Thermoneutral environment improves mouse welfare and reduces stress in metabolic cages.

Metabolic cages (MCs) are often used to collect feces and urine samples. However, the housing of mice in MCs can be stressful, potentially affecting parameters of interest. Here we compare our standard protocol for individual MC housing (4 days at 23 °C: 3 days of permanent acclimatization followed by 24 h sampling (MC23)) with a short-term intermittent acclimatization protocol (3 h of MC housing for 3 days plus 24 h sampling (accMC23)), the provision of a nest (4 days at 23 °C in MC (nest-MC23)) and MC housing at thermoneutrality (4 days at 30 °C, MC30)). C57BL6/N mice were implanted with telemetric transmitters to collect electrocardiograms, blood pressure, core body temperature and activity data. Single-housed mice in the MC at 23 °C had lower core body temperatures and higher heart and respiratory rates than mice in the MC30 group. Mice housed in MCs at 23 °C exhibited increased food consumption and weight loss, combined with significantly increased expression of messenger RNAs of key molecules in brown fat compared with mice housed in MCs at 30 °C. They also showed increased corticosterone levels. Some male mice of the MC23 and accMC23 groups exhibited episodes of reduced core body temperature and reduced blood pressure and heart rate. Our study demonstrates that housing mice in MCs at 23 °C has a substantial impact on their physiology and welfare due to a substantial cold stress. MC housing at thermoneutrality (30 °C) provides a simple solution to improve mouse welfare. Furthermore, the results showed that a single acclimatization period had the same effect as repeated exposure to the MCs and, therefore, provided no additional benefit.