An optimization and refinement of the whole‐gut transit assay in mice

Gastrointestinal motility measurements in mice are currently performed under suboptimal conditions, as these nocturnal animals are measured during light conditions. In addition, other stressors, like individual housing, placement in a new cage during observation, and lack of bedding and cage enrichment cause animal discomfort and might contribute to higher variability. Here we aimed to develop a refined method of the widely‐used whole‐gut transit assay.


| INTRODUC TI ON
Measurement of gastrointestinal motility in laboratory animals is necessary to assess drug dosing and efficacy of new therapeutics, and to aid in the basic understanding of gastrointestinal physiology and pathology. 1 In preclinical studies, terminal methods are available to determine regional motility, by measuring the distance a tracer has traveled since oral gavage. [2][3][4] Alternatively in an ex vivo setup, intestinal segments can be isolated from the animal and motor patterns can be assessed in an organ bath. [5][6][7] However, these methods require killing of the experimental animals. Non-terminal approaches to study gastrointestinal motility often require anesthesia and advanced imaging modalities like scintigraphy, ultrasound, or radiography. 1,8,9 Therefore, the most commonly used in vivo assessment of gastrointestinal motility remains the whole-gut transit assay. In this method, a non-absorbable dye such as carmine red is administered orally and the time until observation of the first dye-containing fecal pellet expelled by the animal is defined as the whole-gut transit time (WGTT). 1,[10][11][12][13][14] Although this is considered the gold standard, 1 there are several limitations to this approach.
Due to the necessity to visually observe the dye-containing fecal pellet, the experiment is usually carried out during the light period, the resting period for nocturnal animals, and thus a physiologically less appropriate period to assess gastrointestinal transit. Another limitation is that the whole-gut transit assay is usually not performed in the home cage, for example, by individually housing in a new cage devoid of bedding. This environmental change induces stress and might affect thermoregulation. [15][16][17] Stress is known to influence gastrointestinal motility, 18 and might confound results even more.
In addition, to refine animal experimentation, procedures should be performed aiming to minimize harm and distress. Therefore, we here evaluate the potential of a refined and optimized method for examining whole-gut transit time. We use a UV-fluorescent dye that is administered to mice on a reversed light-dark schedule, enabling observation during their active period. In addition, mice are socially housed in their home cage to reduce stress. To validate this approach, the refined and standard whole-gut transit method are compared in presence and absence of loperamide to induce a standardized delay of whole-gut transit.

| Animals
Twelve male and twelve female wildtype C57Bl/6J mice (4 weeks old) were obtained from Charles River (Charles River Laboratories

| Experimental design
Mice were 8-10 weeks old at the start of experiments. Four experimental conditions were compared (Table 1): RED, RED + LOP (standard method), DETEX, and DETEX + LOP (refined method) in a crossover design with eight experimental groups, in which conditions were tested in different order (Table S1). Animals were randomly allocated to each group and experimenters were blinded for intervention (with/without loperamide).
The light-dark schedule was gradually reversed by shifting the light period by 1 h every day (phase delay) for 14 days until the light-dark schedule was completely reversed ( Figure 1A), thereby reducing "jet lag". 19 For groups starting with DETEX/DETEX + LOP, the light-dark schedule was first reversed, and later reversed back to normal for RED/RED + LOP conditions ( Figure 1B). Experiments were started after at least 2 days acclimatization.
Conditions RED/RED + LOP were measured during the light period, DETEX/DETEX + LOP were measured in the dark on a reversed light-dark schedule, starting at 9:15 ± 15 min a.m. Food

Key Points
• Whole-gut transit time in mice is currently assessed in suboptimal conditions that limit physiologically-relevant measurements.
• We designed a method that enables measuring wholegut transit in mice during their active period in the dark, and that reduces stress by socially housing mice in their home cage.
• The refined method results in less within-group variation, allowing for a reduction in animal use.
was omitted for 1 h before oral gavage and available ad libitum throughout the procedure in all conditions. For RED/RED + LOP, mice were transferred to individual cages lined with a diaper cloth to absorb urine during the observation period ( Figure 1C). For DETEX/DETEX + LOP, cage changes were not performed later than 24 h preceding the experiment to reduce stress. The oral gavage was given to one of the pairwise housed mice, after which it was returned to its home cage for observation. Part of the cage TA B L E 1 Summary of the four different conditions.  enrichment was removed (cottage, paper strands) to enable observation of fecal pellets ( Figure 1F).
All mice were observed in their cage on a table outside of the ventilation unit with a filter cap to maintain SPF conditions ( Figure 1D,G). Pellet production was monitored every 5-10 min until observation of a dye-containing pellet for each mouse or up to 480 min ( Figure 1E,H). For DETEX/DETEX + LOP conditions, a UV flashlight was used to enable visualization of the Lumitrack® dye.
After expulsion of the first dye-containing fecal pellet, the pellets were collected for 1 h and the cage was returned to the ventilation unit. All fecal pellets were weighed and dried overnight at 75°C to determine their dry weight. Fecal water content was calculated as the difference between wet and dry weight as a percentage of wet weight.

| RE SULTS
The UV-fluorescent DETEX allowed to study the mice in their active period, in the dark. The DETEX provided an excellent alternative to carmine red, as it was clearly visible with a UV flashlight ( Figure 1H). There were no adverse effects on body weight nor observations of discomfort (e.g., altered activity, behavior, or facial expression) after the procedures. To assess the effects of the four conditions (RED, RED + LOP, DETEX, and DETEX + LOP; Table 1) and the eight different groups (cross-over study design) and possible interaction (group*condition) on WGTT, a two-way mixed ANOVA was used. This showed a significant effect of condition on WGTT

| D ISCUSS I ON AND CON CLUS I ON
In this study, we show that oral administration of UV-fluorescent DETEX® allows for a refined method of whole-gut transit assess- in the dark while socially housed in their home cage. This refined method of whole-gut transit assessment detects loperamideinduced delay of transit time equally well and results in less variation compared to the standard method, allowing the detection of alterations in transit time with lower numbers of experimental animals. Therefore, our refined method contributes to two of the 3Rs (Refinement and Reduction), a guiding principle to improve animal experimentation. 20,21 As the refined method differs from the standard method in more than one way, that is, dye (nutrient) composition, light/dark schedule, and housing conditions, it is difficult to attribute the reduced variation and differences in fecal properties to a single factor. The possible disturbance of mice due to exposure to UV light in the dark phase cannot be excluded; however, the refined method allows social interaction, more activity and better thermoregulation, thereby reducing stress and avoiding impact on physiological parameters and disturbance of circadian rhythm. Moreover, biological rhythms, food intake and stress significantly affect gastrointestinal motility, [22][23][24][25][26] affirming the importance of the timing of testing and considerations for experimental housing conditions. Although other optimizations of the whole-gut transit have been described by others, 27,28 our method is, as far as we know, the first to include the influence of housing conditions and social interaction in the experimental design to improve welfare.
In conclusion, we developed an optimized and refined approach to measure WGTT in mice, that allows for reducing experimental animal numbers due to reduced variability within groups.

ACK N OWLED G M ENTS
We would like to acknowledge the help of Kim Smits with the statistical analysis of the data in this manuscript.