Hair Corticosterone Analysis

After cervical dislocation at week 7 (Procedure 1), mice hair of the dorsal zone was shaved using an electric razor. Hair samples were stored in 1.5 ml polypropylene tubes at -20°C. Extraction and analysis of corticosterone concentration were performed according to a previously described protocol (Erickson et al., 2017). Briefly, hair samples were washed with methanol (5 ml) twice rotating for 3 min. After methanol decantation, samples were placed on aluminum foil and dried in a protected hood for 3 days. Dried samples were weighed and transferred to 2 ml polypropylene tubes containing stainless steel grinding beads (2.8 mm Stainless Steel Grinding Balls Pre-Filled Tubes, OPS Diagnostics, Lebanon, NJ) that were placed in a bead beater (Mixermill MM300, Miguel Hernandez University, Alicante, Spain) to produce a powder. Powdered hair samples were incubated with 1.5 ml of methanol for 24 h on slow rotation to extract steroids. Tubes were centrifuged and steroid-containing supernatants were dried in a protected hood for 2–3 days to evaporate methanol. Dry extracts were analysed by a commercial competitive enzyme-linked immunosorbent assay (ELISA) kit (EIACOR, Invitrogen, Spain) following manufacturer instructions.