Validation of Sanitization Practices in Single-use Individually Ventilated Mouse Cages at Standard and Thermoneutral Temperatures

Abstract

Vivarium husbandry practices are based on performance data and adhere to applicable regulatory guidelines. Refinements in husbandry and optimization of sanitization protocols improve animal wellbeing and help standardize the microenvironment, contributing to research reproducibility. The objective of this study was to evaluate the microenvironment to establish performance standards for mouse husbandry and sanitization, including housing at standard and thermoneutral temperatures. Male C57BL/6J mice were housed singly and in groups in disposable IVCs on α-cellulose or corncob bedding and microenvironmental indicators (ammonia, carbon dioxide) were evaluated. In addition, microbial bioburden tests (ATP and RODAC) were performed on cages and cage accessories on days 0, 7, 14 and, 28 to 30 after cage change. Water testing and aerobic culture of the waterspout of bottles containing chlorinated water were performed to determine acceptable replacement schedules. Ammonia levels remained below the National Institute of Occupational Safety and Health 8-h recommended exposure limit for humans (25 ppm) at all time points for all housing conditions through day 21 for group-housed mice, and through day 30 for singly housed mice. Microbial bioburden results for cage accessories and water testing were acceptable up to 28 d after cage change (RODAC less than 50 CFU; ATP less than 100,000 RLU) at both standard and thermoneutral housing temperatures. Mice remained clinically healthy throughout the studies. These results support site operating practices and verify extended sanitization recommendations per the Guide of the Care and Use of Laboratory Animals in this disposable IVC environment: group-housed mice receive bottom cage and water bottle change up to every 14 d with full cage change (including lid and accessories) every 28 d, and singly housed mice receive full cage change every 28 to 30 d or sooner.

Figure 1.

(A) Representative image of air sampling with gas detector unit of ventilated cage. Gas tube for sampling extends from unit through water bottle port in front of cage (red arrow). (B) Representative image of placement of ammonia colorimetric sensor in mouse cage.

Figure 2.

Sampling sites of mouse disposable IVC caging and accessories (cage wall, lid, hopper) for microbiologic monitoring (Blue “X” = ATP, Red “X” = RODAC). Cage and accessory images source credit: Innovive

Figure 3.

Carbon dioxide (CO2, reported in ppm; mean ± SEM) in IVCs with group-housed mice on diced cellulose or corncob bedding (n = 5 each) at days 0, 7, 14, and 28 after cage change under standard temperature conditions.

Figure 4.

Group- and singly housed mouse IVC wall and accessory ATP results (reported in relative light units; RLU; mean ± SEM) at standard (A) and thermoneutral (B) temperature conditions. Symbols used to denote significance of specific sample types between days (*, # or †): for example, *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.