Use and misuse of an imprecise concept: alternative methods in animal experiments

The concept of inhumanity of Russell & Burch in 1959 with proposals for human procedures in experiments on animals by way of replacement, reduction and refinement (the 3 R's) and the revival of these by Smyth (1978) as alternatives to animal experiments are presented. Since then under the name 'alternative methods', replacement of in vivo by in vitro methods has found great public attention and promotion. It is argued that alternative methods are a fallacy, because in the progress of research a continual control and reinvestigation of findings on each system level is required until knowledge is complete, which makes in vivo experiments irreplaceable. True alternatives exist only for refinement within a system level. On the level of the organism, refinement would be in the care of animals and experimental procedure through alleviation of stress. The possibilities which refinement offers for animal welfare have been nearly forgotten and need promotion among researchers, animal technicians and attendants.     

Methods in vascular infusion biotechnology in research with rodents

Infusion of experimental compounds into the vascular system of rodents and the need to collect blood and other biological fluids from small animals comprise an area of emerging importance to biomedical research and drug discovery and development. The advances in the development of transgenic rodents coupled with technical progress in the manufacture and commercial availability of various catheters, swivels, tethers, infusion pumps, and sample collection systems that are described have enabled biomedical scientists to miniaturize vascular infusion and sample collection systems previously used in animal species larger than the rat or mouse. Use of these advanced, miniature vascular infusion systems in rodents is possible only when careful planning of experimental design, expert surgical technique, adequate postoperative care, and fundamental animal welfare considerations are meticulously taken into consideration. Use of these vascular infusion systems in rodents promotes animal welfare and scientific progress through the reduction and refinement of animal models.     

Experimental animal urine collection: a review

Animal urine collection is a vital part of veterinary practice for ascertaining animal health and in scientific investigations for assessing the results of experimental manipulations. Untainted animal urine collection is very challenging, especially with small rodents, and is an almost impossible task under conditions of microgravity. The fundamental aspects of urine collection are: (1) ease of collection, (2) quality of sample, (3) prevention of contamination, (4) severity of procedures used, (5) levels of pain caused to the animal and (6) refinement of methods to reduce stress, pain or distress. This review addresses the collection of urine for qualitative and quantitative purposes from rodents, rabbits, felines, canines, avian species, equines, porcines, ungulates and certain non-human primates, with animal welfare in mind. Special emphasis has been given to rodents, canines and non-human primates, since they are the animals of choice for research purposes. Free catch (voluntary voiding), methods with mild intervention, surgical methods, modified restraint, cage and special requirement methods have been reviewed here. Efforts need to be taken to provide appropriate animal husbandry and to nurture the animals in as natural an environment as possible since experimental results obtained from these research subjects are, to a great extent, dependent upon their well-being. A continuous refinement in the procedures for collecting urine from experimental animals will be the most efficient way of proceeding in obtaining pure urine specimens for obtaining reliable research data.     

The impact of huts on physiological stress: a refinement in post-transport housing of male guineapigs (Cavia porcellus)

The ideal animal model would contribute no confounding variables in experimental science. Variables affect experimental design resulting in increased animal use or repeated studies. We demonstrated a simple refinement which may reduce the number of animals used experimentally while simultaneously improving animal welfare. The objective of this study was to determine if the presence of a hut was an impact on physiological stress levels, as determined by faecal cortisol concentration, during a routine four-day acclimatization period of newly received male Hartley-Outbred guineapigs. We hypothesized that those animals provided with huts would have decreased physiological stress compared with animals not provided with huts. We examined this effect within both paired and single-housed animals. A between-subjects one-way analysis of variance revealed that pair-housed animals with a hut had significantly lower faecal cortisol concentration than pair-housed animals without a hut and the presence and absence of a hut had no significant impact on faecal cortisol concentration in single-housed animals. These findings show that presence of a hut is beneficial in reducing physiological stress when pair housing male guineapigs and does not appear to have an impact when single housing male guineapigs. In addition, we have shown that faecal cortisol, and therefore physiological stress, is still increasing on study day 4 suggesting a longer acclimatization period is necessary. A simple refinement in housing environment and acclimatization time can both reduce the number of animals used experimentally and improve animal welfare.     

Refinement: promoting the three Rs in practice

Refinement of scientific procedures carried out on protected animals is an iterative process, which begins with a critical evaluation of practice. The process continues with objective assessment of the impact of the procedures, identification of areas for improvement, selection and implementation of an improvement strategy and evaluation of the results to determine whether there has been the desired effect, completing the refinement loop and resulting in the perpetuation of good practice. Refinements may be science-driven (those which facilitate getting high-quality results) or welfare-driven or may encompass both groups, but whatever the driver, refinements almost always result in benefits to both welfare and science. Refinements can be implemented in all aspects of animal use: improved methodology in invasive techniques, housing and husbandry, and even statistical analyses can all benefit animal welfare and scientific quality. If refinement is not actively sought, outdated and unnecessarily invasive techniques may not be replaced by better methods as they become available, and thus outdated information is passed down to the next generation, causing perpetuation of old-fashioned methods. This leads to a spiral of ignorance, leading ultimately to poor practice, poor animal welfare and poor-quality scientific data. Refinement is a legal and ethical requirement, yet refinements may not always be implemented. There are numerous obstacles to the implementation of refinement, which may be real or perceived. Either way, in order to take refinement forward, it is important to coordinate the approach to refinement, validate the science behind refinement, ensure there is adequate education and training in new techniques, improve liaison between users and make sure there is feedback on suitability of refinements for use. Overall, refinement requires a coordinated ongoing process of critical appraisal of practice and active scrutiny of resources for likely improvements. In the busy world of biomedical research, this process needs help. In order to develop these themes further, a workshop was held at the LASA Winter Meeting 2006, UK, to assist in identifying potential obstacles to refinement, and then to explore and develop strategies for overcoming these obstacles in key areas. A range of strategies appropriate to different circumstances was identified, which should facilitate the implementation of refinements.     

Survey of Canadian animal-based researchers' views on the Three Rs: replacement, reduction and refinement

The 'Three Rs' tenet (replacement, reduction, refinement) is a widely accepted cornerstone of Canadian and international policies on animal-based science. The Canadian Council on Animal Care (CCAC) initiated this web-based survey to obtain greater understanding of 'principal investigators' and 'other researchers' (i.e. graduate students, post-doctoral researchers etc.) views on the Three Rs, and to identify obstacles and opportunities for continued implementation of the Three Rs in Canada. Responses from 414 participants indicate that researchers currently do not view the goal of replacement as achievable. Researchers prefer to use enough animals to ensure quality data is obtained rather than using the minimum and potentially waste those animals if a problem occurs during the study. Many feel that they already reduce animal numbers as much as possible and have concerns that further reduction may compromise research. Most participants were ambivalent about re-use, but expressed concern that the practice could compromise experimental outcomes. In considering refinement, many researchers feel there are situations where animals should not receive pain relieving drugs because it may compromise scientific outcomes, although there was strong support for the Three Rs strategy of conducting animal welfare-related pilot studies, which were viewed as useful for both animal welfare and experimental design. Participants were not opposed to being offered "assistance" to implement the Three Rs, so long as the input is provided in a collegial manner, and from individuals who are perceived as experts. It may be useful for animal use policymakers to consider what steps are needed to make replacement a more feasible goal. In addition, initiatives that offer researchers greater practical and logistical support with Three Rs implementation may be useful. Encouragement and financial support for Three Rs initiatives may result in valuable contributions to Three Rs knowledge and improve welfare for animals used in science.     

Why modification of the LD50 test will not be enough

During the last 10 years, the 'Three Rs' (reduction, refinement and replacement) concept of alternatives has come to be widely accepted, and new national and international laws require that non-animal procedures should replace animal experimentation wherever possible. Some reduction and refinement of animal use in toxicity testing has been achieved, and non-animal methods are becoming widely used as prescreens. However, even replacing the LD50 test by a modified and validated animal test, the Fixed Dose Procedure, will be a major achievement. In this paper it is argued that this is not good enough, and that more effort must be put into the development, validation, acceptance and use of genuine replacement alternative tests.     

Animal Welfare in Studies on Murine Tuberculosis: Assessing Progress over a 12-Year Period and the Need for Further Improvement

There is growing concern over the welfare of animals used in research, in particular when these animals develop pathology. The present study aims to identify the main sources of animal distress and to assess the possible implementation of refinement measures in experimental infection research, using mouse models of tuberculosis (TB) as a case study. This choice is based on the historical relevance of mouse studies in understanding the disease and the present and long-standing impact of TB on a global scale. Literature published between 1997 and 2009 was analysed, focusing on the welfare impact on the animals used and the implementation of refinement measures to reduce this impact. In this 12-year period, we observed a rise in reports of ethical approval of experiments. The proportion of studies classified into the most severe category did however not change significantly over the studied period. Information on important research parameters, such as method for euthanasia or sex of the animals, were absent in a substantial number of papers. Overall, this study shows that progress has been made in the application of humane endpoints in TB research, but that a considerable potential for improvement remains.     

The refining influence of ethics committees on animal experimentation in Sweden

Mandatory scrutiny of projects by animal ethics committees was introduced in Sweden in 1979. The present study investigated the minutes of meetings held between 1989 and 2000 at which consideration of applications for experimental work in animals resulted in requests for modification (n = 3607). 18.1% of the applications received were approved only after modifications. The majority of the changes requested may be classified as 'Refinement'. The most common requests were for improvement of project design, euthanasia method and housing and husbandry. There was a relative increase in modifications requested by the committees related to anaesthesia, choice of licensed supervisor and the need for licenses or informed consent from animal owners during the period investigated. There was a relative decrease in modifications related to euthanasia, housing and husbandry, and general endpoint assertions. The results suggest that the work of the committees may be perceived as an ongoing process, since several of the applications for which modification was requested were projects that had been approved on a previous occasion but were now up for renewal. In order to have maximal influence on the refinement of scientific protocols it is important that the scientists in the committees are continuously updated on developments in laboratory animal science.     

The search for refinement alternatives: when you've just got to use animals

Refinements to animal-use protocols can help reduce animal pain and distress, thus producing cleaner data. Yet, most researchers neglect a refinement search, despite legal mandates, because the information is not readily available. What's more, IACUCs may have difficulty assessing researchers' refinement searches. The author offers some solutions.     

Training of laboratory-housed non-human primates in the UK

Abstract

Laboratory-housed non-human primates may experience a range of potential stressors, including physical and chemical restraint, venepuncture, injection, catching and cage-change. Training them to co-operate, using positive reinforcement techniques, is one means of significantly reducing the adverse impact of such procedures upon them and, therefore, is a refinement. Furthermore, the additional time that staff spend with the primates, and the need for individual recognition and close observation of animal behavior, mean that the trainer develops a relationship with each individual animal which can be beneficial for animal welfare (e.g., by reducing the occurrence of abnormal behavior and fear of humans). We surveyed use of training in thirteen UK establishments using and breeding primates, utilizing a mixed-mode questionnaire. The survey demonstrated that there is widespread awareness of training as a refinement and appreciation of its diverse benefits, but training is not used as widely or as fully as it might be. This is largely due to real constraints (principally staff and time and a lack of confidence in ability to train), and perceived constraints (such as a supposed lack of information on how to train and assessment of the benefits, and an overestimation of the time investment needed). There is also considerable variation between establishments in the purposes of training and techniques used, with a reliance on negative reinforcement in three. We conclude that there is considerable scope for refinement of common scientific, veterinary and husbandry procedures, and refer to some resources designed to help establishments take action (e.g., Prescott and Buchanan-Smith 2003).     

Management of adverse side-effects after chemotherapy in macaques as exemplified by streptozotocin: case studies and recommendations

The chemotherapeutic streptozotocin is used for induction of diabetes in animal models including non-human primates. Being a cytotoxic nitrosourea compound, it can be associated with adverse events (AEs), mainly nausea and emesis, nephrotoxicity, elevated liver transaminase levels, pulmonary oedema and, most prominently, metabolic acidosis: these can be severe in some cases. The incidence and gravity are to some extent related to the characteristics of the individual animal, diagnostic tools, prompt recognition of symptoms and supportive measures. Careful animal selection, dose adaptation and supportive actions such as renal protective hydration are the main tools in managing AEs, but do not fully eliminate unavoidable and sometimes life-threatening conditions. In our centre we have built experience in a cohort of 78 cynomolgus and rhesus macaques in which six cases manifested severe AEs (8%). This experience has prompted implementation of strategies for early detection and management of adverse effects, together with an animal refinement programme. We present here specific pretreatment regimens, post-infusion laboratory evaluations, and flow charts to assess/treat metabolic acidosis and precipitating factors. Case reports of the six animals with severe AEs are presented to illustrate management of AEs, especially metabolic acidosis, and criteria for early euthanasia where appropriate. We conclude that improved monitoring and validated tools allow for optimal management of adverse effects in an early stage of their manifestation. Reduced morbidity and mortality not only improve individual animal wellbeing but also avoid model-induced confounding that diminishes the translational value of the experimental protocol.     

A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement

The refinement of husbandry and procedures to reduce animal suffering and improve welfare is an essential component of humane science. Successful refinement depends upon the ability to assess animal welfare effectively, and detect any signs of pain or distress as rapidly as possible, so that any suffering can be alleviated. This document provides practical guidance on setting up and operating effective protocols for the welfare assessment of animals used in research and testing. It sets out general principles for more objective observation of animals, recognizing and assessing indicators of pain or distress and tailoring these to individual projects. Systems for recording indicators, including score sheets, are reviewed and guidance is set out on determining practical monitoring regimes that are more likely to detect any signs of suffering. This guidance is intended for all staff required to assess or monitor animal welfare, including animal technologists and care staff, veterinarians and scientists. It will also be of use to members of ethics or animal care and use committees. A longer version of this document, with further background information and extra topics including training and information sharing, is available on the Laboratory Animals website.     

Time- and ensemble-averaged direct NOE restraints

Summary NMR data are collected as time- and ensemble-averaged quantities. Yet, in commonly used methods for structure determination of biomolecules, structures are required to satisfy simultaneously a large number of constrainsts. Recently, however, methods have been developed that allow a better fit of the experimental data by the use of time- or ensemble-averaged restraints. Thus far, these methods have been applied to structure refinement using distance and J-coupling restraints. In this paper, time and ensemble averaging is extended to the direct refinement with experimental NOE data. The implementation of time- and ensemble-averaged NOE restraints in DINOSAUR is described and illustrated with experimental NMR data for crambin, a 46-residue protein. Structure refinement with both time- and ensemble-averaged NOE restraints results in lower R-factors, indicating a better fit of the experimental NOE data.     

On the need to redress an inadequacy in animal welfare science: toward an internally coherent framework

The time is ripe for a greater interrogation of assumptions and commitments underlying an emerging common ground on the ethics of animal research as well on the 3 R (replacement, refinement, reduction) approach that parallels, and perhaps even further shapes, it. Recurring pressures to re-evaluate the moral status of some animals in research comes as much from within the relevant sciences as without. It seems incredible, in the light of what we now know of such animals as chimpanzees, to deny that these animals are properly accorded high moral status. Barring the requirement that they be human, it is difficult to see what more animals such as chimpanzees would have to possess to acquire it. If the grounds for ascribing high moral status are to be non-arbitrary and responsive to our best knowledge of those individuals who possess the relevant features, we should expect that a sound ethical experimental science will periodically reassess the moral status of their research subjects as the relevant knowledge demands. We already can observe this reassessment as scientists committed to humane experimental science incorporate discoveries of enrichment tools and techniques into their housing and use of captive research animals. No less should this reassessment include a critical reflection on the possible elevation of moral status of certain research animals in light of what is discovered regarding their morally significant properties, characteristics or capacities, or so I will argue. To do anything short of this threatens the social and moral legitimacy of animal research.     

On using time-averaging restraints in molecular dynamics simulation

Introducing experimental values as restraints into molecular dynamics (MD) simulations to bias the values of particular molecular properties, such as nuclear Overhauser effect intensities or distances, 3J coupling constants, chemical shifts or crystallographic structure factors, towards experimental values is a widely used structure refinement method. To account for the averaging of experimentally derived quantities inherent in the experimental techniques, time-averaging restraining methods may be used. In the case of structure refinement using 3J coupling constants from NMR experiments, time-averaging methods previously proposed can suffer from large artificially induced structural fluctuations. A modified time-averaged restraining potential energy function is proposed which overcomes this problem. The different possible approaches are compared using stochastic dynamics simulations of antamanide, a cyclic peptide of ten residues.     

Report of the FELASA Working Group on evaluation of quality systems for animal units

This report compares and considers the merits of existing, internationally available quality management systems suitable for implementation in experimental animal facilities. These are: the Good Laboratory Practice Guidelines, ISO 9000:2000 (International Organization for Standardization) and AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International). Good laboratory practice (GLP) is a legal requirement for institutions undertaking non-clinical health and environmental studies for the purpose of registering or licensing for use and which have to be 'GLP-compliant'. GLP guidelines are often only relevant for and obtainable by those institutions. ISO is primarily an external business standard, which provides a management tool to master and optimize a business activity; it aims to implement and enhance 'customer satisfaction'. AAALAC is primarily a peer-reviewed system of accreditation which evaluates the organization and procedures in programmes of animal care and use to ensure the appropriate use of animals, safeguard animal well-being (ensuring state-of-the-art housing, management, procedural techniques, etc.) as well as the management of health and safety of staff. Management needs to determine, on the basis of a facility's specific goals, whether benefits would arise from the introduction of a quality system and, if so, which system is most appropriate. The successful introduction of a quality system confers peer-recognition against an independent standard, thereby providing assurance of standards of animal care and use, improving the quality of animal studies, and contributing to the three Rs-reduction, refinement and replacement.     

Recommendations for improving the home office project licence abstracts

The Freedom of Information Act 2000 prompted the Government to evaluate how public understanding about the use of animals in scientific research can be improved and how animal experimentation is regulated within the UK. This resulted in the publication of anonymous project licence abstracts. Here, the abstracts published between December 2004 and October 2005 have been evaluated, by using a scoring system which takes into account their content and format, in order to assess whether they provide information that will facilitate the increased implementation of reduction, refinement and replacement strategies. The potential impact of freely-available Home Office project licence abstracts on public attitudes to animal testing and on the quality of animal-based research, is also discussed. Although some abstracts scored well, in general, the abstracts were found to be severely lacking in detail about the welfare of experimental animals. Thus, the abstracts tend to present a distorted picture of animal-based research. Many abstracts lack the details which would reveal how the licensing criteria had been met. It is concluded that, in their current form, the project abstracts are not sufficiently informative. A number of recommendations are made, which address this problem.     

Animal welfare and the refinement of neuroscience research methods--a case study of Huntington's disease models

The use of animals in biomedical and other research presents an ethical dilemma: we do not want to lose scientific benefits, nor do we want to cause laboratory animals to suffer. Scientists often refer to the potential human benefits of animal models to justify their use. However, even if this is accepted, it still needs to be argued that the same benefits could not have been achieved with a mitigated impact on animal welfare. Reducing the adverse effects of scientific protocols ('refinement') is therefore crucial in animal-based research. It is especially important that researchers share knowledge on how to avoid causing unnecessary suffering. We have previously demonstrated that even in studies in which animal use leads to spontaneous death, scientists often fail to report measures to minimize animal distress (Olsson et al. 2007). In this paper, we present the full results of a case study examining reports, published in peer-reviewed journals between 2003 and 2004, of experiments employing animal models to study the neurodegenerative disorder Huntington's disease. In 51 references, experiments in which animals were expected to develop motor deficits so severe that they would have difficulty eating and drinking normally were conducted, yet only three references were made to housing adaptation to facilitate food and water intake. Experiments including end-stages of the disease were reported in 14 papers, yet of these only six referred to the euthanasia of moribund animals. If the reference in scientific publications reflects the actual application of refinement, researchers do not follow the 3Rs (replacement, reduction, refinement) principle. While in some cases, it is clear that less-than-optimal techniques were used, we recognize that scientists may apply refinement without referring to it; however, if they do not include such information in publications, it suggests they find it less relevant. Journal publishing policy could play an important role: first, in ensuring that referees seriously consider whether submitted studies were indeed carried out with the smallest achievable negative impact on the animals and, secondly, in encouraging scientists to share refinements through the inclusion of a 3Rs section in papers publishing the results of animal-based research.