3R - Replacement, Reduction and Refinement of animal experiments

Means to use alternatives to live animals whenever possible. This can include:

• In vitro methods (cell cultures, organoids)
• Computer modeling or simulations
• Use of lower-sentience organisms (e.g., invertebrates instead of mammals)

An important control-question to ask yourself is:

• Have there been any developments in your scientific field (including the development/validation of new in vitro or in silico techniques) which would replace some or all of the use of animals?

The animal facility offers dedicated resources for training in practical procedures, including access to specialized equipment and non-animal models to ensure competence before performing live animal experiments.

Means to minimize the number of animals used without compromising scientific validity.

Strategies include:

• Proper statistical planning (power analysis)
• Sharing data and tissues between projects
• Using longitudinal studies instead of multiple cohorts

Control questions:

• Has the project/experimental design been revisited to enable any further reduction in predicted animal use?
• Were the models used still the most appropriate?
• Were the numbers of animals used appropriate for statistical analysis (too many/too few)?
• Could different approaches reduce further animal use?

How can you redcuce animal number?

• Design study so each animal its own control (reduce 50%)
• Control for variation by
  • Standardising the animal (genetics, micobial status)
  • Standardize the environmenatl conditions
  • Control variation in factors that you can explain (like sex, strain, line etc) by use of "blocking-design"

Refinement refers to measures that improve procedures to reduce pain, suffering, and distress, while also optimizing animal welfare.

Examples

• Better anesthesia and analgesia protocols
• Implement Humane endpoints
• Environmental enrichment and improved housing

Refinement not only improves animal welfare but also enhances research quality. By reducing pain and stress, experiments can be conducted more efficiently, with lower costs, less risk, and higher scientific validity.

Refinement supports both good science and good animal welfare.

In your application for animal experimentation, you must document how you have assessed and implemented the 3Rs.

This typically involves:

• Replacement:

  • Explain why alternatives are not sufficient for your research question.

  • If you considered in vitro or computational models, explain why they cannot fully replace animal use.

• Reduction: Provide justification for the number of animals needed by

  • Statistical calculations (e.g., power analysis)
  • Plans to share data or tissues
  • Any design choices that reduce duplication

• Refinement: Detail how you will minimize harm to animals. Examples:

  • Describe anesthesia, analgesia, and monitoring
  • Define humane endpoints
  • Describe enrichment and handling improvements

To ensure your application meets regulatory expectations:

• Use clear headings for each R

  • Structure your application with separate sections for Replacement, Reduction, and Refinement.

• Provide specific, concrete measures

  • ​​​​​​​Avoid general statements. Describe exactly what you will do (e.g., “Power analysis performed to determine minimum animal numbers” or “Use of non-invasive imaging to reduce distress”).

• Reference relevant guidelines

  • ​​​​​​​Cite recognized standards such as FELASA recommendations, AAALAC Reference Rescourses, The EU Directive 2010/63/EU, and national regulations to strengthen your justification.

Preparation of Animals Before Experiments

• Transport & Acclimatization 

  • ​​​​​​​Use climate-controlled vehicles, reduce travel time, and allow a quarantine/acclimatization period (at least 7 days) to enable animals to recover from transport stress. 

• Housing & Enrichment 

  • ​​​​​​​Provide species-appropriate enrichment: nesting materials, shelters, toys, social housing where possible. Ensure cage cleaning minimizes disruption and maintains familiar scents.

• Feeding & Care Routine

  • ​​​​​​​Maintain consistent feeding times, use palatable diets, and schedule non-invasive health checks. Avoid abrupt changes in diet or environment to lower chronic stress. 

Experimental Procedures & Pain-Relief Strategies

• Anesthesia & Analgesia 

  • ​​​​​​​Describe optimized anesthesia protocols (agent, dosage, route). Include multimodal analgesia plans before, during, and after procedures. Adjust dosing based on pilot data or literature.

• Refined Techniques 

  • ​​​​​​​Use less invasive routes (e.g., subcutaneous vs. intraperitoneal). Employ microsurgical tools. Pre-condition animals to handling to decrease stress. 

• Training & Competence 

  • ​​​​​​​Ensure all personnel are properly trained, certified and experienced. Standardize all procedures and document training to minimize variation and risk. 

Experimental Design & Humane Endpoints

• Design Overview 

  • ​​​​​​​State the study’s aim, timeline, and group structure. 

• Humane Endpoints 

  • ​​​​​​​Define early endpoints based on objective parameters liek weight loss (e.g., >15%), or behavior changes. Specify immediate interventions (e.g., euthanasia or treatment) when reached.

• Continuous Monitoring Plan 

  • ​​​​​​​Outline frequency of observation. Use scoring sheets for pain, behavior, grooming, and quantify signs of distress. 

• Intervention Protocols

  • ​​​​​​​Describe responsive actions: adjust analgesia, rehydration, supportive care, or removal from study. Document all responses in welfare logs. 

Welfare Monitoring & Technological Integration

• Use of Non-Invasive Monitoring 

  • ​​​​​​​Include remote video or automated sensing (e.g., cage sensors, activity monitors) to reduce handling stress and detect early welfare changes. 

• Early Diagnostics & Biomarkers 

  • ​​​​​​​Employ real-time physiological measurements (e.g., telemetry, pain biomarker assays) to reduce invasive sampling. 

The FELASA guidelines emphasize minimizing stress and improving welfare during breeding and genotyping.

Key points to include:

• Genotyping Methods

  • Prefer non-invasive or minimally invasive techniques (e.g., ear punch combined with ID, buccal swabs, or hair samples) instead of tail biopsies.
  • Use PCR-based methods that require very small samples.
  • Apply local anesthesia or analgesia for any invasive sampling.

• Genetic Quality Assurance & Monitoring

  • Implement routine checks to avoid unnecessary breeding of incorrect genotypes.
  • Use validated genotyping protocols to reduce repeat sampling.

• Housing & Care

  • Provide enrichment and social housing where compatible.
  • Avoid unnecessary separation of dams and pups; handle gently to reduce stress.

Regulatory authorities require clear evidence that no suitable alternatives exist before approving animal use. A thorough search is critical because it demonstrates due diligence and compliance with ethical and legal standards.

Incomplete or overly narrow searches often lead to delays, as reviewers may request additional documentation or clarification.

To avoid this, use multiple databases beyond PubMed, apply comprehensive keywords related to species, procedures, and research field, and document your search strategy, results, and justification.

This proactive approach ensures transparency and speeds up the approval process.

1. Use Multiple Databases

   • Do not rely only on PubMed (alternatives are poorly represented there).
   • Include specialized 3R resources such as:
     • Norecopa’s 3R Guide (super-search engine combining multiple databases)
     • EURL ECVAM databases
     • ALTEX and other 3R journals
     • Guidelines from FELASA, OECD, and EU Directive 2010/63/EU

2. Choose Comprehensive Keywords

   • Cover animal species/model (e.g., “zebrafish”, “rodent”)
   • Include procedures (e.g., “genotyping”, “imaging”, “toxicity testing”)
   • Add research field terms (e.g., “cardiovascular”, “neurobiology”)
   • Include “alternative”, “in vitro”, “simulation”, “organ-on-chip”, “non-animal method”

3. Document Your Search

   • List databases used, search strings, and date of search
   • Summarize findings and justify why alternatives are not sufficient

• Search strategy: databases, keywords, date
• Results summary: what alternatives exist and why they are not applicable
• Commitment to continuous review: state that you will monitor for new alternatives during the project