Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research.

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.

Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint.

The fish acute toxicity test (TG203; OECD, 2019) is frequently used and highly embedded in hazard and risk assessment globally. The test estimates the concentration of a chemical that kills 50% of the fish (LC50) over a 96 h exposure and is considered one of the most severe scientific procedures undertaken. Over the years, discussions at the Organisation for Economic Co-operation and Development (OECD) have resulted in changes to the test which reduce the number of fish used, as well as the development of a (potential) replacement test (TG236, OECD, 2013). However, refinement of the mortality endpoint with an earlier (moribundity) endpoint was not considered feasible during the Test Guideline's (TG) last update in 2019. Several stakeholders met at a UK-based workshop to discuss how TG203 can be refined, and identified two key opportunities to reduce fish suffering: (1) application of clinical signs that predict mortality and (2) shortening the test duration. However, several aspects need to be addressed before these refinements can be adopted. TG203 has required recording of major categories of sublethal clinical signs since its conception, with the option to record more detailed signs introduced in the 2019 update. However, in the absence of guidance, differences in identification, recording and reporting of clinical signs between technicians and laboratories is likely to have generated piecemeal data of varying quality. Harmonisation of reporting templates, and training in clinical sign recognition and recording are needed to standardise clinical sign data. This is critical to enable robust data-driven detection of clinical signs that predict mortality. Discussions suggested that the 96 h duration of TG203 cannot stand up to scientific scrutiny. Feedback and data from UK contract research organisations (CROs) conducting the test were that a substantial proportion of mortalities occur in the first 24 h. Refinement of TG203 by shortening the test duration would reduce suffering (and test failure rate) but requires a mechanism to correct new results to previous 96 h LC50 data. The actions needed to implement both refinement opportunities are summarised here within a roadmap. A shift in regulatory assessment, where the 96 h LC50 is a familiar base for decisions, will also be critical.

Applying refinement to the use of mice and rats in rheumatoid arthritis research.

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat 'models' of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research.

A 'road map' toward ending severe suffering of animals used in research and testing.

Ending severe suffering is desirable and achievable. In this article we have outlined a 'road map', or series of steps, that can be used to evaluate the extent to which severe suffering occurs within establishments, and some practical principles that can be employed in order to work toward ending it. We urge researchers to act upon these principles, and to begin a journey toward a future where causing severe suffering to animals used in scientific research is a thing of the past.

Harmonisation of education, training and continuing professional development for laboratory animal caretakers, technicians and technologists: Report of the FELASA-EFAT Working Group.

Competent, confident and caring laboratory animal caretakers, technicians and technologists (LAS staff) are vital for good animal welfare, high-quality science and a secure Culture of Care. This requires high-quality education, training, supervision and continuing professional development (CPD) of LAS staff. However, there is a lack of harmonisation regarding how this education and training is conducted among European countries, and nor are there recommendations adapted to Directive 2010/63/EU. Therefore, FELASA and EFAT established a working group with the task of establishing recommendations for education, training and CPD for LAS staff. The working group established five different levels (LAS staff levels 0-4), defining the required level of competence and attitude, as well as suggesting educational requirements for reaching each level. Defining these levels should help to ensure that appropriate educational and CPD activities are in place, and to enable employers and LAS staff to determine the level and career stage attained. Furthermore, proper assessment of competencies and effective CPD schemes for all relevant staff should be established. Regulators should support this by setting standards for competence assessment and ensuring that they are consistently applied. In addition, establishments should involve the LAS staff in defining and developing the Culture of Care. The Animal Welfare Body should be involved and have oversight of education, training and CPD. These recommendations will contribute to harmonisation and increased quality of education, training and CPD, as well as provide clearer career pathways for LAS staff, helping to ensure high standards of animal welfare and science.

Three Rs Approaches in the Production and Quality Control of Fish Vaccines.

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum.

Enrichment for Laboratory Zebrafish-A Review of the Evidence and the Challenges.

Good practice for the housing and care of laboratory zebrafish Danio rerio is an increasingly discussed topic, with focus on appropriate water quality parameters, stocking densities, feeding regimes, anaesthesia and analgesia practices, methods of humane killing, and more. One area of current attention is around the provision of environmental enrichment. Enrichment is accepted as an essential requirement for meeting the behavioural needs and improving the welfare of many laboratory animal species, but in general, provision for zebrafish is minimal. Some of those involved in the care and use of zebrafish suggest there is a 'lack of evidence' that enrichment has welfare benefits for this species, or cite a belief that zebrafish do not 'need' enrichment. Concerns are also sometimes raised around the practical challenges of providing enrichments, or that they may impact on the science being undertaken. However, there is a growing body of evidence suggesting that various forms of enrichment are preferred by zebrafish over a barren tank, and that enriched conditions can improve welfare by reducing stress and anxiety. This review explores the effects that enrichment can have on zebrafish behaviour, physiology and welfare, and considers the challenges to facilities of providing more enrichment for the zebrafish they house.

Refining rodent models of spinal cord injury.

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field.

Refinements in husbandry, care and common procedures for non-human primates: Ninth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement.

Preface Whenever animals are used in research, minimizing pain and distress and promoting good welfare should be as important an objective as achieving the experimental results. This is important for humanitarian reasons, for good science, for economic reasons and in order to satisfy the broad legal principles in international legislation. It is possible to refine both husbandry and procedures to minimize suffering and improve welfare in a number of ways, and this can be greatly facilitated by ensuring that up-to-date information is readily available. The need to provide such information led the British Veterinary Association Animal Welfare Foundation (BVAAWF), the Fund for the Replacement of Animals in Medical Experiments (FRAME), the Royal Society for the Prevention of Cruelty to Animals (RSPCA) and the Universities Federation for Animal Welfare (UFAW) to establish a Joint Working Group on Refinement (JWGR) in the UK. The chair is Professor David Morton and the secretariat is provided by the RSPCA. This report is the ninth in the JWGR series. The RSPCA is opposed to the use of animals in experiments that cause pain, suffering, distress or lasting harm and together with FRAME has particular concerns about the continued use of non-human primates. The replacement of primate experiments is a primary goal for the RSPCA and FRAME. However, both organizations share with others in the Working Group, the common aim of replacing primate experiments wherever possible, reducing suffering and improving welfare while primate use continues. The reports of the refinement workshops are intended to help achieve these aims. This report produced by the British Veterinary Association Animal Welfare Foundation (BVAAWF)/Fund for the Replacement of Animals in Medical Experiments (FRAME)/Royal Society for the Prevention of Cruelty to Animals (RSPCA)/Universities Federation for Animal Welfare (UFAW) Joint Working Group on Refinement (JWGR) sets out practical guidance on refining the husbandry and care of non-human primates (hereinafter primates) and on minimizing the adverse effects of some common procedures. It provides a valuable resource to help understand the physical, social and behavioural characteristics and needs of individual primates, and is intended to develop and complement the existing literature and legislative guidelines. Topics covered include refinements in housing, husbandry and common procedures such as restraint, identification and sampling, with comprehensive advice on issues such as primate communication, assessing and facilitating primate wellbeing, establishing and maintaining social groups, environmental and nutritional enrichment and animal passports. The most commonly used species are the key focus of this resource, but its information and recommendations are generally applicable to other species, provided that relevant individual species characteristics are taken into account.

Applying the 3Rs: A Case Study on Evidence and Perceptions Relating to Rat Cage Height in the UK.

This article investigates the barriers to implementing higher caging in animal research establishments in the UK. The use of animals in research and testing in the UK is regulated by the Animals (Scientific Procedures) Act 1986, which sets out how animal experiments must be licensed and regulated. Within this, the Code of Practice currently allows laboratory rats to be housed in cages that are 20 cm high, even though adults can rear up to 30 cm. Most adult rats therefore cannot stand upright in 'standard' cages. We found that the main factors hindering the implementation of higher caging were classified into five different groups; health and safety, financial, animal welfare, scientific, and 'human'. Suggestions to overcome these barriers are provided, as well as alternative animal welfare changes that can be put into place. We conclude that much of the desired evidence for moving to higher cages is already available, and therefore the focus should be on education and improving access to the existing evidence, in order to encourage facilities to work around existing financial and health and safety concerns.

3Rs-Related and Objective Indicators to Help Assess the Culture of Care.

Within animal research and testing, the need for an effective Culture of Care is widely recognized and described in terms of an establishment-wide commitment to improving the implementation of the 3Rs, animal welfare, scientific quality, care of the staff, and transparency for all stakeholders, including the public. Ideally, each establishment would determine what the Culture of Care means for them, and be able to assess and potentially benchmark their own culture. Some establishments already do this, using various indicators and formal or informal assessments. This paper provides examples of these approaches to assessing the Culture of Care, including surveys and surrogate measures. Many currently-used criteria and indicators tend to be human-centric and subjective, and we suggest using further objective indicators and animal-centric, 3Rs-based criteria. It is preferable to consider each of the 3Rs separately when assessing culture, and some indicators are suggested to facilitate this. Several documents produced by regulators in the UK and European Union are good sources of objective indicators of a good Culture of Care. This concept paper aims to complement the literature on assessing the Culture of Care, providing ideas and sources of information to help identify relevant and measurable criteria.

Report of the 2006 RSPCA/UFAW Rodent Welfare Group meeting.

The RSPCA/UFAW Rodent Welfare Group holds a one-day meeting every autumn to discuss current welfare research and to exchange views on rodent welfare issues. A key aim of the group is to encourage people to think about the lifetime experience of laboratory rodents, ensuring that every potential influence on their well-being has been reviewed and refined. Speakers at the 2006 meeting presented preliminary findings of ongoing studies and discussed regulatory updates. Topics included the housing and husbandry of mice and rats, refining the use of rodents in asthma research, good practice for the euthanasia of rodents using carbon dioxide and achieving reduction by sharing genetically modified mice.

The 9 to 5 Rodent - Time for Change? Scientific and animal welfare implications of circadian and light effects on laboratory mice and rats.

Rodents, particularly rats and mice, are the most commonly used laboratory animals and are extensively used in neuroscience research, including as translational models for human disorders. It is common practice to carry out scientific procedures on rats and mice during the daytime, which is the inactive period for these nocturnal species. However, there is increasing evidence for circadian and light-induced effects on rodent physiology and behaviour which may affect the validity of results obtained from mice and rats in neuroscience studies. For example, testing animals during their inactive periods may produce abnormal results due to cognitive deficits, lack of motivation to perform the task or stress from being disturbed during the resting period. In addition, conducting procedures during an animal's resting period may also pose an animal welfare issue, as procedures may be experienced as more stressful than if these were done during the active phase. In this paper we set out the need to consider the impact of time of day and lighting conditions, when scientific procedures or routine husbandry are performed, on both the welfare of mice and rats used in neuroscience research and on data quality. Wherever possible, husbandry and experimental procedures should be conducted at times of day when the animals would be active, and under naturalistic lighting conditions, to minimise stress and maximise data quality and translatability.

To Group or Not to Group? Good Practice for Housing Male Laboratory Mice.

It is widely recommended to group-house male laboratory mice because they are 'social animals', but male mice do not naturally share territories and aggression can be a serious welfare problem. Even without aggression, not all animals within a group will be in a state of positive welfare. Rather, many male mice may be negatively affected by the stress of repeated social defeat and subordination, raising concerns about welfare and also research validity. However, individual housing may not be an appropriate solution, given the welfare implications associated with no social contact. An essential question is whether it is in the best welfare interests of male mice to be group- or singly housed. This review explores the likely impacts-positive and negative-of both housing conditions, presents results of a survey of current practice and awareness of mouse behavior, and includes recommendations for good practice and future research. We conclude that whether group- or single-housing is better (or less worse) in any situation is highly context-dependent according to several factors including strain, age, social position, life experiences, and housing and husbandry protocols. It is important to recognise this and evaluate what is preferable from animal welfare and ethical perspectives in each case.

Good Science, Good Sense and Good Sensibilities: The Three Ss of Carol Newton.

The Three Rs principle of Replacement, Reduction and Refinement developed by William M. S. Russell and Rex L. Burch in the 1950s has achieved worldwide recognition as a means of reducing the impact of science on animals and improving their welfare. However, application of the Three Rs is still far from universal, and evidence-based methods to implement the Three Rs are still lacking in many areas of laboratory animal science. The purpose of this paper is to create interest in a less well-known but equally useful principle that complements the Three Rs, which was proposed by the American biomathematician Carol M. Newton in the 1970s: the Three Ss-Good Science, Good Sense and Good Sensibilities.

Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care.

A meeting on Contemporary Topics in Zebrafish Husbandry and Care was held in the United Kingdom in 2014, with the aim of providing a discussion forum for researchers, animal technologists, and veterinarians from academia and industry to share good practice and exchange ideas. Presentation topics included protocols for optimal larval rearing, implementing the 3Rs (replacement, reduction, and refinement) in large-scale colony management, and environmental enrichment. The audience also participated in a survey of current practice relating to practical husbandry, cryopreservation, and the provision of enrichment.

A Good Death? Report of the Second Newcastle Meeting on Laboratory Animal Euthanasia.

Millions of laboratory animals are killed each year worldwide. There is an ethical, and in many countries also a legal, imperative to ensure those deaths cause minimal suffering. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. In 2013, an international group of researchers and stakeholders met at Newcastle University, United Kingdom to discuss the latest research and which methods could currently be considered most humane for the most commonly used laboratory species (mice, rats and zebrafish). They also discussed factors to consider when making decisions about appropriate techniques for particular species and projects, and priorities for further research. This report summarises the research findings and discussions, with recommendations to help inform good practice for humane killing.

Refinement of animal models of sepsis and septic shock.

This report aims to facilitate the implementation of the Three Rs (replacement, reduction, and refinement) in the use of animal models or procedures involving sepsis and septic shock, an area where there is the potential of high levels of suffering for animals. The emphasis is on refinement because this has the greatest potential for immediate implementation. Specific welfare issues are identified and discussed, and practical measures are proposed to reduce animal use and suffering as well as reducing experimental variability and increasing translatability. The report is based on discussions and submissions from a nonregulatory expert working group consisting of veterinarians, animal technologists, and scientists with expert knowledge relevant to the field.

Recognizing and assessing pain, suffering and distress in laboratory animals: a survey of current practice in the UK with recommendations.

A survey was undertaken to evaluate how animal pain, suffering and distress are recognized and assessed in UK scientific procedure establishments designated under the Animals (Scientific Procedures) Act 1986. A total of 28 institutions were visited between June 1999 and April 2001, within which 137 people were interviewed including scientists, veterinarians and animal technicians. All 28 establishments use clinical observation sheets to assist the recognition of adverse effects, nine use score sheets and seven use computerized data management systems. Clinical signs used as indicators of potential pain, suffering or distress are largely subjective. The survey also addressed protocols and methods for avoiding and alleviating adverse effects, record keeping, review of policies and protocols and issues relating to team work and training. Respondents use a range of techniques for reducing suffering including analgesia, humane endpoints, ensuring competence and refining husbandry. All establishments review projects regularly but few have the time or resources formally to review adverse effects noted in practice and to compare observations with predictions made in licence applications. Training is very consistent between different establishments and most aim to achieve a 'team approach' for monitoring and assessing animals. Results are summarized in the present, abridged paper and set out in full in a report that can be downloaded at http://www.lal.org.uk/pain/(Hawkins 2002). The present paper and the full report, including its recommendations, are intended to provide a source of information, discussion topics and ideas for all establishments that need to monitor animal well-being.