TOLERAIVCE OF CITELLUS LATERALIS AND C. SPILOSOMA FOR WATER DEPRIVATION

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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 waterdeprivation. -S-. 377-388 in Finnish Academy of Science. AnnalesAcademiae 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 Symposiumgn Natural Manunalian Hibernationr' Helsinki,

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

IY. BIOLOGICA7It27

TOLERAIVCE OF CITELLUSAIVD C. SPILOSOMA FORDEPRIVATIONLATERALISWATER

MARVIN L. 3YRIBDESEL, LAWRENCE R. KLINESTIVERand NANCY R. BBNALLY ----rrBiology Department, University of New Mexico, U.S.A.

HELSINKI I964SUOMALAINEN TIEDEAKATE MIA


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I. IntroiluctionHibernation is not a simple response to a single environmental stress.Environmental stresses described as factors in inducing hibernation includecold, lack of food, lack of water and others. Physiological responses or strainsduring hibernation involve 1) modified temperature regulation, 2) loweredmetabolic rate, 3) decreased heart rate, 4) decreased respiratory rate, 5) limitedor no drinking, and changes in many other processes. The present study isconcerned with dehydration as a factor in hibernation. Initial experimentsare concerned with the capacity of two species of hibernators to withstandwater deprivation. Additional studies have demonstrated the extent of tissue

dehydration during hibernation. Water loss per unit time is undoubtedly verysmall during hibernation when ventilation of the lungs and renal activity arevery low. Nevertheless, the absence of water intake during continuous hiber-nation for a period of several weeks may result in significant net water loss.Changes in the volume of body water compartments during hibernation areimplied by changes in electrolyte concentrations during hibernation andhypothermia (1, 4, 8, 5, 6 and others).The animals selected for this study are of particular interest because theyare closely related and inhabit quite different ecological areas. The Citel-Ius spilosoma marginatus (spotted ground squirrel) inhabits arid regions(average annual rainfall, T-15 inches) of the Lower and Upper SonoranLife Zones (4500-6 000 feet elevation) of New Mexico (2). Citellus lateralisIateralis (golden mantled ground squirrel) lives at higher elevations (8 000 to14 000 feet) between the Transition and Canadian Life Zones and is usuallyfound near meadows and streams (average annual rainfall, 20-+0 inches).

II. Materials anil procedurosThe squirrels were kept in wire bottomed cages for two to eighteen monthsprevious to experimentation. The daily diet consisted of commercial rat pel-lets and mixed cracked grain (corn, wheat, oats, and barley) containing ap-This study has

G-14495.been supported in part by National Science Foundation Grant,


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Ann. Acad. Scienl. Fennicae A.IV. 7112i

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Fig. 1. Weight loss of spraguedawley rats deprived of nater. Fig. 2. Weight loss of Citellus lateralis deprived of rvater. (Winter 1961 -1962.1

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proximately 45 l/o u'ater. The air-conditioned animal quarl-ers maintained anair temperature of 21' C (+ 2'C) and relative humidity of 30 o,/o, rangingfrom a minimum of 15 o/" in winter to 45 gA in summer. Borly weight measure-ments were determined daily on a Model E Mettler, type K, balance rnitha sensitivity of 0.1 g. Body temperature measurements were made on a Leedsand Northrup potentiometer model 8 692, sensitivity of 0.1" C. Copper-con-stantan thermocouples in 18 to 23 gauge hypodermic needles served as probesfor measuring rectal and skin temperatures. Skin temperature measurementsof the dorsal thoracic area were made daily in several experiments. The skintemperature gave an approximation of body temperature with a minimaldisturbance of the animal (3). Tissue weights lvere determined with an ana-lytical balance to the nearest 0.001 g. The rvet weight samples of lung tissueranged 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

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

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Fig. 3. Weight loss of Citellus lateralis Fig. 4. Weight loss of Citellus spilosoma de-deprived of n'ater. (Summer 1962.) prived of water. (Winter 1962-1963.)or decapitation. The method of sacrificing the animals did not affect the data.Drying of tissues during the dissecting and weighing procedure was kept toa minimum by removing the skin from only one portion of the body at atime, wrapping the tissue in aluminum foil as it r,vas dissected, and weighingit immediately. The tissue samples were dried to constant weight in an electricoven at 41'C (+ 2'C).

The term, Dp value)), which is repeatedly used throughout the text refersto the probability that there is no difference other than that due to randomerrors between the mean values. 'fhe p values of less than 5 o/o 'rvere con-sidered significant.III. Results

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


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380 Ann. Acad. Scient. Fennicae A. IY. 711276789rO|12t314--

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Fig. 5. Weight Ioss ol Citellus spilosoma deprived of water. (Summer 1962.)

G--a SummerC. spilosomor n= 3

a- WinterC. spilosomo, n=8G-- Summer

C. lolerolis, n=9r Winter

C. lolerolis, n=8Fig. 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 agiven 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 sig-nificant weight loss within 24 hours, and a near maximum tolerable weight

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M. L. RrnnnsEL et al., Tolerance of C. lateralis and C. spilosoma 38rDoys

No FoodNo WoterNo Food or WoterFig. 7. Weight loss of Citellus lateralis under conditions of no water,

or food. (Summer 1962.)no food and no water

Fig. S. Weight loss of Citettus spilosoma under conditions of no watet, no food and nofood or water. (Summer 1962.)loss in 72 hours (Fig'. 1). The mean rveight loss of the eight rats deprived ofwater for 72 hours was 18.2 g per 100 g initial body weight. The averageinitial body weight was 267 g (range 220 to 320 g). The average of skin tem-perature measurements made daily was 37.1' C (range, 36.2 to 38'0' C). The

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Ann. Acad. Scient. Fennicae A. IV. 71.122water content of blood, spleen, liver, lung, and muscle taken from three ratsdeprived 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 changedby these conditions.Ground squirrels deprived of water tolerated weight losses equivalent to40 o/o of the initial body weight. There rn'as considerable individuai differencein the rate at which the animals lost weight (Fig, 1-5). The weight loss ofa given animal appeared to be due to the extent of activity and body tem-perature. Flowever, body temperatures taken three times daily did not showa statistical correlation with 24 hour weight loss. Apparently, continuousbody temperature measurements will be necessary to demonstrate the cor-relation of body temperature and rate of weight loss.The rate of weight loss by C. Iateralis during the winter and summer rvassimilar for the first four days, but after five and six days of water depriva-tion the animals lost more weight in the summer (Fig. 6). The rate of weightloss by C. spilosoma was similar in the summer and winter. It is of interestthat C. spilosoma have been reported to aestivate during the summer whereasno reports of aestivation have been made on C. lateralls (7). C. lateralis andC. spilosoma lost weight at similar rates with the exception that C. Iateralisconsistently had a higher per cent body weight loss on the first day of r,vaterdeprivation than did C.spilosoma (Fig.7 and 8). When the dehydrated animalswere given water, the food was r,vithdrawn for five hours. After five hours ofaccess to drinking water the mean per cent weight gained by eight C. Iateraliswas 4.6 o/o of the dehydrated body weight and for eight C. spilosoma rn'as3.8 o/" of the dehydrated body weight.The conditions of (a) no rn'ater, (b) no food and (c) no food or rvater de-monstrate the extent to which weight loss during periods of deprivation ofwater is due to decreased food intake (Fig. 7 and 8). The animals regainedbody weight lost during the six to ten days between each of the experimentalconditions. The C. Iateralis lost weight more rapidly when deprived of waterthan when deprived of food. In contrast the C. spilosoma, which inhabitsa more arid environment, lost weight more rapidly when deprived of foodthan when deprived of water (Fig. 7 and 8). The mean per cent weight lossof 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), and25.0 % (SD : 1.8) when deprived of food and water. The mean per centweisht loss of five C. spilosoma deprived of drinking water for four days was13.5 % (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

1 SD : Standard Deviation


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M. L. Rrnonsnl et al., Tolerance of C. Iateralis and C. spilosoma 383

%Ntl2SD

Active, Ts 22"Cwith wolerActive, To 22oCno woler, 36-427o wl. lossHibernoling, To 80C3-4 doys

ts

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

Peroneus BicepsFemorisLUNG MUSCLE

Fig. g. Per cert water content of tissues f.rom Citellus lateralis. (Data taken in part fromKr.rNnsrrvnn 1962, Table 46.)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: Ilusclesbicepsperoneus femoris

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Laboratory ratC. lateralisC. spilosoma

Spleenn:3 7L.2n:5 72.4n:4 74.3Kidney Lung76.6 '17.873.4. 7 4.371.0 '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 squirrelmuscle tissues was similar (p : 0.4) and the spleen tissues from the C' latertilisand laboratory rat had comparable water content (p : 0'3)' The lung tissuesfrom the two ground squinels had similar water content (p - 0.4).The per cent water of tissues taken from c. Iateralis under conditions ofa) active, b) active, no drinking water, and c) hibernating are presented inFig. 9. The time required for the animals to lose 36 to 42 o/o of their initialbody weight following deprivation of water ranged from 6 to 28 days. Thewater content of spleen and kidney taken from C. Iaternlis was not changedappreciably by any of the experimental conditions (Fig. 9). The per cent waterin lung tissue taken from hibernating animals was greater than that takenfrom 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 depriva-tion of $'ater and hibernation (p:0.05-0'1)' The water content of musclefrom dehydrated and hibernating animals was similar (p : 0.2-0.3).

i


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Ann. Acad. Scient,. Fennicae A. rv. 71127Active, To 22"C, n=5with woterAclive, Ts 22"C, n-5no woler, 34-41 "/" wi. lossHibernoting, To28oC, n=lO2-14 doys

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

Table 1Summary of changes in water content of tissues following deprivation of water andhibernation (Citellus lateralis and C. spilosoma\,

Citellus lateralis Citellus spilosomaActive no water

%Nnoo=c

o_

Peroneus BicepsFemorisLUNG MUSCLE

SpleenKidneyLungPeroneusmuscleBicepsfemoris

no changeno changeno changedecreased?

decreased?

no changeno changeincreased?decreased?

decreased?

no changeincreasedincreased?decreased

decreased?

sl. decreaseincreasedincreaseddecreased

decreased

The water content of tissues taken from c. spilosomo showed greaterchanges in response to deprivation of water and hibernation than did thetissues of C. Iateralls. Periods of 8 to 42 days without water resulted in 34to 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. Duringhibernation there was decreased hydration of the spleen (p :0.03). The percent water of the kidney increased during deprivation of water ancl hiberna-tion (p < 0.001). The water content of lung tissue increased during hiberna-tion (p < 0.001) and had a questionable increase over the control values

Hitlernaling I Active no water I Hibernal,ing


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M. L. Rrrnpsnr, et al., Tolerance of C. lateralis and C. spilosomaduring deprivation of water (p:0.05-0.10). The muscle of animals inhibernation and animals deprived of water had a lower water content thantissues taken from the control group (p : 0.04-0.001). The data from figures9 and 10 are summarized in Table 1.

IY. Conelusions antl summarySimilar experiments on responses to deprivation of water conducted underfield conditions may give very different data as suggested by the large indi-vidual differences observed in the present study. Field animals may be ableto maintain body weight on a similar diet containing 45 o/o water as suggestedby the fact that in this study one C. Iateralis survived 28 days and one C. spi-

Iosoma 42 days rvithout water. The similarity of the responses of. C. lateralisand C. spilosoma to water deprivation is of particular interest because theyinhabit very different ecological areas. The renal capacity of C. spilosomo isprobably greater than that of C. Iateralis as evidenced by less weight loss ofC. spilosoma during the first day of water deprivation. A species differencen'as also noted as there rn'as no change in the water content of C. Iateraliskidney tissue whereas per cent water of kidney tissue from dehydrated andhibernating C. spilosoma was greater than control values (Table 1).Hibernation effects redistribution of body water in C. lateralis and C. spl-losoma (Fig. 9). The shifts in body rvater are apparently due to the coolingof tissues rather than loss of total body water since the decreased watercontent was significant after only two days of hibernation. The mechanismscausing water loss or gain are probably determined by the cell membrane orcell metabolism of the particular tissues. Tissue differences in osmotic regula-tion have been demonstrated in other types of studies (9, 6). Most theorieson mechanisms of water transfer through cell membranes associate watertransfer rn'ith electrolytes and neglect the possibility of an active transfer ofrn-ater. Therefore, the loss of water by cells during hibernation is undoubtedlyassociated with the movement of one or more electrolytes, presumably mag-nesium, potassium, and/or sodium.Future work should include studies of the water and electrolyte con-centrations in additional tissues during various stages of hibernation anddehydration. Identification of osmotic regulation of isolated tissues at variedtemperatures would clarify characteristics of the enzyme systems involved inosmotic regulation of each tissue. Radioisotope studies would be helpful inclarifying electrolyte and water distribution during hibernation.


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386 Ann. Acad. Scient. Fennicae A. lv. 7u27References

1. B,tss, D. ll. & A. HnNscHnr,, 1956: Responses of body fluid compartments to heatand 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 groundsquirrels, 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 So-ciety for Experimental Biology 8: Active Transport and Secretion , 202-227.Cambridge Univ.Press, London.5. Rrnrnsnr, M. L., 1960: The internal environment during hibernation. - Bull.Mus.comp.Zool.I{arv. 124, 421 - 435.6. RonrNsox, J. R., 1960: Metabolism of intracellular rvater. - physiol.Rev. 40, 11,2_ 149.7. sunRnr,r,, 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. Arti-ficial hibernation. Also a contribution to chemical physiology of diurnalsleep. - Ann.Acad.Sci.Fenn. A b3, t,iI.9. lvooosunv, D. M., 1956: Effect of acute hyponatremia on distribution of rvaterand 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 themuscles during the inactive period of hibernation? A second queition is,although the hibernation, you say, went on for only a few days, yet this isplenty of time for shifts of rn'ater due to inactivity of ,rcell pumpsu.-We knowthat in isolated tissues, such as slices of kidney, slices of liver,-and so on, ittakes only a few minutes, to demonstrate a swelling of tissue as soon as itis cooled below about 10'. This swelling is very often credited to the coldnarcosis of cellular pumps-probably not pumps for ',vater but pumps forelectrolyte. I v'onder whethir the best controli for Lhe hibernating animalare not either whole animal or isolated tissue which is chillecl for just a fewrn in uLes'.)Rreonsrr,: There is no significant difference between the total tissue massof the active and hibernating animals. Howevero inactivity may be a factor.It is very important to note that tissues from non-hibernaiors (at leastiso.lated tissues) do gain water u'hen in the cold, ancl I think it is of particular interest that the muscle tissue of hibernators very definitely becomesdehydrated. This difference may involve differences in the seniitivity ofenzymes or enzyme systems to temperature.Kar-r,BN: Did you see any difference in the amount of water in the hiber-nating 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


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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 con-tent of these animals was about the same at the beginning and toward theend of hibernation but it dropped about 2 o7o in mid-season. Could this haveany effect on your curves?RrBoesnr-: No, we do not find a change in the water content with dura-tion of hibernation, but I should point out that our periods of hibernationwere short, from 2 to 14 days. Observations were limited to one rbout,r ofhibernation during a single season.Hocx: In the case of the squimel that rn'ent 42 days without rvater, rn'asthis a very fat squirrel? Did the degree of fatness have anything to do withit or did you consider it in this connection?Rrroeser-: Yes, we considered this. In the first place, I should probablysay that they were all fat. There wasn't a thin one in the lot. There was nocorrelation between initial body weight and the extent of a >boutr. One otherpoint I might make with regard to Dr. Adolph's comment and that is thatI think rn'e should look very closely at differences in osmotic regulation indifferent tissues. I was indeed surprised by our own datan rn-hen I realizedthat it meant that within a given animal one sees quite different osmoticregulations in different tissues.TwrNte: Again, this may be due to species differences. We have a rabherlarge colony of Citellus lateralis, some of n'hich have rvater bottles, and somedo not, but we see no significant difference at all, except that they all gainweight, unless they're sick. Although we have had the colony only a feu'months, the data we now have indicate that weight increase during ther>weight-gaining seasonr> is relatively constant regardless as to whether ornot they have water bottles. Their diet consist primarily of sunflorverseeds, pigeon mix, and we give them a carrot about once a week. We alsogive them half an orange soaked in cod liver oil once a rneek. This is the onlyfree water they would get. On the other hand, rn'hen we deprive squirrelsof 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 un-derstand it.RrsoEssr: I think this is a matter of adaptation. These animals havea considerable capacity to adapt to various amounts of water intake. If ananimal has unlimited water available, and the v'ater is suddenly withdralvn,you w-ill observe marked changes (loss of body weight and even death) asopposed to minor or no observable effects rvhen the amount of water avail-able 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 awild ground squirrel take a drink of water. Has anybody ever seen a rn'ildsquirrel take rvater? I don't believe they do.ZruNv: It is of particular interest (along the water line) that up in ouranimal care room, every once in a while there is either a lack of food givento the animals or a lack of rvater. I have animals that are caged either 4 or6 to a cage. They may go along for weeks without any food, but as soonas 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, thatthis is because of the u'ater factor. Sometimes the rn,ater bottle is there. but


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388 Ann. Acad. Scient. Fennicae A. IV. 7r127the water is not draining properly, or sometimes the caretaker has forgottento put a bottle of water in the cage. when this cannibalistic attack takesplace, it is also interesting that they seem to gouge out the eyeballs and gointo 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 neces-sary 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 isyour feeling about that? Did you get a constant weight at 42"? How muchdifference 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, forthose of you in humid environments, this was done in Albuquerque wherethe 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 ana-lyses?RreoBser-: I have, and I think this mav be a good suggestion.

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TOLERAIVCE OF CITELLUS LATERALIS AND C. SPILOSOMA FOR WATER DEPRIVATION