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Riedesel, M. L., Lawrence R. Ktrinestiver and Nancy R. Benally. 7964. Toleranc.e of Cife_llus lateralis and C.

spilosoma for water deprivation. -S-. 377-388 in Finnish Academy of Science. Annales Academiae Scientiarum Fennic"e, S.rius a" W, niotogj-"tl Zt, Paavo Suomalainen, ed" 453 p. Invi ted paper. With discussion.
[Mammalian Hibernation II Proceedings of Second Intrl gn Natural Manunalian Hibernationr' Helsinki,
Symposium

AN]VALES ACADE MI4., SCIENTIARUM FENNIC.4E Serie s A

IY. BIOLOGICA
7It27

TOLERAIVCE OF CITELLUS LATERALIS AIVD C. SPILOSOMA FOR WATER

DEPRIVATION
3Y

MARVIN L. RIBDESEL, LAWRENCE R. ----rr and NANCY R. BBNALLY KLINESTIVER


Biology Department, University of New Mexico, U.S.A.

SUOMALAINEN TIEDEAKATE MIA

HELSINKI I964

I. Introiluction
Hibernation is not a simple response to a single environmental stress. Environmental stresses described as factors in inducing hibernation include cold, lack of food, lack of water and others. Physiological responses or strains during hibernation involve 1) modified temperature regulation, 2) lowered metabolic rate, 3) decreased heart rate, 4) decreased respiratory rate, 5) limited or no drinking, and changes in many other processes. The present study is concerned with dehydration as a factor in hibernation. Initial experiments are concerned with the capacity of two species of hibernators to withstand water deprivation. Additional studies have demonstrated the extent of tissue dehydration during hibernation. Water loss per unit time is undoubtedly very small during hibernation when ventilation of the lungs and renal activity are very low. Nevertheless, the absence of water intake during continuous hibernation for a period of several weeks may result in significant net water loss. Changes in the volume of body water compartments during hibernation are implied by changes in electrolyte concentrations during hibernation and hypothermia (1, 4, 8, 5, 6 and others). The animals selected for this study are of particular interest because they are closely related and inhabit quite different ecological areas. The CitelIus spilosoma marginatus (spotted ground squirrel) inhabits arid regions (average annual rainfall, T-15 inches) of the Lower and Upper Sonoran Life Zones (4500-6 000 feet elevation) of New Mexico (2). Citellus lateralis Iateralis (golden mantled ground squirrel) lives at higher elevations (8 000 to 14 000 feet) between the Transition and Canadian Life Zones and is usually found near meadows and streams (average annual rainfall, 20-+0 inches).

II.

Materials anil proceduros

The squirrels were kept in wire bottomed cages for two to eighteen months previous to experimentation. The daily diet consisted of commercial rat pellets and mixed cracked grain (corn, wheat, oats, and barley) containing ap-

This study
G-14495.

has

been supported

in part by National

Science Foundation Grant,

Ann. Acad. Scienl. Fennicae

A.IV.

7112i

Anrmol

=
!

co

E ': E

Aru C Be D EH G FH H

c---{ ----.
Doys
3

&---{ c--
o o m
-9

a
L o .J

d'

:
0

c
c;

= cn

o 9
,b

.:
_9 f

\\

'l

)
t r;

Animol

rof
I

Number

48
59

(H G--{

.of
,.1

60
77 a2 85

;--{
H D--{ de

t07 tt 4

Fig. 1. Weight loss of sprague


dawley rats deprived of nater.

Fig. 2. Weight loss of Citellus lateralis


prived of rvater. (Winter
1961

-1962.1

air temperature of 21' C (+ 2'C) and relative humidity of 30 o,/o, ranging


from a minimum of 15 o/" in winter to 45 gA in summer. Borly weight measurements were determined daily on a Model E Mettler, type K, balance rnith a sensitivity of 0.1 g. Body temperature measurements were made on a Leeds and Northrup potentiometer model 8 692, sensitivity of 0.1" C. Copper-constantan thermocouples in 18 to 23 gauge hypodermic needles served as probes for measuring rectal and skin temperatures. Skin temperature measurements of the dorsal thoracic area were made daily in several experiments. The skin temperature gave an approximation of body temperature with a minimal disturbance of the animal (3). Tissue weights lvere determined with an analytical balance to the nearest 0.001 g. The rvet weight samples of lung tissue ranged from 0.04 g to 0.4 g; spleen, 0.05 g to 0.480 g; kidney, 0.4 g to 0.9 g; and muscle, 0.2 g to 0.9 g. The animals rn'ere killed by administration of ether

proximately 45 l/o u'ater. The air-conditioned animal quarl-ers maintained an

1\[.

L. RrunnsEL et al., Tolerance of C. lateralis and C. spilosoma

;8 o o

e'' E
An imo
i

Numb er

37H 59 o--4 76H 82H 85 +--r lO7 (H lO8 +--{ lO9 H

.\ \-.. t"F"
\.!

d.^

I
-----

Animol

020

'\

=?4 ci'
?a

Numoer 24(H 38 G--{ 68H 89 .---{ 90 rr-----d 93 a--{ 94 0------0 lol G---c

\
!.

Fig. 3. Weight loss of Citellus deprived of n'ater. (Summer

lateralis Fig. 4. Weight loss of Citellus spilosoma deprived of water. (Winter 1962-1963.) 1962.)

or decapitation. The method of sacrificing the animals did not affect the data. Drying of tissues during the dissecting and weighing procedure was kept to a minimum by removing the skin from only one portion of the body at a

time, wrapping the tissue in aluminum foil

it immediately.
oven at 41'C

as it r,vas dissected, and weighing The tissue samples were dried to constant weight in an electric

(+ 2'C).
Dp value)),

which is repeatedly used throughout the text refers to the probability that there is no difference other than that due to random errors between the mean values. 'fhe p values of less than 5 o/o 'rvere considered significant.

The term,

III. Results
The response of the animals to dehydration rn'as estimated by making daily body weight measurements of animals deprived of drinking rn'ater. This procedure resulted in dehydration as evidenced by the reduced water content of tissues. However, the weight loss was Iargely due to decreased food intake. The tolerance for deprivation of water was determined by general ap-

380

Ann. Acad. Scient. Fennicae

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IY.

71127

6789rO|12t314 -Animol

3
@

12H 34 ;--.
68

-9

o o
o

d'

)
d'

Fig. 5. Weight Ioss ol Citellus spilosoma deprived of water. (Summer

1962.)

3 o

: s
I
o J d

t2

IG

20

G--a
24

Summer

C. spilosomor n= 3

a2a

Winter C. spilosomo, n=8


Summer

G-- r

C. lolerolis, n=9 Winter


C. lolerolis, n=8

Fig. 6. Mean weight loss

of.

Citellus lateralis and C. spilosoma deprived of water.

pearance and activity of each animal when weighing. The weight loss of a given animal appeared to be determined by the gross activity of the animal, and was independent of the initial body weight or average body temperature. Experiments with laboratory rats (Sprague Dawley) demonstrated a significant weight loss within 24 hours, and a near maximum tolerable weight

M. L. RrnnnsEL et al., Tolerance of C. lateralis and C. spilosoma


Doys

38r

-4
! co

E 5r -t

\,'

EI
9 at6
I

3ra

3
No Food No Woter No Food or

Woter

Fig. 7. Weight loss of Citellus lateralis under conditions of no water, no food and no water or food. (Summer 1962.)

0oys

=
@

I
t6

.9

s
o o
c;

z4

G 32

I '6

= ct
E

Fig. S. Weight loss of Citettus

spilosoma under conditions of no watet, no food and no


1962.)

food or water. (Summer

loss in 72 hours (Fig'. 1). The mean rveight loss of the eight rats deprived of water for 72 hours was 18.2 g per 100 g initial body weight. The average initial body weight was 267 g (range 220 to 320 g). The average of skin temperature measurements made daily was 37.1' C (range, 36.2 to 38'0' C). The

Ann. Acad. Scient. Fennicae

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71.122

water content of blood, spleen, liver, lung, and muscle taken from three rats deprived of water for 72 hours was less than values for control animals (p : 0.001 to 0.05). The per cent water in the kidney tissue was not changed

by these conditions.
Ground squirrels deprived of water tolerated weight losses equivalent to of the initial body weight. There rn'as considerable individuai difference in the rate at which the animals lost weight (Fig, 1-5). The weight loss of a given animal appeared to be due to the extent of activity and body temperature. Flowever, body temperatures taken three times daily did not show a statistical correlation with 24 hour weight loss. Apparently, continuous body temperature measurements will be necessary to demonstrate the correlation of body temperature and rate of weight loss. The rate of weight loss by C. Iateralis during the winter and summer rvas similar for the first four days, but after five and six days of water deprivation the animals lost more weight in the summer (Fig. 6). The rate of weight loss by C. spilosoma was similar in the summer and winter. It is of interest that C. spilosoma have been reported to aestivate during the summer whereas no reports of aestivation have been made on C. lateralls (7). C. lateralis and C. spilosoma lost weight at similar rates with the exception that C. Iateralis consistently had a higher per cent body weight loss on the first day of r,vater deprivation than did C.spilosoma (Fig.7 and 8). When the dehydrated animals were given water, the food was r,vithdrawn for five hours. After five hours of access to drinking water the mean per cent weight gained by eight C. Iateralis was 4.6 o/o of the dehydrated body weight and for eight C. spilosoma rn'as 3.8 o/" of the dehydrated body weight. The conditions of (a) no rn'ater, (b) no food and (c) no food or rvater demonstrate the extent to which weight loss during periods of deprivation of water is due to decreased food intake (Fig. 7 and 8). The animals regained body weight lost during the six to ten days between each of the experimental conditions. The C. Iateralis lost weight more rapidly when deprived of water than when deprived of food. In contrast the C. spilosoma, which inhabits a more arid environment, lost weight more rapidly when deprived of food than when deprived of water (Fig. 7 and 8). The mean per cent weight loss of nine C. lateralis deprived of drinking water for four davs u,as 23.3 % (Sn t - 4.8), when deprived of food the loss was 15.2 % (SD : 2.5), and 25.0 % (SD : 1.8) when deprived of food and water. The mean per cent weisht loss of five C. spilosoma deprived of drinking water for four days was 13.5 % (SD : 3.5), when deprived of food the loss was 25.7 9/" (SD : 9.0), and 25.9 % (SD : 1.5) when deprived of food and water for four days. The water content of body tissues were demonstrated to be dependent 40
o/o

1 SD

Standard Deviation

M. L. Rrnonsnl et al., Tolerance of C. Iateralis and C. spilosoma


Active, Ts 22"C

383

with

woler
loss

N
ao-

tl
2SD

Active, To 22oC no woler, 36-427o wl.


Hibernoling, To 80C

3-4

doys

'uf 76i
o

=
c o-

741 721

ts

n=5 n=8 n:5 n=5 SPLEEN


KIDNEY

n=8

Peroneus BicepsFemoris

LUNG

MUSCLE

Fig. g. Per cert water content of tissues f.rom Citellus lateralis. (Data taken Kr.rNnsrrvnn 1962, Table 46.)

in part from

upon the species of the animal, type of tissue, availability of drinking rlv'ater' and hibernation. The per cent watel of a given tissue varied with the species,

for

instance:
Spleen

Iluscles

Laboratory rat
C. lateralis C. spilosoma

n:3 n:5 n:4

7L.2 72.4 74.3

Kidney 76.6 73.4. 71.0

Lung
'17.8
7

peroneus
)

femoris
/ +.O

biceps

6.,/

4.3

12,2
)J,t

71.6
,/4.J

'15.1

Statistical analysis demonstrated the differences to be real (p : 0.01 0.05) with the exception that the per cent watdr of rat and ground squirrel muscle tissues was similar (p : 0.4) and the spleen tissues from the C' latertilis and laboratory rat had comparable water content (p : 0'3)' The lung tissues from the two ground squinels had similar water content (p - 0.4). The per cent water of tissues taken from c. Iateralis under conditions of a) active, b) active, no drinking water, and c) hibernating are presented in Fig. 9. The time required for the animals to lose 36 to 42 o/o of their initial body weight following deprivation of water ranged from 6 to 28 days. The water content of spleen and kidney taken from C. Iaternlis was not changed appreciably by any of the experimental conditions (Fig. 9). The per cent water in lung tissue taken from hibernating animals was greater than that taken from dehydrated (p < 0.01) and hydrated control animals (p : 0.01-0.02). The per cent $'ater in muscle tissue of C. Iateralis decreased during deprivation of $'ater and hibernation (p:0.05-0'1)' The water content of muscle from dehydrated and hibernating animals was similar (p : 0.2-0.3).

Ann. Acad. Scient,. Fennicae


Active, To 22"C, n=5

A.

rv.

71127

with

woter

n
o o

Aclive, Ts 22"C, n-5 no woler, 34-41 "/" wi. loss Hibernoting, To28oC, n=lO
2-14 doys

=
c
o_

Peroneus SPLEEN
KIDNEY

BicepsFemoris

LUNG

MUSCLE

Fig. 10. Per cent $'ater content of tissues trom Citellus spilosoma. (Data taken in part from Kr,rNnsrrvER 1962, Table 46.)

Table

Summary of changes in water content of tissues following deprivation of water and hibernation (Citellus lateralis and C. spilosoma\,
Citellus lateralis Active no water Citellus spilosoma Hibernal,ing

Hitlernaling I Active no water I no change no change


increased?
decreased?

Spleen

no no

change change

Kidney Lung
Peroneus muscle Biceps

no change
decreased?

no change increased increased?


decreased

sl. decrease
increased increased
decreased

femoris

decreased?

decreased?

decreased?

decreased

The water content of tissues taken from c. spilosomo showed greater changes in response to deprivation of water and hibernation than did the tissues of C. Iateralls. Periods of 8 to 42 days without water resulted in 34

to 41 o/o body weight loss (Fig. 10). There was no consistent change in the \ater content of the spleen when c. spilosoma were deprived of water. During hibernation there was decreased hydration of the spleen (p :0.03). The per
cent water of the kidney increased during deprivation of water ancl hibernation (p < 0.001). The water content of lung tissue increased during hibernation (p < 0.001) and had a questionable increase over the control values

M. L. Rrrnpsnr, et al., Tolerance of C. lateralis and C. spilosoma

during deprivation of water (p:0.05-0.10). The muscle of animals in hibernation and animals deprived of water had a lower water content than
tissues taken from the control group (p

0.04-0.001). The data from figures

9 and 10 are summarized in Table

1.

IY.

Conelusions antl summary

Similar experiments on responses to deprivation of water conducted under field conditions may give very different data as suggested by the large individual differences observed in the present study. Field animals may be able to maintain body weight on a similar diet containing 45 o/o water as suggested by the fact that in this study one C. Iateralis survived 28 days and one C. spiIosoma 42 days rvithout water. The similarity of the responses of. C. lateralis and C. spilosoma to water deprivation is of particular interest because they inhabit very different ecological areas. The renal capacity of C. spilosomo is probably greater than that of C. Iateralis as evidenced by less weight loss of C. spilosoma during the first day of water deprivation. A species difference n'as also noted as there rn'as no change in the water content of C. Iateralis kidney tissue whereas per cent water of kidney tissue from dehydrated and hibernating C. spilosoma was greater than control values (Table 1). Hibernation effects redistribution of body water in C. lateralis and C. spllosoma (Fig. 9). The shifts in body rvater are apparently due to the cooling of tissues rather than loss of total body water since the decreased water content was significant after only two days of hibernation. The mechanisms causing water loss or gain are probably determined by the cell membrane or cell metabolism of the particular tissues. Tissue differences in osmotic regulation have been demonstrated in other types of studies (9, 6). Most theories on mechanisms of water transfer through cell membranes associate water transfer rn'ith electrolytes and neglect the possibility of an active transfer of rn-ater. Therefore, the loss of water by cells during hibernation is undoubtedly associated with the movement of one or more electrolytes, presumably magnesium, potassium, and/or sodium.

Future work should include studies of the water and electrolyte concentrations in additional tissues during various stages of hibernation and dehydration. Identification of osmotic regulation of isolated tissues at varied temperatures would clarify characteristics of the enzyme systems involved in osmotic regulation of each tissue. Radioisotope studies would be helpful in clarifying electrolyte and water distribution during hibernation.

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References

ll. & A. HnNscHnr,, 1956: Responses of body fluid compartments to heat and cold. - Physiol.Rev. 36, t2B-i.44. 2. Hrr,r, J.8., L942: Notes on mammals of nortlieastern Nen- Mexico. - J.xlammal. 23, ;9. 3. Kr,rNnsrrvnn, L. R., 1962: A stud;' of body rvater in active and hibernating ground squirrels, Citellus l(ileralis lateralis and Citetlus spilosoma marginatus. - M.S. Thesis, Dept,. of Biology, The University of New Mexico. 4. ll.lrznr,s, M., 1954: Active cation transport in eryttrrocytes, - Symposia of the Society for Experimental Biology 8: Active Transport and Secretion , 202-227.
1. B,tss, D.

Cambridge Univ.Press, London.

5.

Rrnrnsnr, M. L., 1960: The internal environment during hibernation. - Bull.Mus.


comp.Zool.I{arv. 124, 421 435. R., 1960: Metabolism of intracellular rvater.

6. RonrNsox, J. _ 149.
7. sunRnr,r,,

- physiol.Rev. 40, 11,2

F., 1949: A life history study of the ground squirrel citettus spilosoma,, M.S. Thesis, Dept. of Biology, The University of New Mexico. B. Suom.r.r,erNnN, P., 1939: Hibernation of the hedgehog \rI. Serum Mg and Ca. Artificial hibernation. Also a contribution to chemical physiology of diurnal
9.

sleep. - Ann.Acad.Sci.Fenn. A b3, t,iI. lvooosunv, D. M., 1956: Effect of acute hyponatremia on distribution of rvater and electrolytes in various tissues of the rat. - Am.J.phvsiol. 1g5, 2g1 -286.

Discussion following paper presentetl

by Dr. M. Rietlesel

-\oo.pu: Have you some data on act,ual weighLs of tissue? For inslance, in the case of the muscles, was there a significant amount of atrophy of the muscles during the inactive period of hibernation? A second queition is, although the hibernation, you say, went on for only a few days, yet this is plenty of time for shifts of rn'ater due to inactivity of ,rcell pumpsu.-We know that in isolated tissues, such as slices of kidney, slices of liver,-and so on, it takes only a few minutes, to demonstrate a swelling of tissue as soon as it is cooled below about 10'. This swelling is very often credited to the cold narcosis of cellular pumps-probably not pumps for ',vater but pumps for electrolyte. I v'onder whethir the best controli for Lhe hibernating animal are not either whole animal or isolated tissue which is chillecl for just a few
rn in uLes'.)

Rreonsrr,: There is no significant difference between the total tissue mass of the active and hibernating animals. Howevero inactivity may be a factor. It is very important to note that tissues from non-hibernaiors (at least iso.lated tissues) do gain water u'hen in the cold, ancl I think it is of parti cular interest that the muscle tissue of hibernators very definitely becomes dehydrated. This difference may involve differences in the seniitivity of enzymes or enzyme systems to temperature. Kar-r,BN: Did you see any difference in the amount of water in the hibernating animal as related to duration of the hibernation? From Rulot's work (Arch. de Biologie 18,365-375, 1902) on European bats, one can calculate

M. L. Rrnonsnl et al., Tolerance of C. lateralis and C. spilosoma

38;

water content based on fat free body weight; you find that the r,vater content of these animals was about the same at the beginning and toward the end of hibernation but it dropped about 2 o7o in mid-season. Could this have any effect on your curves? RrBoesnr-: No, we do not find a change in the water content with duration of hibernation, but I should point out that our periods of hibernation were short, from 2 to 14 days. Observations were limited to one rbout,r of hibernation during a single season. Hocx: In the case of the squimel that rn'ent 42 days without rvater, rn'as this a very fat squirrel? Did the degree of fatness have anything to do with it or did you consider it in this connection? Rrroeser-: Yes, we considered this. In the first place, I should probably say that they were all fat. There wasn't a thin one in the lot. There was no correlation between initial body weight and the extent of a >boutr. One other point I might make with regard to Dr. Adolph's comment and that is that I think rn'e should look very closely at differences in osmotic regulation in different tissues. I was indeed surprised by our own datan rn-hen I realized that it meant that within a given animal one sees quite different osmotic regulations in different tissues. TwrNte: Again, this may be due to species differences. We have a rabher large colony of Citellus lateralis, some of n'hich have rvater bottles, and some do not, but we see no significant difference at all, except that they all gain weight, unless they're sick. Although we have had the colony only a feu' months, the data we now have indicate that weight increase during the r>weight-gaining seasonr> is relatively constant regardless as to whether or not they have water bottles. Their diet consist primarily of sunflorver seeds, pigeon mix, and we give them a carrot about once a week. We also give them half an orange soaked in cod liver oil once a rneek. This is the only free water they would get. On the other hand, rn'hen we deprive squirrels of rvater after they have become accustomed to drinking from water bottles, they lose weight. We just don't see the same pattern as you do-I don't understand it. RrsoEssr: I think this is a matter of adaptation. These animals have a considerable capacity to adapt to various amounts of water intake. If an animal has unlimited water available, and the v'ater is suddenly withdralvn, you w-ill observe marked changes (loss of body weight and even death) as opposed to minor or no observable effects rvhen the amount of water available is reduced over a period of several weeks. TwnNre: Probably they normally need water - under wild conditions? Rrnoesnr: No. Hocx: I don't think so. In 15 years of study, I have never yet seen a wild ground squirrel take a drink of water. Has anybody ever seen a rn'ild squirrel take rvater? I don't believe they do. ZruNv: It is of particular interest (along the water line) that up in our animal care room, every once in a while there is either a lack of food given to the animals or a lack of rvater. I have animals that are caged either 4 or 6 to a cage. They may go along for weeks without any food, but as soon as they are not getting n'ater, then there is a tendency toward cannibalism. Consequently, I lose a couple. It has ahvays been interesting to me, that this is because of the u'ater factor. Sometimes the rn,ater bottle is there. but

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the water is not draining properly, or sometimes the caretaker has forgotten to put a bottle of water in the cage. when this cannibalistic attack takes place, it is also interesting that they seem to gouge out the eyeballs and go into the brain-looking apparently for some type of fluid. Snarrrl: I was wondering about the use of your drying temperature aL 42". It seems to me that we used to have 84' or something like that, as a necessary value to get the bound water out of the system-admitting some hazards, of course, in losing some of the more volatile fatty acid fractions. what is your feeling about that? Did you get a constant weight at 42"? How much difference did you get if you gave more heat to the system or in vacuum? Rreoesnr-: We did not go to higher temperatures, nor did we try a vacuum. We liked the data the way we got it it was consistent. I might say also, for those of you in humid environments, this was done in Albuquerque where the air is quite dry and we don't have a problem of water accumulation. Sours: Have you thought of using Fisher's reagent for your water analyses?

RreoBser-:

have, and

I think this mav

be a good suggestion.

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