V. Baumans

Recommendations for euthanasia of experimental animals: Part 2:

Working Party: Mrs Bryony Close (Chair), Dr Keith Banister, Dr Vera Baumans, Dr Eva-Maria Bernoth, Dr Niall Bromage, Dr John Bunyan, Professor Dr Wolff Erhardt, Professor Paul Flecknell, Dr Neville Gregory, Professor Dr Hansjoachim Hackbarth, Professor David Morton & Mr Clifford Warwick Correspondence to: Mrs B Close, Battleborough Croft, Battleborough Lane, Brent Knoll, Highbridge. Somerset TA94DS, UK

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 animals species-specific repertoire. When enrichment is introduced into an animals 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.

Effects of Environmental Enrichment for Mice: Variation in Experimental Results

This study focused on the effects of different enriched environments for mice in a number of behavioral and physiological parameters in 2 routine laboratory testing procedures: potency testing for tetanus vaccine and stress-induced hyperthermia. The variability in the results was studied by calculating and analyzing mean absolute deviations. Mice from enriched conditions weighed more and consumed more food than mice from standard housing conditions. However, mice from enriched conditions lost more body weight after being housed individually. Other physiological parameters showed no differences. Mice from standard conditions were more active in an open field, suggesting a tendency to overrespond to various stimuli in a testing environment. Mice from enriched environments were more tranquil and easier to handle. The enrichment did not influence the variability in any of the parameters measured, although earlier results and results of other studies suggest that the effects on the variability in results are parameter dependent. When enrichment does not influence variability, there is no reason for not introducing cage enrichment and by doing so contributing to the animals welfare.

Strength of preference for nesting material as environmental enrichment for laboratory mice

Abstract The present paper describes two experiments in which preferences of laboratory mice for materials which could serve as cage enrichment were investigated. In the first experiment, presence of nesting material (paper towel or tissue) and the presence of a nest box (perforated metal or clear perspex box) were tested against each other. On average, the 47 mice tested spent significantly more time in the cage with the nesting material (more than 69% of their total time, whereas less than 25% of their time in the cage with the nest box). In the second experiment, the preferred nesting material (tissues) was placed in a cage with a grid floor (previously found to be avoided) and the next box (perforated metal) was placed in another cage, connected to the first, with a solid floor covered with sawdust bedding material. In this experiment, 24 female mice were tested and on average they spent more than 67% of their time in the cage with the nesting material, despite the presence of a grid floor. Thus, it is concluded that providing a cage with nesting material (in addition to bedding) may be essential for the well-being of laboratory mice.

Influence of cage enrichment on aggressive behaviour and physiological parameters in male mice

Abstract From welfare perspective group housing of mice is preferred over individual housing. Group housing of male laboratory mice, however, often leads to problems due to excessive aggressive behaviour. In our search for management and housing modifications to decrease aggression in group-housed male laboratory mice, we have tested the effect of two types of environmental enrichment—nesting material and shelter—on aggressive behaviour after cage cleaning and after a 1xa0h isolation period. Severity of wounds, urinary corticosterone levels, body weight, food and water intake and several post-mortem parameters were also monitored. The results indicated that type of enrichment strongly affected both aggressive behaviour and physiological parameters. Overall, nesting material reduced aggressive behaviour, while a shelter increased aggressive behaviour compared to control housing. This effect was also reflected in the number of wounds counted. Furthermore, during shelter housing mice gained less body weight, drank less and showed higher corticosterone levels, while in housing conditions with nesting material, mice ate less. We conclude that providing male mice with nesting material reduces aggression between male mice, and may, thus, be promoted as being beneficial to their physical health and psychological well-being.

Validation of a new system for the automatic registration of behaviour in mice and rats

A newly developed behaviour registration system, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS) for the automatic registration of different behavioural elements of mice and rats was validated. The LABORAS sensor platform records vibrations evoked by animal movements and the LABORAS software translates these into the corresponding behaviours. Data obtained by using LABORAS were compared with data from conventional observation methods (observations of videotapes by human observers). The results indicate that LABORAS is a reliable system for the automated registration of eating, drinking, grooming, climbing, resting and locomotion of mice during a prolonged period of time. In rats, grooming, locomotion and resting also met the pre-defined validation criteria. The system can reduce observation labour and time considerably.

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 animals 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.

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 Freunds 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 virusesl. 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.

Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables:

We compared the behaviours of rats, and measured various blood parameters, after three blood sampling techniques: orbital puncture while they were under diethyl-ether anaesthesia, blood collection by tail vein puncture under O2-N 2O-halothane anaesthesia and puncture of the saphenous vein without anaesthesia. Twelve rats were subjected to the three treatments according to a Latin square design. After each treatment, the behaviour of the rats was automatically monitored using the so-called LABORASTM method, which discriminates between grooming, locomotion and inactivity in rats. Based on excitation scores and urine production, it was found that induction of diethyl-ether anaesthesia combined with orbital puncture caused more distress than did the other two blood sampling techniques. The three techniques had no differential effects on the behaviours of grooming, locomotion and inactivity. Collecting 0.5 ml of blood by orbital puncture was ± 7 times faster than doing so by saphenous vein puncture and ± 15 times faster than collecting blood by tail vein puncture while the rats were under O2-N 2O-halothane anaesthesia. The levels of some haematological and plasma variables differed significantly between the three blood collection techniques. These observations may help to select the most appropriate technique of blood sampling with respect to anticipated discomfort in the animals.