Evaluation of Vibration as an Extrinsic Variable in In Vivo Research.

Vibration is inherent in research animal facilities due to the mechanical systems and practices required for animal care and use. Ample evidence indicates that vibration can change behavior and physiology in multiple species, potentially altering the results of research studies. Although one cannot eliminate environmental vibration, its control is important in research animal environments to decrease the possibility of introducing a research variable due to vibration effects. To assess the potential for a vibration source to alter experimental results and variability, one must understand the principles of vibration, its likely sources, and control methods. The literature regarding the effects of vibration, as it applies in a practical sense, can be challenging to interpret because the vibration frequencies tested to date have often not been within or near the most sensitive ranges of the species being tested. Some previous studies have used unrealistic vibration magnitudes and provided insufficient detail to duplicate or build upon conclusions. Standardization is essential for research examining the effects of vibration on animals to validate knowledge of this extrinsic variable in animal research and identify ways to mitigate the variable in research facilities.

Reporting quality in preclinical animal experimental research in 2009 and 2018: A nationwide systematic investigation.

Lack of translation and irreproducibility challenge preclinical animal research. Insufficient reporting methodologies to safeguard study quality is part of the reason. This nationwide study investigates the reporting prevalence of these methodologies and scrutinizes the reported information's level of detail. Publications were from two time periods to convey any reporting progress and had at least one author affiliated to a Danish University. We retrieved all relevant animal experimental studies using a predefined research protocol and a systematic search. A random sampling of 250 studies from 2009 and 2018 led to 500 publications in total. Reporting of measures known to impact study results estimates were assessed. Part I discloses a simplified two-level scoring "yes/no" to identify the presence of reporting. Part II demonstrates an additional three-level scoring to analyze the reported information's level of detail. Overall reporting prevalence is low, although minor improvements are noted. Reporting of randomization increased from 24.0% in 2009 to 40.8% in 2018, blinded experiment conduct from 2.4% to 4.4%, blinded outcome assessment from 23.6% to 38.0%, and sample size calculation from 3.2% to 14.0%. Poor reporting of details is striking with reporting of the random allocation method to groups being only 1.2% in 2009 and 6.0% in 2018. Reporting of sample size calculation method was 2.4% in 2009 and 7.6% in 2018. Only conflict-of-interest statements reporting increased from 37.6% in 2009 to 90.4%. Measures safeguarding study quality are poorly reported in publications affiliated with Danish research institutions. Only a modest improvement was noted during the period 2009-2018, and the lack of details urgently prompts institutional strategies to accelerate this. We suggest thorough teaching in designing, conducting and reporting animal studies. Education in systematic review methodology should be implemented in this training and will increase motivation and behavior working towards quality improvements in science.

Public consultation in the evaluation of animal research protocols.

One response to calls for increased openness in animal research is to make protocols publicly accessible, but it is unclear what type of input the public would provide if given this opportunity. In this study we invited public responses to five different research projects, using non-technical summaries intended for lay audiences. Our aim was to assess the potential for this type of public consultation in protocol review, and a secondary aim was to better understand what types of animal research people are willing to accept and why. US participants (n = 1521) were asked (via an online survey) "Do you support the use of these (insert species) for this research", and responded using a seven-point scale (1 = "No", 4 = "Neutral", and 7 = "Yes"). Participants were asked to explain the reasons for their choice; open-ended text responses were subjected to thematic analysis. Most participants (89.7%) provided clear comments, showing the potential of an online forum to elicit feedback. Four themes were prevalent in participant reasoning regarding their support for the proposed research: 1) impact on animals, 2) impact on humans, 3) scientific merit, and 4) availability of alternatives. Participant support for the proposed research varied but on average was close to neutral (mean ± SD: 4.5 ± 2.19) suggesting some ambivalence to this animal use. The protocol describing Parkinson's research (on monkeys) was least supported (3.9 ± 2.17) and the transplant research (on pigs) was most supported (4.9 ± 2.02). These results indicate that public participants are sensitive to specifics of a protocol. We conclude that an online forum can provide meaningful public input on proposed animal research, offering research institutions the opportunity for improved transparency and the chance to reduce the risk that they engage in studies that are out of step with community values.

A 3-year evaluation of preclinicaltrials.eu reveals room for improvement in preregistration of animal studies.

In 2018, the first registry dedicated to preregistration of animal study protocols was launched. Despite international support, the overall number of (pre)registered protocols is still low, illustrating the need for pushing the preregistration agenda among researchers and policymakers.

The status and issues of the Institutional Animal Care and Use Committee of Seoul National University: from its establishment to the present day.

The Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU) plays a key role in monitoring and managing the humane use of animals in scientific research. Here, as one of the pioneers of the IACUC in Korea, we reported SNU-IACUC operations and activities including committee establishment and legal formulation, protocol review, and post-approval monitoring of protocols, which the IACUC has undertaken in the last decade. In addition, legal regulations and improvements were also discussed, and encompassed the limited number of committee members and the single IACUC policy in Korea. As of December, 2020, amendments are on the table at the National Assembly. We also emphasized the independent nature of the IACUC in protecting activities, including approval and monitoring animal experiments, and its public role in narrowing the knowledge gap between society and scientists. Thus, the aim of this report is to help society and scientists understand the operations of the SNU-IACUC and its role in animal welfare.

Investigating causality with fecal microbiota transplantation in rodents: applications, recommendations and pitfalls.

In recent years, studies investigating the role of the gut microbiota in health and diseases have increased enormously - making it essential to deepen and question the research methodology employed. Fecal microbiota transplantation (FMT) in rodent studies (either from human or animal donors) allows us to better understand the causal role of the intestinal microbiota across multiple fields. However, this technique lacks standardization and requires careful experimental design in order to obtain optimal results. By comparing several studies in which rodents are the final recipients of FMT, we summarize the common practices employed. In this review, we document the limitations of this method and highlight different parameters to be considered while designing FMT Studies. Standardizing this method is challenging, as it differs according to the research topic, but avoiding common pitfalls is feasible. Several methodological questions remain unanswered to this day and we offer a discussion on issues to be explored in future studies.

Clarification of early end-points for refinement of animal experiments, with specific reference to fish.

Appropriate end-points are integral to the refinement of laboratory animal experiments. Our recent experience has highlighted that ambiguity around end-points is hampering their adoption in experiments that cause severe suffering to fish. In toxicology, the term endpoint (single word) refers to the response variable to the treatment that is measured and analysed. This differs to usage within laboratory animal experimentation, where end-point (hyphenated) refers to the time-point when exposure of the animal(s) to the treatment (and suffering) ends. Within laboratory animal experimentation, standardised terminology is needed for different types of early end-point which are based on the condition of the animal(s) or progress of the experiment. We propose that those involved in regulating and conducting animal experiments consider seven distinct types of early end-point (aim, technical error, biological error, mortality, moribundity, prognostic humane, non-prognostic humane) in addition to the planned experimental end-point (i.e. maximum duration). Moribundity (not morbidity) refers to an animal in a severely debilitated state close to death. Moribundity in fish is not yet defined, so we propose identification via a lack of response to external stimuli, loss of equilibrium (i.e. loss of righting reflex), and a slow opercular ventilation rate. As these clinical signs equate to those of deep/surgical anaesthesia, this moribundity end-point cannot be considered a humane end-point as the fish is likely to be unconscious and have passed the point of maximum suffering. We believe that identification of earlier humane end-points based on clinical signs and wider recognition of other types of early end-point can reduce suffering in experiments.

Meta-analysis of variation suggests that embracing variability improves both replicability and generalizability in preclinical research.

The replicability of research results has been a cause of increasing concern to the scientific community. The long-held belief that experimental standardization begets replicability has also been recently challenged, with the observation that the reduction of variability within studies can lead to idiosyncratic, lab-specific results that cannot be replicated. An alternative approach is to, instead, deliberately introduce heterogeneity, known as "heterogenization" of experimental design. Here, we explore a novel perspective in the heterogenization program in a meta-analysis of variability in observed phenotypic outcomes in both control and experimental animal models of ischemic stroke. First, by quantifying interindividual variability across control groups, we illustrate that the amount of heterogeneity in disease state (infarct volume) differs according to methodological approach, for example, in disease induction methods and disease models. We argue that such methods may improve replicability by creating diverse and representative distribution of baseline disease state in the reference group, against which treatment efficacy is assessed. Second, we illustrate how meta-analysis can be used to simultaneously assess efficacy and stability (i.e., mean effect and among-individual variability). We identify treatments that have efficacy and are generalizable to the population level (i.e., low interindividual variability), as well as those where there is high interindividual variability in response; for these, latter treatments translation to a clinical setting may require nuance. We argue that by embracing rather than seeking to minimize variability in phenotypic outcomes, we can motivate the shift toward heterogenization and improve both the replicability and generalizability of preclinical research.

A Handful of Details to Ensure the Experimental Reproducibility on the FORCED Running Wheel in Rodents: A Systematic Review.

A well-documented method and experimental design are essential to ensure the reproducibility and reliability in animal research. Experimental studies using exercise programs in animal models have experienced an exponential increase in the last decades. Complete reporting of forced wheel and treadmill exercise protocols would help to ensure the reproducibility of training programs. However, forced exercise programs are characterized by a poorly detailed methodology. Also, current guidelines do not cover the minimum data that must be included in published works to reproduce training programs. For this reason, we have carried out a systematic review to determine the reproducibility of training programs and experimental designs of published research in rodents using a forced wheel system. Having determined that most of the studies were not detailed enough to be reproducible, we have suggested guidelines for animal research using FORCED exercise wheels, which could also be applicable to any form of forced exercise.

Implementation of the principles of the 3Rs of animal testing at CDER: Past, present and future.

The safety testing of pharmaceutical candidates has traditionally relied on data gathered from studies in animals, and these sources of information remain a vital component of the safety assessment for new drug and biologic products. However, there are clearly ethical implications that attend the use of animals for safety testing, and FDA fully supports the principles of the 3Rs, as it relates to animal usage; these being to replace, reduce and refine. We provide an overview of some of the events and activities (legal and programmatic) that have had, and continue to have, the greatest impact on animal use in pharmaceutical development, and highlight some ongoing efforts to further meet the challenge of achieving our mission as humanely as possible.

Epidemiology and reporting characteristics of preclinical systematic reviews.

In an effort to better utilize published evidence obtained from animal experiments, systematic reviews of preclinical studies are increasingly more common-along with the methods and tools to appraise them (e.g., SYstematic Review Center for Laboratory animal Experimentation [SYRCLE's] risk of bias tool). We performed a cross-sectional study of a sample of recent preclinical systematic reviews (2015-2018) and examined a range of epidemiological characteristics and used a 46-item checklist to assess reporting details. We identified 442 reviews published across 43 countries in 23 different disease domains that used 26 animal species. Reporting of key details to ensure transparency and reproducibility was inconsistent across reviews and within article sections. Items were most completely reported in the title, introduction, and results sections of the reviews, while least reported in the methods and discussion sections. Less than half of reviews reported that a risk of bias assessment for internal and external validity was undertaken, and none reported methods for evaluating construct validity. Our results demonstrate that a considerable number of preclinical systematic reviews investigating diverse topics have been conducted; however, their quality of reporting is inconsistent. Our study provides the justification and evidence to inform the development of guidelines for conducting and reporting preclinical systematic reviews.

Internal consistency and compatibility of the 3Rs and 3Vs principles for project evaluation of animal research.

Using animals for research raises ethical concerns that are addressed in project evaluation by weighing expected harm to animals against expected benefit to society. A harm-benefit analysis (HBA) relies on two preconditions: (a) the study protocol is scientifically suitable and (b) the use of (sentient) animals and harm imposed on them are necessary for achieving the study's aims. The 3Rs (Replace, Reduce and Refine) provide a guiding principle for evaluating whether the use of animals, their number and the harm imposed on them are necessary. A similar guiding principle for evaluating whether a study protocol is scientifically suitable has recently been proposed: the 3Vs principle referring to the three main aspects of scientific validity in animal research (construct, internal and external validity). Here, we analyse the internal consistency and compatibility of these two principles, address conflicts within and between the 3Rs and 3Vs principles and discuss their implications for project evaluation. We show that a few conflicts and trade-offs exist, but that these can be resolved either by appropriate study designs or by ethical deliberation in the HBA. In combination, the 3Vs, 3Rs and the HBA thus offer a coherent framework for a logically structured evaluation procedure to decide about the legitimacy of animal research projects.

Quality of interventional animal experiments in Chinese journals: compliance with ARRIVE guidelines.

BACKGROUND: In view of the inadequacy and incompleteness of currently-reported animal experiments and their overall poor quality, we retrospectively evaluated the reporting quality of animal experiments published in Chinese journals adhering to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. RESULTS: The databases CNKI, WanFang, VIP, and CBM were searched from inception until July 2018. Two appropriately-trained reviewers screened and extracted articles independently. The ARRIVE guidelines were used to assess the quality of the published reports of animal experiments. The compliance rate of every item was analyzed relative to their date of publication. A total of 4342 studies were included, of which 73.0% had been cited ≤5 times. Only 29.0% (1261/4342) were published in journals listed in the Chinese Science Citation Database. The results indicate that the compliance rate of approximately half of the sub-items (51.3%, 20/39) was less than 50%, of which 65.0% (13/20) was even less than 10%. CONCLUSIONS: The reporting quality of animal experiments in Chinese journals is not at a high level. Following publication of the ARRIVE guidelines in 2010, the compliance rate of the majority of its requirements has improved to some extent. However, less attention has been paid to the ethics and welfare of experimental animals, and a number of specific items in the Methods, Results, and Discussion sections continue to not be reported in sufficient detail. Therefore, it is necessary to popularize the ARRIVE guidelines, advocate researchers to adhere to them in the future, and in particular promote the use of the guidelines in specialized journals in order that the design, implementation, and reporting of animal experiments is promoted, to ultimately improve their quality.

Genomic, microbial and environmental standardization in animal experimentation limiting immunological discovery.

BACKGROUND: The use of inbred mice housed under standardized environmental conditions has been critical in identifying immuno-pathological mechanisms in different infectious and inflammatory diseases as well as revealing new therapeutic targets for clinical trials. Unfortunately, only a small percentage of preclinical intervention studies using well-defined mouse models of disease have progressed to clinically-effective treatments in patients. The reasons for this lack of bench-to-bedside transition are not completely understood; however, emerging data suggest that genetic diversity and housing environment may greatly influence muring immunity and inflammation. RESULTS: Accumulating evidence suggests that certain immune responses and/or disease phenotypes observed in inbred mice may be quite different than those observed in their outbred counterparts. These differences have been thought to contribute to differing immune responses to foreign and/or auto-antigens in mice vs. humans. There is also a growing literature demonstrating that mice housed under specific pathogen free conditions possess an immature immune system that remarkably affects their ability to respond to pathogens and/or inflammation when compared with mice exposed to a more diverse spectrum of microorganisms. Furthermore, recent studies demonstrate that mice develop chronic cold stress when housed at standard animal care facility temperatures (i.e. 22-24 °C). These temperatures have been shown alter immune responses to foreign and auto-antigens when compared with mice housed at their thermo-neutral body temperature of 30-32 °C. CONCLUSIONS: Exposure of genetically diverse mice to a spectrum of environmentally-relevant microorganisms at housing temperatures that approximate their thermo-neutral zone may improve the chances of identifying new and more potent therapeutics to treat infectious and inflammatory diseases.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

Effects of weaning age and housing conditions on phenotypic differences in mice.

Poor reproducibility is considered a serious problem in laboratory animal research, with important scientific, economic, and ethical implications. One possible source of conflicting findings in laboratory animal research are environmental differences between animal facilities combined with rigorous environmental standardization within studies. Due to phenotypic plasticity, study-specific differences in environmental conditions during development can induce differences in the animals' responsiveness to experimental treatments, thereby contributing to poor reproducibility of experimental results. Here, we studied how variation in weaning age (14-30 days) and housing conditions (single versus group housing) affects the phenotype of SWISS mice as measured by a range of behavioral and physiological outcome variables. Weaning age, housing conditions, and their interaction had little effect on the development of stereotypies, as well as on body weight, glucocorticoid metabolite concentrations, and behavior in the elevated plus-maze and open field test. These results are surprising and partly in conflict with previously published findings, especially with respect to the effects of early weaning. Our results thus question the external validity of previous findings and call for further research to identify the sources of variation between replicate studies and study designs that produce robust and reproducible experimental results.

Quality and validity of large animal experiments in stroke: A systematic review.

An important factor for successful translational stroke research is study quality. Low-quality studies are at risk of biased results and effect overestimation, as has been intensely discussed for small animal stroke research. However, little is known about the methodological rigor and quality in large animal stroke models, which are becoming more frequently used in the field. Based on research in two databases, this systematic review surveys and analyses the methodological quality in large animal stroke research. Quality analysis was based on the Stroke Therapy Academic Industry Roundtable and the Animals in Research: Reporting In Vivo Experiments guidelines. Our analysis revealed that large animal models are utilized with similar shortcomings as small animal models. Moreover, translational benefits of large animal models may be limited due to lacking implementation of important quality criteria such as randomization, allocation concealment, and blinded assessment of outcome. On the other hand, an increase of study quality over time and a positive correlation between study quality and journal impact factor were identified. Based on the obtained findings, we derive recommendations for optimal study planning, conducting, and data analysis/reporting when using large animal stroke models to fully benefit from the translational advantages offered by these models.

A semi-structured questionnaire survey of laboratory animal rehoming practice across 41 UK animal research facilities.

If a laboratory animal survives an experiment without lasting compromised welfare, its future must be negotiated. Rehoming may be a consideration. This paper reports on research findings that provide an indication of the uptake of animal rehoming by UK facilities and the associated moral, ethical, practical and regulatory considerations that inform decisions to rehome or not. This research addresses a widely acknowledged gap in the literature to understand both the numbers, and types of animals rehomed from UK research facilities, as well as the main motivations for engaging in the practice, and the barriers for those facilities not currently rehoming. From the ~160 UK research facilities in the UK, 41 facilities completed the questionnaire, giving a response rate of approximately 25%. Results suggest rehoming occurs routinely, yet the numbers are small; just 2322 animals are known to have been rehomed between 2015-2017. At least 1 in 10 facilities are rehoming. There exists a clear preference for the rehoming of some species (mainly cats, dogs and horses) over others (rodents, agricultural animals and primates). Indeed, although 94.15% of species kept in laboratories are rodents, they make up under a fifth (19.14%) of all animals known to be rehomed between 2015-2017. The primary motivation for rehoming is to boost staff morale and promote a positive ethical profile for the facility. Barriers include concern for the animal's welfare following rehoming, high scientific demand for animals that leaves few to be rehomed, and, finally, certain animals (mainly those genetically modified) are simply unsuited to rehoming. The findings of this research will support facilities choosing to rehome, as well as those that are not currently engaging in the practice. By promoting the practice, the benefits to rehoming in terms of improving laboratory animal's quality of life, helping facility staff to overcome the moral stress of killing, and addressing public concern regarding the fate of laboratory animals, can be attained. It is only once an understanding of rehoming from the perspective of UK research facilities has been ascertained, that appropriate policy and support can be provided.