Impact of nesting materials on energy balance in male C57BL/6J mice exposed to different ambient temperatures

Nesting materials are used to enhance animal welfare in laboratory settings, yet their physiological impact, particularly on energy metabolism, remain under-characterized. Here, we investigated the effect of three commonly used nesting materials (nestlet, Enrich-n’Nest®, Bed-r’Nest®) and the presence versus absence of nesting on metabolic parameters in male mice housed at thermoneutral (30°C), standard (22°C), and cold (10°C) temperatures. Using indirect calorimetry, we continuously monitored respiratory exchange ratio (RER), food intake and water intake. Within each thermal condition, the presence of nesting compared with no nesting was associate with reduced oxygen consumption (VO 2 ) at 22°C and an attenuation of thermogenic demand at 10°C, whereas nesting has minimal effects at 30°C. In contrast, the type of nesting material did not differentially affect VO 2 , RER, or ingestive behavior at any temperature. Environmental variables, including temperature stability and relative humidity were closely monitored and remained within standard ranges throughout the experiments. Together, these findings demonstrate that while nesting availability modulates metabolic demand in a temperature-dependent manner, commonly used nesting materials do not introduce meaningful variability in metabolic cage studies. These findings support the continued use of these three nesting materials in metabolic phenotyping and highlight the importance of transparent reporting of housing and enrichment conditions to improve reproducibility in preclinical metabolic research.