Tse labmaster

Energy expenditure, respiratory exchange, and food intake were quantified using the indirect calorimetry system (TSE LabMaster, TSE Systems, Germany) for 3 days.

An automated feeding monitoring system (TSE LabMaster, Project 4261, TSE Systems, Bad Homburg, Germany) was used to analyze feeding patterns (measuring cumulative chow intake [g], meal frequency, meal size [g], and ingestion rate [g/min]) in an olfactory PB cue-enriched environment in both PB taste-naïve (n = 16) and PB taste-familiar (n = 16) rats. This system allows uninterrupted and undisturbed recording of individual meals for each animal. Food hoppers containing regular pelleted chow were suspended on sensors (calibrated prior to starting the experiments) recording food intake to a sensitivity of 1 mg. Rats were transferred into the cages and allowed one-week habituation prior to starting the feeding recordings.

Mice from each experimental group were used as subjects in the TSE LabMaster (TSE Systems, Bad Homburg, Germany), as described previously [63 ]. During the first 24 h, the mice were acclimated and then monitored for 48 h. Every 30 min, data of O₂ consumption and CO₂ production were collected. The calorimeter system provided a standard analysis software allowing the determination of energy expenditure using the collected data. Heat production was calculated using the abbreviated Weir equation ([3.94(VO₂) + 1.11(VCO₂)] 1.44). Other measured parameters during the 48 h were food and water intake and locomotor activity. Thirty min respiratory exchange ratio (RER) values were averaged for each animal at the same time point across 24 h. The median value for each animal was considered baseline, and the deviation to it was calculated, along with the maximal and minimum values.

Oxygen consumption was measured when mice were fed upon HFD for 2 weeks without difference in body weights between groups and physical activity was determined at 12 weeks of age with a TSE LabMaster (TSE Systems, Bad Homburg, Germany), as previously described.^[74] Mice were acclimated to the system maintained at 24 °C in a 12‐h light/dark cycle for 20–24 h, and fasted VO₂ and VCO₂ of each mouse were measured during the next 24 h with food deprivation. Then refed VO₂ and VCO₂ were obtained with free access to food and water. The voluntary activity of each mouse was measured with an optical beam technique (Opto‐M3, Columbus Instruments, Columbus, OH, USA) over 24 h and expressed as 24‐h average activity. Oxygen consumption and energy expenditure data were analyzed using CaIR (version 1.1) (https://calrapp.org/).^[75]

All IntelliCage and PhenoMaster values were divided with the actual time spent in the experiment since minor one-hour differences existed between the experiments. IntelliCage values were divided into hours and PhenoMaster values into days.
Normality was tested using the Kolmogorov–Smirnov test and normality was not observed. Due to the distribution of the data and relatively small sample size, a non-parametric approach was chosen. Data were grouped using the wheel running activity (distance as km) in a metabolic cage. Grouping was done by dividing the population into three equal-sized groups. The Kruskall–Wallis test was used to study differences between all groups and the Mann–Whitney test to study differences between two individual groups. Due to the small sample size, a p-value smaller than 0.10 was chosen as the level of statistical significance. Data were first retrieved from IntelliCage using the NewBehaviorTSE IntelliCage Plus SW, Project 5331, software version 01/2016 and from the PhenoMaster using TSE LabMaster, version 5.7.6, TSE Systems GmbH software in Excel format. Further processing, statistical analysis, and graph generation were done using the IBM SPSS Statistics v.27 and GraphPad Prism (version 9.3.1 for Windows, GraphPad Software, San Diego, CA, USA). All figures are presented as mean and standard deviation.