Unrelieved pain and distress in animals: an analysis of USDA data on experimental procedures

Pain and distress are core issues in the field of animal experimentation and in the controversy that surrounds it. We sought to add to the empirical base of the literature on pain and distress by examining government data on experimental procedures that caused unrelieved pain and distress (UPAD) in animals. Of the species regulated by the U.S. Department of Agriculture (USDA), most of the approximately 100,000 animals subjected to UPAD during the year analyzed (1992) were guinea pigs and hamsters. Most of these animals were used in industry laboratories for various testing procedures, primarily vaccine potency testing. We discuss the limitations of the USDA data and recommend changes to the current reporting system. By identifying experimental procedures that cause UPAD in large numbers of USDA-regulated animals, the present analysis can be viewed as a means of identifying priorities for research and development of alternatives methods (replacements, reductions, and refinements).     

An analysis of the Home Office Statistics of Scientific Procedures on Living Animals, Great Britain 2004

The 2004 Statistics of Scientific Procedures on Living Animals were released by the Home Office in December 2005. They indicate that, for the third year running, there has been a significant increase in the number of laboratory animal procedures undertaken in Great Britain, and that increasing numbers of animals are involved. The overall trends in the use of toxicological and non-toxicological procedures involving animals are described. Particular emphasis is placed on the production and use of genetically modified animals, the production of biological materials, and acute toxicity testing. The use of non-human primates and dogs is also discussed. The implications of these latest statistics are considered with reference to the implementation of the Three Rs and their consequences for animal welfare.     

Diagnosis of ecto- and endoparasites in laboratory rats and mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.     

A report on methods to reduce,refine and replace animal testing in industrial toxicology laboratories

The Committee to Promote Principles of Reduction, Refinement and Replacement of Animal Testing in Industrial Toxicology Laboratories was established in 1987 to work toward industrywide improvements in laboratory animal testing methods. The committee's goals are to gather information about effective nonanimal testing techniques and other methods of conserving and improving the care of laboratory animals, to work toward the systematic validation of nonanimal alternatives, and to disseminate useful information about progressive programs and policies throughout the industrial toxicology community. This is the first in a continuing series of reports the committee plans to produce as part of an ongoing program to promote communication among industrial toxicologists about successful methods of reducing, refining and replacing animal testing. Here are some of the report's major findings: (1) Animal care and use committees charged with the oversight of laboratory animal use are a universal practice at the companies surveyed. (2) Significant reductions in the number of animals used for acute toxicity testing have taken place at all the companies during the last 5- to 10-year period. (3) Structure-activity relationships (predicting a test compound's properties based on the known properties of familiar chemicals with similar structures) are widely used to minimize, but not replace, the use of animals. (4) Tissue and organ culture systems are being used with increasing frequency for screening and mechanistic studies, but are not completely replacing animal evaluations as a final step. (5) There is a pressing need for the systematic and scientifically sound validation of nonanimal alternative techniques to reduce the use of animals in toxicology testing while satisfying requirements for the protection of public safety.     

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

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

Studying primate learning in group contexts: Tests of social foraging, response to novelty, and cooperative problem solving

Learning commonly refers to the modification of behavior through experience, whereby an animal gains information about stimulus-response contingencies from interacting with its physical environment. Social learning, on the other hand, occurs when the same information originates, not from the animal's personal experience, but from the actions of others. Socially biased learning is the 'collective outcome of interacting physical, social, and individual factors' [D. Fragaszy, E. Visalberghi, Learn. Behav. 32 (2004) 24-35.] (see p. 24). Mounting interest in animal social learning has brought with it certain innovations in animal testing procedures. Variants of the observer-demonstrator and cooperation paradigms, for instance, have been used widely in captive settings to examine the transmission or coordination of behavior, respectively, between two animals. Relatively few studies, however, have examined social learning in more complex group settings and even fewer have manipulated the social environment to empirically test the effect of group dynamics on problem solving. The present paper outlines procedures for group testing captive non-human primates, in spacious arenas, to evaluate the social modulation of learning and performance. These methods are illustrated in the context of (1) naturalistic social foraging problems, modeled after traditional visual discrimination paradigms, (2) response to novel objects and novel extractive foraging tasks, and (3) cooperative problem solving. Each example showcases the benefits of experimentally manipulating social context to compare an animal's performance in intact groups (or even pairs) against its performance under different social circumstances. Broader application of group testing procedures and manipulation of group composition promise to provide meaningful insight into socially biased learning.     

Automated auditory recognition training and testing

Laboratory training and testing of auditory recognition skills in animals is important for understanding animal communication systems that depend on auditory cues. Songbirds are commonly studied because of their exceptional ability to learn complex vocalizations. In recent years, mounting interest in the perceptual abilities of songbirds has increased the demand for laboratory behavioural training and testing paradigms. Here, we describe and demonstrate the success of a method for auditory discrimination experiments, including all the necessary hardware, training procedures and freely-available, versatile software. The system can run several behavioural training and testing paradigms, including operant (go-nogo, stimulus preference, and two-alternative forced choice) and classical conditioning tasks. The software and some hardware components can be used with any laboratory animal that learns and responds to sensory cues. The peripheral hardware and training procedures are designed for use with songbirds and auditory stimuli. Using the go-nogo paradigm of the training system, we show that adult zebra finches learn to recognize and correctly classify individual female calls and male songs. We also show that learning the task generalizes to new stimulus classes; birds that learned the task with calls subsequently learned to recognize songs faster than did birds that learned the task and songs at the same time.     

Replacing animal experiments: choices, chances and challenges

Replacing animal procedures with methods such as cells and tissues in vitro, volunteer studies, physicochemical techniques and computer modelling, is driven by legislative, scientific and moral imperatives. Non-animal approaches are now considered as advanced methods that can overcome many of the limitations of animal experiments. In testing medicines and chemicals, in vitro assays have spared hundreds of thousands of animals. In contrast, academic animal use continues to rise and the concept of replacement seems less well accepted in university research. Even so, some animal procedures have been replaced in neurological, reproductive and dentistry research and progress is being made in fields such as respiratory illnesses, pain and sepsis. Systematic reviews of the transferability of animal data to the clinical setting may encourage a fresh look for novel non-animal methods and, as mainstream funding becomes available, more advances in replacement are expected.     

A scientific and animal welfare assessment of the OECD Health Effects Test Guidelines for the safety testing of chemicals under the European Union REACH system

We have assessed each of the OECD Health Effects Test Guidelines (TGs) that were included in an annex to the Internet consultation issued by the European Commission relating to the Registration, Evaluation and Authorisation of Chemicals (REACH) legislation for the testing of new and existing chemical substances. Each guideline has been analysed with respect to its design and its scientific and animal welfare implications, the extent to which it makes use of modern techniques, and its suitability to be used in the REACH system for the testing of large numbers of chemicals. The scientific basis of the test and its justification are considered, as well as the numbers of animals required, and the potential adverse effects on them. The prospects and possibilities for applying the Three Rs (reduction, refinement and replacement) to each of the TGs are also discussed. We have proposed an overall testing strategy for how these TGs and other methods could best be deployed for chemicals testing, should it be necessary to fill data gaps. Certain TGs have been omitted from the strategy, when we have considered them to be unnecessary for chemicals testing. A series of recommendations has been made for improving the TGs with regard to both their scientific content and ways in which they could be better designed in relation to optimising the use of the animals concerned, and minimising adverse welfare consequences to them. Our investigations show that there is an urgent need to update the TGs to reflect modern techniques and methods, and to use current approaches for applying refinement strategies to improve the scientific and animal welfare aspects of the procedures used. Improvements can and should be made in all aspects of toxicity testing, from sample preparation, and animal housing, care and feeding, to dose formulation, test material administration, and the histopathological and clinical analysis of tissue samples. Opportunities for streamlining individual assays are very limited, but testing could be made more efficient by: a) only undertaking studies that provide relevant data; b) making greater use of screens and preliminary testing; c) applying some tests simultaneously to the same animals; d) using one sex; and e) eliminating redundant tests. In conclusion, it is clear that, as they stand, the OECD Health Effects TGs are unsuitable for use in the European Union REACH system, for which potentially very large numbers of laboratory animals will be needed for the testing of a very large number of chemicals.     

Alternatives to animal experimentation for hormonal compounds research

Alternatives to animal testing and the identification of reliable methods that may decrease the need for animals are currently the subject of intense investigation worldwide. Alternative testing procedures are particularly important for synthetic and natural chemicals that exert their biological actions through binding nuclear receptors, called nuclear receptors-interacting compounds (NR-ICs), for which research is increasingly emphasizing the limits of several models in the accurate estimation of the physiological consequences of exposure to these compounds. In particular, estrogen receptor interacting compounds (ER-ICs) have a great impact on human health from the therapeutic, nutritional, and toxicological point of view due to the highly permissive nature of the estrogen receptors towards a large number of natural and synthetic compounds. Similar to in vitro systems, recently generated animal models (e.g., animal models generated for the study of estrogen receptor ligands) may fulfill the 3R principles: refine, reduce, and replace. If used correctly, NR-regulated models, such as reporter mice, xenopus, or zebrafish, and models obtained by somatic gene transfer in reporter systems, combined with imaging technologies, may contribute to strongly decreasing the overall number of animals required for NR-IC testing and research. With these models, flexible and highly standardized parameters and reporter marker quantification can be obtained. Here, we highlight the need for the substitution of currently used testing models with more appropriate ones that can reproduce the features and reactivity of specific mammalian target tissue/organs. We consider the promotion of this advancement a research priority bearing scientific, economic, social, and ethical relevance.     

A scientific and animal welfare assessment of the OECD Health Effects Test Guidelines for the safety testing of chemicals under the European Union REACH system

We have assessed each of the OECD Health Effects Test Guidelines (TGs) that were included in an annex to the Internet consultation issued by the European Commission relating to the Registration, Evaluation and Authorisation of Chemicals (REACH) legislation for the testing of new and existing chemical substances. Each guideline has been analysed with respect to its design and its scientific and animal welfare implications, the extent to which it makes use of modern techniques, and its suitability to be used in the REACH system for the testing of large numbers of chemicals. The scientific basis of the test and its justification are considered, as well as the numbers of animals required, and the potential adverse effects on them. The prospects and possibilities for applying the Three Rs (reduction, refinement and replacement) to each of the TGs are also discussed. We have proposed an overall testing strategy for how these TGs and other methods could best be deployed for chemicals testing, should it be necessary to fill data gaps. Certain TGs have been omitted from the strategy, when we have considered them to be unnecessary for chemicals testing. A series of recommendations has been made for improving the TGs with regard to both their scientific content and ways in which they could be better designed in relation to optimising the use of the animals concerned, and minimising adverse welfare consequences to them. Our investigations show that there is an urgent need to update the TGs to reflect modern techniques and methods, and to use current approaches for applying refinement strategies to improve the scientific and animal welfare aspects of the procedures used. Improvements can and should be made in all aspects of toxicity testing, from sample preparation, and animal housing, care and feeding, to dose formulation, test material administration, and the histopathological and clinical analysis of tissue samples. Opportunities for streamlining individual assays are very limited, but testing could be made more efficient by: a) only undertaking studies that provide relevant data; b) making greater use of screens and preliminary testing; c) applying some tests simultaneously to the same animals; d) using one sex; and e) eliminating redundant tests. In conclusion, it is clear that, as they stand, the OECD Health Effects TGs are unsuitable for use in the European Union REACH system, for which potentially very large numbers of laboratory animals will be needed for the testing of a very large number of chemicals.     

Postapproval monitoring and the IACUC

Monitoring of the use of live vertebrate animals in research, teaching, and testing after approval of their use by an institutional animal care and use committee (IACUC) are receiving increased attention in the laboratory animal community. In this article the author provides his opinions on the value of postapproval monitoring (PAM) to the overall self-regulation that is the responsibility of an IACUC. PAM must never supersede or replace federally mandated IACUC responsibilities, but an efficient PAM process can provide significant additional information that enables an institution to be confident that it is meeting both the letter and the spirit of the federal regulations developed to ensure humane animal care. PAM personnel should be excellent communicators and able to maintain a professional demeanor in challenging circumstances. Their knowledge of laboratory animal care, invasive procedures, and regulations will enable them to align the pursuit of scientific research with adherence to these regulations. An effective PAM program involves knowledgeable individuals who can, on behalf of the IACUC, monitor new procedures and personnel and provide IACUC-mandated training or retraining.     

A sample postapproval monitoring program in academia

The primary goal of an animal care and use program (ACUP) should be to ensure animal well-being while fostering progressive science. Both the Animal Welfare Act (and associated regulations) and the Public Health Service (PHS) Policy require the institutional animal care and use committee (IACUC) to provide oversight of the animal program through continuing reviews to ensure that procedures are performed as approved by the committee. But for many committees the semiannual assessment does not provide an opportunity to observe research procedures being performed. Furthermore, IACUC members are typically volunteers with other full-time commitments and may not be able to dedicate sufficient time to observe protocol performance. Postapproval monitoring (PAM) is a tool that the IACUC can use to ensure that the institution fulfills its regulatory obligation for animal program oversight. When performed by attentive and observant individuals, PAM can extend the IACUC's oversight, management, training, and communication resources, regardless of program size or complexity. No defined PAM process fits all institutions or all situations; rather, the monitoring must match the program under review. Nonetheless, certain concepts, concerns, and conditions affect all PAM processes; they are described in this article. Regardless of the style or depth of PAM chosen for a given program, one thing is sure: failure of the IACUC to engage all available and effective oversight methods to ensure humane, compassionate, efficient, and progressive animal care and use is a disservice to the institution, to the research community and to the animals used for biomedical research, testing, or teaching.     

Laboratory animal science issues in the design and conduct of studies with endocrine-active compounds

The use of rodent models for research and testing on endocrine-active compounds necessitates an awareness of a number of laboratory animal science issues to standardize bioassay methods and facilitate reproducibility of results between laboratories. These issues are not unique to endocrine research but are particularly important in this field due to the complexities and interdependencies of the endocrine system, coupled with the inherently sensitive and variable nature of physiological endpoints. Standardization of animal models and the control of animal environments depend on the establishment of strong scientific partnerships between research investigators and laboratory animal scientists. Laboratory animal care and use programs are becoming increasingly complex and are constantly changing, fueled in part by technological advances, changes in regulations concerning animal care and use, and economic pressures. Since the early 1980s, many institutions have moved to centralization of animal facility operations concomitant with numerous changes in housing systems, barrier concepts, equipment, and engineering controls of the macro- and microenvironment. These and other changes can have an impact on animals and the conduct of endocrine experiments. Despite the potential impact of animal care and use procedures on research endpoints, many investigators are surprisingly naive to the animal facility conditions that can affect in vivo studies. Several key animal care and use issues that are important to consider in endocrine experiments with rodent models are described.     

Alternatives to animal testing in the safety evaluation of products

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.     

Institutional and IACUC responsibilities for animal care and use education and training programs

Training and instruction of personnel are important components of animal care and use programs because they help to ensure the health and welfare of the animals and the integrity of the research or testing results. Training also helps to promote the consideration of alternatives, recognition of animal pain and distress, appropriate use of pain-relieving agents, aseptic technique, pre- and post-procedural care, and personnel health and safety. While individuals who provide the care for or conduct research or testing in laboratory animals should take personal responsibility for ensuring that they have the skills to perform their duties, the institution is ultimately responsible for ensuring their competency. The institution is also responsible for providing the training or instruction that is required by federal legislation, regulations, and policies. The institutional animal care and use committee (IACUC) is responsible for ensuring, as part of their review of research activities, that the personnel are capable of performing the procedures described. The IACUC must also assess the institution's training program as part of their semiannual animal care and use program review and make recommendations regarding training to the institutional official. This article provides a comprehensive overview of the US regulatory mandates for training and personnel qualification.     

Information measures: statistical confidence limits and inference

This paper presents tested statistical methods for reducing sampling bias in, for assigning approximate confidence bounds to, and for testing hypotheses about information-theoretic measures used in the study of animal communication and in behavioral sequence analysis. These procedures are also applicable in fields other than animal behavior, including psychology and ecology. Results of a Monte Carlo evaluation of the methods, including comparison of these techniques with possible alternative procedures, are presented.     

The Development of a Novel Form of Mouse Cage Enrichment

This article describes the design and testing of a novel form of mouse cage enrichment. A cage insert was designed and developed to fulfill a number of enrichment goals pertaining to its effectiveness and practicality (i.e., to improve the environment of mice in laboratories while causing the minimum of interference to the husbandry and care procedures carried out by animal care staff). Behavioral and physical parameters were used over an 8-week period to assess effectiveness, with subjective opinions of cage cleaning staff and physical examinations of the inserts after cleaning being used to assess practicality. Results show that the insert was both a practical and effective form of mouse enrichment which produced a number of enriching effects without adversely affecting the animals, their environment, or the husbandry and care procedures.     

Antiepileptic drugs: detection, quantification, and evaluation

The anticonvulsant potential of chemical substances can be identified with test procedures that act at various biological levels, ranging from subcellular elements to the normal or modified intact animal. All of these procedures modify either some minimal overt threshold electrochemical or neurochemical event or a suprathreshold manifestation such as seizure spread. This suggests that laboratory tests for the detection, quantification, and evaluation of antiepileptic drugs should be designed to identify substances that elevate seizure threshold and/or prevent seizure spread. The s.c. Metrazol (pentylenetetrazol) seizure threshold test and the supramaximal electroshock seizure test are commonly used to achieve this objective. Additional chemoshock tests may be used to delineate further the mechanisms of anticonvulsant action. Numerous variables, such as experimental animals, electroshock apparatus, parameters of electrical and chemical stimulus, and routes of drug administration, must be controlled to ensure accurate, reliable, and reproducible results. The in vivo procedures described are reliable and reproducible, and predict clinical utility of the drugs tested. New models for testing anticonvulsant activity are evaluated against clinically effective antiepileptic drugs originally identified by these same procedures.     

The Development of a Novel Form of Mouse Cage Enrichment

This article describes the design and testing of a novel form of mouse cage enrichment. A cage insert was designed and developed to fulfill a number of enrichment goals pertaining to its effectiveness and practicality (i.e., to improve the environment of mice in laboratories while causing the minimum of interference to the husbandry and care procedures carried out by animal care staff). Behavioral and physical parameters were used over an 8-week period to assess effectiveness, with subjective opinions of cage cleaning staff and physical examinations of the inserts after cleaning being used to assess practicality. Results show that the insert was both a practical and effective form of mouse enrichment which produced a number of enriching effects without adversely affecting the animals, their environment, or the husbandry and care procedures.