“Our story must begin with Charles Hume, for the whole of this grand enterprise began as a twinkle in his eye, which often twinkled” (Russell 1995). Fifty-one years ago, in 1954, besides important achievements in other fields of animal welfare, Hume and the Universities Federation for Animal Welfare (UFAW) had brought out the first edition of The UFAW Handbook on the Care and Management of Laboratory Animals, edited by Alastair Worden (Worden 1947), who was already preparing a greatly enlarged second edition with William Lane-Petter (Worden & Lane-Petter 1957). Hume had already contributed to the first edition of the Handbook an article on statistical analysis, which is clearly relevant to actual experimentation (Hume 1947); he now had the brilliant and totally original idea of starting a general study of humane technique in actual experimentation. The late Rex Burch and I were appointed to undertake this project; the moment Rex walked into my office I knew that here was not only the perfect colleague but also a lifelong friend, and I sadly miss him.

Researchers searching for alternatives to painful procedures that involve animals may find that the dispersed relevant literature and the array of databases make the search challenging and even onerous. This paper addresses a significant gap that exists for researchers, in identifying appropriate databases to use when searching for specific types of information on alternatives. To facilitate the efficient and effective searching by users, and to ensure compliance with new requirements and improved science, we initiate an evolving guide comprising search grids of database resources organised by animal models and topics (http://www.vetmed.ucdavis.edu/Animal_Alternatives/databaseapproach.html). The search grids present organised lists of specific databases and other resources for each animal model and topic, with live links. The search grids also indicate resources that are freely available worldwide, and those that are proprietary and available only to subscribers. The search grids are divided into two categories: ‘animal models’ and ‘topics’. The category ‘animal models’ comprises: animal model selections; mice; rodents — rats/guinea pigs/hamsters; rabbits; dogs, cats; farm animals, sheep, swine; non-human primates; fish, frogs, aquatic; and exotics. The category ‘topics’ comprises: husbandry; behaviour; euthanasia; toxicity; monoclonal antibodies; teaching; endpoints; disease models; analgesia/anaesthesia; emerging technologies; strategies for specific intervention procedures; and standard operating procedures (for example, drawing blood, behavioural training, transportation, handling, restraint and identification). Users are provided with a selected list of linked resources relevant to their particular search. Starting with an appropriate database that covers the type of information that is being sought is the first step in conducting an effective search that can yield useful information to enhance animal welfare.

A technique measuring leukocyte (neutrophil) activity was used to examine differences between stress levels in a breeding colony of rhesus macaques housed in either a traditional caging system or open-rooms. The leukocyte activation test measured the degree to which blood from the two treatment groups could launch a further neutrophil response (superoxide production) to an in vitro challenge. Animals housed in a traditional caging system produced a significantly lower leukocyte response than animals housed in open-rooms, indicating that there was a higher level of stress associated with caged housing than open-room housing. This was not influenced by whether animals were physically restrained or trained to stand for a sedating injection. No differences were found between treatment groups in leukocyte numbers or composition. This study validates the use of the leukocyte activation test to assess physiological stress levels in non-human primates and demonstrates the animal welfare benefits of open-room housing over traditional laboratory caging systems.

The implantation of new biomedical devices into living animals without any previous toxicity or biocompatibility evaluation is possible under current legislation. The HET–CAM (Hen Egg Test–Chorionallantoic Membrane) test offers a partially immunodeficient, borderline in vitro/in vivo test system that allows the simulation of transplantation experiments to obtain biocompatibility data prior to animal testing. A collagen type I/III scaffold, designed for tissue regeneration, was tested for angiogenetic properties and biocompatibility patterns. A significant angiogenetic stimulus caused by the collagen scaffold material was observed. Altering biocompatibility patterns by incubation with the potentially hazardous chemicals acridine orange and ethidium bromide led to severe vessel thrombosis and a foreign body tissue response. CAM testing of biomaterials and tissue engineered products allows selection of the most suitable biomaterial and the elimination of unsuitable materials from animal experiments, leading to a refinement of testing procedures and a reduction in the number of animals required for biocompatibility testing.

At present, animal experimentation remains central to our understanding of human disease-related processes and of the biological effects of many substances. Traditional experiments have relied heavily on invasive techniques to monitor changes in blood biochemistry, tissue structure or function, or to phenotype or genotype genetically modified animals. In some cases, a proportion or all of the animals used during the course of a study may be sacrificed for histopathological assessment. In most cases, this is to track the progression or regression of a disease over time, or to determine the levels of toxicity evident in specific organs or tissues. However, many of these techniques fail to provide details of how a disease develops or how a substance elicits its effects. In recent years there has been a gradual increase in the application of imaging techniques that were originally developed and used in fundamental research or in medicine. These non-invasive techniques allow diseases, and responses to exogenous substances, to be monitored in a temporal and spatial manner, therefore allowing a greater amount of information to be derived from smaller numbers of animals, which in turn, increases the statistical validity of the data by reducing the level of experimental variation. Non-invasive imaging also allows more informative and humane endpoints to be used and, perhaps most importantly, allows functional details to be studied in the context of a living animal. Some of the recent developments within the field of non-invasive imaging and their significance with respect to animal welfare and the understanding of human physiology are discussed.

Growing public concern regarding animal welfare and consumer demand for humanely produced products have placed pressure on the meat, wool and dairy industries to improve and confirm the welfare status of their animals. This has increased the need for reliable methods of assessing animal welfare during commercial farm practices. The measurement of the stress caused by commercial farm practices is a major component of animal welfare assessment. However, a major issue for animal welfare science is that many of the techniques used to measure stress involve invasive procedures, such as blood sampling, which may themselves cause a stress response and therefore affect the measurement of interest. To reduce this problem, a number of non-invasive or minimally invasive methods and devices have been developed to measure stress. These include the measurement of cortisol concentrations in saliva and faeces, and remote devices for recording body temperature, heart rate and the collection of blood samples. This review describes the benefits and limitations of some of these methods for measuring stress. In particular, the review focuses on recent advances and current research in the use of infrared thermography (IRT) for measuring stress. Specific applications for IRT in the dairy and beef industries are also described including an automated, non-invasive system for early diagnosis of infection in cattle. It is essential that non-invasive measures of acute and chronic stress are developed for reliable assessment of animal welfare during standard farm management practices and IRT may be a useful tool for this purpose. IRT may offer advantages over many other non-invasive systems as it appears to be capable of measuring different components of the stress axis, including acute sympathetic and hypothalamic-pituitary-adrenocortical responses.

Russell and Burch's Three Rs principle of replacement, reduction and refinement offers a useful concept for the scientific and ethical evaluation of the use of animals in scientific procedures. Replacement, reduction and refinement are often considered separately, but when applied, one of the Three Rs may have a positive or negative effect on one or both of the other Rs. This paper explores the interplay between the Three Rs and provides examples where the Three Rs have a positive interaction and where they are in conflict with each other. For example, all Three Rs positively interact in the use of cell cultures, but validation studies of replacement techniques may initially increase the numbers of animals used; therefore replacement and reduction are in conflict. Several models of cost-benefit analyses, used by animal ethics committees to justify or reject animal experimentation, contain elements such as quality and significance of the research, the credibility of the research group and the discomfort caused to the animals. Although these models consider the Three Rs, each R is considered independently of the others. Consequently, moral dilemmas may arise when reviewing proposals in which the Three Rs conflict. Currently there is no legal guidance relating to the prioritisation of the Three Rs, but guidance is required to facilitate their use. For example, does a significant reduction in animal numbers justify increased individual suffering? Moral justifications deserve more attention when considering the Three Rs in general, and when considering the application of one or more Rs to a procedure, to a protocol, or to the wider research programme.

Meta-analysis provides a tool to statistically aggregate data from existing randomised controlled animal experiments. The results can then be summarised across a range of conditions and an increased pool of experimental data can be subjected to statistical analysis. New information can be derived, but most frequently the results are a refinement of existing knowledge. By designing experiments and reporting protocols, so that they have the capability of being useful to meta-analyses, maximum benefit can be derived from individual randomised controlled experiments, which may individually have little statistical power, and new avenues for productive research identified. The methodology for meta-analysis is derived from clinical trials in the medical sciences. Now that there is substantial output from animal science experiments, there is an opportunity to apply the technique to these and reduce the need for further experimentation. This paper describes the contribution of meta-analysis to the reduction of animals in research and provides details on data collection, analysis, the models used, and on interpreting and reporting the results. Three applications of meta-analysis to the field of animal science are also briefly described. First, the impact of undernutrition on the production and composition of milk from dairy cows confirmed existing knowledge about partitioning scarce nutrients to milk yield and live weight. Second, increased absorption of cadmium — a widespread toxic element — from organic sources was detected in sheep, which was previously untested. Third, no significant relationships were found between common indicators of undernutrition and weight, and condition score in cattle suggesting that the common indicators used are not suitable as evidence of long term undernutrition. This paper concludes that opportunities exist to increase the information gained from animal experiments by subjecting the results to meta-analysis, particularly if this can be anticipated in advance of study protocols being constructed.

In the past decades the Three Rs concept, famously launched by Russell and Burch in their 1959 book The Principles of Humane Experimental Technique, has gained a prominent place in the landscape of societal and ethical concern about animal use. Important scientific and institutional initiatives have been taken in order to promote replacement, reduction and refinement. It appears, however, that conceptual and ethical thinking about the presuppositions and changing contexts of the Three Rs concept has lagged behind the scientific and practical efforts. In this paper, first, I argue that there is a threefold argument to make for the need to reconsider the moral basis of the Three Rs concept. Second, I outline a number of standard assumptions of the traditional approach to the Three Rs and question the tenability of these assumptions. Third, I propose some elements of a new framework for the Three Rs principle and connect this to a number of developments in science and society. I conclude with four remarks on the future of the ethics of the Three Rs principle.

In many countries scientists planning research that may involve the use of animals are required by law to examine the possibilities for replacement, reduction or refinement (the Three Rs) of these experiments. In addition to the large number of literature databases, there are now many specialist databases specifically addressing the Three Rs. Information centres, with a mandate to assist scientists and lay people locate information on the Three Rs, have also been established. Email discussion lists and their archives constitute another, although less quality-controlled, source of information. Furthermore, guidelines for the care and use of animals in research have been produced both by regulatory bodies and scientific organisations. The growth of the internet has put an enormous amount of data into the public domain, and the problems of accessing relevant information are discussed. Suggestions are also given for search strategies when using these information sources.

This paper highlights the essential need for appropriate statistical design and randomisation in laboratory animal studies. Using an example of a 21 day weight gain study in mice, we show that without the use of an appropriate statistical design and randomisation, incorrect conclusions may have been drawn. We used an experimental design that allowed comparisons to be made between five treatments that were free from systematic error. Two alternative designs that are practically attractive, yet had no statistical basis, are also described in this paper and the potentially incorrect conclusions highlighted. The use of appropriate statistical design is ethical because it results in clear, unambiguous conclusions. Conclusions that may be biased or ambiguous will require verification by further research and this, in the long term, is contrary to the reduction element of the Three Rs.

Six drug regulatory reviewers and 11 pharmaceutical industry scientists were interviewed to explore their perspectives on the obstacles and opportunities for greater implementation of the Three Rs (replacement, reduction, refinement) in drug research and development. Participants generally supported the current level of animal use in the pharmaceutical industry and viewed in vitro methods as supporting, but not replacing, the use of animals. Obstacles to greater use of the Three Rs cited by participants included the lack of non-animal alternatives; requirements for statistical validity; reluctance by industry and regulators to depart from established patterns of animal use; the priority of commercial objectives ahead of the Three Rs; and concern that less animal testing could jeopardise human safety. Opportunities identified for the Three Rs included the development of better animal models including genetically modified (GM) animals; pursuit of more basic knowledge, notably drug action on gene expression; re-use of animals; greater use of pilot studies; using sufficient numbers of animals per test to avoid repeating inconclusive studies; regular review of animal data in regulatory requirements; and following the regulatory option of combining segments of reproductive toxicology studies into one study. In some areas, greater implementation of the Three Rs seemed well aligned with industry priorities, for example, phenotypic characterisation of GM animals and validation of alternative methods. In other areas, wider use of the Three Rs may require building consensus on areas of disagreement including the usefulness of death as an endpoint; the suitability of re-using animals; and whether GM animals and the use of pilot studies contribute to reduction.

Russell and Burch's Three Rs (replacement, reduction and refinement) remain the cornerstone for principles guiding humane experimental research. However, the concept of refinement has evolved considerably since its first inception and there have been numerous interpretations, some of which are regressive from the original definition. In this paper we examine the interpretations of refinement, and propose a harmonised progressive definition that is in line with changes in animal ethics and animal welfare science. Our definition should be applied to all aspects of refinement: those related to housing, husbandry and care, techniques used in scientific procedures, periprocedural care, health and welfare monitoring, and experimental design. We argue not only that the concept should include the avoidance or minimisation of adverse effects experienced at any time during the life of an animal destined for use in a laboratory, but also that it should be applied to the founder animals. Furthermore, we take a proactive stance and argue that refinement should include enhancing well-being through environmental enrichment. The acceptance and application of this new definition by legislative authorities and in guidelines would represent a significant step forward for animal welfare.