How to improve housing conditions of laboratory animals: the possibilities of environmental refinement.

Housing systems for captive animals have often been designed on the basis of economic and ergonomic considerations, such as equipment, costs, space, workload, ability to observe the animals and to maintain a certain degree of hygiene, with little or no consideration for animal welfare. Environmental refinement can be defined as any modification in the environment of captive animals that seeks to enhance the physical and psychological well-being of the animals by providing stimuli which meet the animals' species-specific needs. This article provides an overview of environmental factors that influence the well-being of captive animals with specific reference to the needs of the most common laboratory species. It is important to evaluate environmental refinement in terms of the benefit to the animal, by assessing the use of and preference for certain enrichment, the effect on behaviour, and the performance of species-typical behaviour on physiological parameters. It is also necessary to evaluate the impact of refinement on scientific outcome, including whether and how statistical power is affected. Communication and team work between animal welfare scientists, animal research scientists, institutional animal welfare officers, veterinarians and animal ethics committees, animal facility management and personnel, are essential for success.

Housing environment influences the need for pain relief during post-operative recovery in mice.

The impact of invasive experimental procedures on perceived stress and pain may be dependent on both physical and social environmental conditions. The aim of this study was to evaluate the effects of a physically and a socially enriched environment on the need for pain relief following painful experimental procedures. A non-invasive method to administer analgesics post-operatively is by means of self-administration which is a feasible objective method to measure perceived pain during the post-operative recovery period. In the present study eight groups of mice housed in different conditions underwent the surgical procedure of caecal manipulation or only exposure to anaesthesia. After surgery the mice were given the choice to self-administer an analgesic available in one of their water bottles during two post-operative weeks. It was shown that socially enriched mice drank i.e. self-administered, less from the analgesic containing water than the non-enriched and socially deprived groups. Mice that underwent operation self-administered more analgesic than mice that received only anaesthesia without operation. The findings indicate that the recovery environment can contribute positively to attenuate the need for pain relief in animals submitted to invasive procedures.

Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation.

The use of group-housed rodents in many fields of biomedical research imposes a need to identify individuals in a cage. Few studies have been designed to assess possible negative effects of identification methods of newborn mice on their development and wellbeing. In the present study, three different identification methods were applied to newborn C57BL/6J mice on postnatal day (pnd) 5 (toe clipping, toe tattoo ink puncture and subcutaneous implantation of a small transponder). All identification methods used proved to be effective for long-term marking of individual animals. Newborn mice showed the least reaction to toe clipping followed by toe tattoo ink puncture and transponder implantation was the most distressful individual identification procedure in newborn mice. Importantly, clipped toe tissue proved to be enough for genotyping purposes. No overall consistent differences in somatic and neurological reflex development during the postnatal period were shown as a result of the newborn individual identification procedures used. Further, none of the methods interfered significantly with the adult animals' general normal behaviour (e.g. ability to move, grasp, climb) and sensory-motor functions as assessed with a simplified SHIRPA battery of tests, as well as Rotarod and Elevated Plus Maze tests. Postmortem thymus and adrenal gland weights gave no indication of chronic stress as a consequence of the identification method. We conclude that toe clipping might even be advisable in newborn mice at a very young age, when genotyping is needed. Toe tattoo ink puncture is also a good identification method for newborn mice and transponder implantation should only be used in older newborns or applied at weaning.

The impact of light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats.

Human interaction and physical environmental factors are part of the stimuli presented to laboratory animals everyday, influencing their behaviour and physiology and contributing to their welfare. Certain environmental conditions and routine procedures in the animal facility might induce stress responses and when the animal is unable to maintain its homeostasis in the presence of a particular stressor, the animal's wellbeing is threatened. This review article summarizes several published studies on the impact of environmental factors such as light, noise, cage cleaning and in-house transport on welfare and stress of laboratory rats. The behaviour and physiological responses of laboratory rats to different environmental housing conditions and routine procedures are reviewed. Recommendations on the welfare of laboratory rats and refinements in experimental design are discussed and how these can influence and improve the quality of scientific data.

Influence of environmental enrichment and handling on the acute stress response in individually housed mice.

In this study we investigated the effect of environmental enrichment and handling on the acute physiological stress response caused by short periods of restraint in individually housed female mice. Heart rate (HR) and body temperature (BT) were measured by radiotelemetry and compared with plasma corticosterone (pCORT) levels. Also, postmortem thymus weight and tyrosine hydroxylase (TH) activity were assessed. The acute stress response was seen in both HR and BT. Enrichment and handling were found to increase rather than decrease this stress response, but pCORT values, measured 90 min after restraint, suggested a lower stress response in the enriched groups. No effect was found with thymus weight or TH as parameters.

Murine models of myocardial and limb ischemia: diagnostic end-points and relevance to clinical problems.

Ischemic disease represents the new epidemic worldwide. Animal models of ischemic disease are useful because they can help us to understand the underlying pathogenetic mechanisms and develop new therapies. The present review article summarizes the results of a consensus conference on the status and future development of experimentation in the field of cardiovascular medicine using murine models of peripheral and myocardial ischemia. The starting point was to recognize the limits of the approach, which mainly derive from species- and disease-related differences in cardiovascular physiology. For instance, the mouse heart beats at a rate 10 times faster than the human heart. Furthermore, healing processes are more rapid in animals, as they rely on mechanisms that may have lost relevance in man. The main objective of the authors was to propose general guidelines, diagnostic end points and relevance to clinical problems.

Effect of restraint and injection methods on heart rate and body temperature in mice.

Routine procedures in the laboratory, inducing acute stress, will have an impact on the animals and might thereby influence scientific results. In an attempt to gain more insight into quantifying this acute stress by means of the parameters heart rate (HR) and body temperature (BT), we subjected mice to different restraint and injection methods. We first compared the treatment response of HR and BT, measured by means of radiotelemetry, with the treatment response of plasma corticosterone (pCORT), a common and well-validated parameter for measuring acute stress responses. It was found that HR, and to a lesser extent also BT, parallels pCORT values after subjecting the animals to different methods of restraint. Secondly, the acute stress response caused by different injection methods was evaluated. Again, HR was found to be a more sensitive parameter than BT. We found that, in case of sham injections, the acute stress response after an intraperitoneal (i.p.) injection was more pronounced than after intramuscular (i.m.) or subcutaneous (s.c.) injections, but this difference was found to be inconsistent when saline was used as injection fluid. In a third experiment we investigated if the level of experience of the animal technician influenced the stress response after s.c. injections, but no differences were found. Overall, the results have indicated that HR might be considered as a useful parameter for measuring acute stress responses to routine procedures, but the value of BT seems to be of limited value in this respect.

Neither environmental enrichment nor voluntary wheel running enhances recovery from incomplete spinal cord injury in rats.

Environmental enrichment and exercise may be neuroprotective or promote recovery after different forms of CNS injury. Here, we tested the possible effects of moderate environmental enrichment and voluntary exercise on the outcome of incomplete spinal cord injury in rats. We provided rats in standard cages with basic environmental enrichment (carton house, nesting material, tube, gnawing sticks). We also analyzed the effect of increased activity by housing spinal-cord-injured rats in cages with or without access to running wheels. In a third experiment, we looked at the possible effect of pre-injury training. In all experiments, a battery of behavior tests were used. Enriched environment provided before, after or both before and after injury did not alter the outcome on any of these tests. Similarly, despite excessive running after injury, no differences in terms of recovery and behavior were found in the running experiment. Similarly, running prior to injury did not significantly decrease the degree of functional deficit caused by the injury. Since there were no effects of further enrichment, above the possible effects of being socially housed, and since exercise did not improve the outcome, we conclude that these forms of increased activity do not render the animals significantly less sensitive to spinal cord injury and do not cause robust improvement when initiated after injury. While these results pose a limit to how helpful environmental and physical training programs may be in rodent impact injury models, they do not contradict the fact that voluntary and guided training can be effective tools in human spinal cord rehabilitation.

Science-based assessment of animal welfare: laboratory animals.

The use of animals in experimental research parallels the development of medicine, which had its roots in ancient Greece. The increasing demand for high-standard animal models, together with a critical view of the way animals are used, has led to the development of a multidisciplinary branch of science we now know as 'laboratory animal science'. The guiding principles are replacement, reduction and refinement (the Three Rs), first proposed by Russell and Burch in 1959. When animals are used, the people involved have an obligation to safeguard their welfare and minimise discomfort; this will also generally be beneficial for both the animal and the experimental outcome. The ability of an animal to cope with the environment and exert control over its life seems to be crucial for animal welfare. In this paper, attention is paid to the assessment of welfare, environmental factors affecting welfare, legislative requirements and future trends such as the production and use of genetically modified animals.

Short-term effects of a disturbed light-dark cycle and environmental enrichment on aggression and stress-related parameters in male mice.

In the laboratory setting, environmental factors have a major influence on the well-being of laboratory animals. The present study shows the importance of a semi-natural light-dark cycle. In this experiment one cohort of mice was kept with a continuous lighting for one week. After the first week the artificial light-dark cycle was 12:12 with lights on at 07:00 h. The second cohort of mice was kept with this 12:12 h light-dark cycle from the start. Half of each cohort received environmental enrichment. In order to analyse corticosterone levels, urine samples were collected. To measure agonistic behaviour, the behaviour of the mice was recorded on videotape immediately after cage cleaning. A significant difference in corticosterone levels between cohorts was found during disturbed lighting, but not after lighting conditions were reset to 12:12 h. In the first test week, mice subjected to disturbed lighting also showed a significantly shorter agonistic latency than control mice. This difference had disappeared when in the second test week all mice experienced 12:12 h lighting. No effects of enriched housing were found. This experiment has shown that disturbed lighting for socially-housed male mice caused physiological and behavioural changes indicative of stress, not only leading to much higher levels of corticosterone but also to shorter agonistic latency within the groups.

Use of animals in experimental research: an ethical dilemma?

Mankind has been using animals already for a long time for food, for transport and as companion. The use of animals in experimental research parallels the development of medicine, which had its roots in ancient Greece (Aristotle, Hippocrate). With the Cartesian philosophy in the 17th century, experiments on animals could be performed without great moral problems. The discovery of anaesthetics and Darwin's publication on the Origin of Species, defending the biological similarities between man and animal, contributed to the increase of animal experimentation. The increasing demand for high standard animal models together with a critical view on the use of animals led to the development of Laboratory Animal Science in the 1950s with Russell and Burch's three R's of Replacement, Reduction and Refinement as guiding principles, a field that can be defined as a multidisciplinary branch of science, contributing to the quality of animal experiments and to the welfare of laboratory animals. The increased interest in and concern about animal welfare issues led to legislative regulations in many countries and the establishment of animal ethics committees.

Long-term effects of husbandry procedures on stress-related parameters in male mice of two strains.

In socially unstable groups of male laboratory mice, individuals may experience a chronic stress situation. Previous experiments have shown that the transfer of specific olfactory cues during cage cleaning, and the provision of nesting material decrease aggression and stress in group-housed male mice. In this study, the combined effect of these husbandry procedures were tested for their long-term effect on stress in groups of moderately aggressive (BALB/c) and severely aggressive (CD-1) male mice. The physiological and behavioural stress-related parameters used were body weight, food and water intake, spleen and thymus weight, adrenal tyrosine hydroxylase activity, urine corticosterone levels and behaviour in a cage emergence test. Long-term provision of nesting material and its transfer during cage cleaning was found to influence several stress-related physiological parameters. Mice housed in cages enriched with nesting material had lower urine corticosterone levels and heavier thymuses, and they consumed less food and water than standard-housed mice. Furthermore, marked differences were found between strains. CD-1 mice were less anxious in the cage emergence test, weighed more, ate and drank more, and had heavier thymuses but lighter spleens and lower corticosterone levels than BALB/c mice. We conclude that the long-term provision of nesting material, including the transfer of nesting material during cage cleaning, reduces stress and thereby enhances the welfare of laboratory mice.

Preference for social contact versus environmental enrichment in male laboratory mice.

Due to their aggressive nature, male mice are less frequently used than female mice in biomedical research. When aggressive males are being used, individual housing is common practice. The question arises whether this is an acceptable housing for a social species. The present study was designed to gain more insight into the nature of inter-male social contact and into the potential of a form of environmental enrichment (nesting material) to compensate for the lack of social contact. In a series of tests, we analysed whether male mice of different ages preferred to spend time (1) near a familiar cage mate versus an empty cage, or (2) near to a familiar cage mate versus direct contact with nesting material (tissues). Dwelling time in each of the test cages and sleeping sites was recorded, as was the behaviour of the test mice. Results indicated that when other conditions were similar, male mice preferred to sleep in close proximity to their familiar cage mate. Furthermore, the need to engage in active social behaviour increased with age. Tissues were used to a large extent for sleeping and sleep-related behaviour. It is concluded that single housing in order to avoid aggression between male mice is a solution with evident negative consequences for the animals. When individual housing is inevitable due to excessive aggressive behaviour, the presence of nesting material could partly compensate for the deprivation of social contact.

The importance of learning young: the use of nesting material in laboratory rats.

Unlike mice, adult laboratory rats do not spontaneously build nests when nesting material is offered. As a result, nesting material is often regarded as unsuitable environmental enrichment for laboratory rats. Wild rats and pet rats, however, have been observed to build complex nests from nesting material at hand. It was hypothesized that nest building in rats is an acquired behaviour, rather than genetically predisposed. To test this hypothesis, the progeny of three Wistar rats provided with nesting material (Kleenex tissues) during pregnancy and three standard-housed rats were divided in 34 same-sex couples with access to nesting material: (1) from the age of 8 weeks (n=7); (2) from weaning (n=8); (3) from birth (n=17). The latter were subdivided into two groups after weaning, one provided with Kleenex tissues (n=9), the other with Enviro-dri (n=8). At the age of 12 weeks, all couples were provided with both types of nesting material for one week. Amount, shape, and soiling of the nesting material were scored on weekdays, and behaviour of the rats was scored twice weekly. Results indicated that the older the rats were when first provided with nesting material, the greater the amount of nesting material eaten and soiled, and the nests built were less elaborate. Overall, Enviro-dri was eaten less than Kleenex tissues. It is concluded that rats need to learn the proper use of nesting material. When provided from birth, nesting material is a suitable type of environmental enrichment for laboratory rats.

The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.

Fetal bovine serum (FBS) is a common supplement to in vitro culture media. A workshop was organized to discuss whether or not fetuses might suffer when blood is withdrawn, and to discuss serum replacement methods. When bovine fetuses are exposed after slaughter of the dam, they can suffer only if they inflate their lungs with air and increase their blood oxygen to levels compatible with awareness. Preventing fetuses from breathing air or killing them by an efficient method, according to clearly defined safeguards, ensures that fetal blood collection is humane. Since serum is a supplement of unknown composition, which could be contaminated with unwanted factors, there are scientific and safety reasons for omitting FBS from culture media. Several media have been developed in which minimal or no animal derived components are present. Also, different cell types have been adapted to serum-free media. As yet, no standard serum free media are present, and each cell type requires its own medium composition. Among other recommendations, the establishment of a public database with information on cell types and their serum-free medium composition is proposed.

Male management: Coping with aggression problems in male laboratory mice.

In a laboratory environment, aggressive interactions between male mice may exceed normal levels leading to negative effects both on the well-being of the animals and on the validity of experimental results. In this paper we review results from the literature and our own research with regard to coping with excessive aggressive behaviour in male laboratory mice. Based on this review practical recommendations concerning the housing and care of male laboratory mice are formulated. In short, it is recommended to avoid individual housing, to transfer odour cues from the nesting area during cage cleaning and to apply nesting material as environmental enrichment. Furthermore, group size should be optimized to three animals per cage. Further research, in particular into the effects of frequency, duration, type and severity of disturbances during an experiment on the degree of aggression, is recommended.

Consequences of gene targeting procedures for behavioural responses and morphological development of newborn mice.

In this study the effects of gene targeting procedures on the early behaviour and morphological development of the resulting offspring have been investigated. Six groups of mice, each having undergone a specific aspect of the biotechnological procedure, (including electroporation, microinjection and/or embryo culture) and one control group, were compared. Development of behaviour, morphological characteristics and body weight of the progeny were tested daily from birth to weaning (0-3 weeks) for all groups. No significant differences in behaviour or morphological development were observed. However, the occurrence of increased (perinatal) pup mortality and increased body weight in the procedural groups, indicates that during the production of gene targeted mice, some of the normal physiological and/or developmental processes can be affected. Therefore, gene targeting procedures should always be accompanied by careful monitoring of health and welfare of the resulting offspring.

Use of score sheets for welfare assessment of transgenic mice.

The use of transgenic mice has increased dramatically in recent years and continues to increase further. However, because transgenesis may alter a balanced genotype and produce unpredictable effects, careful monitoring of health and welfare of the transgenic animal is advised. The present study assessed the feasibility of the use of score sheets for monitoring transgenic mice, as part of daily routine, in a transgenic unit. The score sheets used were based on parameters which are sensitive and easy to determine. The score sheets were used by two animal technicians and a thorough evaluation showed that the score sheets, as described in this paper, are useful for routine monitoring in a transgenic unit and may result in the early detection of animal welfare problems. However, notwithstanding the limited number of parameters included and the restricted age-span covered by the screening, the monitoring system was considered to be time consuming. Large-scale implementation of such a scoring system during the first weeks of life would increase daily care time by at least 15-20 min for an average litter of 4-6 pups. Nevertheless, the use of score sheets seems to be a prerequisite for monitoring the animal's welfare in the course of producing transgenic lines.

Behavioral and physiological effects of biotechnology procedures used for gene targeting in mice.

The effects of gene-targeting procedures on the behavior and physiological development of (chimeric) mice have been investigated. We used six groups of mice, each of them undergoing specific aspects of the biotechnological procedure, including electroporation, microinjection, and/or blastocyst culture. Changes in behavior and physiological development of the progeny (age 4-30 weeks) were investigated. Besides increased body weights, no significant difference between the six treatment groups and untreated C57BL/6 controls could be attributed to the biotechnology procedures. Therefore, we conclude that these procedures per se do not induce significant discomfort for the offspring. Differences in behavior, observed for the two groups of chimeric mice [one derived from electroporated embryonic stem (ES) cells and the other from nonelectroporated ES cells] when compared to the other (nonchimeric) groups, are, at least partly, due to the genetic background of the 129/Ola strain from which the ES cells are derived rather than to the biotechnological manipulations of the ES cells and/or blastocysts. The occurrence of hermaphrodites (8%) and some other gross pathologies observed in both groups of chimeric animals seem to indicate that developmental problems may occur when cells from different origin are simultaneously contributing to the development of one individual. This implies that during the production of gene-targeted mice, health and welfare of chimeric animals must be carefully monitored.