I. Anna S. Olsson

Understanding behaviour: the relevance of ethological approaches in laboratory animal science

Applied ethology has traditionally focused on farm animal species, whereas there has been much less research directed at understanding the behaviour of laboratory animals in relation to their use as models in research. In this paper, we identify four areas in which ethological approaches could help improve the welfare of laboratory rodents while at the same time enhancing the validity of research based on them. These areas are: (1) the effects of selective breeding and gene manipulations on the animals’ ability to cope with the laboratory environment; (2) the effects of barren housing conditions on behaviour and the mechanisms underlying normal control of behaviour; (3) the sensory perception of the laboratory environment by the animals; and (4) the applicability of standard behavioural tests and the potential for improving them by taking animals’ species-specific characteristics into account. Given the current increase in the use of rodents in the life sciences, these four areas represent promising areas of future research in applied animal behaviour science. # 2003 Elsevier Science B.V. All rights reserved.

Lower Isoflurane Concentration Affects Spatial Learning and Neurodegeneration in Adult Mice Compared with Higher Concentrations

Background:Volatile anesthetics such as isoflurane are widely used in clinical and research contexts. Concerns have been raised that the effects of these drugs on the central nervous system may result in long-term impairment after surgery or general anesthesia. Hence, this study aimed to detect how different isoflurane concentrations influence spatial learning and cell death in adult mice. Methods:Fifty-two C57BL/6 mice were randomly divided in four groups. Mice in three groups were exposed to different concentrations of isoflurane (1, 1.5, and 2%) for 1 h; the control group was not exposed to anesthesia. Five mice per group were killed 3 h after anesthesia to perform histopathologic and immunohistochemical analyses (hematoxylin-eosin staining; caspase-3 activation). Eight mice per group were used for behavioral tests (open field, T-maze spontaneous alternation, and water maze) on subsequent days. Results:There were no differences between groups in the T-maze spontaneous alternation test or in the open field (no confounding effects of stress or locomotion). The group anesthetized with 1% isoflurane performed worse in the water maze task on day 1 (550.4 ±162.78 cm) compared with the control group (400.1 ± 112.88 cm), 1.5% isoflurane (351.9 ± 150.67 cm), and 2% isoflurane (364.5 ± 113.70 cm; P ≤ 0.05) and on day 3 (305.0 ± 81.75 cm) compared with control group (175.13 ± 77.00 cm) and 2% isoflurane (204.11 ± 85.75 cm; P ≤ 0.038). In the pyramidal cell layer of the region cornu ammonis 1 of the hippocampus, 1% isoflurane showed a tendency to cause more neurodegeneration (apoptosis) (61.4 ± 26.40, profiles/mm2) than the group with 2% of isoflurane (20.6 ± 17.77, profiles/mm2; P = 0.051). Conclusion:Low isoflurane concentration (1%) caused spatial learning impairment and more neurodegeneration compared with higher isoflurane concentrations. Results for mice receiving the latter concentrations were similar to those of control mice.

Evaluation of exploration and risk assessment in pre-weaning mice using the novel cage test

Exploration and risk behaviour (risk assessment/risk taking) are critical to enable mice to cope with novel situations and gain control over their environment. Evaluation of those behaviours would therefore be a useful part of early phenotypic characterization of genetically modified mice, allowing early detection of behavioural phenotypes that require special attention and/or are of scientific interest. This study aimed to evaluate exploration and risk behaviour in pre-weaning mice using the novel cage test, which consists in exploration of a novel, clean, Makrolon type III cage. The results of this test were compared with those obtained in more complex and established tests to which the same mice were subjected as adolescents and young adults. Mice of two inbred strains (129S6/Bkl, n=10; C57BL/6Bkl, n=10) and one hybrid (B6CBAF1/Bkl, n=10) were used for validation of the test. The animals were tested in the novel cage (at weaning), the open field test (at 5 weeks), and from 9 weeks of age in three other tests: the elevated plus-maze, the concentric square field and the rat exposure test. The novel cage test effectively detected strain differences in pre-weaning mice as regards exploration and risk behaviour and the results were largely consistent with those obtained in the established tests later in life. In all tests 129S6 displayed a low locomotion and high risk assessment, while C57BL/6 and B6CBAF1 showed high locomotion and exploration. In addition high levels of risk taking were observed in C57BL/6. The novel cage test is rapid, requires no special equipment and is as discriminatory as more complex tests in detecting strain/genotype differences. This suggests that the novel cage test is a valuable tool for evaluation of exploration, risk assessment and risk taking in juvenile mice.

How “Humane” Is Your Endpoint?—Refining the Science-Driven Approach for Termination of Animal Studies of Chronic Infection

potential human benefits, but if accepting the assumption that human benefits can offset animal suffering, it still needs to be argued that the same benefits could not be achieved with less negative effects on animal welfare. Reducing the animal welfare problems associated with research (‘‘refinement’’ [1]) is therefore crucial in order to render animal-based research less of an ethical problem and to assure public trust in research. Studies that are designed to measure time of death or survival percentages present a particularly challenging situation in which at least some of the animals are made to die from the disease. These studies are frequent in experimental research on severe infections. The scientific community, industry, and regulatory authorities have responded to the ethical concerns over studies in which animals die from severe disease by developing new policies and guidelines for the implementation of humane endpoints as a key refinement measure (e.g., [2–4]). The most widely used definition considers a humane endpoint to be the earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death [5], underlining that ideally such indicators should be identified before the onset of the most severe effects. Euthanizing animals, rather than awaiting their ‘‘spontaneous’’ death, is important to avoid unnecessary suffering in studies in which data on survival is thought to be required for scientific or legal reasons. However, several questions remain open regarding how humane endpoints are to be applied to address real animal welfare problems. We used TB experiments in mice as a case study to highlight the potential to establish biomarkers of disease progress that can replace survival time as a measure of disease severity.

Behaviour of laboratory mice in different housing conditions when allowed to self-administer an anxiolytic

Standard cages prevent mice from performing several natural behaviours for which they are motivated. As a consequence, abnormal behaviours sometimes develop and mice often spend long periods inactive. To improve welfare, cages are sometimes furnished with items such as nesting material, shelters and running wheels. We have previously reported that when allowed to self-administer an anxiolytic, mice in furnished cages consume less anxiolytic than mice in standard cages. This paper presents the results of behaviour studies of the mice in the same experiment. Female C57BL/6J mice (3 per cage) were housed in Standard (n = 10), Unpredictable (n = 10) or Furnished (n = 6) cages. Unpredictable cages were identical to Standard cages, but were exposed to unpredictable events two to three times a week. Furnished cages were double the size of Standard cages and contained nesting material, nest box, tubes, chew blocks and a running wheel. During three consecutive periods, mice had access to only water (control), water or an anxiolytic solution on a daily alternating schedule (forced consumption), and finally, both water and anxiolytic (self-administration). Behaviour was analysed from video recordings taken during the dark phase. The housing type affected behaviour both under the control and the self-administration conditions. Overall, mice in Furnished cages spent less time resting and performing bar-related behaviours and more time on exploratory/locomotory behaviours. Mice in Furnished cages also performed less bar-circling stereotypies than mice in Standard cages. The Unpredictable treatment did not significantly affect behaviour compared to mice in the Standard conditions. There was an overall effect of anxiolytic availability on rest-related behaviours and on exploration–locomotion behaviours, in that mice rested more and spent less time on exploration and locomotion when they were able to self-administer the anxiolytic.

Motor and cognitive deficits in the heterozygous leaner mouse, a Cav2.1 voltage-gated Ca2+ channel mutant

The leaner mutation in mice affects the Ca(v)2.1 voltage-gated calcium channel alpha(1A)-subunit gene (Cacna1a), causing a reduction in calcium currents predominantly in Purkinje cells. This reduction in calcium currents causes severe progressive cerebellar ataxia, beginning around postnatal day 10, in homozygous leaner mice (tg(la)/tg(la)), while their heterozygous littermates (tg(la)/+) present no obvious behavioral deficits. In humans, heterozygous mutations in the Cacna1a orthologous gene produce a broad range of neurological manifestations. To evaluate the phenotypic status of the tg(la)/+ animals, we assessed motor performance and cognition, at different ages, in these mutant mice. We were able to observe age-dependent impairment in motor and cognitive tasks; balance and motor learning deficits were found in demanding tasks on the rotarod and on the hanging wire test, while spatial learning and memory impairment was observed in the Morris water maze. Progressive dysfunction in escape reflexes, indicative of neurological impairment, was also present in tg(la)/+ animals. Although not presenting major motor alterations, tg(la)/+ mice show age-dependent motor and cognitive deficits.

Animal Welfare in Studies on Murine Tuberculosis: Assessing Progress over a 12-Year Period and the Need for Further Improvement

There is growing concern over the welfare of animals used in research, in particular when these animals develop pathology. The present study aims to identify the main sources of animal distress and to assess the possible implementation of refinement measures in experimental infection research, using mouse models of tuberculosis (TB) as a case study. This choice is based on the historical relevance of mouse studies in understanding the disease and the present and long-standing impact of TB on a global scale. Literature published between 1997 and 2009 was analysed, focusing on the welfare impact on the animals used and the implementation of refinement measures to reduce this impact. In this 12-year period, we observed a rise in reports of ethical approval of experiments. The proportion of studies classified into the most severe category did however not change significantly over the studied period. Information on important research parameters, such as method for euthanasia or sex of the animals, were absent in a substantial number of papers. Overall, this study shows that progress has been made in the application of humane endpoints in TB research, but that a considerable potential for improvement remains.

Examining why ethics is taught to veterinary students: a qualitative study of veterinary educators' perspectives

Although it is widely agreed that veterinary students need to be introduced to ethics, there is limited empirical research investigating the reasons why veterinary ethics is being taught. This study presents the first extensive investigation into the reasons for teaching veterinary ethics and reports data collected in semi-structured interviews with educators involved in teaching undergraduate veterinary ethics at three European schools: the University of Copenhagen, the University of Nottingham, and the Technical University of Lisbon (curricular year 2010-2011). The content of the interview transcripts were analyzed using Toulmins argumentative model. Ten objectives in teaching veterinary ethics were identified, which can be grouped into four overarching themes: ethical awareness, ethical knowledge, ethical skills, and individual and professional qualities. These objectives include recognizing values and ethical viewpoints, identifying norms and regulations, developing skills of communication and decision making, and contributing to a professional identity. Whereas many of the objectives complement each other, there is tension between the view that ethics teaching should promote knowledge of professional rules and the view that ethics teaching should emphasize critical reasoning skills. The wide range of objectives and the possible tensions between them highlight the challenges faced by educators as they attempt to prioritize among these goals of ethics teaching within a crowded veterinary curriculum.

Pup mortality in laboratory mice - infanticide or not?

BackgroundDespite being the most commonly used mammal in biomedical research, problems with perinatal mortality in mice have received little attention and the causes of pup death are still poorly known. Females are often housed alone with their litters and since the lost pups are generally eaten, it is commonly assumed that the mother has killed them. However, more detailed observations than have been reported previously in the literature are required to establish if the cause of death is infanticide. Litter loss can only be prevented efficiently after underlying causes have been carefully investigated and interpreted. The aim of this study was to investigate if females actively kill their pups by observing the behaviour of females and pups in litters that later were lost. We used video recordings of females that lost their entire litter to observe females in detail from parturition until the pups died. In total, 10 C57BL/6 females (wildtype and the knockouts Hfe−/− and β2m−/−) were studied, housed in Makrolon II cages with or without access to a small amount of nesting material.ResultsThree of the females had pups that were never seen moving, and another three females had one or two pups that never moved, indicating that some pups were most likely still-born. In five females with live-born pups, detailed observations from the time when a pup was last seen moving until it died were possible to carry out. We observed females eating dead offspring and interacting with both moving and dead pups. However, we never observed a pup stop moving when manipulated by the female, nor were any wounds seen in the pups. Hence, we found no evidence of infanticide when studying females that had lost their entire litter.ConclusionThese results suggest that other causes than infanticide plays a major role in mouse pup death, and stress the need for more systematic and careful investigations of the causality of litter loss.

Improving transparency and ethical accountability in animal studies

The use of animals for research remains a highly controversial issue that receives much public attention and comment. This was illustrated, for example, by the participation in the European Commissions internet consultation on the revision of Directive 86/609/EEC, which regulates their use in this way. Moreover, scientists using animals in their work still face fierce, sometimes violent opposition that includes death threats or the vandalism of research facilities by anti‐vivisectionist and animal rights groups who want to see all use of animals in research completely banned. Given the polarizing nature of the issue, it is not always easy for the public and lawmakers to effectively weigh the benefits of biomedical research against the deaths of the animals used. > …it is not always easy for the public and lawmakers to effectively weigh the benefits of biomedical research against the deaths of the animals used To address public resistance to animal experiments, governments and scientific entities have called for increased transparency and an informed dialogue between regulators, scientists and the concerned public. As a part of this endeavour, several European countries, notably Denmark and the UK, have begun to post summaries of approved animal experiments on the websites of responsible authorities. The rationale is that more transparency will increase public confidence in the appropriate conduct and regulation of animal research and therefore help to maintain public acceptance. However, this approach towards greater transparency leaves out one crucial piece of information, which is of both ethical and scientific relevance: the link between approved experiments and the results thus generated. The scientific community also misses an important opportunity to bring together information from prospective ethics reviews—which contain detailed information about animal procedures and ‘3R’ considerations (replacement, refinement and reduction), but little scientific information—with retrospective peer‐reviewed results that provide extensive scientific information but say very …