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The 3Rs principle of replacement, reduction, and refinement has increasingly been endorsed by legislators and regulatory bodies as the best approach to tackle the ethical dilemma presented by animal experimentation in which the potential benefits for humans stand against the costs borne by the animals. Even when animal use is tightly regulated and supervised, the individual researcher’s responsibility is still decisive in the implementation of the 3Rs. Training in laboratory animal science (LAS) aims to raise researchers’ awareness and increase their knowledge, but its effect on scientists’ attitudes and practice has not so far been systematically assessed. Participants (n = 206) in eight LAS courses (following the Federation of European Laboratory Animal Science Associations category C recommendations) in Portugal were surveyed in a self-administered questionnaire during the course. Questions were related mainly to the 3Rs and their application, attitudes to animal use and the ethical review of animal experiments. One year later, all the respondents were asked to answer a similar questionnaire (57% response rate) with added self-evaluation questions on the impact of training. Our results suggest that the course is effective in promoting awareness and increasing knowledge of the 3Rs, particularly with regard to refinement. However, participation in the course did not change perceptions on the current and future needs for animal use in research. [ABSTRACT FROM AUTHOR]

Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Applying evidence-based veterinary medicine (EBVM) is considered a Day One competence for veterinary graduates. Furthermore, the increasing interest in the use of complementary and alternative (non-conventional) veterinary therapies (NCTs) must be grounded on EBVM principles. Few studies have mapped the teaching of EBVM and of NCTs and assessed their content. This study analyses the official curricula of six (out of eight) Portuguese veterinary schools in terms of EBVM and NCTs, using the self-evaluation documents submitted to the National Agency for Assessment and Accreditation of Higher Education (A3ES) (2014-2015). Results show that, with few exceptions, veterinary education in Portugal follows a traditional, clinically-driven approach to evidence, with concepts taught mostly from an empirical and experiential perspective instead of a systematic one. Core EBVM topics, such as placebo effect , methodological validity , PICO , cognitive bias , and systematic review are either absent or insufficiently covered. Moreover, the teaching of NCTs was found in three out of the six curricular programs, namely acupuncture, phytotherapy, homeopathy, traditional Chinese medicine, aromatherapy, Bach flower remedies, ayurveda, energetic healing (reiki) , and massage. We found no evidence that these therapies are being taught under the principles of EBVM. Taken together, these results highlight the need for more explicit and targeted teaching of EBVM-related topics, namely regarding the critical appraisal of scientific literature and the integration of best evidence into clinical decision-making. Results can also be useful to inform the accreditation process by the A3ES and by education quality assurance agencies in other jurisdictions.

This article argues the need for education and training of researchers carrying out animal studies on the fundamentals of experimental design (ED), as a key means of improving the reliability and reproducibility of preclinical results. The current landscape in ED education in Europe is presented, and we make the case for dedicated tutor-guided teaching of ED. With less than a day dedicated to it in many courses effective techniques for communicating key issues are needed. We have developed two approaches that transfer to experimental design teaching the case-study, problem-solving techniques known to be effective in other fields. They use realistic research scenarios to provoke discussion and engage learning. In one the scenario is for group discussion or informal or formal assessment with subsequent tutor-led discussion of key points. For this each scenario needs a clear statement of the purpose of the research study, simplified text outlining the comparisons and procedures, and a statement of the outcome measure. In the other approach, the scenario is used with freely-available software with a good graphical output to explore the sizing of experiments and the use of both sexes. Trainee feedback and informal assessment show that these approaches can make for interesting and memorable sessions and offer a useful contribution to improvement in experimental design teaching so that it produces meaningful learning that can translate into better practice.

Background: There is an increased interest in Non-Conventional Therapies (NCTs), often referred to as complementary and alternative medicines, in veterinary clinical practice. Aim: To map the bibliometric outputs of NCTs in veterinary medicine, and identify which are most prevalent, and the extent to which their publishing has increased. Methods: Text mining algorithms were applied to detect 17 NCTs-related terms ( acupuncture , ayurveda/ayurvedic , traditional Chinese medicine , traditional medicine, chiropractic , electroacupuncture , essential oil , plant extract, ethnopharmacology , herbal medicine , homeopathy , low-level laser therapy , medicinal plant, natural product , osteopathy , phytotherapy , and massage ) in the title, abstract or keywords of all retrievable literature until 2020 under the PubMed MeSH term 'veterinary' ( N  = 377 556). Point prevalence, incidence by decade and cumulative incidence were calculated. Results: Bibliometric trend analysis revealed an overall increase in NCTs-related terms over the last 20 years, with a substantial growth of studies mentioning plant extracts , essential oils and medicinal plants. Traditional Chinese medicine , herbal medicine and natural product have also increased in the same period, although their numbers remain low. Conversely, reference to acupuncture has decreased in the last decade when compared with the previous decade, whereas references to homeopathy , electroacupuncture , osteopathy and chiropractic remained scarce, suggesting that their use in veterinary clinical practice may not be based on published evidence. Conclusion: Further reviews to explore this issue are warranted, differentiating secondary from primary literature, and assessing relevance and methodological quality of individual studies, following the principles of evidence-based veterinary medicine.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA 's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro , in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA 's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU . They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Stress-induced hyperthermia (SIH) is a physiological response to acute stressors in mammals, shown as an increase in core body temperature, with redirection of blood flow from the periphery to vital organs. Typical temperature assessment methods for rodents are invasive and can themselves elicit SIH, affecting the readout. Infrared thermography (IRT) is a promising non-invasive alternative, if shown to accurately identify and quantify SIH. We used in-house developed software ThermoLabAnimal 2.0 to automatically detect and segment different body regions, to assess mean body (T body ) and mean tail (T tail ) surface temperatures by IRT, along with temperature (T sc ) assessed by reading of subcutaneously implanted PIT-tags, during handling-induced stress of pair-housed C57BL/6J and BALB/cByJ mice of both sexes (N = 68). SIH was assessed during 10 days of daily handling (DH) performed twice per day, weekly voluntary interaction tests (VIT) and an elevated plus maze (EPM) at the end. To assess the discrimination value of IRT, we compared SIH between tail-picked and tunnel-handled animals, and between mice receiving an anxiolytic drug or vehicle prior to the EPM. During a 30 to 60 second stress exposure, T sc and T body increased significantly ( p < 0.001), while T tail ( p < 0.01) decreased. We did not find handling-related differences. Within each cage, mice tested last consistently showed significantly higher ( p < 0.001) T sc and T body and lower ( p < 0.001) T tail than mice tested first, possibly due to higher anticipatory stress in the latter. Diazepam-treated mice showed lower T body and T sc , consistent with reduced anxiety. In conclusion, our results suggest that IRT can identify and quantify stress in mice, either as a stand-alone parameter or complementary to other methods.

Although there is a wide range of animal models of type 2 diabetes mellitus (T2DM) used in research; we have limited evidence on their translation value. This paper provides a) a comparison of preclinical animal and clinical results on the effect of five dipeptidyl peptidase-4 (DPP4) inhibitors by comparing the pharmaceutical caused glucose changes, and b) an evaluation of methodological and reporting standards in T2DM preclinical animal studies. DPP4 inhibitors play an important role in the clinical management of T2DM: if metformin alone is not sufficient enough to control the blood sugar levels, DPP4 inhibitors are often used as second-line therapy; additionally, DPP-4 inhibitors are also used in triple therapies with metformin and sodium-glucose co-transporter-2 (SGLT-2) inhibitors or with metformin and insulin. In our analysis of 124 preclinical studies and 47 clinical trials, (1) we found no evidence of species differences in glucose change response to DPP4 inhibitors, which may suggest that, for this drug class, studies in mice and rats may be equally predictive of how well a drug will work in humans; and (2) there is good reporting of group size, sex, age, euthanasia method and self-reported compliance with animal welfare regulations in animal studies but poor reporting of justification of group size, along with a strong bias towards the use of male animals and young animals. Instead of the common non-transparent model selection, we call for a reflective and evidenced-based assessment of predictive validity of the animal models currently available.

Body temperature changes in laboratory mice are often assessed by invasive and stressful methods, which may confound the measurement. Infrared thermography is a possible non-invasive alternative, but the cost of standard thermal cameras, lack of dedicated software for biomedical purposes, and labour-intensiveness of thermal image analysis have limited their use. An additional limitation lies on the scarcity of research on the causing factors of differences between body surface and core body temperature. We propose a method for automatic assessment of mean body surface temperature in freely-moving mice, using dedicated software for thermal image analysis. While skin surface temperature may not necessarily be linearly correlated with core body temperature (in itself an imprecise concept), under standardized environmental conditions, such as those in which laboratory animals are kept, mean body surface temperature can provide useful information on their thermal status (i.e. deviations from normothermia, namely hypo- and hyperthermia). We developed a publicly available software that includes an imaging analysis workflow/algorithm for automatic segmentation of the pixels associated with the animal from the pixels associated with the background, removing the need for manually defining the area of analysis. A batch analysis mode is also available, for automatic and high-throughput analysis of all image files located in a folder. The software is compatible with the most widespread thermal camera manufacturer, 'FLIR Systems', as well as with the low-cost 'Thermal Expert TE-Q1' miniaturized high-resolution thermal camera used for this study. Furthermore, the software has been validated in a mouse model expressing non-transient hypothermia, where the thermal analysis results were compared with readings from implanted thermo-sensitive passive integrated transponders tags. Thermography allows for thermal assessment of laboratory animals without the effect of handling stress on their physiology or behaviour. Our automatic image analysis software also removes observer errors and bias, while speeding up the data processing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Infrared thermography has been suggested as a non-invasive, objective tool to evaluate animal welfare. In this study, we investigated: 1) how body temperature, measured through thermal imaging, is affected by different mild stressors frequently experienced by laboratory mice; 2) which methodology to use for assessing temperature variations with infrared thermography; 3) whether the chosen stressors cause anxiety in mice. Eighty C57BL/6 male mice were included in the study. The mice were allocated to either a control group or one of three groups being subjected to a mild stressor once daily for 4 days: 1) anaesthesia with isoflurane for 10 min; 2) handling by scruffing; 3) intraperitoneal injection of 0.2 ml 0.9% saline. On all four intervention days, thermal images were obtained in all groups and all animals were assessed for fur status and body weight. On day five, all animals were tested in the elevated-plus-maze for 5 min. From the thermal images, the maximum eye temperature, the maximum tail base temperature and the average body temperature were obtained. Ten minutes of anaesthesia with isoflurane led to a decrease in maximum eye temperature, average body temperature and maximum tail base temperature. The animals recovered from this drop in temperature within 10 min. No drop in temperature was seen after scruffing or intraperitoneal injection of saline. Based on the number of missing values, intra-rater and inter-rater agreement, the average body temperature was found most ideal for measuring body temperature variations in mice. Finally, the elevated plus maze did not reveal any differences in anxiety between the groups and the body weight did not decrease at any time point during the study., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Animal use in biomedical research is generally justified by its potential benefits to the health of humans, or other animals, or the environment. However, ethical acceptability also requires scientists to limit harm to animals in their research. Training in laboratory animal science (LAS) helps scientists to do this by promoting best practice and the 3Rs. This study evaluated scientists' awareness and application of the 3Rs, and their approach to other ethical issues in animal research. It was based on an online survey of participants in LAS courses held in eight venues in four European countries: Portugal (Porto, Braga), Germany (Munich, Heidelberg), Switzerland (Basel, Lausanne, Zurich), and Denmark (Copenhagen). The survey questions were designed to assess general attitudes to animal use in biomedical research, Replacement alternatives, Reduction and Refinement conflicts, and harm-benefit analysis. The survey was conducted twice: immediately before the course ('BC', N = 310) and as a follow-up six months after the course ('AC', N = 127). While courses do appear to raise awareness of the 3Rs, they had no measurable effect on the existing low level of belief that animal experimentation can be fully replaced by non-animal methods. Most researchers acknowledged ethical issues with their work and reported that they discussed these with their peers. The level of an animal's welfare, and especially the prevention of pain, was regarded as the most pressing ethical issue, and as more important than the number of animals used or the use of animals as such. Refinement was considered more feasible than Replacement, as well as more urgent, and was also favoured over Reduction. Respondents in the survey reversed the 'hierarchy' of the 3Rs proposed by their architects, Russell and Burch, prioritizing Refinement over Reduction, and Reduction over Replacement. This ordering may conflict with the expectations of the public and regulators., Competing Interests: The authors have declared that no competing interests exist.

Directive 2010/63/EU introduced requirements for the classification of the severity of procedures to be applied during the project authorisation process to use animals in scientific procedures and also to report actual severity experienced by each animal used in such procedures. These requirements offer opportunities during the design, conduct and reporting of procedures to consider the adverse effects of procedures and how these can be reduced to minimize the welfare consequences for the animals. Better recording and reporting of adverse effects should also help in highlighting priorities for refinement of future similar procedures and benchmarking good practice. Reporting of actual severity should help inform the public of the relative severity of different areas of scientific research and, over time, may show trends regarding refinement. Consistency of assignment of severity categories across Member States is a key requirement, particularly if re-use is considered, or the safeguard clause is to be invoked. The examples of severity classification given in Annex VIII are limited in number, and have little descriptive power to aid assignment. Additionally, the examples given often relate to the procedure and do not attempt to assess the outcome, such as adverse effects that may occur. The aim of this report is to deliver guidance on the assignment of severity, both prospectively and at the end of a procedure. A number of animal models, in current use, have been used to illustrate the severity assessment process from inception of the project, through monitoring during the course of the procedure to the final assessment of actual severity at the end of the procedure (Appendix 1).

Animal research is not only regulated by legislation but also by self-regulatory mechanisms within the scientific community, which include biomedical journals' policies on animal use. For editorial policies to meaningfully impact attitudes and practice, they must not only be put into effect by editors and reviewers, but also be set to high standards. We present a novel tool to classify journals' policies on animal use-the EXEMPLAR scale-as well as an analysis by this scale of 170 journals publishing studies on animal models of three human diseases: Amyotrophic Lateral Sclerosis, Type-1 Diabetes and Tuberculosis. Results show a much greater focus of editorial policies on regulatory compliance than on other domains, suggesting a transfer of journals' responsibilities to scientists, institutions and regulators. Scores were not found to vary with journals' impact factor, country of origin or antiquity, but were, however, significantly higher for open access journals, which may be a result of their greater exposure and consequent higher public scrutiny.

The use of non-human animals in biomedical research has given important contributions to the medical progress achieved in our day, but it has also been a cause of heated public, scientific and philosophical discussion for hundreds of years. This review, with a mainly European outlook, addresses the history of animal use in biomedical research, some of its main protagonists and antagonists, and its effect on society from Antiquity to the present day, while providing a historical context with which to understand how we have arrived at the current paradigm regarding the ethical treatment of animals in research.

Refinement measures to improve animal welfare can ease the ethical dilemma between human benefit and animal harm in research with animal models of neurodegenerative diseases. To evaluate the potential for refinement, as well as its implementation in research, we analysed papers on murine models of Huntington's disease (HD) published between 1999 and 2009 (n = 233). Each study was classified according to a four-level severity scale, in terms of the disease stage that animals were allowed to reach, the execution of invasive procedures, and the implementation of refinement. Reports of ethical approval, and regulatory compliance in general, followed a clear rising trend over the years reviewed (p <0.001). However, the proportion of high-severity studies did not change over that period. Also, of the studies for which approval was reported (n = 120), 36% were attributed the highest severity level. The observed discrepancy between the rising motivation to affirm regulatory compliance, and the unaltered proportion of studies allowing animals to reach severely distressful stages, raises both ethical and methodological issues, which we discuss in this paper.

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.