Optimal Cage Cleaning Frequency for Laboratory Mice: Balancing Hygiene and Welfare

The cage environment plays a crucial role in the welfare of laboratory mice, as it is where they eat, drink, and excrete. Over time, waste accumulation, particularly urine and feces, can lead to increased concentrations of harmful gases like ammonia. Regular cage cleaning is essential for maintaining a safe and hygienic environment, but excessive cleaning may disrupt mice behavior and physiology, affecting both their welfare and experimental outcomes. This article explores the balance between maintaining cage hygiene and minimizing stress from frequent cage changes.

Key Contaminants in Cage Environments

The two main gaseous contaminants affecting mice in enclosed environments are carbon dioxide (CO?) and ammonia (NH?):

• CO?: While CO? levels in mouse cages can rise, studies indicate that concentrations typically remain well below the harmful threshold of 15,000 ppm, even after nine days without bedding replacement. Thus, CO? is generally not the primary concern for mice welfare.
• NH?: Ammonia is produced when urease enzymes in bedding or fecal bacteria break down urea from urine. High concentrations of ammonia can damage the nasal mucosa, olfactory tissues, and respiratory system in mice, and it is also an irritant to human researchers. Managing ammonia levels is, therefore, a critical component of cage cleaning protocols.

Impact of Frequent Cage Cleaning

Excessive cage cleaning can disrupt the natural behavior and physiology of mice:

• Sleep Disruption: Mice, being nocturnal, rest during the day. Cleaning activities during this time interrupt their sleep and increase anxiety.
• Scent Disruption: Mice rely heavily on scent for communication. Frequent cage changes create an unfamiliar environment, heightening stress and potentially leading to stereotypic or aggressive behavior.
• Reproductive Challenges: For breeding and nursing females, frequent cage changes can increase instances of pup abandonment or cannibalism.

Establishing an Optimal Cleaning Frequency

The optimal cleaning frequency must balance maintaining hygiene while minimizing stress:

• Regulatory Guidelines: The 2011 Guide for the Care and Use of Laboratory Animals recommends cage cleaning at least every two weeks, but acknowledges that the frequency should depend on specific conditions, such as cage type and ammonia accumulation.
• Ammonia Thresholds: While specific thresholds for ammonia exposure in mice are not universally established, the U.S. Occupational Safety and Health Administration (OSHA) guidelines for humans suggest a maximum exposure of 50 ppm, with 25 ppm as the average for an 8-hour workday. Many studies adopt these values as reference thresholds for rodents.

Factors Influencing Ammonia Accumulation

• Ventilation:

1. Static cages accumulate ammonia quickly due to limited airflow, often exceeding 25 ppm within 7 days.
2. Individually ventilated cages (IVCs) maintain lower ammonia levels for longer periods. For example, in IVCs housing two to three mice, ammonia levels remained below 25 ppm for up to 14 days.

• Animal Density: Higher densities increase waste accumulation, accelerating ammonia buildup.
• Sex Differences: Male mice generate ammonia more rapidly than females, with concentrations reaching 25 ppm in about 10 days compared to 16 days for females in similar conditions.

Recommendations for Cage Cleaning

To establish an optimal cleaning frequency:

• Monitor Ammonia Levels: Measure the rate of ammonia accumulation in the facility under typical housing conditions. Set a cleaning schedule that ensures ammonia levels remain below established thresholds (e.g., 20–25 ppm).
• Minimize Stress: Avoid excessive cleaning by extending intervals whenever safe, particularly in ventilated cages. This reduces environmental disruption and promotes stable physiological conditions.
• Tailored Protocols: Adjust cleaning frequency based on cage type, animal density, and sex of the housed mice, as these factors significantly influence ammonia buildup.

Conclusion

Managing cage cleaning frequency is a critical aspect of laboratory animal welfare. By balancing the need for hygiene with the goal of minimizing stress and physiological disruptions, researchers can improve the welfare of laboratory mice while ensuring reliable experimental results. Establishing protocols based on ammonia accumulation dynamics and specific housing conditions offers a practical and effective approach.