Narketan

All of the animals were humanely euthanized 24 h after the last treatment by exsanguination under Xylapan/Narketan anaesthesia (Narketan, Vetoquinol UK Ltd., Towcester, UK, 80 mg/kg b. w.; Xylapan, Vetoquinol UK Ltd., 12 mg/kg b. w., i.p.). A licensed veterinarian at IMROH examined gross pathological changes of the internal organs in all of the animals.
Testes and epididymis were dissected, cleaned from adhering matters, and rinsed in cold PBS buffer (without Ca²⁺ and Mg²⁺). The tissues were symmetrically bisected and weighed. The left testis/epididymis was stored in cryo-tubes without additional media or cryoprotectants, while the right testis/epididymis was symmetrically halved and stored separately. One-half was used for the measurements of antioxidant status parameters and the other one for element analysis. The tubes were immediately frozen in liquid nitrogen and stored at −80 °C until use.

At the end of the experiment, all animals were humanely euthanized by exsanguination under intraperitoneal Xylapan/Narketan anesthesia (Xylapan, Vetoquinol UK Ltd., Towcester, UK, 12 mg/kg bw i. p./Narketan, Vetoquinol UK Ltd., 80 mg/kg bw). A licensed veterinarian at IMROH examined the animals for gross pathological changes of the internal organs. The blood samples were collected in heparinized vacutainers by a dissection of the carotid artery under general anesthesia and further processed. One portion of whole blood was immediately used to prepare agarose microgels for the alkaline comet assay while the portion used for biochemical analyses was centrifuged (980× g, 10 min, at +4 °C) to separate red blood cells and plasma that were stored at −20 °C until further processing. Livers, kidneys, and brains were dissected out and weighted. Based on the obtained values, relative liver, kidney, and brain weight were calculated using the following formula:
Tissues were rinsed in cold PBS buffer (without Ca²⁺ and Mg²⁺), weighed, and divided into two portions intended for biochemical analyses and the comet assay. The samples used for biochemical analyses were immediately frozen in liquid nitrogen and stored at −80 °C until further processing while the tissue samples used for comet assay were prepared as described later in the text.

Pregnant dams were euthanized with i.p. injection of ketamine (0.8 mL/kg, Narketan; Vetoquinol, Bern, Switzerland) and xylazine (0.6 mL/kg, Xylapan; Vetoquinol, Lure. Cedex, France) on GD 15 or GD 20. Placentas were isolated, weighted (all 352), and fixed in St. Marie solution (96% EtOH with 1% glacial acetic acid), dehydrated, and embedded in paraffin. Serial sections (5 µm) were cut for histology or immunohistochemistry on a Leica microtome.
DNA was isolated in TE buffer pH9 with 0.1 mg/mL of Proteinase K and 0.25% of Nonidet P40 (both from (Sigma, St. Louis, MO, USA) at 56 °C for 24 h, heated for 10 min at 95 °C to inactivate Proteinase K, spun and the supernatant was then frozen at −20 °C. DNA concentration and quality were measured with the NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA).