Metabolic measurements

Energy expenditures were assessed using an indirect open-circuit calorimeter Oxylet Physiocage System (LE1305 Physiocage 00; LE405 O2/CO2 Analyzer; LE400 Air Supply and Swithching; Panlab, Cornella, Spain), equipped with a laser absorption O₂ sensor and an infrared technology CO₂ sensor. The experiment took 2 days, the first day for acclimation and the second day for testing. Indirect calorimetry recording was performed with room air flowing through each chamber at a rate of 450 ml/min. The O₂ and CO₂ levels were measured during 3 min sampling periods triggered every 30 min, and data were then analyzed using the METABOLISM software (v2.2.01). Oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were expressed in milliliter per minute per kilogram. The RER was determined by the ratio VCO₂/VO₂. Energy expenditure (EE) was calculated according to the formula EE (kcal/day per kg) = VO₂ × 1.44 × [3.815 + (1.232 × respiratory quotient)]. The O₂/CO₂ analyzer was calibrated with purified gas standards, and the food hoppers were replenished every day at the beginning of the light phase. The mean values for RER and EE of dark cycle and light cycle were compared for each group.