Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents

Dosing of experimental animals and the removal of blood are two of the most frequent procedures performed in biomedical research using live animals. Despite the apparently simple nature of these procedures, they can, if not correctly carried out, have significant effects on the welfare of the animals and the scientific value of the results. There are several methods by which research staff may obtain training in the administration of substances. These include practical demonstrations during teaching courses; observation of techniques; videos and educational computer programs and practising on recently killed animal cadavers or plastic animal models. Each method has its own advantages and disadvantages. A common factor encountered during training is the difficulty in assessing competency. This paper reports a pilot study on the use of bioluminescent imaging technology to assess competency in the administration of substances to rodents. Bioluminescence was rapidly detected after dosing of animals with a bioluminescent substance. However, living animals were required for a signal to be generated. The data presented suggest that this technology is ideal for use as a teaching aid and may also prove valuable in assessing the effectiveness of 'complex' and novel administration routes in 'realtime'.     

Survey of the Quality of Experimental Design,Statistical Analysis and Reporting of Research Using Animals

For scientific, ethical and economic reasons, experiments involving animals should be appropriately designed, correctly analysed and transparently reported. This increases the scientific validity of the results, and maximises the knowledge gained from each experiment. A minimum amount of relevant information must be included in scientific publications to ensure that the methods and results of a study can be reviewed, analysed and repeated. Omitting essential information can raise scientific and ethical concerns. We report the findings of a systematic survey of reporting, experimental design and statistical analysis in published biomedical research using laboratory animals. Medline and EMBASE were searched for studies reporting research on live rats, mice and non-human primates carried out in UK and US publicly funded research establishments. Detailed information was collected from 271 publications, about the objective or hypothesis of the study, the number, sex, age and/or weight of animals used, and experimental and statistical methods. Only 59% of the studies stated the hypothesis or objective of the study and the number and characteristics of the animals used. Appropriate and efficient experimental design is a critical component of high-quality science. Most of the papers surveyed did not use randomisation (87%) or blinding (86%), to reduce bias in animal selection and outcome assessment. Only 70% of the publications that used statistical methods described their methods and presented the results with a measure of error or variability. This survey has identified a number of issues that need to be addressed in order to improve experimental design and reporting in publications describing research using animals. Scientific publication is a powerful and important source of information; the authors of scientific publications therefore have a responsibility to describe their methods and results comprehensively, accurately and transparently, and peer reviewers and journal editors share the responsibility to ensure that published studies fulfil these criteria.     

Computer simulation studies and the scientific method

The scientific method is the formal procedure for all acceptable scientific endeavors. With this methodology, there is a continual interaction between theory, in the form of an hypothesis, and objective, experimental analysis. There is a new step in the scientific method that involves the use of computer models and simulation studies. When computer models are incorporated into hypothesis formulation, they can be used in simulation studies to test ideas before they are tried experimentally. An iterative feedback between these tests and current ideas allows for a preliminary refinement of hypotheses and development of more intelligent research protocols. In this way, computer simulation studies can serve as an intermediate step in the scientific method, reducing the number of animals used in biomedical experimentation. In this article we also explore other ways that computer simulation studies could limit the use of animals in biomedical research and education.     

Anesthesia, analgesia, and euthanasia of invertebrates

Invertebrate animals have long played an important role in biomedical research in such fields as genetics, physiology, and development. However, with few exceptions, scientists, veterinarians, and technicians have paid little attention to the anesthesia, analgesia, and euthanasia of these diverse creatures. Indeed, some standard research procedures are routinely performed without anesthesia. Yet various chemical agents are available for the immobilization or anesthesia of invertebrates, ranging from gases or volatile liquids that can be pumped into either an anesthetic chamber (for terrestrial species) or a container of water (aquatic species), to benzocaine and other substances for fish. Many invertebrates are not difficult to immobilize or anesthetize and the procedures recommended in this article appear to be safe; however, none should be considered totally risk-free. Analgesia of invertebrates is as yet a largely unexplored field; until scientific data are available, other measures can promote the well-being of these animals in the laboratory. For euthanasia, various methods (physical or chemical or a combination of both) have been recommended for different taxa of invertebrates, but most have not been properly studied under laboratory conditions and some can be problematic in the context of research procedures and tissue harvesting. Furthermore, relevant data are scattered, sometimes available only in languages other than English, and there is no international approach for seeking and collating such information. In this article I review various methods of anesthesia, analgesia, and euthanasia for terrestrial and aquatic invertebrates, as well as areas requiring further research.     

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.     

Reducing sample size in experiments with animals: historical controls and related strategies

Reducing the number of animal subjects used in biomedical experiments is desirable for ethical and practical reasons. Previous reviews of the benefits of reducing sample sizes have focused on improving experimental designs and methods of statistical analysis, but reducing the size of control groups has been considered rarely. We discuss how the number of current control animals can be reduced, without loss of statistical power, by incorporating information from historical controls, i.e. subjects used as controls in similar previous experiments. Using example data from published reports, we describe how to incorporate information from historical controls under a range of assumptions that might be made in biomedical experiments. Assuming more similarities between historical and current controls yields higher savings and allows the use of smaller current control groups. We conducted simulations, based on typical designs and sample sizes, to quantify how different assumptions about historical controls affect the power of statistical tests. We show that, under our simulation conditions, the number of current control subjects can be reduced by more than half by including historical controls in the analyses. In other experimental scenarios, control groups may be unnecessary. Paying attention to both the function and to the statistical requirements of control groups would result in reducing the total number of animals used in experiments, saving time, effort and money, and bringing research with animals within ethically acceptable bounds.     

Issues to consider for preparing ferrets as research subjects in the laboratory

The domestic or European ferret (Mustela putorius furo) has been domesticated for thousands of years. Ferrets have been used for hunting and fur production, as pets, and as models in biomedical research. Despite the relatively small numbers used in the laboratory, ferrets have some unique applications including study of human influenza and severe acute respiratory syndrome (SARS)-associated corona virus. They have served as models for peptic ulcer disease, carotenoid metabolism, cystic fibrosis, and drug emesis screening, among others. Most research ferrets are males, due to estrus-related health problems in females. They may be housed conventionally and are easy to care for when their biology and behavior are understood. Due to the small number of ferret suppliers, animals are often shipped long distances, requiring air transport and intermediate handlers. It is important to minimize shipment stress, especially with weanling and pregnant animals. Additional expertise is required for success with pregnant and whelping ferrets and for rearing of neonates. The animals have specific dietary requirements, and proper nutrition is key. Successful housing requires knowledge of ferret behaviors including social behavior, eating habits, a general inquisitive nature, and a species-typical need to burrow and hide. Regular handling is necessary to maintain well-being. A ferret health care program consists of physical examination, immunization, clinical pathology, and a working knowledge of common ferret diseases. Various research methodologies have been described, from basic procedures such as blood collection to major invasive survival surgery. Ferrets have a distinct niche in biomedical research and are hardy animals that thrive well in the laboratory.     

Common husbandry-related variables in biomedical research with animals

Common, often overlooked, variables in biomedical research with animals are reviewed. The barren primary enclosure is an abnormal living environment for laboratory animals. Species-appropriate enrichment attenuates some of the distress resulting from chronic understimulation. Social deprivation distress of individually-caged social animals is best mitigated by the provision of compatible companionship. Biotelemetry and positive reinforcement training avoid or minimize stress reactions that typically occur when animals are forcibly restrained during procedures. The variables, 'light' and 'position of living quarters' are inherent in the multi-tier caging system. To date there is no satisfactory alternative other than the single-tier cage arrangement that eliminates both variables. Removing test animals from their familiar home environment and from their cage mates for procedures introduces stress as an avoidable influential variable. Music may become an important variable if not all subjects are exposed to it. Disturbance time cannot be controlled as an extraneous variable but it should at least be mentioned to explain possible incongruities of data. A positive relationship between animal care personnel and research subjects is a key requisite to minimize stress as a data-confounding variable.     

Reduction strategies in animal research: a review of scientific approaches at the intra-experimental, supra-experimental and extra-experimental levels

When discussing animal use and considering alternatives to animals in biomedical research and testing, the number of animals required gets to the root of the matter on ethics and justification. In this paper, some reduction strategies are reviewed. Many articles and reports on reduction of animal use focus mostly on the experimental level, but other approaches are also possible. Reduction at the intraexperimental level probably offers the greatest scope for reduction, as the design and statistical analysis of individual experiments can often be improved. Supra-experimental reduction aims to reduce the number of animals by a change in the setting in which a series of experiments take place--for example, by improved education and training, reduction of breeding surpluses, critical analysis of test specifications, and re-use of animals. At the extra-experimental level, reduction is a spin-off of other developments, rather than the direct goal. Through improved research or production strategies, aimed at better quality, consistency and safety, reduction in the number of animals used can be substantial. A revised definition of reduction is proposed, which does not include the level of information needed, as in some cases reduction in the number of animals resulting in less information or data, is still acceptable.     

Infrared thermal imaging associated with pain in laboratory animals

The science of animal welfare has evolved over the years, and recent scientific advances have enhanced our comprehension of the neurological, physiological, and ethological mechanisms of diverse animal species. Currently, the study of the affective states (emotions) of nonhuman animals is attracting great scientific interest focused primarily on negative experiences such as pain, fear, and suffering, which animals experience in different stages of their lives or during scientific research. Studies underway today seek to establish methods of evaluation that can accurately measure pain and then develop effective treatments for it, because the techniques available up to now are not sufficiently precise. One innovative technology that has recently been incorporated into veterinary medicine for the specific purpose of studying pain in animals is called infrared thermography (IRT), a technique that works by detecting and measuring levels of thermal radiation at different points on the body’s surface with high sensitivity. Changes in IRT images are associated mainly with blood perfusion, which is modulated by the mechanisms of vasodilatation and vasoconstriction. IRT is an efficient, noninvasive method for evaluating and controlling pain, two critical aspects of animal welfare in biomedical research. The aim of the present review is to compile and analyze studies of infrared thermographic changes associated with pain in laboratory research involving animals.     

The fourth EC report on the statistics of laboratory animal use: trends, recommendations and future prospects

At the beginning of 2005, the European Commission published its fourth report on the statistics of the number of animals used for experimental and other scientific purposes. A total of 10.7 million animals were used within the Member States of the European Union (EU) in 2002, an increase of almost a million animals since the 1999 report. France, Germany and the UK continue to be the largest users of animals for scientific purposes, and mice, rats, fish and birds remain the most commonly-used animals. For the first time, all 15 Member States used the standardised EU tables, as had been agreed in 1998. This has made it easier to identify areas on which Three Rs initiatives should be focused. Nevertheless, the reporting system still has a number of serious deficiencies. In particular, there are insufficient data on the numbers of animals that are kept or bred for research purposes, the numbers of transgenic animals, and the severity of procedures that are applied.     

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.     

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.     

Preparing chimpanzees for laboratory research

The chimpanzee is the only representative of the Great Apes that is extensively involved in biomedical research in primate laboratories. These apes are used as animal models in a variety of studies, including research on infectious disease, parasitic disease, pharmacokinetic studies, neuroscience, cognition, and behavior. Chimpanzees used in biomedical research in the United States reside largely in six specialized research and holding facilities, and most of the research with them is conducted at these sites. Given the relatively small population of chimpanzees and its importance to biomedical research, it is imperative that we carefully manage the care, production, and use of these animals in biomedical research studies. Selection criteria and preparation techniques are reviewed in this article in an effort to begin a discussion on best practices for choosing and handling chimpanzees participating in biomedical research. The use of routine health assessment information is described for subject selection, as are behavioral issues to be considered. Due to the relatively small number of chimpanzees available, issues related to experimental design and multiple uses of chimpanzees are discussed. Practices related to the transportation and acclimation of chimpanzees are described. Finally, behavioral conditioning procedures are discussed, including habituation, desensitization, and positive reinforcement training that have been applied to reduce animal distress and improve the quality of the science being conducted with chimpanzee subjects.     

Variability of human blood pressure with reference mostly to the non-chronobiologic literature

The voluminous literature on the variability encountered in 24-h recordings of human blood pressure in health is here presented by reference to selected clinical articles. Most references are cited by number for the sake of brevity, with a few cited by author when this appears to be of particular interest. Reports of work on laboratory animals are included when the findings are directly pertinent as background to studies on human beings. Results from semiautomatic and automatic direct and indirect measurements are briefly reviewed and aligned with results from work in which blood pressure was self-measured or measured conventionally by staff. The considerable and not generally recognized range of human blood pressure variability is thus extracted from the literature. An apparently limited extent of variation is shown to result mostly from the averaging of data from individuals constituting the groups investigated. Once variation is overwhelmingly documented and recognized as a fact, the different ways in which variations are presented and utilized by different author-investigators gain in importance. In a number of studies, methods of time series analysis are used. Thus, major attention can be paid to the extent to which predictable changes, so-called rhythms, characterize the data. Circadian rhythms are found to be quite prominent. By the assessment of these rhythms and about-yearly (circannual) ones, one quantifies health and individualized risk as well as disease. Otherwise 'unmanageable' variability, reviewed herein, can be resolved by relatively simple inferential statistical procedures as a set of new endpoints. A formidable foe thus becomes a powerful friend: the rhythm characteristics can be used in cardiovascular physiology and epidemiology, and preventive and curative medicine. Long-term blood pressure monitoring is no longer a mere research tool and a curiosity for the practitioner of medicine. Results from such monitoring should immediately be used in the clinic, in the school and at home. Automatic blood pressure monitoring, cost-effectively used in combination with self-measurement, as needed, may become a routine procedure if data collection can be wedded to appropriate analyses yielding new endpoints as sensitive gauges of health.     

Guidelines for the design and statistical analysis of experiments using laboratory animals

For ethical and economic reasons, it is important to design animal experiments well, to analyze the data correctly, and to use the minimum number of animals necessary to achieve the scientific objectives---but not so few as to miss biologically important effects or require unnecessary repetition of experiments. Investigators are urged to consult a statistician at the design stage and are reminded that no experiment should ever be started without a clear idea of how the resulting data are to be analyzed. These guidelines are provided to help biomedical research workers perform their experiments efficiently and analyze their results so that they can extract all useful information from the resulting data. Among the topics discussed are the varying purposes of experiments (e.g., exploratory vs. confirmatory); the experimental unit; the necessity of recording full experimental details (e.g., species, sex, age, microbiological status, strain and source of animals, and husbandry conditions); assigning experimental units to treatments using randomization; other aspects of the experiment (e.g., timing of measurements); using formal experimental designs (e.g., completely randomized and randomized block); estimating the size of the experiment using power and sample size calculations; screening raw data for obvious errors; using the t-test or analysis of variance for parametric analysis; and effective design of graphical data.     

To pluck or not to pluck: scientific methodologies should be carefully chosen,not ‘one size fits all’

McDonald and Griffith (2011) raise important points in their critique of reliance on feathers as a source of DNA for scientific research. Although those authors are right about many details, their one‐size‐fits all approach (i.e. prescribing blood draws for avian DNA analyses) obscures bigger picture issues that are of extraordinary relevance to avian biology. We introduce four points to provide alternative perspectives on their commentary. In particular, we feel that a) scientific goals should determine methodologies; b) stress to animals is context specific and blood sampling is not always less stressful to birds than feather plucking; c) feather DNA is too valuable to be ignored, especially when coupled with other analyses that require feathers; and d) logistical and other concerns often preclude blood sampling. A one size fits all approach to science is generally short‐sighted, be it in regard to the collection of genetic or other samples from birds, or to a suite of other research problems.     

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.