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.

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.

The rat vertex-middle latency auditory-evoked potential as indicator of anaesthetic depth: a comparison with evoked-reflex testing.

We investigated whether components from the rat Vx-MLAEP could be used to assess depth of anaesthesia induced by propofol. Propofol decreased MLAEP amplitudes and increased latencies. We propose that the P(16)-N(22) wave in the rat MLAEP is similar to the human P1, and that recovery of this wave during propofol anaesthesia correlates with behavioural measures of the regaining of consciousness.

Impact of transgenic procedures on behavioral and physiological responses in postweaning mice.

This study evaluates the effects of biotechnological procedures involved in the process of microinjection-induced transgenesis in the mouse by comparing four groups of C57BL/6 mice that differ in their transgenic background (transgenics after integration of a functional corticotropin-releasing factor (CRF) gene construct, transgenics after integration of a nonfunctional CRF gene construct, nontransgenics after transgenic procedures, and controls). These four groups have been tested in various behavioral paradigms. Moreover, the development in growth, morphological characteristics, and clinical appearance of the animals have been recorded from 4 till 30 weeks of age. Differences in behavior, weight gain, and morphology were found between Group 1 (transgenic CRF animals) and Group 4 (control animals). For Group 2 (animals with a noncoding construct) and Group 3 (nontransgenic animals after transgenic procedures), no significant differences from control animals were found. This indicates that, under the present conditions, the biotechnological procedures related to transgenesis (microinjection, in vitro culture, and embryo transfer) have no significant effect on the normal development of the mice in the postweaning period. These results substantiate previous findings on these animals, obtained by screening them in the preweaning period (Days 0-21). However, before general conclusions as to what extent the technique of transgenesis affects the welfare of the animals can be drawn, more and different transgenic lines should be studied in this or a similar way.

Modulation of aggression in male mice: influence of group size and cage size.

Aggression in group-housed male mice is known to be influenced by both cage size and group size. However, the interdependency of these two parameters has not been studied yet. In this study, the level of aggression in groups of three, five, or eight male BALB/c mice housed in cages with a floor size of either 80 or 125 cm(2)/animal was estimated weekly after cage cleaning for a period of 14 weeks. Furthermore, urine corticosterone levels, food and water intake, body weight, and number of wounds were measured weekly. At the end of the experiment, tyrosine hydroxylase (TH) activity, testosterone levels, and weight of spleen, thymus, testes, and seminal vesicles were determined. Results indicate a moderate increase of intermale aggression in larger cages when compared to the smaller cages. Aggression in groups of eight animals was considerably higher than in groups of three animals. The increase of agonistic behavior was observed both in dominant and subordinate animals. Physiological parameters indicate differences in stress levels between dominant and subordinate animals. It is concluded that aggressive behavior in group-housed male BALB/c mice is best prevented by housing the animals in small groups of three to five animals, while decreasing floor size per animal may be used as a temporary solution to decrease high levels of aggression in an existing social group.

Nesting material as environmental enrichment has no adverse effects on behavior and physiology of laboratory mice.

Environmental enrichment may improve the quality of life of captive animals by altering the environment of animals so that they are able to perform more of the behavior that is within the range of the animal's species-specific repertoire. When enrichment is introduced into an animal's environment, it is important to evaluate the effect of the enrichment program and to assess whether the animal continues to use the enrichment in the long-term. Groups of mice were housed under either standard or enriched conditions for several weeks. Nesting material which was highly preferred in previous studies was used as enrichment. During the period of differential housing several behavioral parameters (behavioral tests and handling) and physiological parameters (urine and plasma corticosterone, food and water intake, body and adrenal weight) were monitored to determine the impact of environmental enrichment. Observations were made to determine whether or not the mice continued to use the enrichment. The results indicated that throughout the study all mice used the nesting material to build nests and that mice from enriched conditions weighed more than mice housed under standard conditions, although the latter consumed more food. No major differences for behavioral and physiological parameters were found between the groups of mice housed under different conditions. Therefore it is not likely that supply of nesting material will jeopardize the outcome of experiments.

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.

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.

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 use of repeated treatment with Ivomec and Neguvon spray in the control of murine fur mites and oxyurid worms.

In a previous study, a single external treatment with Ivomec appeared to be more effective than Neguvon treatment. In this study the antiendoparasitic qualities of external application of Ivomec were investigated, together with the effectiveness of a combined Neguvon and Ivomec treatment. After 3 treatments, all mice were mite- and worm-free: they remained free of ectoparasites until 18 weeks after the last treatment; eggs of endoparasites reappeared 9 weeks after the last treatment.

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.

The effectiveness of Ivomec and Neguvon in the control of murine mites.

The effectiveness of different methods of external applications of Neguvon and Ivomec for treating infestations with Myobia musculi and Myocoptes spp. in conventional colonies of laboratory mice was investigated. Ivomec appeared to be effective over a longer period of time than Neguvon. There was no difference in the results obtained with different methods of application, but spraying seems to be more practical for large colonies of mice than individual dipping.

On the use of T61 for euthanasia of domestic and laboratory animals; an ethical evaluation.

A number of experiments was carried out to determine the sequence of events leading to death following administration of the euthanizing agent T61. Simultaneous recordings of the EMG, EEG, ECG and end-tidal CO2 (dogs only) were obtained in acutely instrumented rabbits and dogs. Results show that following T61 administration the loss of consciousness and loss of muscle activity occurred simultaneously. Vocalization and increased muscle movement occurred in the initial phase of the injection in 3 of 8 dogs, injected with T61 or butyramide. From this study it was concluded that the presence of the muscle relaxant does not pose an ethical problem for the use of T61 as an euthanizing agent, but our results suggest that the use of T61 may have some emotionally unpleasant side-effects.

Preferences of mice and rats for types of bedding material.

The type of bedding material has been reported to affect the environment in both the animal cage and animal room. It has an impact on the health and well-being of the animals and may cause biased experimental results. Requirements for bedding materials, particularly those regarding animal comfort are poorly supported by experimental data. In this study, various types of bedding material were evaluated using preference tests with mice and rats. It was found that beddings consisting of relatively small particles (< or = 1.2 x 1.6 mm2) were generally avoided, whereas beddings consisting of large fibrous particles were preferred. The characteristics of the bedding materials were further investigated by scanning the size and shape of the particles, and by the assessment of ultrasound produced by the moving of the beddings. The results seem to indicate that size manipulability are among the main determinants of the appreciation of bedding particles by laboratory mice and rats, and larger particles are preferred.

Assessment of side effects induced by injection of different adjuvant/antigen combinations in rabbits and mice.

We evaluated the side effects induced by injection of Freund's adjuvant (FA) and alternative adjuvants combined with different antigens. Rabbits and mice were injected subcutaneously, intramuscularly (rabbits) and intraperitoneally (mice) with different adjuvants (FA, Specol, RIBI, TiterMax, Montanide ISA50) in combination with several types of antigens (synthetic peptides, autoantigen, glycolipid, protein, mycoplasma or viruses). The effects of treatment on the animals' well-being were assessed by clinical and behavioural changes (POT and LABORAS assays) and gross and histopathological changes. In rabbits, treatment did not appear to induce acute or prolonged pain and distress. Mice showed behavioural changes immediately after (predominantly secondary) immunization. Injection of several adjuvant/antigen mixtures resulted in severe pathological changes, depending on adjuvant, type of antigen, animal species used and route of injection. Both rabbits and mice showed pathological changes ranging from marked to severe after injection of FA, and ranging from minimal to marked after Specol and Montanide injections. Pathological changes after RIBI injections were severe in rabbits, though slight in mice. After TiterMax injections, pathological changes were moderate in rabbits, though severe in mice. In conclusion, injection of FA according to present guidelines resulted mostly in severe pathological changes, whereas only very few clinical and behavioural signs indicated prolonged severe pain. Our findings indicate that Montanide ISA50 and Specol induce acceptable antibody titres, and cause fewer pathological changes than FA. Thus they are effective alternatives to FA.

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.

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.