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The effects of short and prolonged cold exposure on arousal in the free-tailed bat, Tadarida brasiliensis cynocephala (Le Conte)
Camp. Biochem. Physiol., 1972, Vol. 42A, pp. 559 to 567. Pergomon Press. Printed in Great Britain
THE EFFECTS OF SHORT AND PROLONGED COLD EXPOSURE ON AROUSAL IN THE FREE-TAILED BAT, TADARIDA BRASILIENSIS CYNOCEPHALA (LE CONTE) JOHN
F. PAGELS”
Department of Biology, Tulane University, New Orleans, Louisiana 70118 (Received
3 September
1971)
Tadarida brasiliensis cynocephala (Le Conte) was resident in New Orleans, Louisiana, in the winters 1967-68 and 1968-69. The bats fed periodically during winter months; however on cold days, the bats were inactive and often torpid. 2. Laboratory studies to determine the effects of variable periods of cold exposure on the ability of Tadarida to rewarm to flight temperature demonstrated that the total number of warmups plays an important role in the animal’s ability to arouse to flight temperature on subsequent attempts. 3. The effect of warmups on successive evenings was observed to be greater than the effects of prolonged cold exposure. 4. In all arousal experiments, the body weights of animals that rewarmed were greater than the body weights of animals that did not rewarm. 5. The total amount and proportions of interscapular brown fat were greater in animals that rewarmed than in animals that did not rewarm.
Abstract-l.
INTRODUCTION
ALTHOUGH the western form of the Mexican free-tailed bat, Tadarida bra.siZiensis mexicana, is migratory (Villa & Cockrum, 1962), the eastern subspecies, Tadarida brasiliensis cynocephala, is resident in New Orleans, Louisiana. In the absence of caves in New Orleans, Tudarida roosts in homes, warehouses and other suitable structures. Winter temperatures in New Orleans are quite variable and (within a single winter month) ambient temperatures as low as freezing and as high as 26°C are not uncommon. The daily range of maximum-minimum temperatures in the winter is also relatively great in New Orleans. Because of the nature of the roost sites the bats are often directly exposed to changes in ambient temperatures. It is not uncommon to find Tadarida in a torpid condition with a very low body temperature on cold winter days. The torpid condition apparently occurs infrequently in the cave-dwelling migratory form, T. b. mexicana. Herreid (1963a) found that T. b. mexicana maintained a resting body temperature between 32 and 42°C when cave temperatures ranged from 12 to 32°C. When exposed to low temperatures in the laboratory T. b. mexicana in a postabsorptive state dropped to near ambient * Present
address : Department
of Biology,
Richmond, Virginia 23220. 559
Virginia
Commonwealth
University,
560
JOHN
F. PAGELS
temperature in several hours (Herreid, 1963b). Herreid (1963~) also noted that a reduction in body temperature does increase survival of Tadarida, but that survival was not as great as in hibernating forms placed under similar conditions. During cold exposure in the laboratory some T. b. mexicana roused spontaneously from low body temperatures similar to hibernating forms while others did not (Herreid, 1963d). The objective of this study initiated in early fall, 1968, and terminated in midwinter was to determine the effect of short and prolonged cold exposure on the ability of T. b. cynocephala to arouse from hypothermia to flight temperature. The body weights and amounts and proportions of interscapular brown fat in relation to length of exposure and arousability were also considered. MATERIALS
AND METHODS
Bats used in the arousal studies were collected at a warehouse in downtown New Orleans. Bats were always collected in the morning. The animals roosted between a steel “I” beam and the brick wall of the warehouse. With the use of an extension ladder placed on the dock, bats could be easily reached and extracted from the narrow roost area with a pair of long forceps. For cold exposure in each experiment, the bats were put in pint cardboard cans and placed in a cold unit set at 6-7°C. Rectal temperatures were recorded with a YSI (44TD) thermistor tele-thermometer. On 30 September 1968, eighteen Taduridu (twelve females, six males) were collected. The bats were weighed and divided into three groups of four females and two males each and placed in the cold chamber. After 36 hr, one of the groups (Group 1) of six was removed from the cold chamber and placed at room temperature. Immediately upon removal from the cold chamber, the rectal temperatures of the bats were recorded. For the first 2 hr, rectal temperatures were recorded at O-S-hr intervals. After 1.0 additional hr, the temperatures were again recorded. The six animals were then weighed and returned to the cold chamber. On the second evening, 2 October, the first group of six and a second group of six (Groups 1 and 2) were removed from the cold chamber and the aforementioned procedure was repeated. On the third evening, 3 October, the first, second and last groups (Groups 1, 2 and 3) of six bats were removed from the cold chamber and the procedure was repeated. On 4 October, all three groups were again removed and the procedure repeated for the last time. The animals were sacrificed and weighed on 5 October. The percentage that reached flight temperature was calculated for each group. I found from numerous laboratory and field samplings of rectal temperatures and corresponding flight abilities that 30°C represents the lowest body temperature at which T. b. cynocephala is able to fly in a maneuverable fashion. Hence 30°C was used in all calculations involving flight temperature. Herreid (1963a, b) reported body temperature necessary for flight of T. b. mexicuna as 3l”C, or (Herreid, 1963d) “about 31°C” (29-32°C). On 11 November, thirty female Tudurida were collected. Ten bats were killed immediately, weighed and examined for the presence and development of interscapular brown fat. The twenty other bats were weighed and placed in the cold chamber. The next evening, 12 November, all twenty animals were removed from the cold chamber and the rectal temperatures were recorded immediately. The temperatures were again recorded at 0.5, l-0 and 3.0 hr after removal from the cold chamber. As in all experiments I handled the bats in the same order each time that the temperatures were measured. All bats were then weighed and returned to the cold unit. This procedure was repeated on each evening through 18 November. Rectal temperatures were not measured after 2f hr on 17 November. At the end of the 3-hr period on 18 November the bats were killed, weighed and the interscapular brown fat was removed and weighed. It has been demonstrated that brown fat provides
THE
EFFECTS
OF COLD
EXPOSURE
ON
AROUSAL
IN
THE
FREE-TAILED
BAT
561
quick energy to animals during arousal from low body temperature (Smalley & Dryer, 1963 ; Smith & Hock, 1963; Hayward et al., 1965; Bruce & Wiebers, 1966; Hayward Sz Lyman, 1967; Hayward, 1968). The percentage of bats in the second experiment that reached flight temperature on each evening was calculated. The third warmup experiment was initiated to compare the effect of prolonged cold exposure as opposed to the nightly warmups in the first two studies. On 27 November, twenty-nine female Tadarida were collected. Ten animals were killed immediately, body weight was recorded and interscapular brown fat was removed and weighed. The other nineteen bats were weighed and placed in the cold chamber. On the evening of 1 December, nine of the bats were removed from the cold chamber and the rectal temperatures were recorded immediately. Rectal temperatures were subsequently recorded 0.5 and 1 .O hr after initial removal from the cold chamber. In this experiment temperatures were not recorded later than 1-O hr after removal, since the earlier experiments demonstrated that bats that were going to rewarm to flight temperature did so in the first hour. Before returning the bats to the cold unit they were weighed. On 5 December the first nine bats, plus the other ten were removed from the cold unit and the procedure of 1 December was repeated. This procedure was carried out on the evenings of 1, 5, 8, 12, 15, 19 and 22 December. After the regular procedure was completed on 22 December, the bats were killed, total weight recorded and the interscapular brown fat was removed and weighed. The percentage that reached flight temperature on each date was calculated. On 6 January 1969, twenty-eight female Tadarida were collected for a fourth warmup experiment. Eight bats were killed immediately, body weight was recorded and the interscapular brown fat was removed and weighed. The other twenty bats were placed in the cold unit. Ten of the bats were removed each evening from 7 January through 15 January. Except for the number of days between warmups, the same procedure was used for this experiment as in the previous study. The other ten bats were taken out of the cold unit on each night only to record body weights. On the evening of 15 January all twenty bats (nine warmups and one warmup) were removed from the cold unit and given an opportunity to rewarm. At the end of the procedure the bats were killed, weighed and interscapular brown fat was removed and weighed. In addition to determining SE., experimental groups were compared by means of unpaired t-tests. A 5 per cent level of significance was used in all statistical tests. RESULTS
The percentage of animals that reached flight temperature in each of the four warmup experiments is given in Fig. 1. In the early October experiment the percentages are the same in each group based on the number of times the animals had been checked for warmup ability, and the percentages apparently are not related to the length of time the bats had been in the cold chamber. For example, all bats in each group reached flight temperature on the first attempt, whether first taken from the cold unit on 1, 2 or 3 October. In November, after the second warmup the percentage of bats that reached flight temperature on successive days began to decline. The bats were sacrificed on the eighth day of cold exposure after the seventh warmup attempt when 55 per cent of the animals reached flight temperature. In the December experiment with 3- to 4-day intervals between warmups and 25 days of cold exposure, the percentage of bats that reached flight temperature on the seventh attempt was the same as in the seventh attempt of the November experiment, 55 per cent.
562
JOHN
F.
PACELS
In the January experiment, all ten bats removed from the cold unit on successive evenings reached flight temperature through the sixth evening. Fifty per cent of the animals reached flight temperature on the eighth warmup. On the ninth evening, a bat reached flight temperature that had not done so on the previous I
Group
2
100
50
0
llnll_L -
u_bL
I
2
3
4
2
3
4
3
4
October
2 ,”
50
z
0
::
I2
13
14
15
16
17
I8
November
I
8
5
12
15
19
22
13
14
December 100
50
0 7
8
9
10
II
January
FIG.
l-Percentage
12
I5
15 0llly
of bats that reached flight temperature on each date in each experiment.
evening. Of the seventy-seven bats exposed to cold in the four warmup experiments, this was the only animal that warmed to flight temperature on an evening subsequent to an evening on which it had not. Eight of the ten animals checked One of the two that did not reonly on 15 January reached flight temperature. warm was dead. Data on the body weights of bats at the initiation and end of the last three experiments are given in Table 1. Note that the initial body weights of the bats collected in early November (November experiment) and in late November (December experiment) were very similar, and that the animals collected in January weighed considerably less. In each of the three experiments, the final body weights of the animals that did not rewarm on the last check were less than
THE EFFECTS OF COLD EXPOSURE ON AROUSAL TABLE
~-RELATIONSHIPS TO
IN THE FREE-TAILED BAT
563
BETWEEN BODY WEIGHTS (g) AND THE ABILITY OF BATS REWARM TO FLIGHT TEMPERATURE
Mean body weight No. of warm-ups
7
6-7
9
Days between warm-ups
Start of experiment
End of experiment
Warmed up last time
Did not warm up last time
Started 11 November 1968 3 of total: 16.2 11.9 16.5 (0.27) 12.1 (0.28) 15.8 (0.36)
11.7 (0.20)
Started 27 November 1968 3-4 P of total: 15.8 10.9 16.2 (0.29) 11 *o (0.07) 15.2 (0.52)
10.7 (0.35)
1
1
15 January only
Started 6 January Z of total: 13.4 11.2 13.9 (0.25) 12.6 (0.22) D of total: 14.3 14.6 (0.39) 13.1 (0)
11.5 (0.30) 10.8 (0.50)
11.5 11.8 (0.22) 10.3 (0.50)
Numbers in parentheses designate 1 S.E.M.
the final body weights of animals that rewarmed. Although a definite trend is apparent, in only one case is the difference significant. Final weights of the bats that rewarmed only on 15 January are significantly different than the final weights of the animals that did not rewarm (t = 2.923, d.f. = 8). At the end of the experiments determinations of the initial body weights of the animals that did and did not warm up on the last attempt also revealed differences in body weight. The initial body weights of animals that did not warm up on the last attempt were less than the initial body weights of the bats that did warm up on the last attempt. These differences are not significant at the 5 per cent level. Data on the weights of interscapular brown fat are given in Table 2. Similar to body weights, the weights of brown fat in animals that did not rewarm were less than in animals that rewarmed. The differences are significant in the December experiment (t = 3.984, d.f. = 17) and for the animals rewarmed only once in the January experiment (t = 2.870, d.f. = 8). No significant difference was detected for animals rewarmed on nine successive evenings in the January experiment. When weights of brown fat of all animals that rewarmed in the January experiment (after nine and one warmup attempts) are compared with all animals that did not rewarm the difference is significant (t = 3,041, d.f. = 18). More importantly,
1
3-4
1 1.5 January only
7
6-7
9 1
OF INTERSCAPUIARBRO~ FLIGHT TEMPERATURE.
121.0 (12.0)
124.0 (14.0) 137.0 (9.0)
101.0 (80)
Started 27 November 1968 0.94 Started 6 January 1969 0.87
110.0 (12*0)
Started 11 November 1968 -
-
150.0 (11.0)
Brown fat weight
Brown fat/ body weight
1.17 1.16
o-92
0‘90
Brown fat/ body weight
Warmed up last time
TO
97.0 (12.0) 82.0 (4.0)
58.0 (4.0)
89.0 (16.0)
Brown fat
0.90 0.79
0.53
o-77
Brown fat/ body weight
Did not warm up last time
Experimental animals
FAT (mg) ANDTHEABILITYOFBAT~TORJ~WARM
Brown fat weight
Animals sacrificed start of experiment
THE AMOUNT
The numbers in parentheses designate 1 S.E.M.
Days between warm-ups
No. of warm-ups
TABLED--RELATIONSHIPS BETWEN
2 8 t;
‘;; g r
THE EFFECTS OF COLD EXPOSURE ON AROUSAL IN THE FREE-TAILED
BAT
565
however, not only did the animals that rewarmed the last time weigh more and have more interscapular brown fat, but in each experiment the proportion of brown fat in relation to body weight was also greater in animals that rewarmed. DISCUSSION
A likely effect of the variable winter temperatures in New Orleans and the effect of cold induced torpidity on the ability of Tad&da to warm up was demonstrated in these studies. The greater the number of arousals (without feeding) the less likely it was that an animal would reach flight temperature on a subsequent attempt. On the basis of its responses to warmups on successive evenings, Tadarida demonstrated a fall to winter change in its ability to arouse to flight temperature. The data from the present study suggest that in the winter Tadarida could warm up on a greater number of days without feeding and go back into torpor a greater number of times with less danger of not again rewarming than in early fall. In the present study there was a progressive increase in the percentage of animals that reached flight temperature after a given number of warmups as the experiments progressed from fall to winter. A similar fall to winter change in response was demonstrated by Tadarida in its ability to resist low ambient temperatures. Tadarida collected in New Orleans better resisted hypothermia in the winter than in early fall and summer when exposed to low ambient temperatures in the laboratory (Pagels, unpublished). Bats that were exposed to cold for 3-4 days between warmups demonstrated an ability to rewarm to flight temperature after a greater number of days of cold exposure than bats that had rewarmed the same number of times on successive days. These data collected in the laboratory suggest that the torpid condition may indeed be of survival value to Tadarzihz during periods of temperature stress. Indeed Tadarida was often found torpid on cold winter days in New Orleans. It was hoped that the role of brown fat in arousal could at least be partially evaluated on the basis of the amounts present in the interscapular depot. Although the evidence gained in this study regarding body weight and amounts of interscapular brown fat in relation to warmup ability was not conclusive, several trends were observed. As noted, animals that did not warm up weighed less than animals that did. The body weights of Tadarida at the termination of each of the experiments apparently reflected the overall fitness of the animals. The cold unit was kept very humid, but some weight loss was probably due to dehydration. Most weight loss was due to the use of white adipose tissue. This was easily recognized when the animals were examined following the studies. Except for two or three bats that were thin at the initiation of some of the experiments, it is doubtful that the lengths of the various experiments had a substantial effect on actual mortality. Female Tadmida collected in mid-December in New Orleans lived 40-60 days at 6-7°C without food or water (Pagels, unpublished). The initial stress afforded the animals in the experimental regimes of this study and under similar regimes in nature, would not be mortality but sooner the weakened animals inability to arouse
566
JOHN F. PAGELS
above ambient temperatures to flight temperature. This study measured only the success or failure of Tadarida to arouse in relation to body weight and amounts of interscapular brown fat, not the rate of arousal. However, Heldmaier (1969) found that heavy Myotis myotis with large amounts of brown fat was able to arouse much faster than light bats with small amounts of brown fat. Although body weight and weights of brown fat seemed to be important factors in the ability of Tadarida to arouse to flight temperature, it seems these would be of less importance than the proportion of brown fat present, expressed herein as percent body weight. Since bats that did not rewarm had less brown fat per g body weight than animals that rewarmed lends credence to the suggestion that the role of brown fat in the thermogenesis may be evaluated on the basis of the amount present. Of the thirteen deposits of brown fat in the little brown bat, Myotis Zucifugus, the interscapular deposit is the largest (Rauch & Hayward, 1969). Total brown fat constitutes about 5 per cent of total body weight of a hibernator, Eptesicus fwcus (Hayward & Lyman, 1967). In the present experiments weight of brown fat dissected from the interscapular region of Tadarida weighed only about 1 per cent of the total body weight. Mejsnar & Jansky (1970) reported that the weight of interscapular brown fat removed from M. myotis taken in February in Slovakia averaged 306 mg. Myotis myotis weighs approximately twice as much as T. b. cynocephala. Based on an average body weight (24 g) M. myotis had approximately 20-30 per cent more brown fat in the interscapular deposit than T. b. cynocephala in the present study. The appearance of the interscapular brown fat was markedly changed in Tadarida at the end of several warmups, especially in bats that had been unable to In such animals the bilobed structure was much more arouse to flight temperature. distinct, and appeared much darker and somewhat stringy. In conclusion, the results demonstrated that arousals on successive evenings decreased the animal’s ability to warm up on subsequent attempts sooner than when there was extended cold periods between warmups. The regimes of the present study resulted in the depletion of both white and brown fat deposits. Based on the rest&s of this study, although supportive, it is difficult to demonstrate that the brown fat deposits were reduced because of thermogenesis, and not cold exposure and subsequent starvation alone.
Acknowledgements-This work was a portion of a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy. Thanks are due to Drs. Clyde Jones and Norman C. Negus for their advice and support. Dr. R. D. Suttkus is gratefully acknowledged for his encouragement and also for support from National Institutes of Health Environmental Training Grant (S-TOlES0027) to Dr. Suttkus. Support was also received from a National Aeronautics and Space Administration Fellowship to the author. For assistance in the collection of animals I thank Frank Thomas, Frederick Jannett, Robert Cashner and especially Keith Grisham. Drs. John S. Hayward and James R. Reed read the original manuscript and offered several helpful criticisms.
THEEFFECTS OF COLDEXPOSURE ON AROUSAL IN THEFREE-TAILED BAT
567
REFERENCES BRUCED, S. & WIEBERSJ. E. (1946) T issue oxygen consumption in the hibernating and active bat Myotis Eucifugus. Physiol. Z&l. 39, 237-243. HAYWARDJ. S. (1968) The magnitude of noradrenaI~e-induced thermogenesis in the bat (Mjotis Zu~f~g~s) and its relation to arousal from hibe~ation. C’an.J. Physio2. Pharmac. 46, 713-718. HAYWARDJ. S. & LYMAN C. P. (1967) Nonshivering heat production during arousal from hibernation and evidence for the contribution of brown fat. In Mammalian Hibernation (Edited by FISHERK. C., DAWE, A. R., LYMAN, C. P., SCHBNBAUM, E. & SOUTH,F. E. Jr.), Vol. III, pp. 346-355. American Elsevier, New York. HAYWARDJ. S., LYMAN C. P. & TAYLOR C. R. (1965) The possible role of brown fat as a source of heat during arousal from hibernation. Ann. N. Y. Acad. Sci. 131, 441446. HELDMAIERG. (1969) Die Thermogenese der Mausohrfledermaus (Myotis myotis Borkh.) beim Erwachen aus dem Winterschiauf. 2. nergl. Physiol. 63, 59-84. HERREIDC. F., II. (1963a) Temperature regulation of Mexican free-tailed bats in cave habitats. J. cabal. 44, 560-573. HERREIDC. F., II. (1963b) Temperature regulation and metabolism in Mexican freetail bats. Science, N. Y. 142, 1573-l 574. HERREIDC. F., II. (1963c) Survival of a migratory bat at different temperatures. J. Mammal. 44,431433. HERREIDC. F., II. (1963d) Metabolism of the Mexican free-tailed bat. J. cell. camp. Physiol. 61, 201-207. MEJSNARJ. & JANSKY L. (1970) Shivering and nonshivering thermogenesis in the bat (Myotis myotis Borkh.) during arousal from hibernation. Cnn. J. Physiol. Pharmac. 48, 102-106. PAGELSJ. F. (1970) The dynamics of temperature responses of the free-tailed bat, Z’adarida byas~li~s~ cy~oc~h~u (Le Conte). Unpublished Ph.D. thesis, TuIane University. RAUCHJ. C. & HAYWARDJ. S. (1969) Topography and vascdarization of brown fat in a hibernator (Little brown bat, Myotis lac$agas). Can.J. 2001, 47, 1315-1323. SMALLEYR. L. & DRYER R. L. (1963) Brown fat: thermogenic effect during arousal from hibernation in the bat. Science, N. Y. 140, 1333-1334. SMITH R. E. & HOCK R. J. (1963) Brown fat: thermogenic effector of arousal in hibernators. Science, N. Y. 140, 199-200. VILLA R. & COCKRUME. L. (1962) Migration in the guano bat, Tadarida brasiliensis mexicana (Saussure). J. Mammal. 43, 43-64. Key Word Index-Cold exposure ; bats; Tadarida brasiliensis cynocephala; hypothermia; brown fat; body weight of bats.
THE EFFECTS OF SHORT AND PROLONGED COLD EXPOSURE ON AROUSAL IN THE FREE-TAILED BAT, TADARIDA BRASILIENSIS CYNOCEPHALA (LE CONTE) JOHN
F. PAGELS”
Department of Biology, Tulane University, New Orleans, Louisiana 70118 (Received
3 September
1971)
Tadarida brasiliensis cynocephala (Le Conte) was resident in New Orleans, Louisiana, in the winters 1967-68 and 1968-69. The bats fed periodically during winter months; however on cold days, the bats were inactive and often torpid. 2. Laboratory studies to determine the effects of variable periods of cold exposure on the ability of Tadarida to rewarm to flight temperature demonstrated that the total number of warmups plays an important role in the animal’s ability to arouse to flight temperature on subsequent attempts. 3. The effect of warmups on successive evenings was observed to be greater than the effects of prolonged cold exposure. 4. In all arousal experiments, the body weights of animals that rewarmed were greater than the body weights of animals that did not rewarm. 5. The total amount and proportions of interscapular brown fat were greater in animals that rewarmed than in animals that did not rewarm.
Abstract-l.
INTRODUCTION
ALTHOUGH the western form of the Mexican free-tailed bat, Tadarida bra.siZiensis mexicana, is migratory (Villa & Cockrum, 1962), the eastern subspecies, Tadarida brasiliensis cynocephala, is resident in New Orleans, Louisiana. In the absence of caves in New Orleans, Tudarida roosts in homes, warehouses and other suitable structures. Winter temperatures in New Orleans are quite variable and (within a single winter month) ambient temperatures as low as freezing and as high as 26°C are not uncommon. The daily range of maximum-minimum temperatures in the winter is also relatively great in New Orleans. Because of the nature of the roost sites the bats are often directly exposed to changes in ambient temperatures. It is not uncommon to find Tadarida in a torpid condition with a very low body temperature on cold winter days. The torpid condition apparently occurs infrequently in the cave-dwelling migratory form, T. b. mexicana. Herreid (1963a) found that T. b. mexicana maintained a resting body temperature between 32 and 42°C when cave temperatures ranged from 12 to 32°C. When exposed to low temperatures in the laboratory T. b. mexicana in a postabsorptive state dropped to near ambient * Present
address : Department
of Biology,
Richmond, Virginia 23220. 559
Virginia
Commonwealth
University,
560
JOHN
F. PAGELS
temperature in several hours (Herreid, 1963b). Herreid (1963~) also noted that a reduction in body temperature does increase survival of Tadarida, but that survival was not as great as in hibernating forms placed under similar conditions. During cold exposure in the laboratory some T. b. mexicana roused spontaneously from low body temperatures similar to hibernating forms while others did not (Herreid, 1963d). The objective of this study initiated in early fall, 1968, and terminated in midwinter was to determine the effect of short and prolonged cold exposure on the ability of T. b. cynocephala to arouse from hypothermia to flight temperature. The body weights and amounts and proportions of interscapular brown fat in relation to length of exposure and arousability were also considered. MATERIALS
AND METHODS
Bats used in the arousal studies were collected at a warehouse in downtown New Orleans. Bats were always collected in the morning. The animals roosted between a steel “I” beam and the brick wall of the warehouse. With the use of an extension ladder placed on the dock, bats could be easily reached and extracted from the narrow roost area with a pair of long forceps. For cold exposure in each experiment, the bats were put in pint cardboard cans and placed in a cold unit set at 6-7°C. Rectal temperatures were recorded with a YSI (44TD) thermistor tele-thermometer. On 30 September 1968, eighteen Taduridu (twelve females, six males) were collected. The bats were weighed and divided into three groups of four females and two males each and placed in the cold chamber. After 36 hr, one of the groups (Group 1) of six was removed from the cold chamber and placed at room temperature. Immediately upon removal from the cold chamber, the rectal temperatures of the bats were recorded. For the first 2 hr, rectal temperatures were recorded at O-S-hr intervals. After 1.0 additional hr, the temperatures were again recorded. The six animals were then weighed and returned to the cold chamber. On the second evening, 2 October, the first group of six and a second group of six (Groups 1 and 2) were removed from the cold chamber and the aforementioned procedure was repeated. On the third evening, 3 October, the first, second and last groups (Groups 1, 2 and 3) of six bats were removed from the cold chamber and the procedure was repeated. On 4 October, all three groups were again removed and the procedure repeated for the last time. The animals were sacrificed and weighed on 5 October. The percentage that reached flight temperature was calculated for each group. I found from numerous laboratory and field samplings of rectal temperatures and corresponding flight abilities that 30°C represents the lowest body temperature at which T. b. cynocephala is able to fly in a maneuverable fashion. Hence 30°C was used in all calculations involving flight temperature. Herreid (1963a, b) reported body temperature necessary for flight of T. b. mexicuna as 3l”C, or (Herreid, 1963d) “about 31°C” (29-32°C). On 11 November, thirty female Tudurida were collected. Ten bats were killed immediately, weighed and examined for the presence and development of interscapular brown fat. The twenty other bats were weighed and placed in the cold chamber. The next evening, 12 November, all twenty animals were removed from the cold chamber and the rectal temperatures were recorded immediately. The temperatures were again recorded at 0.5, l-0 and 3.0 hr after removal from the cold chamber. As in all experiments I handled the bats in the same order each time that the temperatures were measured. All bats were then weighed and returned to the cold unit. This procedure was repeated on each evening through 18 November. Rectal temperatures were not measured after 2f hr on 17 November. At the end of the 3-hr period on 18 November the bats were killed, weighed and the interscapular brown fat was removed and weighed. It has been demonstrated that brown fat provides
THE
EFFECTS
OF COLD
EXPOSURE
ON
AROUSAL
IN
THE
FREE-TAILED
BAT
561
quick energy to animals during arousal from low body temperature (Smalley & Dryer, 1963 ; Smith & Hock, 1963; Hayward et al., 1965; Bruce & Wiebers, 1966; Hayward Sz Lyman, 1967; Hayward, 1968). The percentage of bats in the second experiment that reached flight temperature on each evening was calculated. The third warmup experiment was initiated to compare the effect of prolonged cold exposure as opposed to the nightly warmups in the first two studies. On 27 November, twenty-nine female Tadarida were collected. Ten animals were killed immediately, body weight was recorded and interscapular brown fat was removed and weighed. The other nineteen bats were weighed and placed in the cold chamber. On the evening of 1 December, nine of the bats were removed from the cold chamber and the rectal temperatures were recorded immediately. Rectal temperatures were subsequently recorded 0.5 and 1 .O hr after initial removal from the cold chamber. In this experiment temperatures were not recorded later than 1-O hr after removal, since the earlier experiments demonstrated that bats that were going to rewarm to flight temperature did so in the first hour. Before returning the bats to the cold unit they were weighed. On 5 December the first nine bats, plus the other ten were removed from the cold unit and the procedure of 1 December was repeated. This procedure was carried out on the evenings of 1, 5, 8, 12, 15, 19 and 22 December. After the regular procedure was completed on 22 December, the bats were killed, total weight recorded and the interscapular brown fat was removed and weighed. The percentage that reached flight temperature on each date was calculated. On 6 January 1969, twenty-eight female Tadarida were collected for a fourth warmup experiment. Eight bats were killed immediately, body weight was recorded and the interscapular brown fat was removed and weighed. The other twenty bats were placed in the cold unit. Ten of the bats were removed each evening from 7 January through 15 January. Except for the number of days between warmups, the same procedure was used for this experiment as in the previous study. The other ten bats were taken out of the cold unit on each night only to record body weights. On the evening of 15 January all twenty bats (nine warmups and one warmup) were removed from the cold unit and given an opportunity to rewarm. At the end of the procedure the bats were killed, weighed and interscapular brown fat was removed and weighed. In addition to determining SE., experimental groups were compared by means of unpaired t-tests. A 5 per cent level of significance was used in all statistical tests. RESULTS
The percentage of animals that reached flight temperature in each of the four warmup experiments is given in Fig. 1. In the early October experiment the percentages are the same in each group based on the number of times the animals had been checked for warmup ability, and the percentages apparently are not related to the length of time the bats had been in the cold chamber. For example, all bats in each group reached flight temperature on the first attempt, whether first taken from the cold unit on 1, 2 or 3 October. In November, after the second warmup the percentage of bats that reached flight temperature on successive days began to decline. The bats were sacrificed on the eighth day of cold exposure after the seventh warmup attempt when 55 per cent of the animals reached flight temperature. In the December experiment with 3- to 4-day intervals between warmups and 25 days of cold exposure, the percentage of bats that reached flight temperature on the seventh attempt was the same as in the seventh attempt of the November experiment, 55 per cent.
562
JOHN
F.
PACELS
In the January experiment, all ten bats removed from the cold unit on successive evenings reached flight temperature through the sixth evening. Fifty per cent of the animals reached flight temperature on the eighth warmup. On the ninth evening, a bat reached flight temperature that had not done so on the previous I
Group
2
100
50
0
llnll_L -
u_bL
I
2
3
4
2
3
4
3
4
October
2 ,”
50
z
0
::
I2
13
14
15
16
17
I8
November
I
8
5
12
15
19
22
13
14
December 100
50
0 7
8
9
10
II
January
FIG.
l-Percentage
12
I5
15 0llly
of bats that reached flight temperature on each date in each experiment.
evening. Of the seventy-seven bats exposed to cold in the four warmup experiments, this was the only animal that warmed to flight temperature on an evening subsequent to an evening on which it had not. Eight of the ten animals checked One of the two that did not reonly on 15 January reached flight temperature. warm was dead. Data on the body weights of bats at the initiation and end of the last three experiments are given in Table 1. Note that the initial body weights of the bats collected in early November (November experiment) and in late November (December experiment) were very similar, and that the animals collected in January weighed considerably less. In each of the three experiments, the final body weights of the animals that did not rewarm on the last check were less than
THE EFFECTS OF COLD EXPOSURE ON AROUSAL TABLE
~-RELATIONSHIPS TO
IN THE FREE-TAILED BAT
563
BETWEEN BODY WEIGHTS (g) AND THE ABILITY OF BATS REWARM TO FLIGHT TEMPERATURE
Mean body weight No. of warm-ups
7
6-7
9
Days between warm-ups
Start of experiment
End of experiment
Warmed up last time
Did not warm up last time
Started 11 November 1968 3 of total: 16.2 11.9 16.5 (0.27) 12.1 (0.28) 15.8 (0.36)
11.7 (0.20)
Started 27 November 1968 3-4 P of total: 15.8 10.9 16.2 (0.29) 11 *o (0.07) 15.2 (0.52)
10.7 (0.35)
1
1
15 January only
Started 6 January Z of total: 13.4 11.2 13.9 (0.25) 12.6 (0.22) D of total: 14.3 14.6 (0.39) 13.1 (0)
11.5 (0.30) 10.8 (0.50)
11.5 11.8 (0.22) 10.3 (0.50)
Numbers in parentheses designate 1 S.E.M.
the final body weights of animals that rewarmed. Although a definite trend is apparent, in only one case is the difference significant. Final weights of the bats that rewarmed only on 15 January are significantly different than the final weights of the animals that did not rewarm (t = 2.923, d.f. = 8). At the end of the experiments determinations of the initial body weights of the animals that did and did not warm up on the last attempt also revealed differences in body weight. The initial body weights of animals that did not warm up on the last attempt were less than the initial body weights of the bats that did warm up on the last attempt. These differences are not significant at the 5 per cent level. Data on the weights of interscapular brown fat are given in Table 2. Similar to body weights, the weights of brown fat in animals that did not rewarm were less than in animals that rewarmed. The differences are significant in the December experiment (t = 3.984, d.f. = 17) and for the animals rewarmed only once in the January experiment (t = 2.870, d.f. = 8). No significant difference was detected for animals rewarmed on nine successive evenings in the January experiment. When weights of brown fat of all animals that rewarmed in the January experiment (after nine and one warmup attempts) are compared with all animals that did not rewarm the difference is significant (t = 3,041, d.f. = 18). More importantly,
1
3-4
1 1.5 January only
7
6-7
9 1
OF INTERSCAPUIARBRO~ FLIGHT TEMPERATURE.
121.0 (12.0)
124.0 (14.0) 137.0 (9.0)
101.0 (80)
Started 27 November 1968 0.94 Started 6 January 1969 0.87
110.0 (12*0)
Started 11 November 1968 -
-
150.0 (11.0)
Brown fat weight
Brown fat/ body weight
1.17 1.16
o-92
0‘90
Brown fat/ body weight
Warmed up last time
TO
97.0 (12.0) 82.0 (4.0)
58.0 (4.0)
89.0 (16.0)
Brown fat
0.90 0.79
0.53
o-77
Brown fat/ body weight
Did not warm up last time
Experimental animals
FAT (mg) ANDTHEABILITYOFBAT~TORJ~WARM
Brown fat weight
Animals sacrificed start of experiment
THE AMOUNT
The numbers in parentheses designate 1 S.E.M.
Days between warm-ups
No. of warm-ups
TABLED--RELATIONSHIPS BETWEN
2 8 t;
‘;; g r
THE EFFECTS OF COLD EXPOSURE ON AROUSAL IN THE FREE-TAILED
BAT
565
however, not only did the animals that rewarmed the last time weigh more and have more interscapular brown fat, but in each experiment the proportion of brown fat in relation to body weight was also greater in animals that rewarmed. DISCUSSION
A likely effect of the variable winter temperatures in New Orleans and the effect of cold induced torpidity on the ability of Tad&da to warm up was demonstrated in these studies. The greater the number of arousals (without feeding) the less likely it was that an animal would reach flight temperature on a subsequent attempt. On the basis of its responses to warmups on successive evenings, Tadarida demonstrated a fall to winter change in its ability to arouse to flight temperature. The data from the present study suggest that in the winter Tadarida could warm up on a greater number of days without feeding and go back into torpor a greater number of times with less danger of not again rewarming than in early fall. In the present study there was a progressive increase in the percentage of animals that reached flight temperature after a given number of warmups as the experiments progressed from fall to winter. A similar fall to winter change in response was demonstrated by Tadarida in its ability to resist low ambient temperatures. Tadarida collected in New Orleans better resisted hypothermia in the winter than in early fall and summer when exposed to low ambient temperatures in the laboratory (Pagels, unpublished). Bats that were exposed to cold for 3-4 days between warmups demonstrated an ability to rewarm to flight temperature after a greater number of days of cold exposure than bats that had rewarmed the same number of times on successive days. These data collected in the laboratory suggest that the torpid condition may indeed be of survival value to Tadarzihz during periods of temperature stress. Indeed Tadarida was often found torpid on cold winter days in New Orleans. It was hoped that the role of brown fat in arousal could at least be partially evaluated on the basis of the amounts present in the interscapular depot. Although the evidence gained in this study regarding body weight and amounts of interscapular brown fat in relation to warmup ability was not conclusive, several trends were observed. As noted, animals that did not warm up weighed less than animals that did. The body weights of Tadarida at the termination of each of the experiments apparently reflected the overall fitness of the animals. The cold unit was kept very humid, but some weight loss was probably due to dehydration. Most weight loss was due to the use of white adipose tissue. This was easily recognized when the animals were examined following the studies. Except for two or three bats that were thin at the initiation of some of the experiments, it is doubtful that the lengths of the various experiments had a substantial effect on actual mortality. Female Tadmida collected in mid-December in New Orleans lived 40-60 days at 6-7°C without food or water (Pagels, unpublished). The initial stress afforded the animals in the experimental regimes of this study and under similar regimes in nature, would not be mortality but sooner the weakened animals inability to arouse
566
JOHN F. PAGELS
above ambient temperatures to flight temperature. This study measured only the success or failure of Tadarida to arouse in relation to body weight and amounts of interscapular brown fat, not the rate of arousal. However, Heldmaier (1969) found that heavy Myotis myotis with large amounts of brown fat was able to arouse much faster than light bats with small amounts of brown fat. Although body weight and weights of brown fat seemed to be important factors in the ability of Tadarida to arouse to flight temperature, it seems these would be of less importance than the proportion of brown fat present, expressed herein as percent body weight. Since bats that did not rewarm had less brown fat per g body weight than animals that rewarmed lends credence to the suggestion that the role of brown fat in the thermogenesis may be evaluated on the basis of the amount present. Of the thirteen deposits of brown fat in the little brown bat, Myotis Zucifugus, the interscapular deposit is the largest (Rauch & Hayward, 1969). Total brown fat constitutes about 5 per cent of total body weight of a hibernator, Eptesicus fwcus (Hayward & Lyman, 1967). In the present experiments weight of brown fat dissected from the interscapular region of Tadarida weighed only about 1 per cent of the total body weight. Mejsnar & Jansky (1970) reported that the weight of interscapular brown fat removed from M. myotis taken in February in Slovakia averaged 306 mg. Myotis myotis weighs approximately twice as much as T. b. cynocephala. Based on an average body weight (24 g) M. myotis had approximately 20-30 per cent more brown fat in the interscapular deposit than T. b. cynocephala in the present study. The appearance of the interscapular brown fat was markedly changed in Tadarida at the end of several warmups, especially in bats that had been unable to In such animals the bilobed structure was much more arouse to flight temperature. distinct, and appeared much darker and somewhat stringy. In conclusion, the results demonstrated that arousals on successive evenings decreased the animal’s ability to warm up on subsequent attempts sooner than when there was extended cold periods between warmups. The regimes of the present study resulted in the depletion of both white and brown fat deposits. Based on the rest&s of this study, although supportive, it is difficult to demonstrate that the brown fat deposits were reduced because of thermogenesis, and not cold exposure and subsequent starvation alone.
Acknowledgements-This work was a portion of a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy. Thanks are due to Drs. Clyde Jones and Norman C. Negus for their advice and support. Dr. R. D. Suttkus is gratefully acknowledged for his encouragement and also for support from National Institutes of Health Environmental Training Grant (S-TOlES0027) to Dr. Suttkus. Support was also received from a National Aeronautics and Space Administration Fellowship to the author. For assistance in the collection of animals I thank Frank Thomas, Frederick Jannett, Robert Cashner and especially Keith Grisham. Drs. John S. Hayward and James R. Reed read the original manuscript and offered several helpful criticisms.
THEEFFECTS OF COLDEXPOSURE ON AROUSAL IN THEFREE-TAILED BAT
567
REFERENCES BRUCED, S. & WIEBERSJ. E. (1946) T issue oxygen consumption in the hibernating and active bat Myotis Eucifugus. Physiol. Z&l. 39, 237-243. HAYWARDJ. S. (1968) The magnitude of noradrenaI~e-induced thermogenesis in the bat (Mjotis Zu~f~g~s) and its relation to arousal from hibe~ation. C’an.J. Physio2. Pharmac. 46, 713-718. HAYWARDJ. S. & LYMAN C. P. (1967) Nonshivering heat production during arousal from hibernation and evidence for the contribution of brown fat. In Mammalian Hibernation (Edited by FISHERK. C., DAWE, A. R., LYMAN, C. P., SCHBNBAUM, E. & SOUTH,F. E. Jr.), Vol. III, pp. 346-355. American Elsevier, New York. HAYWARDJ. S., LYMAN C. P. & TAYLOR C. R. (1965) The possible role of brown fat as a source of heat during arousal from hibernation. Ann. N. Y. Acad. Sci. 131, 441446. HELDMAIERG. (1969) Die Thermogenese der Mausohrfledermaus (Myotis myotis Borkh.) beim Erwachen aus dem Winterschiauf. 2. nergl. Physiol. 63, 59-84. HERREIDC. F., II. (1963a) Temperature regulation of Mexican free-tailed bats in cave habitats. J. cabal. 44, 560-573. HERREIDC. F., II. (1963b) Temperature regulation and metabolism in Mexican freetail bats. Science, N. Y. 142, 1573-l 574. HERREIDC. F., II. (1963c) Survival of a migratory bat at different temperatures. J. Mammal. 44,431433. HERREIDC. F., II. (1963d) Metabolism of the Mexican free-tailed bat. J. cell. camp. Physiol. 61, 201-207. MEJSNARJ. & JANSKY L. (1970) Shivering and nonshivering thermogenesis in the bat (Myotis myotis Borkh.) during arousal from hibernation. Cnn. J. Physiol. Pharmac. 48, 102-106. PAGELSJ. F. (1970) The dynamics of temperature responses of the free-tailed bat, Z’adarida byas~li~s~ cy~oc~h~u (Le Conte). Unpublished Ph.D. thesis, TuIane University. RAUCHJ. C. & HAYWARDJ. S. (1969) Topography and vascdarization of brown fat in a hibernator (Little brown bat, Myotis lac$agas). Can.J. 2001, 47, 1315-1323. SMALLEYR. L. & DRYER R. L. (1963) Brown fat: thermogenic effect during arousal from hibernation in the bat. Science, N. Y. 140, 1333-1334. SMITH R. E. & HOCK R. J. (1963) Brown fat: thermogenic effector of arousal in hibernators. Science, N. Y. 140, 199-200. VILLA R. & COCKRUME. L. (1962) Migration in the guano bat, Tadarida brasiliensis mexicana (Saussure). J. Mammal. 43, 43-64. Key Word Index-Cold exposure ; bats; Tadarida brasiliensis cynocephala; hypothermia; brown fat; body weight of bats.