Influence of animal husbandry practices on void spot assay outcomes in C57BL/6J male mice

Abstract

Aims Mice are increasingly being used as models to investigate aspects of urinary dysfunction that humans with lower urinary tract symptoms (LUTS) experience. One method used to examine voiding function is the spontaneous void spot assay. The purpose of this study was to characterize and identify animal husbandry conditions that might confound results of the spontaneous void spot assay in male C57Bl/6J mice. Methods Mice were placed in cages lined with filter paper for four hours and urine was visualized with UV transillumination. Voiding parameters including urine spot number, spot size, total urine area, primary void area, corner and center voiding were quantified. Results Adult male mice void more frequently with advancing age and a subpopulation (5–10%) display a frequent spotting pattern at 6–9 weeks of age. Voiding was not significantly different in male mice weaned to group housing (4–6 per cage) versus single housing, and was not altered when they were used as breeders. Voiding was changed upon transferring group housed adult males to single density cages, which decreased total urine area. Repeated assays of male voiding behavior over three consecutive days increased primary void area by the third day of monitoring and revealed that voiding behavior is impacted by routine cage changes and time of day. Conclusions Together these results identify housing and husbandry practices that influence male voiding behaviors in the spontaneous void spot assay and will inform voiding behavior analyses conducted with male C57Bl/6J mice.

Evaluation of voiding assays in mice: impact of genetic strains and sex

“…However, as with all studies entailing live animals, technical factors can substantially affect results. We have recently reported that husbandry factors, including time of day of testing, cage changes within 24 h of testing, as well as age and body weight, significantly affected the results of VSA conducted with male C57BL/6J mice (15). However, we also observed that repeated testing of male mice over 3 consecutive days produced nearly identical results, particularly when testing was consistently performed at the same time of day (e.g., morning, afternoon, or night) (15).…”

“…All protocols conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. VSA Standardized procedures were followed to mimic those used in a previous study (31) and to minimize the impact of husbandry on results (15). Mice were allowed free access to water until placed in cages for testing.…”

Impact of a folic acid-enriched diet on urinary tract function in mice treated with testosterone and estradiol

“…Void spot assays were conducted at 6 (before TϩE2 implant), 10, and 14 wk of age and analyzed as described previously (18,39). Mice were placed in a clean empty cage lined with 3MM Whatman filter paper (057163W, Fisher Scientific).…”

“…Assays were conducted in the same quiet location and time of day unless indicated otherwise. Filter papers were imaged using UV light on a transilluminator and analyzed using Image J Software (Version 1.46r) as described previously (18). Spot number, total urine area, spot size, area within the primary (largest) void, % total urine area in primary void, % total area in the corner of the paper, and % total area in the center of the paper were analyzed as described previously (18,39).…”

“…Filter papers were imaged using UV light on a transilluminator and analyzed using Image J Software (Version 1.46r) as described previously (18). Spot number, total urine area, spot size, area within the primary (largest) void, % total urine area in primary void, % total area in the corner of the paper, and % total area in the center of the paper were analyzed as described previously (18,39). The specific variables of urine spot number, urine area, primary void area, and % area in corners were shown previously to correlate with body mass (18) and were normalized accordingly.…”