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Behavioral thermoregulatory responses of single- and group-housed mice1
Physiology & Behavior, Vol. 65, No. 2, pp. 255–262, 1998 © 1998 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/98 $19.00 1 .00
PII S0031-9384(98)00148 – 6
Behavioral Thermoregulatory Responses of Single- and Group-Housed Mice1 CHRISTOPHER J. GORDON,2 PEGGY BECKER AND JOSEPH S. ALI Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA Received 29 January 1998; Accepted 29 April 1998 GORDON, C. J., P. BECKER AND J. S. ALI. Behavioral thermoregulatory responses of single- and group-housed mice. PHYSIOL BEHAV 65(2) 255–262, 1998.2The ambient temperature (Ta) to house and study laboratory rodents is critical for nearly all biomedical studies. The ideal Ta for housing rodents and other animals should be based on their thermoregulatory requirements. However, fundamental information on the behavioral thermoregulatory responses of single- and group-housed rodents is meager. To address this issue, thermoregulatory behavior was assessed in individual and groups of CD-1 mice housed in a temperature gradient. Mice were housed in groups of five or individually while selected Ta and motor activity were monitored. Single- and group-housed mice displayed a circadian oscillation of selected Ta and motor activity with relatively warm Tas of ;29°C selected during the light phase; during the dark phase selected Ta was reduced by 4°C, whereas motor activity increased. Selected Ta of aged (11 months old) mice housed individually was ;1.0°C warmer than the group-housed mice. Thermal preference of younger mice (2 months old) was similar for single- and group-housed animals. The operative Ta of mice housed in standard facilities was estimated by measuring the cooling rate of “phantom” mice modeled from aluminum cylinders. The results show that the typical housing conditions for single- and group-housed mice are cooler than their Ta for ideal thermal comfort. © 1998 Elsevier Science Inc. Behavioral thermoregulation
Motor activity
Animal housing
THE domesticated mouse (Mus musculus) has become one of the most widely used species in physiology, pharmacology, and toxicology investigations. The growing number of breeds and strains, development of genetic knockouts, and relatively low cost are some of the main reasons that have contributed to the popularity of the mouse as a test species. The ambient temperature (Ta) to house and study laboratory rodents is a critical issue in most biomedical studies (1,4). Ambient conditions (e.g., temperature, humidity, and wind speed) that stress the metabolic and other thermoregulatory effector systems of a species should be avoided. Thus, the Ta for housing the animals should be set, in most instances, at a level where stress is minimal. The optimal thermal environment is also an important issue in the construction and operation of animal housing facilities. The environment of animal housing facilities is generally designed around 1) the requirements for ideal thermal comfort of the personnel and 2) the limitations and costs for operating the air handling mechanisms of the facility (e.g., cooling, heating, and dehumidification). The thermoregulatory requirements of the animals have historically not been the primary factor in the design and operation of animal facilities. It is important to understand the ideal thermal environment of mice and other rodents. The Ta for housing mice has marked effects on most physiological and behavioral processes, such as
metabolism, motor activity, growth and development, food and water consumption, sensitivity to toxicants and other chemical agents, immune response, cardiovascular function, and many others (4,11). The ideal thermal environment for autonomic and behavioral thermoregulatory responses of individual mice has been well documented. For example, when placed in a temperature gradient permitting selection from a wide range of Tas, individual mice prefer a Ta of about 30 –31°C (3). This Ta range is equal to the mouse’s thermoneutral zone where metabolic rate and evaporative heat loss are near basal levels (3,4). Animal housing facilities and testing facilities are generally maintained at much cooler Ta values of around 22°C. This would lead one to expect that mice are subjected to cold stress when housed and/or tested in typical laboratory environments (e.g., Ta ' 22°C). However, mice are often housed in groups of five or more and, because they huddle and usually maintain close body contact throughout the day, it has been assumed that thermal comfort for groups of mice housed under standard conditions is not compromised. To evaluate these assumptions, it is necessary to compare the thermoregulatory behavior of single- and group-housed mice. Allowing animals to remain in a temperature gradient for several days has revealed that single-housed hamsters and rats display a circadian rhythm of selected Ta with relatively warm Ta values selected during the light phase and cooler Ta values selected during
1
This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 2 To whom correspondence should be addressed. E-mail: [email protected]
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the dark phase (5). Moreover, the selected Ta rhythm of individual rodents was found to increase at the same time as motor activity was reduced (4,5). It remains to be shown if groups of mice housed in a temperature gradient will exhibit a similar pattern of warm selected Ta values coupled with reduced motor activity during the light phase or will alter their thermoregulatory behavior because of huddling. A related issue is to determine the operative Ta selected by mice when allowed to behaviorally thermoregulate. Past studies from this laboratory and others (4), have not considered how the measurement of air temperature in a temperature gradient system reflects the operative Ta the animal encounters when housed in various cage systems. Furthermore, since metabolism and motor activity and other functions decline with age in mice and other species, measuring thermoregulatory behavior of young and aged mice allows the comparison of thermoregulatory behavior in the same species but with markedly different thermoregulatory requirements (4,7,10). Hence, there were several key objectives of the current study: 1) measure the thermoregulatory behavior of young and aged mice when housed individually or in groups in a temperature gradient; 2) determine the impact of motor activity on thermoregulatory behavior of single- and group- housed mice; and 3) to compare the thermal preferences of the mice to the thermal environments they encountered when housed in various laboratory cage systems, by calculating the operative Ta of the mice in a temperature gradient. MATERIALS AND METHODS
Animals used in this study were female mice of CD-1 strain obtained from Charles River Laboratories (Raleigh, NC, USA). The mice were obtained at 30245 days of age and housed in groups of 526 in standard acrylic cages with pine shaving as bedding material. Mice were maintained at a Ta of 22°C, relative humidity of 50%, and a 12:12 L:D photoperiod. Two age groups of mice were tested in this study; young mice were tested at 2 months of age (mean body weight 5 28 g) and another group of older mice were tested at approximately 11 months of age (mean body weight 5 52 g). Temperature gradient. The gradient construction, operation, and data analysis for the current experiments were modified slightly from earlier studies published by this laboratory (6). The gradient consisted of a copper tube 2 m length, 20.3 cm inside diameter, 0.63 cm wall thickness. The tube was split longitudinally and a hinge was secured on one side to allow the gradient to be opened for access and cleaning. One end of the gradient was heated, and the other end cooled by circulating water through copper tubing. A runway to house the animals in the temperature gradient was made of galvanized 1.3-cm wire-mesh on the sides and bottom, with a 0.31-cm thick Plexiglas top. A tray was placed beneath the runway to collect animal wastes. The spigot of a water bottle and a food dispenser were placed near the center of the runway. A series of low voltage lamps at 10-cm intervals were placed above the runway to illuminate the interior of the gradient (12:12 L:D photoperiod). Photocells were placed at 10-cm intervals along the length of the runway (total of 18 photocells in gradient) that served to track the position of the mice. A copper-constantan thermocouple was positioned immediately above each photocell. The thermocouples were placed within ;2 mm of the runway cage so that the temperature could be measured while preventing animals from interfering with the ancillary wiring of the photocells and thermocouples. The gradient had a temperature range of 12237°C. These gradient temperature were converted to operative Tas selected by the mice (see below). Data acquisition in the temperature gradient was altered from
previous studies so that more than one animal could be monitored simultaneously. All blocked photocells and their corresponding temperatures were recorded with a PC-based acquisition system (Dianachart) every 15 s. The time constant of data acquisition was increased to a value of 2 (normally set to 0 for larger species), assuring that if a mouse momentarily blocked a photocell its position would be more likely to be recorded. It should be noted that if two mice blocked one photocell at the same time, the system could not distinguish between one or two animals at that particular position. Protocol. An individual mouse or group of five mice were weighed and then placed in the runway at approximately 1500 hours with food and water provided ad lib. Data were continuously recorded for the next two nights. The mice were removed from the gradient at approximately 0800 hours, weighed, and returned to the animal facility. Thus, mice were left in the gradient for approximately 40 h. The first night in the gradient was considered to be an acclimation period. The 24-h period beginning from 0600 to 0559 hours the following day was used for statistical analysis. Data Analysis. The raw data from the gradient were stored in a ASCII format and then reprocessed to a new summary file. The position and temperature of each blocked photocell were determined. If more than one photocell was blocked, the temperatures at each blocked location were averaged and written to the summary file. The range of the positions of the blocked photocells (if more than one was blocked) was calculated and written to the summary file. The average of the position of the animal(s) was also determined. The change in average position at 15 sec intervals was calculated and stored in the summary file. The change in average position per unit time (i.e., m/h) was calculated, yielding a measure of motor activity. It is important to note that motor activity calculated for groups of mice is a measure of the movement of the group as a whole unit and is not a quantitative measure of the movement of individuals within the group. Thus, it is not possible to compare the levels of motor activity between single- and grouphoused mice in the gradient. However, the motor activity parameter does yield a qualitative measure of the group’s relative level of activity. The data in the summary file (i.e., 240 measurements per h) were averaged into 1-h bins for statistical analysis. The selected Ta and motor activity data were subjected to repeated measures ANOVA to determine the effect of time. Two-way ANOVAs were used to compare the single- and group-housed selected Ta values monitored over a 24 h period in the temperature gradient. Twelve hour means of the selected Ta during the light and dark phases were also calculated and compared with each age and housing condition using a two-tailed t-test. Calculation of operative Ta in temperature gradient. In past studies from this laboratory and others, the air temperature measured at the animal’s position within the temperature gradient was deemed the animal’s selected Ta or (i.e., thermopreferendum or preferred Ta). This assumption has been reevaluated in this study because it is recognized that, due to the complexities and nonlinearity of the temperature gradient, the measurement of air temperature is not necessarily equal to the animal’s operative Ta (see appendix A). The operative Ta was determined by comparing the rate of heat loss of a “phantom” mouse in the temperature gradient to the heat loss when maintained at fixed “black box” temperatures. The “phantom” mouse was constructed from aluminum cylinder stock (6.35 cm long 3 2.54 cm diameter; 60.8 cm2 surface area). Two brass legs made of 3.1-mm square stock were secured to the bottom of the cylinder to prevent movement during testing. A hole drilled through the center of the cylinder allowed for the insertion of a thermocouple to measure the internal temperature of the
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FIG. 1. Averages of selected Ta and motor activity 60-min time course of single- and group-housed young and aged mice plotted over a 24-h period. Data plotted as the mean 6 S.E. n 5 4. Horizontal bar indicates period of darkness.
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GORDON ET AL. TABLE 1a SELECTED Ta MEAN 6 S.E. OF FEMALE CD-1 MICE HOUSED INDIVIDUALLY OR IN GROUPS OF 5 IN A TEMPERATURE GRADIENT AS A FUNCTION OF TIME OF DAY SELECTED Ta
Period
light dark 24 h
Young
Aged
Individual
Group
Individual
Group
27.7 6 .3 26.5 6 .6 27.1 6 .3
27.4 6 0.2 26.2 6 .1a 26.8 6 .1
29.5 6 .3 27.6 6 .4a 28.6 6 .3b
28.4 6 .1 27.0 6 .1a 27.7 6 .1c
p , 0.01 compared to light phase for same age group; p 5 0.03 compared to young individuals over 24 h; c p , 0.01 compared to young group over 24 h. a
b
TABLE 1b MOTOR ACTIVITY Period
Young Individual
light dark 24 h
12.1 6 .5a 19.1 6 3.7 15.7 6 1.9b
Aged Group
30.1 6 2.4a 43 6 2.8a 36.6 6 1.2a
Individual
6.3 6 1 13.1 6 1.7 9.7 6 1.4
Group
17.2 6 1.4 30.3 6 1 23.8 6 1
a p , 0.01 or b p , 0.05 compared to aged mice at same time period; dark phase motor activity was significantly greater than light phase for all groups (p , 0.01).
cylinder (Tcyl). The cylinder was spray painted with flat black acrylic paint. The operative Ta was defined by measuring the cooling rate of the cylinder after being heated to a specified temperature and then being allowed to cool in a defined environment. The “defined environment” was arbitrarily selected where the heat from the “phantom” mouse would dissipate primarily by radiation and convection under still air conditions in an “ideal” thermal environment where the surface area of the surrounding environment was very large relative to the surface area of the “phantom”. The ideal thermal environment consisted of a box (35.6 3 35.6 3 35.6 cm) made from aluminum sheet metal. The ratio of surface area of the cylinder to that of the black box was 1:125. The inside of the box was spray painted flat black. Two thin supports made of 3.1-mm thick Plexiglassy were placed in the middle of the box that served to suspend the cylinder in the center of the box. The box was placed inside an environmental chamber that was maintained at Tas of 10236°C. Thermocouples measuring air (Tair) and wall (Twall) temperatures were placed inside the box. The box was not hermetically sealed; however, the enclosed box assured that no air turbulence such as from the fan of the environmental chamber would affect the convective heat loss of the “phantom” mouse. Tair and Twall of the black box and Tcyl were continuously recorded at 15-s intervals using a PC-based data acquisition system. The temperature of the environmental chamber was stabilized to one of several Ta values. The cylinder was placed in a plastic bag that was immersed in a water bath stabilized to 44°C. This temperature was selected because it was anticipated that it would
be approximately 5°C higher than the warmest temperature in the gradient. Care was taken to prevent moisture from contacting the surface of the cylinder. After the cylinder was heated uniformly to 44°C, it was quickly positioned in the center of the black box. Tcyl was recorded at 15-s intervals until it decreased below 38°C. Over a Tcyl range of 42239°C the change in temperature as a function of time was nearly linear with a coefficient of correlation that was consistently greater than 0.99. The rate of cooling over this specified temperature range was calculated with the cylinder placed in the black box and maintained at Ta values of 10236°C. A calibration curve of the mean black box temperature (i.e., Tair 1 Twall/2) versus the cooling rate of the cylinder was developed. The cooling rate of the cylinder was then evaluated in the temperature gradient. The cylinder was heated as described above and then placed lengthwise and equidistant between the two sides of the gradient runway such that the center of the “phantom” blocked the light from a photocell. Ta of the gradient where the cylinder was located along with Tcyl was recorded at 15 sec intervals. This procedure was performed at different positions in the gradient and a calibration curve of the gradient Ta versus cooling rate of the cylinder was calculated. Because the rate of cooling of the cylinder as a function of Ta in the black box and the gradient was linear, a linear formula could be used to convert the gradient Ta to operative Ta. The equation to convert gradient Ta to operative Ta was: operative Ta 5 0.84*gradient Ta-0.16. The slope and intercept will vary as a function of the size of the cylinder. For example, tests with a larger cylinder (12.7 cm length 3 5.08 cm diameter, 242.5 cm2) to mimic the approximate size of a laboratory rat yielded the following conversion: operative Ta 5 0.81*gradient Ta 1 2.5. With a much smaller cylinder (4.44 cm length x 1.9 cm diameter; 32.3 cm2) the conversion was: operative Ta 5 0.84*gradient Ta- 1.8. Additional experiments were performed to estimate the operative Ta encountered by individual mice when housed in a variety of laboratory cage environments. The “phantom” mouse was heated as described above and then placed in various locations of a standard acrylic rodent cage (46 cm length 3 24 cm width 3 20.3 cm depth) fitted with a standard wire top or filter top cover. The bottom of the cage was bare acrylic, wire-screen, layered with wood shavings, or layered with wood chips (“Beta-Chip”; laboratory grade, heat treated, hardwood animal bedding; Northeastern Products, Warrensburg, NY, USA). The mice used in the present study had been housed in cages layered with wood shavings. On the other hand, rodents are often housed in cages lined with beta chips or on wire-screen floors. The wire top was of a standard size designed to hold the food and water bottle. The filter top consisted of an acrylic frame with filter paper which is commonly used in most animal facilities. When testing the wire-screen bottom, care was taken to prevent air from blowing directly on the cylinder. All cages were tested in an environmental chamber maintained at a Ta of 22°C. The cylinder cooling rate was evaluated in three positions in the cage: 1) center; 2) side; and 3) corner. In the cage lined with wood shavings, the cylinder was either placed on the surface of the shavings or was covered with shavings to mimic the way mice burrow into this type of bedding. RESULTS
It is important to note that all selected Tas reported henceforth are operative Ta values. The selected Ta and motor activity of single- and group-housed mice displayed a 24-h rhythm (Fig. 1). At the start of the light phase, the selected Ta of young, singlehoused mice was a relatively low 24.5°C, which then rapidly increased to 29°C, concomitant with a reduced motor activity.
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FIG. 2. Selected Ta and position range time course of a group of five aged mice housed in a temperature gradient. Data points plotted at 15-s intervals represent the mean of selected Ta and range of the group of mice. Horizontal bar indicates period of darkness.
Three hours before the start of the dark phase, there was an abrupt rise in motor activity and a drop in selected Ta. Selected Ta decreased to 23°C in the middle of the dark phase and then increased over the next 4 h to 29°C. Groups of young mice showed a similar pattern, with a relatively low Ta of 24°C selected at the start of the light phase. Four hours before the dark phase, the selected Ta of the groups underwent a gradual reduction at the same time the motor activity increased. Selected Ta reached a minimum of 24°C during the middle of the dark phase and then rapidly increased over a 4-h period to 28.5°C. Two-way ANOVA analysis indicated no significant effect of single versus group housing on selected Ta of the young mice (Table 1a,b). Selected Ta of single-housed, aged mice reached a peak of 30°C at 1400 hours. In the transition from light to dark, the
selected Ta decreased abruptly by 1.5°C. During the dark phase, it reached its lowest point of 26°C at 0300 hours. The selected Ta of the aged, group-housed mice reached a peak of 29°C at 1300 hours. There was an abrupt decrease of 1.0°C during the last hour of the light phase. During the dark phase, the selected Ta of the group-housed mice reached a nadir of 24°C at 2000 hours, rose slowly for the next several hours and then decreased again as the end of the dark phase approached. Overall, single-mice in the aged group selected significantly warmer Tas than the group-housed mice: the selected Ta was 1.0°C warmer during the light phase and 1.5°C warmer during the dark phase [treatment, F(1, 7) 5 6.8, p 5 0.04; treatment-time, F(23, 138) 5 2.2, p 5 0.008] (Table 1a). The range in position of the group-housed animals was directly related to their motor activity. For example, in a typical time-
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GORDON ET AL.
FIG. 3. Motor activity and selected Ta correlation in single- and group- housed young and aged mice. Each data point represents average of selected Ta and motor activity averaged over 1 h of a 24-h cycle.
course for one group of five mice, their position range varied between 0 and 170 cm (Fig. 2). In general, the position range of the groups of mice was a minimal 20 cm during the period when selected Ta was the warmest and a maximum of 70 cm when selected Ta was the coolest. Motor activity varied inversely with selected Ta in both singleand group-housed animals (Fig. 3). There was a near linear correlation between these variables in both housing conditions. The slopes of the regression lines for young and aged mice were similar for both single- and group-housed mice. In most cases, motor activity averaged over the light and dark phases was significantly higher in the younger mice compared to the aged mice when housed individually or in groups (Table 1b). However, the difference in activity of individual young and aged mice during the dark phase was not significant. Motor activity of young and aged mice displayed robust elevations during the dark phase. The cage micro environment had marked effects on the heat loss of the “phantom” mouse (Table 2). Operative Ta was highest (30°C) when the “phantom” was buried in the wood shavings in a cage with a filter top and lowest (19.2°C) when placed on the bare acrylic surface with a wire top. The presence of a filter top resulted in consistently warmer operative Ta values compared to the wire
top. A layer of beta chip bedding resulted in operative Ta values that were ;2°C higher than Ta of the bare acrylic floor.
TABLE 2 EFFECT OF VARIOUS CAGE MICRO ENVIRONMENTS ON THE OPERATIVE Ta OF THE “PHANTOM” MOUSE IN A CAGE MAINTAINED AT A Ta OF 22°C. DATA REPRESENT MEAN 6 S.E. OF OPERATIVE Ta MEASURED IN THREE POSITIONS (CENTER, SIDE, AND CORNER OF CAGE) Cage conditions
Wood shavings; wire top; phantom on surface Wood shavings; wire top; phantom buried Wood shavings; filter top; phantom on surface Wood shavings; filter top; phantom buried Beta chips; wire top; phantom on surface Beta chips; filter top; phantom on surface No bedding; wire top No bedding; filter top No bedding; wire top; wire bottom No bedding; filter top; wire bottom
Operative Ta, °C
25.8 6 0.3 30.0 6 0.8 27.5 6 0.3 30.5 6 0.2 23.8 6 0.6 24.3 6 0.5 19.2 6 1.4 21.2 6 1.7 20.9 6 0.9 20.5 6 0.7
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DISCUSSION
One of the goals of this study was to compare the operative Ta selected by single- and group-housed mice. To the best of our knowledge, this has not been addressed in previous behavioral thermoregulatory studies. Single- and group-housed mice displayed a 24-h rhythm of selected Ta, preferring relatively warm Tas during the middle of the light phase concomitant with minimal motor activity. Cooler Ta values and higher levels of motor activity were exhibited during the early and late dark phase. A surprising observation is that the selected Ta of group-housed mice was just slightly below that of individual mice. Depending upon the time of day and number of animals housed in the gradient, the selected Ta is 228°C above the Ta used in typical animal facilities ('22°C) Single-housed mice normally acclimated to standard room temperatures of 22°C, when placed in a temperature gradient, prefer much warmer Ta values of approximately 30°C (2,3). The selected Ta of the mouse is approximately equal to its zone of thermoneutrality. Thus, mice appear to select Ta values that result in minimal metabolic expenditure. It follows that mice maintained at 22°C are probably subjected to varying amounts of cold stress. On the other hand, groups of mice are gregarious and frequently form a huddle during the day. The close body contact between mice in a huddle would lead one to suspect that thermal comfort of groups of mice under standard housing conditions is not compromised as with single mice housed at 22°C. The results of the present study show that group-housed mice remain in close contact during the light phase while selecting a Ta which was only ;1.0°C lower than that selected by single-housed mice. It is of interest to note that the optimal Ta for reproduction, development, and growth of mice maintained in standard acrylic cages with wood shavings ranges from 20226°C (11). It will be of interest to re-evaluate these Ta ranges in view of the thermal preferences of single- and grouphoused mice. For example, it would be important to determine if the mouse’s behavioral thermal preferences of single- and grouphoused mice match the Ta values for optimal reproduction and growth. The differences in selected Ta of young and aged mice may be explained by their patterns of motor activity. The younger mice tended to be more active for longer durations during the nocturnal phase as compared to the aged animals. Because the slopes of the correlations between motor activity and selected Ta were similar in the two age groups (Fig. 3), greater levels of activity in the younger mice would result in lowered selected Ta values as compared to the older, less active mice. The measurement of heat loss from the “phantom” mouse in various cage configurations suggests that the operative Ta and the thermal comfort of mice can be markedly affected depending on the method of housing (Table 2). These data allow a comparison between the operative Tas measured in the temperature gradient and that of standard methods for housing rodents. Wood shaving bedding appears to provide an optimal range of thermal environ-
ments for mice because they can remain on the surface or burrow into the wood shavings and change their operative Ta from 252 over 30°C when the cage is maintained under standard housing conditions of 22°C. Beta chip bedding, which is frequently used in animal housing facilities, does not provide mice or other species with as varied a thermal environment. That is, mice cannot burrow into the beta chip bedding to increase their operative Ta as occurs with wood shaving bedding. In addition, the beta chip bedding provides an increase in operative Ta of only ;2°C. Not surprisingly, wire screen floors provide little insulation. Wire screen tops reduce the operative Ta whereas filter tops increase the operative Ta. Comparing the operative Ta values with the “phantom” and real mice suggests that housing the mice on wood shaving bedding in an acrylic cage provides an ideal thermal environment, permitting a selection of thermal environments that can match the behaviorally regulated thermal preference. It is acknowledged that the aluminum cylinder is an extreme simplification of the heat loss of the mouse. However, we believe that these model data can provide an important framework for future studies in rodent thermal physiology in terms of coupling the operative Ta of the housing environment to the animal’s ideal zone of thermal comfort. ACKNOWLEDGMENTS
We thank Drs. Abraham Haim and Farhad Memarzadeh for their review of the manuscript. APPENDIX A-PRINCIPLES OF HEAT EXCHANGE
The exchange of radiant energy between an animal and its immediate surrounding (i.e., temperature gradient chamber) is determined by the temperature and emissivities of the surfaces of the animal and chamber (9). The relative size of surface areas between the organism and chamber is also critical. Basically, ideal radiant heat exchange is achieved when the surface area of the environmental chamber is relatively large in relation to the size of the animal. When the walls of the chamber have low emissivities and the chamber surface area is relatively small, radiant energy from the chamber walls is reflected back to the animal, resulting in a reduction in the organism’s net heat loss at a given air temperature. The surface area of the temperature gradient system is relatively small in relationship to the animal and constitutes a nonuniform thermal environment. It is constructed of materials with varying thermal emissivities (copper, wire-screen, Plexiglas) and has undefined patterns of air flow. Thus, it is important to determine an “operative” Ta that the animal encounters when placed in various positions in the gradient. Operative Ta is defined as “The temperature of a uniform (isothermal) ‘black’ enclosure in which a solid body or occupant would exchange the same amount of heat by radiation and convection as in the actual nonuniform environment” (8).
REFERENCES 1. Burger, G. T.; Miller, L. C. Animal care and facilities. In: Hayes, A.W., ed. Principles and methods of toxicology, 2nd ed. New York: Raven Press; 1989:521–552. 2. Eedy, J. W.; Ogilvie, D. M. The effect of age on the thermal preference of white mice (Mus musculus) and gerbils (Meriones unguiculatus). Can. J. Zool. 48:1303–1306; 1970. 3. Gordon, C. J. Relationship between autonomic and behavioral thermoregulation in the mouse. Physiol. Behav. 34:687– 690; 1985. 4. Gordon, C. J. Temperature regulation in laboratory rodents. New York: Cambridge University Press; 1993.
5. Gordon, C. J. Twenty-four hour rhythms of selected ambient temperature in rat and hamster. Physiol. Behav. 53:257–263; 1993. 6. Gordon, C. J.; Lee, K. A.; Chen, T. A.; Killough.; P.; Ali, J. S. Dynamics of behavioral thermoregulation in the rat. Am. J. Physiol. 261:R705–R711; 1991. 7. Hoffman-Goetz, L.; Keir, R. Body temperature responses of aged mice to ambient temperature and humidity stress. J. Gerontol. 39:547–551; 1984. 8. IUPS, 1987; Glossary of terms for thermal physiology, 2nd ed. Revised by The Commission for Thermal Physiology of the International
262 Union of Physiological Sciences IUPS Thermal Commission. Pflugers Arch. 410:567–587; 1987. 9. Porter, W. Thermal radiation in metabolic chambers. Science 166:15– 117; 1969. 10. Talan, M. Body temperature of C57BL/6J mice with age. Exp. Gerontol. 19:25–29; 1984.
GORDON ET AL. 11. Yamauchi, C., Fujita, S.; Obara. T; Ueda, T. Effects of room temperature on reproduction, body and organ weights, food and water intakes, and hematology in mice. Dikken Dobutsu. 32:1–11; 1983.
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Behavioral Thermoregulatory Responses of Single- and Group-Housed Mice1 CHRISTOPHER J. GORDON,2 PEGGY BECKER AND JOSEPH S. ALI Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA Received 29 January 1998; Accepted 29 April 1998 GORDON, C. J., P. BECKER AND J. S. ALI. Behavioral thermoregulatory responses of single- and group-housed mice. PHYSIOL BEHAV 65(2) 255–262, 1998.2The ambient temperature (Ta) to house and study laboratory rodents is critical for nearly all biomedical studies. The ideal Ta for housing rodents and other animals should be based on their thermoregulatory requirements. However, fundamental information on the behavioral thermoregulatory responses of single- and group-housed rodents is meager. To address this issue, thermoregulatory behavior was assessed in individual and groups of CD-1 mice housed in a temperature gradient. Mice were housed in groups of five or individually while selected Ta and motor activity were monitored. Single- and group-housed mice displayed a circadian oscillation of selected Ta and motor activity with relatively warm Tas of ;29°C selected during the light phase; during the dark phase selected Ta was reduced by 4°C, whereas motor activity increased. Selected Ta of aged (11 months old) mice housed individually was ;1.0°C warmer than the group-housed mice. Thermal preference of younger mice (2 months old) was similar for single- and group-housed animals. The operative Ta of mice housed in standard facilities was estimated by measuring the cooling rate of “phantom” mice modeled from aluminum cylinders. The results show that the typical housing conditions for single- and group-housed mice are cooler than their Ta for ideal thermal comfort. © 1998 Elsevier Science Inc. Behavioral thermoregulation
Motor activity
Animal housing
THE domesticated mouse (Mus musculus) has become one of the most widely used species in physiology, pharmacology, and toxicology investigations. The growing number of breeds and strains, development of genetic knockouts, and relatively low cost are some of the main reasons that have contributed to the popularity of the mouse as a test species. The ambient temperature (Ta) to house and study laboratory rodents is a critical issue in most biomedical studies (1,4). Ambient conditions (e.g., temperature, humidity, and wind speed) that stress the metabolic and other thermoregulatory effector systems of a species should be avoided. Thus, the Ta for housing the animals should be set, in most instances, at a level where stress is minimal. The optimal thermal environment is also an important issue in the construction and operation of animal housing facilities. The environment of animal housing facilities is generally designed around 1) the requirements for ideal thermal comfort of the personnel and 2) the limitations and costs for operating the air handling mechanisms of the facility (e.g., cooling, heating, and dehumidification). The thermoregulatory requirements of the animals have historically not been the primary factor in the design and operation of animal facilities. It is important to understand the ideal thermal environment of mice and other rodents. The Ta for housing mice has marked effects on most physiological and behavioral processes, such as
metabolism, motor activity, growth and development, food and water consumption, sensitivity to toxicants and other chemical agents, immune response, cardiovascular function, and many others (4,11). The ideal thermal environment for autonomic and behavioral thermoregulatory responses of individual mice has been well documented. For example, when placed in a temperature gradient permitting selection from a wide range of Tas, individual mice prefer a Ta of about 30 –31°C (3). This Ta range is equal to the mouse’s thermoneutral zone where metabolic rate and evaporative heat loss are near basal levels (3,4). Animal housing facilities and testing facilities are generally maintained at much cooler Ta values of around 22°C. This would lead one to expect that mice are subjected to cold stress when housed and/or tested in typical laboratory environments (e.g., Ta ' 22°C). However, mice are often housed in groups of five or more and, because they huddle and usually maintain close body contact throughout the day, it has been assumed that thermal comfort for groups of mice housed under standard conditions is not compromised. To evaluate these assumptions, it is necessary to compare the thermoregulatory behavior of single- and group-housed mice. Allowing animals to remain in a temperature gradient for several days has revealed that single-housed hamsters and rats display a circadian rhythm of selected Ta with relatively warm Ta values selected during the light phase and cooler Ta values selected during
1
This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 2 To whom correspondence should be addressed. E-mail: [email protected]
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the dark phase (5). Moreover, the selected Ta rhythm of individual rodents was found to increase at the same time as motor activity was reduced (4,5). It remains to be shown if groups of mice housed in a temperature gradient will exhibit a similar pattern of warm selected Ta values coupled with reduced motor activity during the light phase or will alter their thermoregulatory behavior because of huddling. A related issue is to determine the operative Ta selected by mice when allowed to behaviorally thermoregulate. Past studies from this laboratory and others (4), have not considered how the measurement of air temperature in a temperature gradient system reflects the operative Ta the animal encounters when housed in various cage systems. Furthermore, since metabolism and motor activity and other functions decline with age in mice and other species, measuring thermoregulatory behavior of young and aged mice allows the comparison of thermoregulatory behavior in the same species but with markedly different thermoregulatory requirements (4,7,10). Hence, there were several key objectives of the current study: 1) measure the thermoregulatory behavior of young and aged mice when housed individually or in groups in a temperature gradient; 2) determine the impact of motor activity on thermoregulatory behavior of single- and group- housed mice; and 3) to compare the thermal preferences of the mice to the thermal environments they encountered when housed in various laboratory cage systems, by calculating the operative Ta of the mice in a temperature gradient. MATERIALS AND METHODS
Animals used in this study were female mice of CD-1 strain obtained from Charles River Laboratories (Raleigh, NC, USA). The mice were obtained at 30245 days of age and housed in groups of 526 in standard acrylic cages with pine shaving as bedding material. Mice were maintained at a Ta of 22°C, relative humidity of 50%, and a 12:12 L:D photoperiod. Two age groups of mice were tested in this study; young mice were tested at 2 months of age (mean body weight 5 28 g) and another group of older mice were tested at approximately 11 months of age (mean body weight 5 52 g). Temperature gradient. The gradient construction, operation, and data analysis for the current experiments were modified slightly from earlier studies published by this laboratory (6). The gradient consisted of a copper tube 2 m length, 20.3 cm inside diameter, 0.63 cm wall thickness. The tube was split longitudinally and a hinge was secured on one side to allow the gradient to be opened for access and cleaning. One end of the gradient was heated, and the other end cooled by circulating water through copper tubing. A runway to house the animals in the temperature gradient was made of galvanized 1.3-cm wire-mesh on the sides and bottom, with a 0.31-cm thick Plexiglas top. A tray was placed beneath the runway to collect animal wastes. The spigot of a water bottle and a food dispenser were placed near the center of the runway. A series of low voltage lamps at 10-cm intervals were placed above the runway to illuminate the interior of the gradient (12:12 L:D photoperiod). Photocells were placed at 10-cm intervals along the length of the runway (total of 18 photocells in gradient) that served to track the position of the mice. A copper-constantan thermocouple was positioned immediately above each photocell. The thermocouples were placed within ;2 mm of the runway cage so that the temperature could be measured while preventing animals from interfering with the ancillary wiring of the photocells and thermocouples. The gradient had a temperature range of 12237°C. These gradient temperature were converted to operative Tas selected by the mice (see below). Data acquisition in the temperature gradient was altered from
previous studies so that more than one animal could be monitored simultaneously. All blocked photocells and their corresponding temperatures were recorded with a PC-based acquisition system (Dianachart) every 15 s. The time constant of data acquisition was increased to a value of 2 (normally set to 0 for larger species), assuring that if a mouse momentarily blocked a photocell its position would be more likely to be recorded. It should be noted that if two mice blocked one photocell at the same time, the system could not distinguish between one or two animals at that particular position. Protocol. An individual mouse or group of five mice were weighed and then placed in the runway at approximately 1500 hours with food and water provided ad lib. Data were continuously recorded for the next two nights. The mice were removed from the gradient at approximately 0800 hours, weighed, and returned to the animal facility. Thus, mice were left in the gradient for approximately 40 h. The first night in the gradient was considered to be an acclimation period. The 24-h period beginning from 0600 to 0559 hours the following day was used for statistical analysis. Data Analysis. The raw data from the gradient were stored in a ASCII format and then reprocessed to a new summary file. The position and temperature of each blocked photocell were determined. If more than one photocell was blocked, the temperatures at each blocked location were averaged and written to the summary file. The range of the positions of the blocked photocells (if more than one was blocked) was calculated and written to the summary file. The average of the position of the animal(s) was also determined. The change in average position at 15 sec intervals was calculated and stored in the summary file. The change in average position per unit time (i.e., m/h) was calculated, yielding a measure of motor activity. It is important to note that motor activity calculated for groups of mice is a measure of the movement of the group as a whole unit and is not a quantitative measure of the movement of individuals within the group. Thus, it is not possible to compare the levels of motor activity between single- and grouphoused mice in the gradient. However, the motor activity parameter does yield a qualitative measure of the group’s relative level of activity. The data in the summary file (i.e., 240 measurements per h) were averaged into 1-h bins for statistical analysis. The selected Ta and motor activity data were subjected to repeated measures ANOVA to determine the effect of time. Two-way ANOVAs were used to compare the single- and group-housed selected Ta values monitored over a 24 h period in the temperature gradient. Twelve hour means of the selected Ta during the light and dark phases were also calculated and compared with each age and housing condition using a two-tailed t-test. Calculation of operative Ta in temperature gradient. In past studies from this laboratory and others, the air temperature measured at the animal’s position within the temperature gradient was deemed the animal’s selected Ta or (i.e., thermopreferendum or preferred Ta). This assumption has been reevaluated in this study because it is recognized that, due to the complexities and nonlinearity of the temperature gradient, the measurement of air temperature is not necessarily equal to the animal’s operative Ta (see appendix A). The operative Ta was determined by comparing the rate of heat loss of a “phantom” mouse in the temperature gradient to the heat loss when maintained at fixed “black box” temperatures. The “phantom” mouse was constructed from aluminum cylinder stock (6.35 cm long 3 2.54 cm diameter; 60.8 cm2 surface area). Two brass legs made of 3.1-mm square stock were secured to the bottom of the cylinder to prevent movement during testing. A hole drilled through the center of the cylinder allowed for the insertion of a thermocouple to measure the internal temperature of the
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FIG. 1. Averages of selected Ta and motor activity 60-min time course of single- and group-housed young and aged mice plotted over a 24-h period. Data plotted as the mean 6 S.E. n 5 4. Horizontal bar indicates period of darkness.
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GORDON ET AL. TABLE 1a SELECTED Ta MEAN 6 S.E. OF FEMALE CD-1 MICE HOUSED INDIVIDUALLY OR IN GROUPS OF 5 IN A TEMPERATURE GRADIENT AS A FUNCTION OF TIME OF DAY SELECTED Ta
Period
light dark 24 h
Young
Aged
Individual
Group
Individual
Group
27.7 6 .3 26.5 6 .6 27.1 6 .3
27.4 6 0.2 26.2 6 .1a 26.8 6 .1
29.5 6 .3 27.6 6 .4a 28.6 6 .3b
28.4 6 .1 27.0 6 .1a 27.7 6 .1c
p , 0.01 compared to light phase for same age group; p 5 0.03 compared to young individuals over 24 h; c p , 0.01 compared to young group over 24 h. a
b
TABLE 1b MOTOR ACTIVITY Period
Young Individual
light dark 24 h
12.1 6 .5a 19.1 6 3.7 15.7 6 1.9b
Aged Group
30.1 6 2.4a 43 6 2.8a 36.6 6 1.2a
Individual
6.3 6 1 13.1 6 1.7 9.7 6 1.4
Group
17.2 6 1.4 30.3 6 1 23.8 6 1
a p , 0.01 or b p , 0.05 compared to aged mice at same time period; dark phase motor activity was significantly greater than light phase for all groups (p , 0.01).
cylinder (Tcyl). The cylinder was spray painted with flat black acrylic paint. The operative Ta was defined by measuring the cooling rate of the cylinder after being heated to a specified temperature and then being allowed to cool in a defined environment. The “defined environment” was arbitrarily selected where the heat from the “phantom” mouse would dissipate primarily by radiation and convection under still air conditions in an “ideal” thermal environment where the surface area of the surrounding environment was very large relative to the surface area of the “phantom”. The ideal thermal environment consisted of a box (35.6 3 35.6 3 35.6 cm) made from aluminum sheet metal. The ratio of surface area of the cylinder to that of the black box was 1:125. The inside of the box was spray painted flat black. Two thin supports made of 3.1-mm thick Plexiglassy were placed in the middle of the box that served to suspend the cylinder in the center of the box. The box was placed inside an environmental chamber that was maintained at Tas of 10236°C. Thermocouples measuring air (Tair) and wall (Twall) temperatures were placed inside the box. The box was not hermetically sealed; however, the enclosed box assured that no air turbulence such as from the fan of the environmental chamber would affect the convective heat loss of the “phantom” mouse. Tair and Twall of the black box and Tcyl were continuously recorded at 15-s intervals using a PC-based data acquisition system. The temperature of the environmental chamber was stabilized to one of several Ta values. The cylinder was placed in a plastic bag that was immersed in a water bath stabilized to 44°C. This temperature was selected because it was anticipated that it would
be approximately 5°C higher than the warmest temperature in the gradient. Care was taken to prevent moisture from contacting the surface of the cylinder. After the cylinder was heated uniformly to 44°C, it was quickly positioned in the center of the black box. Tcyl was recorded at 15-s intervals until it decreased below 38°C. Over a Tcyl range of 42239°C the change in temperature as a function of time was nearly linear with a coefficient of correlation that was consistently greater than 0.99. The rate of cooling over this specified temperature range was calculated with the cylinder placed in the black box and maintained at Ta values of 10236°C. A calibration curve of the mean black box temperature (i.e., Tair 1 Twall/2) versus the cooling rate of the cylinder was developed. The cooling rate of the cylinder was then evaluated in the temperature gradient. The cylinder was heated as described above and then placed lengthwise and equidistant between the two sides of the gradient runway such that the center of the “phantom” blocked the light from a photocell. Ta of the gradient where the cylinder was located along with Tcyl was recorded at 15 sec intervals. This procedure was performed at different positions in the gradient and a calibration curve of the gradient Ta versus cooling rate of the cylinder was calculated. Because the rate of cooling of the cylinder as a function of Ta in the black box and the gradient was linear, a linear formula could be used to convert the gradient Ta to operative Ta. The equation to convert gradient Ta to operative Ta was: operative Ta 5 0.84*gradient Ta-0.16. The slope and intercept will vary as a function of the size of the cylinder. For example, tests with a larger cylinder (12.7 cm length 3 5.08 cm diameter, 242.5 cm2) to mimic the approximate size of a laboratory rat yielded the following conversion: operative Ta 5 0.81*gradient Ta 1 2.5. With a much smaller cylinder (4.44 cm length x 1.9 cm diameter; 32.3 cm2) the conversion was: operative Ta 5 0.84*gradient Ta- 1.8. Additional experiments were performed to estimate the operative Ta encountered by individual mice when housed in a variety of laboratory cage environments. The “phantom” mouse was heated as described above and then placed in various locations of a standard acrylic rodent cage (46 cm length 3 24 cm width 3 20.3 cm depth) fitted with a standard wire top or filter top cover. The bottom of the cage was bare acrylic, wire-screen, layered with wood shavings, or layered with wood chips (“Beta-Chip”; laboratory grade, heat treated, hardwood animal bedding; Northeastern Products, Warrensburg, NY, USA). The mice used in the present study had been housed in cages layered with wood shavings. On the other hand, rodents are often housed in cages lined with beta chips or on wire-screen floors. The wire top was of a standard size designed to hold the food and water bottle. The filter top consisted of an acrylic frame with filter paper which is commonly used in most animal facilities. When testing the wire-screen bottom, care was taken to prevent air from blowing directly on the cylinder. All cages were tested in an environmental chamber maintained at a Ta of 22°C. The cylinder cooling rate was evaluated in three positions in the cage: 1) center; 2) side; and 3) corner. In the cage lined with wood shavings, the cylinder was either placed on the surface of the shavings or was covered with shavings to mimic the way mice burrow into this type of bedding. RESULTS
It is important to note that all selected Tas reported henceforth are operative Ta values. The selected Ta and motor activity of single- and group-housed mice displayed a 24-h rhythm (Fig. 1). At the start of the light phase, the selected Ta of young, singlehoused mice was a relatively low 24.5°C, which then rapidly increased to 29°C, concomitant with a reduced motor activity.
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FIG. 2. Selected Ta and position range time course of a group of five aged mice housed in a temperature gradient. Data points plotted at 15-s intervals represent the mean of selected Ta and range of the group of mice. Horizontal bar indicates period of darkness.
Three hours before the start of the dark phase, there was an abrupt rise in motor activity and a drop in selected Ta. Selected Ta decreased to 23°C in the middle of the dark phase and then increased over the next 4 h to 29°C. Groups of young mice showed a similar pattern, with a relatively low Ta of 24°C selected at the start of the light phase. Four hours before the dark phase, the selected Ta of the groups underwent a gradual reduction at the same time the motor activity increased. Selected Ta reached a minimum of 24°C during the middle of the dark phase and then rapidly increased over a 4-h period to 28.5°C. Two-way ANOVA analysis indicated no significant effect of single versus group housing on selected Ta of the young mice (Table 1a,b). Selected Ta of single-housed, aged mice reached a peak of 30°C at 1400 hours. In the transition from light to dark, the
selected Ta decreased abruptly by 1.5°C. During the dark phase, it reached its lowest point of 26°C at 0300 hours. The selected Ta of the aged, group-housed mice reached a peak of 29°C at 1300 hours. There was an abrupt decrease of 1.0°C during the last hour of the light phase. During the dark phase, the selected Ta of the group-housed mice reached a nadir of 24°C at 2000 hours, rose slowly for the next several hours and then decreased again as the end of the dark phase approached. Overall, single-mice in the aged group selected significantly warmer Tas than the group-housed mice: the selected Ta was 1.0°C warmer during the light phase and 1.5°C warmer during the dark phase [treatment, F(1, 7) 5 6.8, p 5 0.04; treatment-time, F(23, 138) 5 2.2, p 5 0.008] (Table 1a). The range in position of the group-housed animals was directly related to their motor activity. For example, in a typical time-
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FIG. 3. Motor activity and selected Ta correlation in single- and group- housed young and aged mice. Each data point represents average of selected Ta and motor activity averaged over 1 h of a 24-h cycle.
course for one group of five mice, their position range varied between 0 and 170 cm (Fig. 2). In general, the position range of the groups of mice was a minimal 20 cm during the period when selected Ta was the warmest and a maximum of 70 cm when selected Ta was the coolest. Motor activity varied inversely with selected Ta in both singleand group-housed animals (Fig. 3). There was a near linear correlation between these variables in both housing conditions. The slopes of the regression lines for young and aged mice were similar for both single- and group-housed mice. In most cases, motor activity averaged over the light and dark phases was significantly higher in the younger mice compared to the aged mice when housed individually or in groups (Table 1b). However, the difference in activity of individual young and aged mice during the dark phase was not significant. Motor activity of young and aged mice displayed robust elevations during the dark phase. The cage micro environment had marked effects on the heat loss of the “phantom” mouse (Table 2). Operative Ta was highest (30°C) when the “phantom” was buried in the wood shavings in a cage with a filter top and lowest (19.2°C) when placed on the bare acrylic surface with a wire top. The presence of a filter top resulted in consistently warmer operative Ta values compared to the wire
top. A layer of beta chip bedding resulted in operative Ta values that were ;2°C higher than Ta of the bare acrylic floor.
TABLE 2 EFFECT OF VARIOUS CAGE MICRO ENVIRONMENTS ON THE OPERATIVE Ta OF THE “PHANTOM” MOUSE IN A CAGE MAINTAINED AT A Ta OF 22°C. DATA REPRESENT MEAN 6 S.E. OF OPERATIVE Ta MEASURED IN THREE POSITIONS (CENTER, SIDE, AND CORNER OF CAGE) Cage conditions
Wood shavings; wire top; phantom on surface Wood shavings; wire top; phantom buried Wood shavings; filter top; phantom on surface Wood shavings; filter top; phantom buried Beta chips; wire top; phantom on surface Beta chips; filter top; phantom on surface No bedding; wire top No bedding; filter top No bedding; wire top; wire bottom No bedding; filter top; wire bottom
Operative Ta, °C
25.8 6 0.3 30.0 6 0.8 27.5 6 0.3 30.5 6 0.2 23.8 6 0.6 24.3 6 0.5 19.2 6 1.4 21.2 6 1.7 20.9 6 0.9 20.5 6 0.7
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DISCUSSION
One of the goals of this study was to compare the operative Ta selected by single- and group-housed mice. To the best of our knowledge, this has not been addressed in previous behavioral thermoregulatory studies. Single- and group-housed mice displayed a 24-h rhythm of selected Ta, preferring relatively warm Tas during the middle of the light phase concomitant with minimal motor activity. Cooler Ta values and higher levels of motor activity were exhibited during the early and late dark phase. A surprising observation is that the selected Ta of group-housed mice was just slightly below that of individual mice. Depending upon the time of day and number of animals housed in the gradient, the selected Ta is 228°C above the Ta used in typical animal facilities ('22°C) Single-housed mice normally acclimated to standard room temperatures of 22°C, when placed in a temperature gradient, prefer much warmer Ta values of approximately 30°C (2,3). The selected Ta of the mouse is approximately equal to its zone of thermoneutrality. Thus, mice appear to select Ta values that result in minimal metabolic expenditure. It follows that mice maintained at 22°C are probably subjected to varying amounts of cold stress. On the other hand, groups of mice are gregarious and frequently form a huddle during the day. The close body contact between mice in a huddle would lead one to suspect that thermal comfort of groups of mice under standard housing conditions is not compromised as with single mice housed at 22°C. The results of the present study show that group-housed mice remain in close contact during the light phase while selecting a Ta which was only ;1.0°C lower than that selected by single-housed mice. It is of interest to note that the optimal Ta for reproduction, development, and growth of mice maintained in standard acrylic cages with wood shavings ranges from 20226°C (11). It will be of interest to re-evaluate these Ta ranges in view of the thermal preferences of single- and grouphoused mice. For example, it would be important to determine if the mouse’s behavioral thermal preferences of single- and grouphoused mice match the Ta values for optimal reproduction and growth. The differences in selected Ta of young and aged mice may be explained by their patterns of motor activity. The younger mice tended to be more active for longer durations during the nocturnal phase as compared to the aged animals. Because the slopes of the correlations between motor activity and selected Ta were similar in the two age groups (Fig. 3), greater levels of activity in the younger mice would result in lowered selected Ta values as compared to the older, less active mice. The measurement of heat loss from the “phantom” mouse in various cage configurations suggests that the operative Ta and the thermal comfort of mice can be markedly affected depending on the method of housing (Table 2). These data allow a comparison between the operative Tas measured in the temperature gradient and that of standard methods for housing rodents. Wood shaving bedding appears to provide an optimal range of thermal environ-
ments for mice because they can remain on the surface or burrow into the wood shavings and change their operative Ta from 252 over 30°C when the cage is maintained under standard housing conditions of 22°C. Beta chip bedding, which is frequently used in animal housing facilities, does not provide mice or other species with as varied a thermal environment. That is, mice cannot burrow into the beta chip bedding to increase their operative Ta as occurs with wood shaving bedding. In addition, the beta chip bedding provides an increase in operative Ta of only ;2°C. Not surprisingly, wire screen floors provide little insulation. Wire screen tops reduce the operative Ta whereas filter tops increase the operative Ta. Comparing the operative Ta values with the “phantom” and real mice suggests that housing the mice on wood shaving bedding in an acrylic cage provides an ideal thermal environment, permitting a selection of thermal environments that can match the behaviorally regulated thermal preference. It is acknowledged that the aluminum cylinder is an extreme simplification of the heat loss of the mouse. However, we believe that these model data can provide an important framework for future studies in rodent thermal physiology in terms of coupling the operative Ta of the housing environment to the animal’s ideal zone of thermal comfort. ACKNOWLEDGMENTS
We thank Drs. Abraham Haim and Farhad Memarzadeh for their review of the manuscript. APPENDIX A-PRINCIPLES OF HEAT EXCHANGE
The exchange of radiant energy between an animal and its immediate surrounding (i.e., temperature gradient chamber) is determined by the temperature and emissivities of the surfaces of the animal and chamber (9). The relative size of surface areas between the organism and chamber is also critical. Basically, ideal radiant heat exchange is achieved when the surface area of the environmental chamber is relatively large in relation to the size of the animal. When the walls of the chamber have low emissivities and the chamber surface area is relatively small, radiant energy from the chamber walls is reflected back to the animal, resulting in a reduction in the organism’s net heat loss at a given air temperature. The surface area of the temperature gradient system is relatively small in relationship to the animal and constitutes a nonuniform thermal environment. It is constructed of materials with varying thermal emissivities (copper, wire-screen, Plexiglas) and has undefined patterns of air flow. Thus, it is important to determine an “operative” Ta that the animal encounters when placed in various positions in the gradient. Operative Ta is defined as “The temperature of a uniform (isothermal) ‘black’ enclosure in which a solid body or occupant would exchange the same amount of heat by radiation and convection as in the actual nonuniform environment” (8).
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GORDON ET AL. 11. Yamauchi, C., Fujita, S.; Obara. T; Ueda, T. Effects of room temperature on reproduction, body and organ weights, food and water intakes, and hematology in mice. Dikken Dobutsu. 32:1–11; 1983.