Ambient temperature limits and stability of temperature regulation in telemetered male and female rats

Pergamoo J. therm. Biol. Vol. 21, No. S/6, pp. 353-363, 1996

Published by Elsevier Science Ltd Printed in Great Britain.

PII: so306-4565(!X$ooo21-6 0306-4565/96 $15.00 + 0.00

AMBIENT TEMPERATURE LIMITS AND STABILITY OF TEMPERATURE REGULATION IN TELEMETERED MALE

AND FEMALE RATS

YING YANG’~* and CHRISTOPHER J. GORDON’* ‘Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S.

Environmental Protection Agency, Research Triangle Park, NC 27711, U.S.A. and 2Visiting scientist, Institute of Food Safety Control and Inspection, Ministry of Public Health, Beijing, China

(Received 23 March 1996; accepted 8 June 1996)

Abstract-Knowing the ambient temperature (2;) limits of normothermia in laboratory rodents is important because their thermoregulatory responses are useful in studies of physiology, pharmacology and toxicology. The present study assessed the T limits of normothermia using radiotelemetry to monitor core temperature (9, heart rate (HR), and motor activity (MA) in unrestrained, male and female Long-Evans rats over a 24 h period. Rats were housed individually in acrylic cages with wire-screen tops and bottoms and maintained at T’s ranging from 12 to 33S”C for 24 h with food and water provided ad libirum on a 12:12 L:D photoperiod. The limits of normothermia (i.e. where there was no significant change in Q were < 12-29.5”C for females and 14%29S”C for males. T of males at T’s of 12, 32, and 33.5”C increased significantly above the baseline T. Female rats had a lower z than males at the warmest and coldest T’s HR and MA were generally higher in females at all rs. Males appeared to be poorly adapted to thermoregulate at T’s above 30°C as based on their excessively high T’s, low MA, and marked weight loss compared to that of the females. Within the limits of normothermia the stability of T regulation (i.e. [(A?& / dYfJ x 100]/2) was + 1.3 and f 0.9% for males and females, respectively, over a 24 h period. These data on the stability of I; in the male and female rat provide a valuable framework to study the acute and chronic effects of drugs, chemicals and other agents that affect temperature regulation. Published by Elsevier Science Ltd.

Key Word Index: Temperature regulation; radiotelemetry; estrous cycle; ambient temperature; heart rate; motor activity; sex difference

INTRODUCI’ION

The thermoregulatory system has evolved as a means to maintain a stable body (i.e. core) temperature over a relatively wide range of ambient temperatures (x) as well as during exercise. The efficacy of temperature regulation in rodents is important to the biomedical disciplines of physiology, pharmacology, toxicology, nutrition and others (Zeisberger et al., 1994; Gordon et al., 1988; Gordon, 1993a; Clark and Clark, 1981; Watkinson and Gordon, 1993). That is, an altered body temperature of laboratory rodents is often used as an endpoint to determine the effectiveness and/or toxicity of many drugs, chemical toxicants, and other agents. Conventionally, a change in body temperature considered significant by most investigators was usually greater than 1 .O”C. There has been no attempt to comprehend the relevance of small, albeit statistically significant changes in body temperature

*To whom correspondence should be addressed.

in the rat and other species. In other words, should a 0.1, 0.5 or l.O”C change in core temperature be considered pathological, or does it fall within the range of ‘noise’ found in normal regulatory patterns?

To determine if a change in body temperature is significant, the stability of the thermoregulatory system when faced with heat and cold stress must be assessed. Our knowledge of the limits of normothermia in the rat comes from short-term studies where core temperature is usually measured with a colonic probe in animals exposed to a range of T’s for several hours (for review, see Gordon, 1993a). This approach has drawbacks because stress from the initial period of exposure, handling in a new environment, and repeated probing of the animals can lead to artifactual changes in body temperature (Gallaher et al., 1985; Kluger et al., 1987). Ideally, the limits of normothermia should be determined in animals that are adjusted psychologically to a novel environment (e.g. 3 h or more) but do not remain in the

353

354 Ying Yang and Christopher J. Gordon

altered ambient conditions for too long such that they acclimate physiologically. Moreover, because the stability of core temperature regulation may vary from day to night (Refinetti and Menaker, 1992), it follows that the limits of normothermia should be studied over a 24 h time period.

Radiotelemetry is a valuable tool to study thermoregulation and other systems because data can be collected 24 h per day from unrestrained animals in their home cages, thereby eliminating effects of handling and other sources of stress. Heart rate and motor activity, important physiological variables directly influenced by core temperature and T,, are also easily monitored with telemetry. The purpose of this study was to assess the K limits of normothermia and stability of core temperature as well as the effects of T, on heart rate and motor activity in male and female rats.

MATERIALS AND METHODS

Animals

Male and female rats of the Long-Evans strain were obtained from Charles River Laboratories (Raleigh, NC) at an age of 60 days. They were housed individually in the same room in acrylic cages lined with wood shavings at a T of 22”C, 50% relative humidity, and a 12:12 L:D photoperiod (lights on at 0600 h).

Surgery

Surgical implantation of the radiotelemetry unit to monitor core temperature, heart rate, and motor activity (Data Sciences, model TAl lCTA-F40) was performed using an aseptic technique. Briefly, the rats (n = 6 per sex) were anesthetized with sodium pentobarbital (-50 mg/kg; ip) and the abdominal area was shaved. The ECG leads of the transmitter were tunneled under the skin over the chest, and the body of the transmitter containing the temperature sensor was implanted into the abdominal cavity. The incision in the abdominal muscle was sutured with 4-O silk and the skin was closed with wound clips. Each rat was given 30,000 units of penicillin (im) following surgery. The temperature precision of the transmitters was determined prior to implantation and again after the animals were terminated. The transmitter calibration data provided by the manu- facturer was found to be within O.l”C of a NIST-traceable thermometer prior to implantation and after extraction from the rat.

Apparatus. The rats were allowed at least 10 days’ recovery from surgery before testing. Exposures were performed in a temperature-controlled cabinet

(Sure-Temp, Apex, NC) that regulated T to within + 05°C. Four animals (two of each sex) were tested at one time in the system. It was decided to test males and females simultaneously in the same chamber since they were housed in a similar manner in the animal facility. Each cage was positioned on a shelf with a fluorescent lamp positioned 26 cm from the center of the cage. The light intensity in the center of the cage varied depending upon orientation; 980 lx facing towards the light; 68 lx facing away from the light; 70 lx facing the back of the chamber; and 23 lx facing the front of chamber (mean intensity = 249 lx). The animals were housed in cages made of acrylic walls with wire-screen bottoms and tops. Wire-screen bottoms were used instead of wood shavings in order to prevent the animals from building nests which could alter the effective T, within the cage. Food and water were provided ad libitum throughout the test period. A plastic tray containing absorbant wood chips was placed beneath each cage to collect urine and fecal material. T, and relative humidity were continuously monitored inside the environmental chamber. Fresh air was vented into the chamber; dew point temperature was proportional to x, ranging from 8 to 15°C (Table 1).

Protocol

The rats were brought to the laboratory, weighed, and their telemetry units were activated by actuating a magnetic switch. The rats were placed in the environmental chamber at approximately 1500 h on the day before testing to allow psychological adaptation to the novel environment. It was estimated that 3-6 h would be needed to assure psychological adaptation as based on various reports in the literature (Singer et al., 1986; Gordon, 1993a). Core temperature, heart rate, and motor activity data were collected at 5 min intervals and stored on disk for later analysis (Gordon, 1993b). The z order of testing was 22,29.5, 14.5,32,26, 12, 33.5 and a repeat of 22°C. The animals were left undisturbed until 0800 h the day following testing. During the first test at 22°C the female’s core temperature was higher

Table 1. Ambient temperature (Q, relative humidity (RH), and dew point temperature (I&) of environmental chamber

T, “C RH, % ZprOC

12 82 8.8 14.5 66.4 8.2 22 60.4 13.9 26 42.5 12.2 29.5 35.8 12.8 32 33.5 13.8 33.5 33.8 15.3

Limits of temperature regulation in rats 355

than expected. This response turned out to be inconsistent with the subsequent T. tests and it was decided to use the repeat 22°C experiment in the statistical analysis of the data.

Body weight was determined before and after each T, exposure. Mean body weight over the 9 week test period increased from 409 to 526 g in males and 249 to 284 g in females. After weighing the females, the stage of the estrous cycle was determined by examining vaginal lavage fluids (Cooper et al., 1993). The rats were then returned to the animal facility and were not tested again for at least 6 days.

A follow-up study was performed with the female rats housed in their home cages in the animal facility to determine the effect of the estrous cycle on the telemetry data. The stage of the estrous cycle was determined daily at 0900 h, after which the females

39.0

were returned to cages with clean bedding material. Core temperature, heart rate, and motor activity were recorded as described above. This procedure was repeated for 7 days to assure that each rat had completed at least one estrous cycle.

Data analysis

For most of the analysis the 5 min telemetry data were averaged over 1 h intervals. Twelve hour averages of responses during the light and dark phases were also calculated for statistical analysis. These data were then subjected to repeated measures analysis of variance (ANOVA) to determine the effect of x;;, time of day, and sex on each of the telemetry variables (GB-Stat, Silver Spring, MD). Significant ANOVAs were followed with Tukey’s protected t-tests. Plots of the 5 min measurements of core

38.5

38.0

o T, = 33.W

?? T, = 32.O”C

A T, = 29.5”C

A T, = 26.O”C

0 T, = 22.O”C

?? T, = 14.5”C

?? T, = 12.O”C

6am 12N 6pm

Time (hr)

Fig. 1 (A). Caption overleaf.


temperature were also made to determine the minimum and maximum temperatures over 4 h time periods at each I;;. The minimum and maximum temperatures were averaged into light and dark phase groups and then analyzed with repeated measures ANOVA. Most statistical analyses of T, were made in reference to a x of 22°C which reflects the K to which the rats were housed in the animal facility. In the follow-up study, the 24 h measurements for each telemetry variable were categorized according to the stage of the estrous cycle and then subjected to repeated measures ANOVA as described above.

RESULTS

The time-course of core temperature over a 24 h period showed a qualitatively similar pattern at all z ‘s between sexes (Fig. 1A). The regulation of core

(B)

temperature was unaffected at T’s of 14.5-29.X. Over this range of z ‘s core temperature at the start of the light cycle was slightly elevated and then decreased, reaching a nadir at approximately 1200 h. At this point there was a gradual elevation of g O.l”C/h over the next 5 h. An abrupt 0.X elevation then occurred during the transition from light to dark. This pattern was significantly different at the warmest and coldest T, ‘s. At the warm K ‘s of 32 and 33S”C, there was a parallel increase in core temperature during the light and dark phases, the shift being especially marked in the male rats (Fig. 1A). At the cold T of 12°C core temperature remained elevated during the early phase of the light cycle, decreased to a nadir similar to that of the other z’s at 1200 h, and then underwent a gradual increase over the latter part of the light phase. Five out of six male rats and all female rats were able to regulate a

I I I I

male 0 T, = 33.K

m T, = 32.O”C

A T, = 29.YC

A T, = 26.0°C

0 T, = 22.O’C

?? T, = 14.5’C

0 Ta = 12.O”C

6am 12N 6pm

Time (hr)

Fig. 1 (B).

6am

Limits of temperature regulation in rats

I , I I male

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0

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4 T, = 26.0°C

0 T, = 22.O”C

??T, = 14S”C

* T, = 12.O”C

6am 12N 6pm

Time (hr)

6am

Fig. 1. Time-course of core temperature, heart rate, and motor activity in unrestrained male and female rats maintained at Ts of 12 - 33S”C. n = 6 per group for core temperature and motor activity with exception to males at T of 32 and 33S”C where n = 5. Horizontal bar indicates period of darkness.

stable core temperature at the highest To. On the other hand, the core temperature of one male remained at z 40°C over the course of the light phase at a T of 33S”C. This animal was removed from the chamber because it was not certain that it would survive the full 24 h period of warm T exposure.

Averaging the telemetry variables over the light, dark, and 24 h periods facilitates comparison of effects of T, and gender (Fig. 2A; see appendix for statistics). When compared to a z of 22°C the core temperature of male rats was significantly elevated at T’s of 12, 32, and 33S”C; core temperature of females was also higher at the two warmest cs but was unaffected at the coldest z. The rise in core temperature of females was significantly less than that of males at the two warmest rs. At every I;; tested the 24 h mean core temperature of females

was below that of the males; however, the only significant gender differences were at the two warmest and coldest T,‘s.

At most of the T,‘s heart rate was minimal at 1200 h followed by a gradual elevation over the remainder of the light phase (Fig. 1B). Heart rate was markedly dependent on z (Fig. 2B). Significant elevations and reductions in heart rate were noted at rs below and above 22°C respectively. The heart rate of females was significantly higher that that of males at T,‘s of 22 and 26°C. Motor activity was increased in males and females at the colder T,‘s of 12 and 14S”C (Fig. 2C). There was a marked gender difference in motor activity at a T, of 12°C. Attenuation of the nocturnal elevation in motor activity was noted in males housed at the two warmest T’s

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Minimum/maximum core temperatures

Maximum core temperature (X - max) was similar in both sexes at K’s of 26 and 29.5”C (Fig. 3). I;: - max was significantly higher in males at E’s < 22°C and > 32°C. r - max during the light phase at the warmest E of 33.5”C was nearly l.O”C higher in males. The minimum core temperature (x - min) was the same in both sexes at a T, of 29.5”C in the light phase; in the dark phase the r - min of females was consistently below that of males at all z’s. z - min was nearly l.O”C higher in males at the warmest z. Two-way repeated measures ANOVAs for sex, T,, and their interactions were significant factors on the z - min and r - max parameters (P < 0.001 in all tests).

Body weight

Male rats experienced significant reductions in body weight of 26.8 g (6%) and 40.5 g (8.3%) after exposure to K’s of 32 and 33.5”C (Fig. 4). Body weight was unaffected by T. in the female rats.

Estrous cycle

The stage of the estrous cycle was determined in females after exposure to each T,. In a preliminary analysis, core temperature and motor activity over a z range of 14.5 to 29.5”C were sorted based on the stage of estrous. Core temperature was elevated by

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36.5 1 10 I5 20 25 30 35

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Fig. 3. Mean &- S.E. of the maximum and minimum core temperatures during the light and dark phases. Sample sizes

are the same as Fig. 1.

0 male

-50 ’ ’ 12 14.5 22 26 29.5 32 33.5

Ambient temperature (“C)

Fig. 4. Effect of z on the change in body weight in male and female rats. Samples sizes same as Fig. 1. *indicates significant difference between males and females

at given T.

z 0.4”C during the dark phase of the last 5 h of proestrus compared to other stages of the estrous cycle (data not presented).

In a another study with the same female rats, heart rate, motor activity and core temperature were grouped as a function of the estrous cycle at a T, of 22°C (Fig. 5). During the night, motor activity was relatively stable during metestrus, diestrus, and estrus; however, there was a marked elevation during proestrus. Core temperature at night was approximately 0.2”C higher during proestrus as compared to diestrus. Core temperature during the night of estrus was also significantly higher than during diestrus. Heart rate during the night was unaffected by the stage of the estrous cycle. Core temperature during the light phase was also unaffected by the stage of estrous. However, it should be noted that the elevation in core temperature resulting from daily cage change and vaginal lavage technique probably eliminated the ability to detect a subtle difference caused from the estrous cycle.

DISCUSSION

A main goal of this study was to determine the ambient limits of normothermia for the unrestrained male and female rat over a 24 h period. The mean core temperature during the light and dark phases was stable over a T, range of 14.5-29.5”C in the male rat and < 12-29.5”C in the female rat. The group mean core temperature varied by only 0.04 and 0.07”C in male and female rats, respectively, over their T. ranges of normothermia. On the other hand, the mean variation in core temperature of individual animals was greater, varying from 0.28 to 0.46”C, depending upon the time of day and gender. Assuming that the thermoregulatory system’s


principal function is the regulation of core temperature over a wide range of x’s, the stability of the system can be expressed with a simple calculation of the percent change in core temperature divided by the change in K: stability ( + %) = ((d core temperature/ A K)* 100)/2

Thus, when exposed to a continuous 24 h warm or cold z challenge that does not exceed the limits of normothermia, the rat thermoregulatory system regulated core temperature to within + 0.9 to f 1.3% in female and male rats, respectively, when stability is expressed in terms of the 24 h mean of core temperature in individual animals (Table 2).

a

37.8 _

.z ‘”

0 80-

z 70-

Estrous cycle stage

Fig. 5. Effect of the stage of the estrous cycle on the core temperature, heart rate, and motor activity of female rats during the dark phase. Rats housed at a T of 22°C in cages lined with wood chip bedding. Abbreviations: M, metestrus; D, diestrus; P, proestrus; E, estrus. Abbreviations: a, significantly different from all other stages of estrous cycle; b, significant compared to diestrus. n = 6 for metestrus, 15

for diestrus, 7 for proestrus, and 10 for estrus.

Heart rate is generally proportional to metabolic demands and thus varied inversely with z. Motor activity was also affected by K with marked reductions when exposed to z’s above thermoneutrality, presumably a response to lower heat production and avoid elevations in core temperature. While core temperature was relatively stabile over a T range of 14.S29S”C, heart rate and motor activity were more variable. That is, over a T, range of 14.S29.5”C, heart rate varied by 26% for both males and females; motor activity varied by 65 and 3 1% for males and females, respectively.

The upper z limit of thermoregulatory control was clearly discerned in this and other studies (Gordon, 1993a). It would appear that thermoregulatory control in the rat ‘breaks down’ within a narrow range of warm K’s, On the other hand, it is difficult to ascertain a z where core temperature is significantly below baseline because rats are relatively well adapted to resist cold exposure. It is likely that at least several days of exposure at a given cold T, are required before a significantly reduced core temperature is detectable.

The female rat’s core temperature was below that of males and was more stable. Gender differences in temperature regulation were more delineated in the measurement of r - min and 7; - max. Past studies have found female rats to have higher metabolic rates and body temperatures compared to males (Marrone et al., 1976; Castella and Alemany, 1985; Fujii and Ohtaki, 1985); however, these studies employed relatively brief periods of monitoring using colonic probes. Telemetric monitoring eliminates many sources of stress which cause artifactual elevations in temperature. The better stability of body temperature in females when exposed to warm T’s may be attributed to their smaller body size. The smaller mass of the females provides a greater surface area/body mass ratio. Hence, females have a higher metabolic rate and heart rate compared to males and are also expected to have a higher zone of thermoneutrality (for review, see Gordon, 1993a). A higher thermoneutral zone should be associated with an elevation in the upper T, limit of normothermia. In future studies, the measurement of heat loss and heat production in male and female rats at warm T,‘s should be made to verify this hypothesis. It will also be of interest to compare the thermal stability of ‘weight-matched’ male and female animals to clarify the role of gender on thermal stability.

The significant weight loss and higher core temperatures of male rats at T,‘s of 32 and 33~5°C suggests that females rats were better suited to maintain thermal homeostasis at the warmer z’s This may be attributed to differences in gonadal


Table 2. Mean + S.E. of the individual variations in core temperature averaged over the light phase, dark phase, and 24 h period over a 1; range either exceeding or within the limits of normothemria of male and female rats

Period of analysis

Sex T. range, “C Light Dark

Male 12-33.5 1.38 + 0.29”C 1.18 f 0.27”c (exceed limits) ( f 3.2%) ( + 2.7%)

Female 12-33.5 0.86 + 0.15”C 0.87 + 0.14”c (exceed limits) ( + 2.0%) ( + 2.0%)

Male 14.5-29.5 0.46 + 0.09”C 0.39 + 0.09”C (within limits) (& 1.5%) ( f 1.3%)

Female 12-29.5 0.31 f 0.08”C 0.40 + 0.04C (within limits) (* 1.0%) ( * 1.3%)

Numbers in parentheses represent stability of thermoregulatory control (see Discussion).

24 h

1.26 f 0.28”C ( f 2.9%)

0.79 + 0.16”C ( f 1.9%)

0.39 &- 0.05”C ( f 1.3%)

0.28 k 0.05”C ( + 0.9%)

function. Administering estrogen to ovariectomized Sprague-Dawley (SD) rats improves their ability to lose heat by evaporation and regulate a lower core temperature during exposure to acute heat stress (Baker et al., 1994). Female SD rats displayed a greater resistance to acute heat stress as determined by their survival time to exposure to a T, of 425°C (Furuyama, 1982). This contradicts an earlier finding that female rats were less capable of thermoregulating at an extremely warm T, of 44°C (Hainsworth, 1967). Interestingly, saliva spreading to increase evaporative heat loss was initiated at a T, of 32°C in male SD rats but at a x of 36°C in females (Hainsworth, 1967), suggesting that female rats are less sensitive to the initial effects of heat stress. Other studies have shown that estrogen-treated, ovariectomized rats have a lower body temperature (Wilkinson et al., 1980); however, estrogen can have complex effects on heat production and heat loss which are difficult to interpret (Laudenslager et al., 1982). Furthermore, while estrogen may be considered an agent to lower core temperature, progesterone is considered to stimulate thermogenesis (Freeman et al., 1970).

At the coldest z of 12°C the core temperature of male rats increased above basal levels while that of females was stable. Cold-induced elevation in core temperature may result from an overcompensation of autonomic thermoregulatory effecters (Morimoto et al., 1986; Watkinson et al., 1995). Such an overcompensation would be expected over a period of a few hours, but it is surprising to find that male rats maintained the elevated core temperature in the cold for over 24 h. Female rats have an attenuated thermogenic response to norepinephrine, a response reversed by ovariectomy (Doi and Kuroshima, 1982). Male rats have been shown to have greater shivering response to cold exposure compared to female rats when exposed to a & of 5°C. It is possible that differences in vasomotor tone in male and female rats may be responsible for the differences in core

temperature during cold exposure. In a thorough study, Herrington (1940) found that the colonic temperature of rats measured 6 h after exposure to a wide range of K’s was minimal at a x of 25°C and increased sharply at T,‘s above 28°C. As T, was lowered below 25°C core temperature peaked at a T, of 17.5”C then decreased as T, was lowered to 13°C. It is not clear why female rats did not show the cold-induced rise in core temperature. With telemetric monitoring of core temperature, we found better stability and a greater range of normothermia compared to that of Herrington (1940).

A concern of this study was what impact the stages of the estrous cycle would have on the comparison of thermoregulatory responses between male and female rats. The thermoregulatory pattern of female rats on running wheels is modulated as a function of the estrous cycle with a 0.26”C higher core temperature (monitored by telemetry) and higher wheel running activity on the night of proestrus (Kent et al., 1991). During the night between proestrus and estrus, core temperature measured by rectal probe was 0.54”C higher compared to the other stages of the estrous cycle (Yanase et al., 1989). In rats maintained at T,‘s of 14.5-29.5”C, we found core temperature during the last 3 h of the dark phase in proestrus to be x 0.5”C higher relative to the other stages of estrous cycle. In rats maintained at 22°C in the follow-up study, motor activity was 60% higher during the night of proestrus and there was a 0.2”C elevation in core temperature. The presence of a running wheel appears to accentuate the effects of motor activity on core temperature during proestrus (Kent et al., 1991). Overall, the stage of estrous appears to have little effect on accuracy of the thermoregulatory system during the day. On the other hand, a 0.2”C elevation in the dark phase core temperature during proestrus is relatively large compared to the variation in core temperature (0.4”C) over the T, limits of normothermia (cf. Table 2).


To summarize, it is important to document the stability of thermoregulation as a function of T,, time of day, and sex of the animal. Within the range of normothermia (E = 14.5-29.5”C), the core temperature of individual rats varied by approximately + 1.0% (Table 2). These stability data provide a valuable framework to study the acute and chronic effects of drugs, chemicals, and other agents that affect thermoregulation. For example, treatment with a drug which causes a l.O”C reduction in core temperature as T, is changed from 30 to 20°C (i.e. a 10% change in accuracy of regulation) is relatively large compared to the normal stability of core temperature reported here. Acute drug and chemical treatments lead to marked deviations in the accuracy of thermoregulatory control which easily exceed 10% (Clark and Clark, 1981; Gordon, 1993a). Chronic drug treatments or pathological sequelae leading to permanent alterations in core temperature regulation are beginning to be assessed in rodents. For example, we have recently found that the offspring of pregnant rats exposed to dioxin regulate core temperature at approximately 0.5”C below that of controls (Gordon et al., 1995). Considering the temperature stability data of the present study, it is clear that dioxin’s effect on the core temperature is quite marked.

A9-THC, CNS depressants and stimulants, hormones, inorganic ions, gases, 2,4-DNP and miscellaneous agents. Neurosci. Biobehav. Rev. 5, 1-136.

Cooper, R.L., Goldman, J.M., and Vandenbergh, J.G., 1993, Monitoring of the estrous cycle in the laboratory rodent by vaginal lavage. In: Methods in Toxicology, Vol. 3B, pp. 45-56, Academic Press, New York.

Doi K. and Kuroshima A. (1982) Sexual difference in thermoregulatory ability of rats exposed to cold and heat. J. Therm. Biol. 7, 99-105.

Freeman M. E., Crissman J. K. Jr, Louw G. N., Butcher R. L. and Inskeep E. K. (1970) Thermogenic action of progesterone in the rat. Endocrinology 86, 717-720.

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Acknowledgements-We thanks Drs P. Rowsey and W. P. Watkinson for their critical review of the manuscript.

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APPENDIX A

Table Al. Repeated measures ANOVA results of light, dark, and 24 h analysis of core temperature, heart rate, and motor activity

Parameter Time period

Sex F value

T Sex-z Sex T Sex-T Probability

Core temperature Core temperature Core temperature Degrees of freedom Heart rate Heart rate Heart rate Degrees of freedom Motor activity Motor activity Motor activity Degrees of freedom

Light Dark 24 h

Light Dark 24 h

Light 1.6 Dark 4.3 24 h 3.3

12.7 13.6 18.9 1,lO 11.5 7.1 9.5

16.2 2.4 17.5 1.5 13.9 1.3 6,60 6,60 164.1 19.1 64.8 14.5 106.4 16.7 1,lO 7,70 8.2 3.1 6.7 3.4 9.1 3.8 1,lO 7,70

0.0052 < 0.0001 0.04 0.0042 < 0.0001 0.2 0.0015 < 0.0001 0.25

0.0068 0.023 0.011 7,70 0.22 0.6 0.09 7,70

< 0.0001 < 0.0001 < 0.0001

< 0.0001 0.0066 < 0.0001 0.0034 < 0.0001 0.0016

< 0.0001 < 0.0001 < 0.0001

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Ambient temperature limits and stability of temperature regulation in telemetered male and female rats