Table 2 Summary of key components of the 3R-Refinement principles discussed in the paper

Arrival and Acclimatization of Animals

Proper unpacking protocols, including disinfection and health monitoring, ensure animal welfare upon arrival. Gradual acclimatization to new environments and gentle handling techniques, reduce stress and foster positive relationships with caretakers.

[10,11,12,13,14,15]

Housing and Enrichment Environment

Enriched housing, including large cage spaces and social groupings, promotes natural behaviors and reduces stress. Incorporating various enrichment materials and observation boxes creates stimulating living conditions while facilitating research on animal behavior.

[16,17,18,19,20,21,22,23,24]

Handling and Training Methods

Non-restraint handling techniques, such as tunnel handling, promote animal welfare and cooperation during experimental procedures. Gentle handling and the use of soft bedding materials establish trust and comfort, enhancing scientific observations and ethical treatment.

[25,26,27,28,29,30,31,32,33,34,35,36]

Dosing and Sampling Techniques

Refinement in dosing and sampling methods, such as soft oral feeding tubes and non-restraint injections, prioritizes animal comfort and minimizes distress. Capillary micro-sampling techniques reduce blood volume requirements and uphold ethical considerations.

[37,38,39,40,41,42,43,44]

Assessment and Management of Pain

Effective pain assessment and management strategies, including the use of analgesics and non-invasive monitoring techniques, mitigate animal suffering during experimental procedures. Establishing humane endpoints and employing swift euthanasia procedures further uphold ethical standards.

[45,46,47,48,49,50]

Animal Welfare Monitoring

Regular monitoring of animal welfare indicators, both behavioral and physiological, allows for early detection of distress and timely intervention.

[51,52,53]

Establishment of humane endpoints

Monitoring animal behavior and physiological parameters. Setting thresholds to avoid extreme distress or suffering, and taking appropriate actions when endpoints are reached.

[54,55,56]

Euthanasia Procedures

Swift and humane methods for euthanasia. Consultation with specialized veterinarians for method selection along with design of preliminary studies to determine appropriate euthanasia methods. Consideration of biosecurity and environmental impact in carcass disposal.

[57, 58, 7]

Collaboration with Animal Care and Ethics Committees

Review and approval of research protocols so that they follow ethical standards and regulations. Facilitation of training and education initiatives. Dissemination of best practices and knowledge sharing. Contribution to the advancement of ethical animal research practices.

[59, 60]