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.     

Review: Assessing fish welfare in research and aquaculture,with a focus on European directives

The number of farmed fish in the world has increased considerably. Aquaculture is a growing industry that will in the future provide a large portion of fishery products. Moreover, in recent years, the number of teleost fish used as animal models for scientific research in both biomedical and ecological fields has increased. Therefore, it is increasingly important to implement measures designed to enhance the welfare of these animals. Currently, a number of European rules exist as requirements for the establishment, care and accommodation of fish maintained for human purposes. As far as (teleost) fish are concerned, the fact that the number of extant species is much greater than that of all other vertebrates must be considered. Of further importance is that each species has its own specific physical and chemical requirements. These factors make it difficult to provide generalized recommendations or requirements for all fish species. An adequate knowledge is required of the physiology and ecology of each species bred. This paper integrates and discusses, in a single synthesis, the current issues related to fish welfare, considering that teleosts are target species for both aquaculture and experimental models in biological and biomedical research. We first focus on the practical aspects, which must be considered when assessing fish welfare in both research and aquaculture contexts. Next, we address husbandry and the care of fish housed in research laboratories and aquaculture facilities in relation to their physiological and behavioural requirements, as well as in reference to the suggestions provided by European regulations. Finally, to evaluate precisely which parameters described by Directive 2010/63/EU are reported in scientific papers, we analysed 82 articles published by European researchers in 2014 and 2015. This review found that there is a general lack of information related to the optimal environmental conditions that should be provided for the range of species covered by this directive.     

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.     

Ten practical realities for institutional animal care and use committees when evaluating protocols dealing with fish in the field

Institutional Animal Care and Use Committee’s (IACUCs) serve an important role in ensuring that ethical practices are used by researchers working with vertebrate taxa including fish. With a growing number of researchers working on fish in the field and expanding mandates of IACUCs to regulate field work, there is potential for interactions between aquatic biologists and IACUCs to result in unexpected challenges and misunderstandings. Here we raise a number of issues often encountered by researchers and suggest that they should be taken into consideration by IACUCs when dealing with projects that entail the examination of fish in their natural environment or other field settings. We present these perspectives as ten practical realities along with their implications for establishing IACUC protocols. The ten realities are: (1) fish are diverse; (2) scientific collection permit regulations may conflict with IACUC policies; (3) stakeholder credibility and engagement may constrain what is possible; (4) more (sample size) is sometimes better; (5) anesthesia is not always needed or possible; (6) drugs such as analgesics and antibiotics should be prescribed with care; (7) field work is inherently dynamic; (8) wild fish are wild; (9) individuals are different, and (10) fish capture, handling, and retention are often constrained by logistics. These realities do not imply ignorance on the part of IACUCs, but simply different training and experiences that make it difficult for one to understand what happens outside of the lab where fish are captured and not ordered/purchased/reared, where there are engaged stakeholders, and where there is immense diversity (in size, morphology, behaviour, life-history, physiological tolerances) such that development of rigid protocols or extrapolation from one species (or life-stage, sex, size class, etc.) to another is difficult. We recognize that underlying these issues is a need for greater collaboration between IACUC members (including veterinary professionals) and field researchers which would provide more reasoned, rational and useful guidance to improve or maintain the welfare status of fishes used in field research while enabling researchers to pursue fundamental and applied questions related to the biology of fish in the field. As such, we hope that these considerations will be widely shared with the IACUCs of concerned researchers.     

Stress and welfare in ornamental fishes: what can be learned from aquaculture?

The ornamental fish trade is estimated to handle up to 1·5 billion fishes. Transportation and handling of fishes imposes a range of stressors that can result in mortality at rates of up to 73%. These rates vary hugely, however, and can be as low as 2%, because they are generally estimated rather than based on experimental work. Given the numbers of ornamental fishes traded, any of the estimated mortality rates potentially incur significant financial losses and serious welfare issues. Industry bodies, such as the Ornamental Aquatic Trade Association (OATA), have established standards and codes of best practice for handling fishes, but little scientific research has been conducted to understand the links between stress, health and welfare in ornamental species. In aquaculture, many of the same stressors occur as those in the ornamental trade, including poor water quality, handling, transportation, confinement, poor social and physical environment and disease and in this sector directed research and some resulting interventions have resulted in improved welfare standards. This review considers the concept of welfare in fishes and evaluates reported rates of mortality in the ornamental trade. It assesses how the stress response can be quantified and used as a welfare indicator in fishes. It then analyses whether lessons from aquaculture can be usefully applied to the ornamental fish industry to improve welfare. Finally, this analysis is used to suggest how future research might be directed to help improve welfare in the ornamental trade.     

Expanding the moral circle: farmed fish as objects of moral concern

Until recently fish welfare attracted little attention, but international and national legislation and standards of fish welfare are now emerging and an overview of these developments is presented in this study. Whereas animal welfare legislation is based on public morality, animal ethics does not automatically accept public morality as normative and elaborates arguments regarding the way humans should treat animals (referred to as moral standards). In this study we present the most common animal ethics theories. For most of these, sentience is considered a demarcation line for moral concern: if an animal is sentient, then it should be included in the moral circle, i.e. receive moral consideration in its own right and some basic welfare should be ensured. As for fish, research has revealed that the sensory system of teleosts can detect noxious stimuli, and that some kind of phenomenal consciousness, allowing the fish to feel pain, seems to be present. This raises the ethical question as to how much evidence we need in order to act on such indications of fish sentience. A simple risk analysis shows that the probability that fishes can feel pain is not negligible and that if they do indeed experience pain the consequences in terms of the number of suffering individuals are great. We conclude that farmed fish should be given the benefit of the doubt and we should make efforts that their welfare needs are met as well as possible. Finally, the way forward is briefly discussed: efforts must be made to understand what fish welfare means in practical fish farming. This will involve the development of research and education, greater accountability and transparency, compliance with and control of policies, and quality assurance schemes.     

实验用鱼类福利的发展现状

伴随着鱼类作为科研重要材料的迅猛发展,实验用鱼类福利引起了人们的关注。本文旨在阐述国内外鱼类福利发展的历史和现状,剖析鱼类福利实施困难的因素,提供提高福利的参考对策,以期帮助科研工作者进一步地了解实验用鱼类福利,促进在实验过程中鱼类福利的发展。     

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 Brazilian legal framework on the scientific use of animals

Brazil has an exceptionally dynamic research sector in Latin America in health, biotechnology, and pharmacology, backed by defined government policies on science and technology and a health research agenda focusing on important neglected diseases: malaria, leishmaniasis, Chagas disease, turberculosis, leprosy, and dengue. The Brazilian health research policy promotes partnerships and networks among scientists in academic institutions in both wealthy industrialized and disease-endemic countries, and in these efforts the government's guidelines for animal use in biomedical research are considered fundamental to guarantee both animal welfare and the quality of research. Given international discussions of animal experimentation regulations and guidelines, in this article we describe current Brazilian legislation governing the use of animals in scientific investigations. We conclude that, despite advances in the implementation of the 3Rs (reduction, refinement, replacement), the new regulatory framework does not sufficiently incorporate ethical considerations, lacking explicit reference to the 3Rs as well as measures for their full application. The more humane use of animals in research will depend on the approach adopted by Brazil's National Council for the Control of Animal Experimentation to promote the 3Rs and to improve internal regulations as well as data collection and analysis in research institutions. In Brazil as elsewhere, one of the greatest challenges to policymakers is to harmonize the myriad and intertwined legal provisions without hindering biomedical research.     

Understanding behaviour to improve the welfare of an ornamental fish

Some common practices in aquaculture, ornamental trade and fish facilities may disturb the behavioural repertoire of fish and its natural adaptive value, reducing welfare and impairing fish production. Hence, it is necessary to understand fish behaviour, as well as the factors affecting it, to improve the quality of fish's life under artificial environment. Here, we reviewed the behaviour of the angelfish Pterophyllum scalare, an Amazonian cichlid used worldwide both as an ornamental fish and as a fish model in scientific research. We characterized social, reproductive and feeding behaviour, as well as the amazing cognitive ability of the angelfish. In addition, we reviewed the effects of environmental enrichment and suggested some important variables that need to be considered for rearing P. scalare. In this review, we show for the first time a synthesis on behaviour and a best practice overview to improve the welfare of angelfish as a target species. Nonetheless, most topics reviewed fit a broader set of fish species, particularly ornamental ones. This synthesis can therefore open a path for further behavioural research applied to the welfare of angelfish and bring insights to other fish species.     

The use of non-human primates in biological and medical research: evidence submitted by FRAME to the Academy of Medical Sciences/Medical Research Council/Royal Society/Wellcome Trust Working Group

The Academy of Medical Sciences, the Medical Research Council, the Royal Society and the Wellcome Trust are undertaking a study into the use of non-human primates in biological and medical research. An independent working group of scientific experts, led by Sir David Weatherall, aims to produce a report summarising the findings of this study, early in 2006. The trends in primate research, and the nature and effects of recent and proposed changes in the global use of non-human primates in research, will be investigated. The associated ethical, welfare and regulatory issues, and the role and impact of the Three Rs principles of refinement, reduction and replacement will also be reviewed. As part of this study, a call for evidence was made. The evidence submitted by FRAME emphasised that the use of non-human primates for fundamental research or for regulatory testing still fails to take into account the fact that, although non-human primates are anatomically and physiologically similar to humans, they are not necessarily relevant models for studies on human disease or human physiology. FRAME continues to believe that we have a duty to ensure that these animals are not used without overwhelming evidence that they are the only suitable and relevant models for use in work of undeniable significance.     

Acoustic deterrents to manage fish populations

Finding effective ways to direct native fish away from anthropogenic hazards and limit the spread of invasive species, without physical intervention, harming non-target fishes or interrupting aquatic commerce is a major challenge for fisheries management. One option is to target fish sensory systems to manipulate behavior using attractive or repulsive cues. Many, if not all species of fish, use sound as part of their behavioral repertoire and display varying degrees of phonotaxis. Sound has inherent advantages over other sensory stimuli such as light or odor as underwater sound attenuates slowly, is highly directional and is unimpeded by low light or water turbidity. This review details the use of acoustics to deter and guide fish movements for a wide variety of fishes, before critically assessing the benefits and limitations of the technology. No single method of fish deterrence is a “one size fits all”, and therefore this review will assist both managers and researchers attempting to use acoustic deterrents for different fish orders.

     

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.     

南极鱼类多样性和适应性进化研究进展

南极地区是地球上唯一未被人类活动大量影响的地区, 其极端寒冷的环境为南极生物的进化提供了“温床”。过去三千万年间, 南极鱼亚目鱼类在南极海洋逐渐变冷的过程中快速进化, 从一个温暖海域的底栖祖先分化成南极海域最为多样化的鱼类类群。由于其在南极圈内和南极圈外的各种温度区间都有分布, 因而成为研究鱼类适应性进化和耐寒机制的良好生物模型。本文综述了有关南极海域鱼类区系组成与物种多样性现状, 南极鱼亚目鱼类适应低温的一系列特化的生物学性状及其关键的遗传进化机制。现有研究表明: 南极鱼类在几千万年零度以下低温环境的进化中发生了大量基因的大规模扩增和基因表达的改变, 如铁调素、卵壳蛋白和逆转座子等118个基因发生了显著的扩增。另外, 有些从南极鱼中获得的抗寒基因已经用于提高动植物低温抗性的研究并取得了良好的效果。在今后的几年中, 将会有多个南极鱼物种的全基因组得到破译, 在低温适应相关基因的功能和进化方面的研究也会更加深入, 这些研究将深入揭示低温压力下基因组的进化规律以及鱼类低温适应的分子机制。     

Synchrony between parasite development and host behaviour change

Teleost fish are commonly used as model species in laboratory studies of behaviour and ecology. In comparison to other groups of vertebrates used routinely in such studies, however, relatively little attention has been paid to their environmental requirements from a welfare perspective. Fish naturally inhabit a wide variety of aquatic habitats that differ enormously in the range of light environments they provide, and light regime has enormous potential to affect behaviour. Yet the level and quality of illumination (in terms of intensity and wavelength spectrum) provided in experimental studies of fish behaviour is generally designed to maximize ease of recording by the observer. In addition, display or home aquaria provide illumination that maximizes the 'viewing pleasure' of the observer, and specialist lighting tubes are available to stimulate rapid plant growth and to 'show off' the colours of fish, rather than to provide 'natural' light environments. Here we present the results of three studies designed to examine the effects of light intensity, wavelength spectrum and their interactions on the behaviour of a model species commonly used in behavioural studies, the three‐spined stickleback Gasterosteus aculeatus . Our aims are to determine whether unnatural light environments, generated by manipulating light intensity and wavelength spectrum, affect behaviour in ways that may lead to concern for the welfare of fish as research animals or pets.     

The welfare of fish

Our interactions with fish cover a wide range of activities including enjoying them as pets to consuming them as food. I propose that we confine the consideration of the welfare of fish to their physiology, and not join the discussion on whether fish can feel pain and suffering, as humans. A significant proportion of the papers on animal welfare center on whether non-human animals can feel pain, and suffer as humans. This is a question that never can be answered unequivocally. The premise of the present paper is that we have an ethical responsibility to respect the life and wellbeing of all organisms. Thus, we should concentrate on the behavioural, physiological, and cellular indicators of their well-being and attempt to minimize a state of stress in the animals that we have in our care or influence.     

“What’s wrong with my monkey?” Ethical perspectives on germline transgenesis in marmosets

The birth of the first transgenic primate to have inherited a transgene from its parents opens the possibility to set up transgenic marmoset colonies, as these monkeys are small and relatively easy to keep and breed in research facilities. The prospect of transgenic marmoset models of human disease, readily available in the way that transgenic laboratory mice are currently, prompts excitement in the scientific community; but the idea of monkeys being bred to carry diseases is also contentious. We structure an ethical analysis of the transgenic marmoset case around three questions: whether it is acceptable to use animals as models of human disease; whether it is acceptable to genetically modify animals; and whether these animals’ being monkeys makes a difference. The analysis considers the prospect of transgenic marmoset studies coming to replace transgenic mouse studies and lesion studies in marmosets in some areas of research. The mainstream, broadly utilitarian view of animal research suggests that such a transition will not give rise to greater ethical problems than those presently faced. It can be argued that using marmosets rather than mice will not result in more animal suffering, and that the benefits of research will improve with a move to a species more similar in phylogenetic terms to humans. The biological and social proximity of monkeys and humans may also benefit the animals by making it easier for scientists and caretakers to recognize signs of suffering and increasing the human motivation to limit it. The animal welfare and research impacts of the transition to marmoset use will depend very much on the extent to which researchers take these issues seriously and seek to minimize animal harm and optimize human benefit.     

Thirty years after Brambell: whither animal welfare science?

It has been just over 30 years since the Brambell Committee issued its clarion call for research on the welfare of animals used in agriculture. What progress has farm animal welfare science made in those 30 years, and what challenges will it face in the next 30? In this article, I discuss the ways in which the Brambell report, with its emphasis on behavioral needs and suffering, both propelled and constrained the development of animal welfare science. The role that economic factors play and the ways in which they have imposed other kinds of constraints on scientists working with farm animals are also mentioned. Despite these constraints, animal welfare science has contributed to a number of significant improvements in the welfare of farm animals. There is, however, a growing sense that animal welfare science has reached an impasse and that ethical and scientific questions about animal welfare have become hopelessly entangled. In this context, I address what I view to be the principal challenge facing farm animal welfare science, namely to move the issue of beyond suffering to an evaluation of broader quality-of-life questions and their application to improvements in welfare.     

Control, choice, and assessments of the value of behavioral management to nonhuman primates in captivity

Many people have devoted considerable effort to enhancing the environments of nonhuman primates in captivity. There is substantial motivation to develop experimental, analytical, and interpretational frameworks to enable objective measurements of the value of environmental enrichment/behavioral management efforts. The consumer-demand approach is a framework not frequently implemented in studies of nonhuman primate welfare but profitably used in studies of the welfare of nonhuman animals in agriculture. Preference studies, in which primates can voluntarily choose to socialize or to participate in training, may be the best current examples of a consumer-demand-like approach to assessing the effects of captive management strategies on primate welfare. Additional work in this area would be beneficial; however, there are potential ethical constraints on purposefully subjecting primates to adverse circumstances to measure their demand for a resource. Primate welfare researchers need to design consumer-demand studies with obstacles that will help measure the relative value of resources to captive primates without compromising the welfare they are attempting to evaluate and enhance.     

Current status of sperm cryopreservation in biomedical research fish models: zebrafish, medaka, and Xiphophorus

Aquarium fishes are becoming increasingly important because of their value in biomedical research and the ornamental fish trade, and because many have become threatened or endangered in the wild. This review summarizes the current status of sperm cryopreservation in three fishes widely used in biomedical research: zebrafish, medaka, and live-bearing fishes of the genus Xiphophorus, and will focus on the needs and opportunities for future research and application of cryopreservation in aquarium fish. First, we summarize the basic biological characteristics regarding natural habitat, testis structure, spermatogenesis, sperm morphology, and sperm physiology. Second, we compare protocol development of sperm cryopreservation. Third, we emphasize the importance of artificial fertilization in sperm cryopreservation to evaluate the viability of thawed sperm. We conclude with a look to future research directions for sperm cryopreservation and the application of this technique in aquarium species.