS parameters were optimized for every compound. Linearity was tested with a calibration curve including 6 levels. The optimal analytical conditions for the tested mycotoxins are presented in Table 5.

**Table 5.** Optimized conditions for the tested mycotoxins [15].


#### 5.4.5. CF

Carry-over toxicity takes place when an organism is able to survive under exposure to low doses of mycotoxins. Mycotoxins can exert a negative effect on tissues or organ function [56] and modify their activity [10,37]. The carry-over was determined in the examined tissues when the daily dose of zearalenone (5, 10, or 15 µg ZEN/kg BW) administered to each animal was equivalent to 560-32251.5 µg zearalenone/kg of the complete diet, depending on daily feed consumption. Mycotoxin contents in tissues were expressed in terms of the dry matter content of the samples.

The CF was calculated as follows:

CF = toxin concentration in tissue [ng/g]/toxin concentration in diet [ng/g]

#### 5.4.6. Statistical Analysis

Data were processed statistically at the Department of Discrete Mathematics and Theoretical Computer Science, of the Faculty of Mathematics and Computer Science of the University of Warmia and Mazury in Olsztyn, Poland, as described previously [37].

#### *5.5. Toxicological Studies of Blood*

#### Blood samples collection

Blood was sampled in vivo from 5 gilts from every group on each analytical date. The first analytical date was exposure day 7 (D1); the second analytical date was exposure

day 14 (D2); the third analytical date was exposure day 21 (D3); the fourth analytical date was exposure day 28 (D4); the fifth analytical date was exposure day 35 (D5), and the sixth analytical date was exposure day 42 (D6). Blood samples of 20 mL each were collected from all gilts (blood was sampled within 20 seconds after immobilization [57]) directly before slaughter by jugular venipuncture with a syringe containing 0.5 mL of heparin solution. The collected blood was centrifuged at 3000 rpm for 20 minutes at 4 ◦C. The obtained plasma samples were stored at −18 ◦C until estradiol (E2) and progesterone (P4) concentration analyses were performed.
