• Develop refined procedures for capture, immobilisation, marking, and release or re-homing.
• Develop refined procedures for substance administration, sampling, sedation and anaesthesia, surgery and other techniques.

The division of labour and responsibility between the facility and scientists regarding the recording of observations on the animals must be discussed at a very early stage. Novel routines for these observations, including the production of scoring sheets, may have to be developed. Noting the time of day and order of sampling, not just the date, is important. Previous experience from similar studies should be sought and built upon. Procedures must be described in sufficient detail to enable them to be assessed. Descriptions of the monitoring methods and welfare assessment schemes to be used will be important aspects of this planning phase, since these records will facilitate the reporting of actual severity where this is a legal requirement. All of the techniques involved in the procedure need to be identified, and efforts made to ensure that the most refined methods are used.

Some examples of specific action points include the following:

Capture, immobilisation, marking, release re-use or re-homing

• The likely effects on the animals
• The potential danger to personnel and observers
• The use of different personnel than those performing procedures on the animals or assessing their status, to avoid bias
• The possibility of using alternative, non-invasive techniques, such as biometric methods of identification
• Welfare and scientific considerations of tattooing and ear tagging for mouse identification

Administration techniques

• Consideration of species-specific guidelines for administration of substances, including the likely stress caused by handling and immobilisation. Traumatic injuries to the skin are particularly common in fish. Where at all possible, substances should be administered in connection with a pleasurable activity such as feeding, rather than as an isolated negative event.
• Evaluation of the potential effect at the injection site, including methods to refine the injection such as the use of smaller needles, local anaesthetic cream, buffered solutions, liquids at body temperature and the avoidance of incorrect deposition of the injectate
• An assessment of the likely effect of the injection volume on circulatory, renal and pulmonary function
• The timing, order of treatment and length of the administration process, to avoid introducing temporal artefacts and treatment bias caused by the animal's temperament, particularly if multiple measurements are to be made in the presence of other animals. The effects of performing tasks on different weekdays, when they may be very different routines and activity levels in a facility, should also be considered, since animals adapt quickly to these routines.
• The possible effects of differences in competence levels between personnel
• The possible secondary effects of a treatment, such as loss of body weight or muscle mass, and interference from co-habitants
• The appearance of sham-treated animals, to avoid observer bias

Links to resources on administration and sampling

General or collective guidance:

• Guidance on training animals from the NC3Rs
• A good practice guide to the administration of substances and removal of blood, including routes and volumes
• Films and slide shows of handling, injection and blood sampling techniques
• Guidelines for handling research animals
• A collection of guidelines on procedures
• Understanding and selecting surgical suture and needle
• The re-use of needles
• Single use needles: putting refinement into practice
• Animal Technician Hub from the NC3Rs
• Refining procedures for the administration of substances
• Blood, sweat and tears: a review of non-invasive DNA sampling

Species-specific guidance:

• Clicker training of mice and rats
• Preclinical validation of the micropipette-guided drug administration (MDA) method in the maternal immune activation model of neurodevelopmental disorders (mice)
• Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems (Schalbetter et al., 2021) (mice)
• A spoonful of sugar helps the medicine go down: a novel technique to improve oral gavage in mice
• Voluntary ingestion of antiparasitic drugs emulsified in honey represents an alternative to gavage in mice
• A table of scientific papers summarising the research published to date on tunnel handling and cupping methods for handling mice
• Handling method alters the hedonic value of reward in laboratory mice
• The welfare impact of gavaging laboratory rats
• Videos of administration to rodents by gavage (Instech Laboratories)
• An improved method of continuous infusion in mice
• A Noninvasive Ocular (Tear) Sampling Method for Genetic Ascertainment of Transgenic Mice and Research Ethics Innovation (Balafas et al., 2019)
• Training Rats Using Water Rewards Without Water Restriction (Reinagel, 2018)

Blood sampling techniques

• Knowledge of the total circulating blood volume of the animal
• Consideration of species-specific guidelines for blood sampling and choice of the most refined method
• Assessment of the likely consequences of blood removal (including the stress of handling)
• Consideration of steps that can be taken to minimise residual bleeding (within or outside the animal) after the sample has been taken.

Links to resources on blood sampling

• General guidance on blood sampling
• Links to more resources on bleeding animals
• Videos of automated blood sampling techniques (Instech Laboratories)
• Orbital sinus blood sampling in rats: effects upon selected behavioural variables (van Herck et al., 2000)
• Guidance on microsampling from the NC3Rs
• Microsampling: considerations for its use in pharmacological drug discovery and development

Sedation, anaesthesia and analgesia

• There are major concerns about the lack of details on pain management in papers reporting experiments involving survival surgery. Justification should be provided for the drugs to be used, or for withholding analgesia or anaesthesia. The case for using traditional techniques must be weighed against novel ones which may improve animal welfare. The effects of withholding sedatives, anaesthetics and analgesics on the quality of collected data, procedural severity, ease of animal handling and welfare must be considered before any study where this is contemplated. The water quality of the anaesthetic or analgesic bath must be monitored in experiments on aquatic species.
• An assessment must be made of the risk of physical danger to the animals and observers, particularly during induction and recovery, and of inhalation or self-injection of anaesthetics
• Guidelines for procedures in emergency situations and out-of-hours, when less experienced personnel may be available, must be provided
• An assessment must be made of the need for specialised personnel and equipment, and for adequate staffing in the post-operative phase when pain assessment and pain management are critical
• Evaluation of methods of housing and care which can help to alleviate pain and suffering, such as the provision of soft bedding and food, and floor feeding, must be considered.
• Analgesia and anaesthesia of fish are discussed in a separate section, as is the subject of pain and suffering in these species.

Other resources

• Examples and references from the NC3Rs
• How serious are we about sepsis for rodent surgery? (Paul Flecknell)
• Refining rodent stereotactic surgeries, webinar (Amanda Novak)
• Guidance on toe clipping in mice
• Guidance on anaesthesia, analgesia, and surgical research, including that for fish and other aquatic species
• LASA Guiding Principles for Preparing for and Undertaking Aseptic Surgery (2nd edition, 2017)
• Guidance on fasting (food deprivation) in rodents
• Fasting of mice: a review (Jensen et al., 2013)
• Guidelines for the use of animals in cancer research
• Guidelines on severity classification or procedures and recognition of pain and distress (e.g. using Grimace Scales)
• Guidelines for neuroscience research
• Avoiding mortality in animal research and testing
• Animal models - theme issues of the ILAR journal
• Training rats using water rewards without water restriction
• Refining Housing, Husbandry and Care for Animals Used in Studies Involving Biotelemetry
• Refinements in telemetry procedures
• Body temperature measurement in mice during acute illness: implantable temperature transponder versus surface infrared thermometry
• Eye, body or tail? Thermography as a measure of stress in mice
• State of the art in vivo imaging techniques for laboratory animals
• Forskrift om utførelse av arbeid, bruk av arbeidsutstyr og tilhørende tekniske krav
• Opportunities for refinement in neuroscience: Indicators of wellness and post-operative pain in laboratory macaques (Descovich et al., 2019)
• Pathology in Biomedical Research: A Mission-Critical Specialty for Reproducibility and Rigor in Translational Research (special issue of the ILAR Journal) 
• Pathology Study Design, Conduct, and Reporting to Achieve Rigor and Reproducibility in Translational Research Using Animal Models, (Everitt et al., 2019)
• Rigor and reproducibility in rodent behavioral research (Gulinello et al., 2019) 
• Guidelines for the use of animals in preclinical studies within psychopharmacology, published by the Journal of Psychopharmacology.
• VCRACW Statement on the forced swim test (Porsalt Swim Test)
• Experimenter effects on behavioral test scores of eight inbred mouse strains under the influence of ethanol
• Confusing preclinical (predictive) drug screens with animal ‘models’ of psychiatric disorders, or ‘disorder-like’ behaviour, is undermining confidence in behavioural neuroscience (Stanford, 2017)
• Repeatability analysis improves the reliability of behavioral data (Rudeck et al., 2020)

The principles embodied in the WHO Guidelines for Safe Surgery are highly recommended, even though they were developed for human surgery. See also The Checklist Manifesto by Atul Gawande about the development of these guidelines. This book also contains a useful checklist for constructing checklists.

See also the other sections of the PREPARE guidelines in the menu at the top of this page. They include Section 9 on Test substances and procedures, Section 10 on Experimental Animals and Section 12 on Housing and Husbandry.