13-Experimental procedures

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
• Picking up mice after sanitizing your hands can affect mouse behaviour
• A collection of guidelines on procedures
• Collection of biological material from hair follicles
• Understanding and selecting surgical suture and needle
• The re-use of needles
• Single use of needles:
• Single use of needles: how AWERBs can support refinements in 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
• Effects of restraint and injection methods on heart rate and body temperature in mice
• Baran et al. (2022): Emerging Role of Translational Digital Biomarkers Within Home Cage Monitoring Technologies in Preclinical Drug Discovery and Development

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) An SOP for this technique from the University of Zurich can be downloaded here.
• 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
• Practical advice on tunnel handling (8 minute video from Swiss 3RCC)
• 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)
• A new group-housing approach for non-human primate metabolism studies (Stow et al., 2021)

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.
• The TextBase database which is part of Norecopa's website includes citations of many textbooks on anaesthesia and analgesia, including Laboratory Animal Anaesthesia by Paul Flecknell.
• eLearning modules on anaesthesia and analgesia developed by Newcastle University and FLAIRE consultants
• Navarro et al. (2021): Mouse anesthesia: the art and the science
• Crisis management in anaesthesia: What can we learn from airline pilots? (recording of a presentation by Colin Dunlop & Nathan Koch)
• The PPRECISE guidelines for ensuring transparency and minimisation of methodological bias in preclinical pain research (Andrews et al., 2016)

Advice on reducing hypothermia in small rodents

• Celeste et al. (2021): Effects of cling film draping material on body temperature of mice during surgery
• Klune et al. (2020): Hypothermia during general anesthesia interferes with pain assessment in laboratory rats (Rattus norvegicus)
• Rufiange et al. (2020): Prewarming followed by active warming is superior to passive warming in preventing hypothermia for short procedures in adult rats (Rattus norvegicus) under isofluorane anesthesia

Other resources

• Examples and references from the NC3Rs
• Reliability of common mouse behavioural tests of anxiety: A systematic review and meta-analysison the effects of anxiolytics (Rosso et al., 2022)
• The Behaviour Forum - A place to discuss experimental methods in animal behaviour
• Development and Application of Home Cage Monitoring in Laboratory Mice and Rats: A Systematic Review (Kahnau et al., 2023)
• It's a height thing: Tumour Volume Estimation in Cancer Research (presentation by Verinnogen)
• Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how). (Little et al., 2023)
• Focus on novel approaches: Home-cage monitoring of laboratory mice (Bartelik et al., 2023)
• Animal Research Advisory Committee Guidelines on Animal Care and Use Procedures from the NIH, USA
• How serious are we about sepsis for rodent surgery? (Paul Flecknell)
• Introduction to Aseptic Techniques (workshop organised by The Learning Curve)
• Refining rodent stereotactic surgeries, webinar (Amanda Novak)
• Refining the use of head fixation and fluid control in rodents
• Guidance on toe clipping in mice
• Measuring Endogenous Corticosterone in Laboratory Mice - a Mapping Review, Meta-Analysis, and Open Source Database (van der Mierden et al., 2020)
• 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)
• A Commentary on Fasting of Nonclinical Research Animals (Gauvin et al., 2023)
• 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
• Improving Stereotaxic Neurosurgery Techniques and Procedures Greatly Reduces the Number of Rats Used per Experimental Group - A Practice Report (Ferry & Gervasoni, 2021)
• 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.
• Advice on the forced swim test from the UK Animals in Science Committee (June 2023)
• 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)
• Defensor et al. (2021): Biomonitoring and Digital Data Technology as an Opportunity for Enhancing Animal Study Translation
• The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias (Curtis et al., 2013)
• Accelerating drug discovery for Alzheimer's disease: best practices for preclinical animal studies (Shineman et al., 2011)
• Kurien et al. (2004): Experimental animal urine collection: a review

Please see the section on Educational Courses, including a link to WASP Science.

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.