Serum corticosterone
Blood was collected from the catheter implanted into the femoral artery. The first blood sample was collected to measure the baseline value of the hormone corticosterone (basal measurement). Fifteen minutes after the onset of restraint stress, a new sample was taken (stress measurement). The volume obtained for each sample was 0.3 ml. To obtain the serum, the samples remained at room temperature for 1 h to allow clot retraction and were subsequently centrifuged at 2000 xg for 15 min. After centrifugation, the serum was separated and stored in a -80 °C freezer for further analysis. Serum corticosterone was measured by immunoenzymatic assay (ELISA) using a commercially available kit (Cayman Chemical, Michigan, USA).