URINE MARKING OF CAGES AND VISUAL ISOLATION AS POSSIBLE SOURCES OF ERROR IN BEHAVIOURAL STUDIES OF SMALL MAMMALS

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1 LaboratoryAnimals (1971) 5, URINE MARKING OF CAGES AND VISUAL ISOLATION AS POSSIBLE SOURCES OF ERROR IN BEHAVIOURAL STUDIES OF SMALL MAMMALS by ANNE 1. DAGG, W. L. BELL AND D. E. WINDSOR Department of Zoology, University of Guelph, Guelph, Ontario, Canada SUMMARY Chipmunks (Tamias striatus), jumping mice (Zapus hudsonius), voles (Microtus pennsylvanicus), deer mice (Peromyscus maniculalus), hamsters (Mesocricetus auratus), house mice (Mus musculus) and gerbils (Meriones unguiculalus) were individually confined in dark cages. A corner of each cage had been marked with dried urine. Animals tended (311 of 461 tests) to urinate in the marked corner. Animals did not usually urinate (except adult gerbils) or defecate (except hamsters) in their nests. The effect of visual isolation of individual mice was not apparent in organ weights, but did lead to altered behaviour. MARKIERUNG VON KAFIGEN MIT URIN UND VISUELLE ISOLlERUNG ALS MOGLlCHE IRRTUMSQUELLEN IN VERHALTENSSTUDIEN VON KLEINEN SAUGETIEREN Chipmunks (amerikanische Hornchen, Tamias stria/us), Springmause (Zaplls hlldsonius), Wi.ihlmause (Microtus pennsylvanicus), Hirschmause (Peromysclls maniculatus), Hamster (Mesocricetus auratus), Hausmiiuse (Mus musculus) und Gerbils (Rennmause, Meriones unguiculatus) wurden einzeln in dunklen Kafigen gehalten. Ein Winkel jedes Kiifigs war mit getrocknetem Urin markiert worden. Die Tiere neigten dazu, in dem markierten Winkel Urin abzusetzen (in 311 von 461 Versuchen). Gewohnlich setzten die Tiere keinen Urin (ausser erwachsenen Gerbils) bzw. Kot (mit Ausnahme von Hamstern) in ihren Nestern abo Die Wirkung der visuellen Isolierung von einzelnen Miiusen zeigte sich nicht im Gewicht der Organe, fuhrte jedoch zu Anderungen im Verhalten. LE MARQUAGE DES CAGES PAR L'URINE ET L'ISOLEMENT VISUEL COMME SOURCES POSSIBLES D'ERREURS DANS LES ETUDES SUR LE COMPORTEMENT DES PETITS MAMMIFERES Des tamias (Tamias striatus), des zapodes (Zapus hudsonius), des campagnols (Microtus pennsylvanicus), des Peromyscus (Peromyscus maniculalus), des

2 164 ANNE I. DAGG, W. L. BELL AND D. E. WINDSOR hamsters (Mesocricetus aurafus), des souris (Mus musculus) et des gerbilles (Meriones unguiculafus) ont ete places dans des cages individuelles sans lumiere. Dans chacune des cages, un coin avait ete marque avec de I'urine dessechee. Les animaux (311 sur 461 tests) avaient tendance it uriner dans Ie coin marque. Generalement ils n'urinaient pas (excepte les gerbilles adultes) et ne defequaient pas (excepte les hamsters) dans leurs nids. L'isolement visuel des souris en cages individuelles n'avait aucun elfet sur Ie poids des organes mais modifiait, par contre, Ie comportement. URINE MARKING OF CAGES While testing the olfactory sensitivity of gerbils (Dagg & Windsor, ] 97]), we noticed that their sense of smell was less acute on the days on which their bedding had not been changed, even though the gerbils were removed from the bedding and placed in a scrubbed maze for the testing. Apparently the smell of their own urine and faeces affected their subsequent behaviour. We have now investigated if and how the scent of dried urine affects 7 species of small mammals under relatively normal conditions, using wildcaught chipmunks, Tamias striatus (10' 3 ~~), jumping mice, Zapus hudsonius (10' 1~), voles, Microtus pennsylvanicus (2 0'0' 2~~), and deer mice, Peromyscus maniculatus (2~~); and laboratory-bred mice, Mus musculus (50'0' 6~~), gerbils, Meriones unguiculatus (100'0' 1O~~), and hamsters, Mesocricetus auratus (60'0' 6~~). Method For each test an individual was placed in a dark cage in which a corner had been marked with its own or con specific dried urine. The urine covered an area of 12 em 2, 1/12 the area of the polystyrene cage floor. The cage was also provided with small pieces of sterile cotton and food pellets spread at random, plus a bottle of water. After 22 hours in the cage the individual was removed, and the areas where it had urinated, defaecated and nested were noted. No animal was tested on 2 concurrent days, although each was tested 10 times if possible. Between tests the cages and metabolic chambers were scrubbed with a strong cleanser. Results For all of the tests the male and female members of each species reacted similarly to comparable stimuli, so that the test results were pooled for each species. Since the area marked with urine was much smaller than the area of the cage floor, the chance that an animal at random would urinate, defaecate or nest in some area other than the marked one was high unless the individual was affected by the urine scent. It would be onerous to prove the urine scent repelled these activities, but not that it stimulated them.

3 SCENT, ISOLATION & BEHAVIOURAL STUDIES 165 Its own urine smell or that of a con specific usually stimulated an individual to urinate in that corner-chipmunks (20 tests out of 28), voles (29 of 35), jumping mice (9 of 12), deer mice females (5 of 6), laboratory mice (56 of 89), hamsters (60 of 113), and gerbils (132 of 178). Only for the gerbil was this reaction stronger for its own than for a conspecific's urine (p<o.oi). Animals did not usually build their nests over the dried urine scent (34 % did so, N=453, range 12-66%) or defaecate there (25% did, N=340, range 3-70 %), although in all species this occurred at least twice. With the exception of the adult gerbils (82 tests out of 105), these species, including juvenile gerbils (12 tests out of 66), usually did not urinate in the nests which they had constructed from the cotton pieces. Nor did they usually defaecate in them, with the exception of the hamsters (114 tests out of 119). Discussion The experiment confirmed our expectation that mice and other small mammals are affected by the scent of their own or conspecific urine, and it is essential that this reaction be taken into account for all behavioural studies. Preferably no urine should be present during any behavioural testing, especially if olfactory sensitivity is involved. VISUAL ISOLATION In recent years, behavioural experiments with small mammals have sometimes been preceded by periods of visual isolation for the subjects (as Bailey, ] 969). This isolation was felt to stabilize the animals' physiology. However, it seems that such isolation itself may be stressful to a normally social species such as the laboratory mouse Mus musculus. Method We planned a pilot study in which 8 male mice were to be exposed to constant mouse noises to determine if this stimulus caused changes in testes, adrenals and spleen weights and, therefore, was a source of stress to the animals. The mice were visually isolated in 8 separate cylindrical plastic containers 21 cm in diameter, with paper covering their outer sides to a height of 22 cm. The mice were to remain this way, with ample food and water, for 21 days. However, we were unable to record suitable mouse noises to act as the stimulus and the mice remained visually isolated in the containers for 60 days.

4 166 ANNE 1. DAGG, W. L. BELL AND D. E. WINDSOR Fig. 1. Self-inflicted lesion on a mouse visually isolated for 60 days. Results Towards the end of the period, 6 of the mice had developed crescent areas across their upper backs (above a scapula) on which all the hair was missing (Fig. 1). In 3 of the animals this area was bleeding. Also, 4 mice had developed the habit of jumping up to 60 times in succession, to a height of about] 9 cm. This may have been to try to see over the paper lining the sides of the container, as it was never done by mice that could See out. To determine the cause of these abnormalities, 1 of the mice was autopsied. There was no parasitic infestation or mycotic infection in the skin, but there was hair in the stomach. Because of this and the symmetrical distribution of the lesion, the autopsy report suggested that a behavioural disturbance of the mouse had caused it to injure itself by pulling and swallowing hair from the area on its back that it could reach with its teeth. To determine if the stress of such isolation was reflected in changes in organ weight, we weighed the testes, adrenals and spleen, dried to constant weight at 65 C, of the 7 remaining mice. These weights were similar to those of 8 male laboratory control mice that had been kept in a group. Discussion It seems that even though the stress of being visually isolated is not reflected in changes of various organ weights in mice, it can result in behavioural ab-

5 SCENT, ISOLATION & BEHAVIOURAL STUDIES 167 normalities. We feel that it is inadvisable to isolate visually mice that are to be used in behavioural experiments. ACKNOWLEDGEMENTS We are grateful to Dr J. D. Schroder and Dr P. 1. Lusis of the Ontario Veterinary College, who prepared the autopsy report, and to the National Research Council of Canada who supported this research financially. REFERENCES Bailey, E. D. (1969). Immigration and emigration as contributory regulators of populations through social disruption. Canadian Journal 0/ Zoology 47, Dagg, A. I. & Windsor, D. E. (1971). Olfactory discrimination in gerbils. Canadian Journal o/zoology 49,283.

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