4.7. Urine Corticosterone Sampling

Urine samples were collected daily for corticosterone levels, by gently removing each rat to metabolic cages for 30 min, as described previously [74]. Animals were placed in these cages from their home cage and, once the procedure was complete, the animal was returned to its home cage. Rats were allowed to acclimate to the metabolic cage for 7 days before urine collection. Metabolic cages are regular cages with grooves along the floor, allowing for urine collection in suspended calibrated cylinders. All samples were immediately frozen (−80 °C) after collection. Samples were taken 60–90 min following exposure (between 12:30 and 14:00 h). Corticosterone concentrations were measured with a DSL-10-81000 ELISA kit according to the instructions of the manufacturer (Diagnostic Systems Laboratories, Webster, TX, USA) by a person blind to experimental procedures. All samples were measured in duplicate.