Seasonal torpor and normothermic energy metabolism in the Eastern chipmunk (Tamias striatus)

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1 J Comp Physiol B (2010) 180: DOI /s x ORIGINAL PAPER Sesonl torpor nd normothermic energy metbolism in the Estern chipmunk (Tmis stritus) Dnielle L. Levesque Æ Glenn J. Tttersll Received: 5 April 2009 / Revised: 5 August 2009 / Accepted: 18 August 2009 / Published online: 16 September 2009 Ó Springer-Verlg 2009 Abstrct To ssess the chnges in thermoregultory chrcteristics tht ccompny the sesonl expression of torpor we mesured sesonl differences in body mss djustments, body temperture (T b ) nd metbolic rte (MR) in both summer- nd winter-cclimted individuls from species of food-storing hiberntor, the Estern chipmunk (Tmis stritus). Torpor occurred only in the winter nd ws ssocited with lower normothermic T b, during inter-bout rousl periods thn in the summer. Chipmunks incresed body mss before the initition of torpor in winter, nd stedily lost mss s the hiberntion seson progressed. Torpor expression ws correlted to initil mss gin, with the individuls who showed the lrgest mss increse in the fll showing the highest degree of torpor. Acclimtion to winter-like conditions produced decline in normothermic MR t ll mbient tempertures exmined. The findings indicte tht torpor expression is ccompnied by decrese in T b nd MR during normothermy, indicting tht conservtion of energy metbolism occurs, not only in torpor, but lso during the inter-bout rousl periods. Keywords Sesonlity Cold-cclimtion Metbolism Hiberntor Torpor Body temperture Acclimtistion Abbrevitions C Wet Wet therml conductnce (W C -1 kg -1 ) C Dry Dry therml conductnce (W C -1 kg -1 ) Communicted by H. V. Crey. D. L. Levesque (&) G. J. Tttersll Deprtment of Biologicl Sciences, Brock University, St Cthrines, ON L2S 3A1, Cnd e-mil: dnielle.l.levesque@gmil.com E Evportive cooling (W kg -1 ) MR Metbolic rte (W kg -1 ) BMR Bsl metbolic rte (W kg -1 ) EWL Rte of evportive wter loss (mg kg -1 h -1 ) f R Brething rte (min -1 ) T Ambient temperture (chmber temperture) T b Core body temperture VO _ 2 Volumetric rte of oxygen consumed by the _ VCO 2 RER WVD Introduction niml (mlo 2 kg -1 h -1 ) volumetric rte of crbon dioxide produced by the niml (mlco 2 kg -1 h -1 ) Respirtory exchnge rtio (rtio of VCO _ 2 : VO _ 2 ) Wter vpour density (bsolute humidity) of the ir leving the chmber (mg H 2 OmL -1 ) In generl, mmmls dpt to the cold by incresing the cpcity to produce het, or decresing the rte of het loss to the environment (Hrt 1971; Brtholomew 1972). However, the smll size of mny mmmls, especilly hiberntors, prevents lrge sesonl djustments in insultion (Gordon, 1993). This reltive incpcity to increse insultion, coupled with the high costs of mintining elevted rtes of metbolism (MR) in the cold, long with sesonl fluctutions in food vilbility, re considered to be the primry fctors which promote the widespred use of sesonl torpor expression in smll mmmls, which consists of periodic djustments in MR nd body temperture (T b ) over the hiberntion period (Lymn et l. 1982; Heldmier et l. 2004). Most thermoregultory studies on hiberntors, however, focus on MR nd T b control during

2 280 J Comp Physiol B (2010) 180: torpor (Brnes nd Buck 2000), or on chrcterising the costs of rousl nd the subsequent normothermic periods (Wng 1978). While sesonlity in MR hs been demonstrted for some heterothermic species the Djungrin hmster (Heldmier et l. 1982) the mrmot (Ortmnn nd Heldmier 2000) for the most prt, thermoregultory control, thermogenic cpcity, nd bsl metbolic turnover rtes (BMR) of hiberntors during their normothermic periods in winter re reltively unknown. Knowledge of the rtes of MR during the normothermic stte cn provide criticl insights into the physiologicl mechnisms t ply which support nd prepre the nimls for torpor, nd is, therefore, n importnt stte to consider. Estern chipmunks (Tmis stritus, Linneus, 1758) provide n interesting model for studying mmmlin thermoregultion. They re one of the few species of hiberntors tht subsist primrily on stored food nd not internl ft stores during the winter hiberntion period (Humphries et l. 2003b; Munro et l. 2008), nd unlike some mmmls (e.g., hmsters), do not require food deprivtion to initite torpor. Although their hiberntion ptterns re reltively similr to those of ft storing hiberntors, food storing hiberntors exhibit shorter torpor bout length nd longer inter-bout rousl times (Humphries et l. 2003b; Munro et l. 2005; Lndry-Cuerrier et l. 2008). This is, in prt, becuse they need to et during rousls to ssimilte the energy required to sustin them in hiberntion until their next rousl (Willis 1982; Humphries et l. 2003b). Recent studies hve lso found tht the length nd depth of torpor bouts in chipmunks re dependent both on the mount of stored food nd the ftty cid composition of the food hord (Geiser nd Kengy 1987; French 2000; Humphries et l. 2003; Munro et l. 2005; Lndry-Cuerrier et l. 2008) generlly, nimls with lrger food hords expressed less torpor (French 2000; Humphries et l. 2003; Humphries nd Rodgers 2004; Lndry-Cuerrier et l. 2008), highlighting tht chipmunk hiberntion is conditionl on predictive energy vilbility. Torpor expression ppers to be purely sesonl occurrence; there hve been no reported cses of torpor outside of winter (Wng nd Hudson 1971; Pivorun 1976). The bility to store most of the energy needed for hiberntion in the form of food, nd not ft, mkes Estern chipmunks nerly unique mong hiberntors in tht they do not increse in mss drmticlly (\25% compred to % body mss chnge in relted Sciuride) prior to the strt of winter (Pnusk 1959; Geiser nd Kengy 1987; Trombulk 1989; Michener nd Lockler 1990; Ortmnn nd Heldmier 2000; Humphries et l. 2003b; Kuffmn et l. 2004). In theory, this mkes them well-suited for sesonl comprisons of metbolic trits becuse the confounds of chnging body size nd body ft composition on mss-dependent trits re minimised (Singer et l. 1995). In ddition, longer inter-bout rousl periods provide greter opportunities to chrcterize thermoregultion nd energetics in normothermic nimls, nd ssess the presence of diurnl fluctutions common to circdin regultion of homeosttic processes. Chipmunks, therefore, provide good model from which to determine whether cclimtion to winter-like conditions leds to metbolic nd thermoregultory djustments outside of torpor expression (i.e., during normothermic periods). We hypothesized tht sesonl torpor expression, mnifested s frequent entries into nd rousls from torpor, would influence the MR nd T b of normothermic nimls throughout the hiberntion seson. Bsed on the premise tht metbolic djustments tht promote torpor crry throughout the hiberntion period, we predicted tht nimls undergo torpor would hve reduced MR expenditure in the normothermic stte. In order to ssess this, we chrcterised the energetics nd thermoregultion in both summer- nd winter-cclimted Estern chipmunks by recording body mss nd dily T b in both summer- nd winter-cclimted nimls nd mesuring MR over rnge of mbient tempertures (T ). Mterils nd methods Animls, husbndry, nd sesonl cclimtion To ssess potentil sesonl differences in the vribles tested, seprte cohorts of nine individul Estern chipmunks were collected in the summer nd the winter. Animls were collected from Te Lke Cmpsite nd the Swn Lke Forest Reserve in Algonquin Provincil Prk, Ontrio ( N, W). During the summer (June August 2007), nine chipmunks (five mles, four femles) were housed indoors t the Wildlife Reserch Sttion in Algonquin Prk. The chipmunks were fed diet of stndrd rt chow nd sunflower seeds which were hidden throughout the cge s form of enrichment. Temperture in the room fluctuted ccording to outside tempertures, but ws not llowed to drop below 15 C or rise bove 30 C. Lighting ws provided vi overhed fluorescent tubes, mintined on light cycle coinciding with nturl light periods (pprox 05:30 21:00 h, 16 h light:8 h drk) chnging throughout the study period to mtch dily nturl light drk rhythms. For the winter phse (October 2007 Mrch 2008), nine nimls (four mles, five femles) were housed in n environmentl control room t Brock University. The room ws kept t tempertures mimicking the known sesonl chnges in burrow tempertures (rnging from 4 to 20 C throughout the winter) of chipmunks from Southern Quebec (D. Munro personl communiction; Lndry-Cuerrier et l.

3 J Comp Physiol B (2010) 180: ). Light cycles were mintined ccording to sunrise nd sunset times in Algonquin Prk to mimic nturl light:drk cycles. Rt chow ws provided up to mid- November, period in which chipmunks experience more mixed diet in the wild (Humphries et l. 2001), fter which they were exclusively fed sunflower seeds until the end of the hiberntion period in April when their feed ws once gin supplemented with rt chow. Body mss ws recorded t the strt of ech experiment, or once week during cge clening. Certin individuls in the winter were weighed less frequently due to deep periods of torpor when they were left undisturbed. Dt collection lsted from the end of June to the 3rd of September in the summer nd from the first week of Jnury to the first week of April in the winter. To ensure tht the housing setup ws sufficient to induce torpor in the chipmunks, preliminry study ws undertken in the fll of Ten nimls (four femles nd six mles) were housed under identicl conditions to the winter-cclimted nimls. Only body mss chnges were collected from these individuls. All nimls were implnted with temperture-sensitive telemeters (single-stge rdio trnsmitters, Sirtrck TM, Hvelock North, NZ) tht were coted with biologiclly inert enmel nd weighed no more thn 3 g (\3% body mss). In the summer phse, nimls were given week to djust to cptivity before undergoing surgery for telemeter implnttion. Due to erly fll immergence dtes for this species (Humphries et l. 2002), it ws necessry to cpture the nimls for the winter phse few months before the hiberntion period. The limited bttery life of the telemeters (4 months) prevented them from being implnted throughout the entire October Mrch cptivity period, therefore surgery ws undertken in lte-november to ensure tht the telemeters would lst throughout the experimentl period (Jnury Mrch). Before implnttion, ech telemeter ws individully clibrted ginst rnge of tempertures (5 42 C, NIST stndrdised) to determine the correct formul ( 5th order polynomil) to convert the pulse intervl into temperture (precision is less thn or equl to ±0.1 C). The telemeters were implnted into the peritonel cvity to provide ccurte redings of core T b throughout the study. Additionl detils on housing nd the methods used for ssessing T b with telemeter implnts re outlined in Levesque nd Tttersll (2009). Series I: spontneous fluctutions in T b Telemeter implnts llowed for the recording of dily T b fluctutions throughout the entire period of study, except during periods when the recording equipment ws utilised by the experiments in Series II. A rdio receiver (R1000 Receiver, Communictions Specilists Inc., Ornge, CA, USA) ws progrmmed to scn between chnnels, preprogrmmed to ech telemeter s frequency t 60 s intervls, llowing for 1 min recordings from ech individul. The period intervl between pulses (s) ws determined using the Tch3 Intelligent Tchometer (Sble Systems, Ls Vegs, NV, USA) nd recorded to computer (Expedt softwre, v1.0.18, Sble Systems). The period vlues were converted into T b using 5th order polynomil determined for ech telemeter. Dt nlysis: Series I The dt from the week following surgeries were excluded from ll T b nlyses to ensure full recovery. To determine if there were diurnl ptterns in T b in the summer nd the winter, T b vlues recorded for ech individul were binned into hlf-hour segments ccording to time of dy. The sesonl comprison of T b ws divided into two prts. The first compred ll T b s from the summer to those from the winter, including torpid vlues. The second ws direct comprison of normothermic vlues, therefore the torpor bouts were excluded from the winter dt. A spredsheet ws designed to seprte the torpid T b from the normothermic vlues. The cut-off T b for designting torpor (\35.5 C) ws chosen s described in Willis (2007); this vlue ws the T b t which resting metbolic rte ws observed to drop below bsl metbolic rte for three individuls tht entered torpor during respirometry experiments t 8 nd 15 C (dt not shown). A sesonl comprison ws mde between vrious T b prmeters. The first ws between the bsolute mximum nd minimum T b vlues recorded from ech niml. To compre vlues tht were more representtive of overll trends, the dily mximum nd minimum vlues from the hlf-hour segments (i.e., the time of dy t which the individul hd the highest nd lowest verge T b, respectively) were used. Confidence (95%) limits of T b for summer nd winter normothermic periods, s well s winter torpor periods, were clculted for ech niml. Torpor ptterns were nlysed by isolting ech individul torpor bout nd recording: the time t which the nimls entered nd roused from torpor, the length of the torpor bout, nd the length of the preceding normothermic (interbout) intervl. Only bouts for which precise entry nd exit times recorded were included in the verges. All nimls occsionlly showed brief periods (generlly less thn 1 h) where T b declined to no less thn 32 C. These bouts were considered short bouts nd the length of these bouts were excluded from the clcultion of verge bout length. Series II: respirometry experiments Flow-through respirometry ws used to obtin vlues for oxygen consumption ( _ VO 2 ), crbon dioxide production

4 282 J Comp Physiol B (2010) 180: ( VCO _ 2 ), evportive wter loss (EWL) nd therml conductnce during both sesons. Experiments were run over rnge of T to record chnges in thermoregultory chrcteristics nd minimum therml conductnce between sesons. Trils were conducted t T s of 8, 15, 22 nd 29 C; t lest three of these T s (8, 15 nd 22 C) fll below the reported therml neutrl zone for this species (28 36 C; Neumnn 1967; C; Wng nd Hudson 1971). Animls were fsted for 8 h before the experiment strted to ensure post-bsorptive stte. Experimentl times were chosen to coincide with the time of dy when the nimls were most likely to be t rest. During the summer phse this ws between 21:00 nd 04:00 h but ws more vrible in the winter where the time of dy t which ech individul ws most likely to rest ws different. Erlier strt times ( h) were necessry for the few individuls tht went torpid ech night, nd lter times ( h) for those tht remined ctive lter on in the dy. Ech experiment consisted of period of 4 h in which the niml ws plced in cylindricl respirometery chmber (700 ml, 8.5 cm dimeter Animl Chmber, Qubit Systems, Kingston, ON, Cnd). The chmber ws plced inside temperture-controlled environmentl chmber consisting of cooler (Rubbermid Ò ), in which the T ws controlled using wter bth connected to n internlly mounted het exchnger/fn ssembly. Dry CO 2 - free ir ws pumped into the chmber t rte of 400 ml min -1. The incurrent ir ws scrubbed of H 2 O nd CO 2 using column contining lyer of Drierite TM, lyer of sod-lime, nd finl lyer of Drierite TM, necessry for bsorbing H 2 O vpour relesed by the sod lime. A subsmple of the ir from the respirometer ws pulled through the O 2, CO 2, H 2 O nlysers t 180 ml min -1 using Sble PP2 Dul Pump System, controlled vi n nlog mss flow controller. The ir ws first routed through reltive humidity nlyser (Model RH200, Sble Systems), to record the H 2 O vpour density (lg ml -1 ). The respirometer ir ws subsequently diverted through tube contining Drierite TM prior to entering crbon dioxide nlyser (Model CD-3A; AEI Technologies, Nperville, IL, or CA-2; Sble Systems). From there, the ir smple ws pssed through tube contining sod lime followed by Drierite TM nd finlly through the oxygen nlyser (FC-1B O 2 Anlyser, Sble Systems). The following were recorded to dt cquisition system (Expedt, Sble Systems) t one smple per second: the frctionl concentrtions of O 2 nd CO 2, nd WVD in the ir leving the chmber, the incurrent flow rte, respirometer T, environment chmber T, nd T b telemeter pulse rte. T in the environmentl chmber nd in the respirometry chmber were monitored using thermocouple meter (Model TC-2000; Sble Systems). All thermocouples were pre-clibrted by the provider (Sble Systems) to 0.2 C; clibrtions were verified to within 0.1 C by submerging them in ice wter nd in boiling wter. The positive side of differentil pressure trnsducer (Vlidyne model DP4510, Northridge, CA, USA) ws lso connected to the respirometer chmber, permitting the continuous recording of pressure within the chmber, which, though not clibrted to provide tidl volume, provided brething rte (f R ). To ensure ccurte detection of f R, higher smple rte, 100 smples s -1, ws necessry. Therefore, dt were collected from the pressure trnsducer into Biopc Ó MP150 nd recorded in Acq- Knowledge (v , BIOPAC Systems, Golet, CA, USA). The O 2 nlyser ws regulrly clibrted using CO 2 - free, dried ir (20.95% O 2 ). The CO 2 nlyser ws similrly clibrted using pure nitrogen s zero vlue nd 1% CO 2 mixed gs (certified) s spn gs. The H 2 O nlyser ws lso clibrted regulrly using pure nitrogen s the zero vlue, nd ir bubbled through wter of known temperture, nd therefore known WVD, s the spn gs. Empiriclly determined CO 2 :O 2 vlues using the combustion of ethnol yielded vlues of , suggesting tht the clibrtions were ccurte ssessments of niml CO 2 production nd O 2 consumption. To ensure tht the incurrent O 2,CO 2 nd WVD remined constnt throughout the experiment, bseline mesurement, consisting of dry crbon-dioxide free ir, ws set up to record for 3 min every 20 min. To do so, gs flow distributor (Sble Systems, RM8 Intelligent Multiplexer) ws plced just fter the respirometer nd progrmmed to control which ir strem (the respirometer or the bseline) entered the gs nlysers. Dt nlysis: Series II The respirometry files were nlysed initilly by correcting ll frctionl gs concentrtions for nlyser drift throughout the experimentl period using the collected bseline vlues. Vlues for VO _ 2, VCO _ 2, EWL, T b, C WET nd RER were clculted using equtions dpted from pge 456 of Withers (2001), by inputting the recorded vlues for O 2 nd CO 2, WVD, flow rte, T nd telemeter pulse rte into spredsheet. To obtin stedy-stte vlues, pre-recorded mcro ws used to locte multiple 3-min sections of dt with the most stble trce in resting stte. The lowest of these vlues ws used s the resting rte for tht T. Experiments in which the niml did not rest for the entire 4 h period were re-ttempted t different dte. Some nimls would not rest t the lowest T s (two t 8 C, one t 15 C) nd vlues for these nimls were omitted from the nlysis. To ccount for potentil effects of slight differences in body mss on the metbolic vribles mesured, ll mss-dependent vlues were divided by body per

5 J Comp Physiol B (2010) 180: mss -1 (Blxter 1989). Initil nlyses hd been performed using mss -0.75, however, using mss -1 did not chnge ny sttisticl outcomes nd therefore, the ltter (mss -1 scling) ws retined for simplicity in dt nlysis nd reporting. VO _ 2 nd EWL (in mlo 2 kg -1 h -1 nd mg kg -1 h -1 ) were trnsformed into MR (W kg -1 ) nd E (W kg -1 ) by multiplying them by energetic equivlence of oxygen, s determined by the RER (Blxter 1989), nd the ltent het of vporiztion (McNb 2002), respectively. Wet therml conductnce (C WET ) ws clculted using Eqution 3 from McNb (1980). Stedy stte vlues of f R (min -1 ) obtined from the sme periods of time where MR obtined were clculted by tking the inverse of the verge time the niml required for ten breths nd multiplying this by 60-sec. Due to the loss of function of some of the telemeters before the end of the study period, vlues for T b nd C WET hd smller smple sizes thn MR, VO _ 2, VCO _ 2, RER, E nd f R (Tble 4). Mesuring wter loss t 8 C proved to be impossible becuse of excess condenstion in the chmber, suggesting fully sturted ir. Similrly, if the niml urinted during the experiment, which hppened regulrly t the lower T, ccurte vlues for evportion could not be recorded; these vlues were omitted from the clcultion nd nlysis of E. Sttisticl nlysis All sttistics were performed using SigmStt or Systt 12 (Systt Softwre, Inc., Point Richmond, CA, USA) nd resultnt p vlues were compred to n -vlue of 0.05 unless otherwise stted. One-wy ANOVAs were used to nlyse vlues for bsolute mximum nd minimum body mss s well s rtes of mss chnge (% mximum mss dy -1 ) for summer nd winter nimls. For this nlysis, the winter group included mss dt from the Fll of 2006 nd ws seprted into two groups nimls who consistently entered torpor (winter torpid) nd those who remined normorthermic (winter normothermic). Summer vlues for T b, s well s the time t which the mximum nd minimum vlues were recorded were compred to overll winter T b (including torpor), normothermic winter T b nd torpid winter T b using multiple t tests or, if the dt were non-norml, Mnn Whitney U tests. To compenste for the use of multiple t tests on dt derived from similr vlues, the lph vlue for these tests ws Bonferroni-corrected to 0.01 for the summer/winter comprison nd 0.01 for the comprison of normothermic vlues between sesons. Summer nd winter vlues for MR, VCO _ 2, T b, C WET, RER, E nd f R were compred using two-wy repeted mesures ANOVA (RMANOVA), with seson nd T s the two fctors tested. When significnt differences were observed, the Holm Sidk post hoc method for multiple pir wise comprisons ws used. If the dt filed to meet the ssumptions of normlity or homoscedsticity, trnsformtions (log, squre-root or reciprocl) were used. Results With the exception of single bout in the summer (where T b dropped s low s 30 C for period of 2 dys) torpor ws restricted to the winter months (December April) nd showed high level of inter-individul vrition in terms of degree nd mgnitude (Fig. 1; Tbles 1, 2). While only eight of out of the eleven individuls from the Fll- Winter of 2006 expressed torpor, ll but one (8/9) of the individuls from the Winter of 2007 expressed torpor (Tbles 1, 2). Sesonl vritions in body mss Animls in both sesons hd vrible msses t dte of cpture ( g) nd tended to gin pproximtely 10 g during the first few weeks in cptivity. A slight increse in mss ws observed in ll individuls over the summer seson, with some of the inter-individul vribility Men Body Temperture Men Body Mss (g) Jun Aug Oct Dec Feb Apr Dte Fig. 1 Men (±SD) body mss nd men mximum (filled circles) nd minimum (open circles) body tempertures of summer- nd winter-cclimted chipmunks over time. Stndrd devition is only presented for the body mss for visul clrity. Summer nimls were relesed round the 1st of September 2007 nd the dt s of October 2007 re from different group of individuls. The shded res indicte the dtes over which the nimls were used in the respirometry studies. The telemeter implnt dtes re indicted by the dshed lines

6 284 J Comp Physiol B (2010) 180: Tble 1 Dtes of the initition nd termintion of torpor expression in Estern chipmunks in the winter Individul Dte of onset of torpor End of torpor Proportion of time spent torpid 1 30 November 2007 fter 2 Februry December Mrch Jnury Jnury December Mrch December 2007 After 3 Mrch 2008 b December 2007 After 3 Mrch 2008 b December April December April Finl dte of torpor unknown becuse the telemeter s bttery died erly, 2 Februry 2008; however, the niml ws observed to be torpid fter tht dte b The btteries in both nimls telemeters died round the 3 Mrch 2008; subsequent bouts of torpor in both individuls were observed Tble 2 Chrcteriztion of torpor prmeters in chipmunks from the winter phse Individul No. of short bouts No. of long bouts Averge bout length (h) Mximum bout length (h) Minimum bout length (h) Averge interbout rousl time (h) Minimum torpid body temperture Averge torpid body temperture Men ± SD 9 ± 5 51 ± ± ± ± ± ± ± 3.3 An niml ws considered to be torpid whenever T b dropped below 35.5 C for more thn 30 min. All bouts of torpor lsting more thn 30 min nd less thn 2 h in which T b remined bove 32 C were clssified s short bouts. The remining bouts, those lsting longer thn 2 h, where clssified s long bouts. Only long bouts for which the precise entry nd exit times were known were included in the clcultion of the verge bout length becoming dmpened s the seson progressed (Fig. 1). Body mss in the summer rnged between 90 nd 110 g. The minimum vlue for mss of the summer nimls ws recorded t the dte of cpture (91.4 ± 11.3 g), nd the mximum ws recorded in mid-august (106.7 ± 7.4 g). Mss ws much more vrible in the winter, both t dte of cpture ( g) nd throughout the seson (Fig. 1). A generl trend, however, ws still pprent with ll nimls initilly incresing in body mss, nd reching pek verge of.7 ± 14.9 g ner the end of October, before declining stedily until the end of Mrch when most individuls cesed hiberntion nd commenced re-gining mss. At the time of relese, April 2008, mss reched levels similr to individuls cught in the summer ([90 g). The downwrd trend in mss in the winter begn before the implnt surgeries. An RMANOVA performed on the mss tken from ech niml just before the metbolic experiments (Series II) indicted tht, in spite of n initil mss increse in the fll, winter nimls hd lower body mss thn summer nimls (F 1,17 = 9.0, p = 0.007). Rte of mss loss differed in nimls tht regulrly expressed torpor (n = 15), nd ws greter thn the summer (n = 9) nd winter nimls (n = 4) tht remined normothermic throughout the period of study (Fig. 2; F 2,26 = 42.2, p \ 0.001). Similrly, nimls tht expressed torpor regulrly hd both higher mximum (Fig. 2b; F 2,26 = 12.6, p \ 0.001) nd lower minimum mss vlues (F 2,26 = 9.3, p \ 0.001). Series I: Torpor expression nd spontneous vrition in T b Significnt differences were observed between the summer nd the winter vlues for bsolute mximum nd minimum T b, s well s verged dily mximum nd minimum vlues (p \ 0.001, t tests nd Mnn Whitney tests). T b ws consistently lower in the winter even during periods of normothermy (p \ 0.01, t tests nd Mnn Whitney U tests;

7 J Comp Physiol B (2010) 180: Rte of Mss Loss (% dy -1 ) Mss (g) b Summer Winter Normothermic Fig. 1, Tble 3). The time of dy t which the mximum dily T b ws observed ws lso different; 06:37 h ± 38 min in the summer compred to 14:48 h ± 202 min in the winter (T 8,8 = 99, p \ 0.001). However, no differences were observed between the times t which the minimum T b occurred; 00:27 h ± 49 min in the summer 01:33 h ± 189 min in the winter (T 8,8 = 61, p = 0.5). All of the vlues recorded during the winter phse showed higher inter-individul vrition thn the summer vlues (Fig. 3). Cler diurnl ptterns in T b were pprent in nimls from the summer phse (Fig. 3); there ws visible distinction between nocturnl T b ( h), nd diurnl T b ( h). In the summer, T b reched pek round 07:00 h coinciding with observed bouts of ctivity. T b declined grdully throughout the fternoon, reching plteu just before nightfll, fter which it declined to night-time vlues. In ddition, the verge dily mximum T b differed from the verge minimum (t 8 = 20.88, p \ 0.001). Similr ptterns were less pprent in the winter; lthough the dily mximum nd minimum T b were different for normothermic c Winter Torpid Fig. 2 Men (±SD) rte of mss chnge per dy (clculted s the slope of mss chnge per dy divided by mximum body mss nd multiplied by 100; % mximum mss dy -1 ) from summer-cclimted Estern chipmunks nd winter-cclimted nimls tht did not exhibit torpor (winter normothermic) or went torpid (winter torpid) throughout the experimentl period. Indictes significnt difference between groups. b Men (±SD) of the mximum (white brs) nd minimum (blck brs) for body mss from summer, winter normothermic nd winter torpid chipmunks. Different symbols represent sttisticlly significnt difference for mximum or minimum vlues between groups b b vlues (T 8,8 =-36, p = 0.008) no similr difference could be observed in the torpid vlues (t 4 = 3.32, p = 0.029, = 0.01). Series II: respirometry A lower MR ws mesured in winter-cclimted nimls thn in summer-cclimted ones (F 1,16 = 6.9, p = 0.017) nd MR incresed t lower T s(f 3,46 = 515.7, p \ 0.001; Tble 4, Fig. 4) during both sesons. VCO _ 2 levels were similrly ffected by seson (F 1,16 = 5.9, p = 0.026) nd T (F 3,45 = 357.0, p \ 0.001). While no differences were observed in RER between sesons (F 1,17 = 3.1, p = 0.09), T hd n effect (F 3,45 = 8.6, p \ 0.001); RER vlues for 29 C were higher thn those for the other T s nd T -seson interction ws mesured t 29 C (F 3,45 = 3.4, p = 0.027); where the summer vlues were higher thn the winter ones. Effects of seson nd T on f R were mesured (F 1,16 = 34.2, p \ 0.001; nd F 3,43 = 101.1, p \ 0.001, respectively), lthough there ws no T -seson interction (F 3,43 = 1.1, p = 0.34). f R ws consistently lower in the winter thn in the summer, nd during both sesons incresed s T decresed. Additionlly, seson ffected E (F 1,11 = 9.1, p = 0.006), which ws consistently lower in the winter thn in the summer, lthough no differences were mesured in E mong different T (F 2,11 = 0.63, p = 0.55). Sesonl differences were lso pprent in T b during the respirometry experiments; T b ws ffected by seson (F 1,13 = 12.8, p = 0.003) nd T (F 3,28 = 3.3, p = 0.037), lthough no seson-t interction ws observed (F 3,28 = 1.4, p = 0.25). As with the dily T b dt, T b during these experiments ws lower in the winter thn in the summer. The only within seson difference ws found between vlues from 8 C nd those from 29 C, lthough the generl trend ws for T b to increse with decresing T (Fig. 4). C wet did not chnge sesonlly (F 1,13 = 2.4, p = 0.144) nd the only difference between T vlues ws found t 29 C which ws higher thn ll other recorded vlues for therml conductnce (F 3,27 = 20.5, p \ 0.001). C dry equlled C wet t ll T s, except for 29 C nd is therefore not reported for the lower tempertures. At 29 C, C dry ws equl to the miniml levels found t the lower T s ( W C -1 kg -1 ). Correltions between torpor expression nd energetics Since torpor expression vried in the winter chipmunks, we were ble to estblish correltions between vribles of interest. Averge torpor bout length ws strongly nd inversely correlted with the verge torpid T b (F 1,6 = 30.8, p = 0.001, r 2 = 0.81; Fig. 5). This tight correltion llowed for the estimtion of Q 10 for torpor

8 286 J Comp Physiol B (2010) 180: Tble 3 Men ± SD of the dily mximum nd minimum T b vlues from summer- nd winter-cclimted Estern chipmunks Summer Winter (ll vlues) Winter (normothermic vlues only) Minimum Mximum Minimum Mximum Minimum Mximum Averge 37.9 ± ± ± ± ± ± 0.5 Time of dy t verge 00:00 h 06:30 h 01:00 h 14:00 h 21:30 h 12:00 h 95% confidence limits Lower Upper Lower Upper Lower Upper 37.1 ± ± ± ± ± ± 0.6 Averge vlues refer to the time of dy (when binned in hlf hour increments) tht hd the highest, or lowest, body temperture. The verge 95% confidence limits of the body temperture derived from ech individul re lso presented. The second group of winter vlues (normothermic vlues only) were tken solely from the periods of normothermy between torpor bouts T b b mss loss nd the proportion of time tht the individuls spent in torpor (see next section for torpor ssessment); greter degree of torpor expression correlted to higher rtes of mss chnge (F 1,6 = 8.1, p = 0.029, r 2 = 0.50; Fig. 5b). BMR (MR from chipmunks t 29 C) ws negtively correlted with the number of torpor bouts exhibited over the course of the entire winter (F 1,6 = 9.0, p = 0.024, r 2 = 0.53; Fig. 5c). In ssocition with chnges in BMR, the normothermic T b exhibited by chipmunks during their bouts of rousls ws lso negtively correlted with the degree of torpor expression (F 1,6 = 15.3, p = 0.008, r 2 = 0.67; Fig. 5d). 40 T b T b c Locl time (h) Fig. 3 Diurnl ptterns in T b in the two sesons of the study, the plots represent the mens (±SD) of the T b for ech hlf hour of the dy from the summer phse (), normothermic only T b vlues from the winter individuls (b), nd torpid vlues only from the winter individuls (c) durtion (4) by clculting the slope for ln (bout length -1 ) versus T -1 (K -1 ) nd deriving the ctivtion energy. There ws lso correltion between the rte of Discussion The chipmunks in this study showed yerly rhythms in both mss nd T b. Mss incresed over the fll in preprtion for hiberntion, nd decresed s hiberntion progressed, nd, in the winter, ws elevted in nimls tht spent greter proportion of time in torpor. Concurrent with the decline in mss, T b fluctuted to greter extent in the winter, with the mjority of the nimls expressing torpor throughout the seson. Torpor expression lso resulted in chnges to the diurnl pttern of T b regultion; dily peks nd ndirs in T b were diminished in the winter nimls, whether in torpor or during their periods of normothermi. As result, even while normothermic, winter nimls hd lower T b thn the summer nimls. This decrese in T b ws concurrent with decline in MR with the sesonl difference becoming greter t lower T.In sum, the sesonl chnges ssocited with hiberntion in the chipmunk result in n energy spring response during periods of normothermic rousl tht ppers to be fuelled by diminished diurnl chnges in T b, s well s decrese in metbolic turnover (i.e. BMR). Sesonlity in body mss Chnges in body mss over the period of study were similr to those observed in other lbortory bsed studies

9 J Comp Physiol B (2010) 180: Tble 4 A summry of thermoregultory vribles from the summer (S) nd winter (W) phses T (8C) MR (W kg -1 ) S ± 1.31(9) ± 1.19 (9)b 9.27 ± 0.98 (9)c 6.45 ± 0.50 (9)d W ± 1.53(8) ± 1.26(8)b 8.94 ± 0.38 (9)c 6.22 ± 0.28 (9)d VO _ 2 (mlo 2 h -1 kg -1 ) S 3,561 ± 239 (9) 2,745 ± 237 (9)b 1,687 ± 190 (9)c 1,131 ± 86 (9)d W 3,303 ± 273(8) 2,568 ± 231(8)b 1,627 ± 74 (9)c 1,124 ± 52 (9)d VCO _ 2 (mlco 2 h -1 kg -1 ) S 2,444 ± 190 (9) 1,814 ± 191 (9)b 1,203 ± 107 (9)c 989 ± 62 (9)d W 2,291 ± 216 (8) 1,792 ± 168 (8)b 1,161 ± 69 (9)c 826 ± 47 (9)d RER S 0.69 ± 0.03 (9) 0.68 ± 0.03 (9) 0.72 ± 0.04 (9) 0.84 ± 0.14 (9) W 0.69 ± 0.02 (8) 0.70 ± 0.02 (8) 0.71 ± 0.04 (9) 0.74 ± 0.03 (9) T b (8C) S 38.6 ± 0.6 (7) 37.9 ± 0.5 (5)b 37.9 ± 0.8 (7)b 37.5 ± 0.5 (5)b W 38.1 ± 0.7 (7) 37.2 ± 0.8 (5)b 38.0 ± 0.4 (7)b 37.3 ± 0.4 (7)d C WET (W C -1 kg -1 ) S 0.65 ± 0.05 (8) 0.65 ± 0.07 (7) 0.58 ± 0.07 (8) 0.78 ± 0.07 (7)b W 0.61 ± 0.04 (4) 0.63 ± 0.09 (4) 0.60 ± 0.03 (5) 0.78 ± 0.06 (7)b E (W kg -1 ) S n/ 0.71 ± 0.13 (2) 0.86 ± 0.21 (4) 0.86 ± 0.12 (7) W n/ 0.69 ± 0.04 (3) 0.64 ± 0.07 (7) 0.59 ± 0.09 (7) f R (min -1 ) S 120 ± 29 (9) 104 ± 39 (9)b 63 ± 15 (9)c 45 ± 10 (9)d W 74 ± 16 (8) 57 ± 13 (8)b 37 ± 11 (9)c 25 ± 7 (9)d All dt presented re mens ± SD (n) Significnt differences between mbient tempertures re indicted by different letters Significnt difference between sesons on Estern chipmunks. Individuls from the summer were slightly hevier thn the verge wild-cught summer niml, which generlly weighs less thn 100 g (Pidduck nd Flls 1973). This mss gin is consistent with generl increse in the body mss observed in some cptive species (Kengy 1981; Lrcombe nd Withers 2007 etc.), including Estern chipmunks (Pnusk 1959; Forbes 1966). More extreme mss gins were observed in the fll, during the beginning of winter, consistent with the prehiberntion mss gin in wild chipmunks (Forbes 1966; Scott nd Fisher 1972; Pivorun 1977). It is not surprising tht the 15 20% mss gin before the onset of hiberntion differs from the extreme, % mss gin observed in ft-storing hiberntors (Pnusk 1959; Trombulk 1989; Michener nd Lockler 1990; Ortmnn nd Heldmier 2000; Kuffmn et l. 2004), since chipmunks rely primrily on their food hord to provide enough energy to sustin them for the winter (Humphries et l. 2003b). However, despite the fct tht the nimls were provided food d libitum throughout the winter phse, stedy decrese in the mss of the nimls regulrly exhibiting torpor ws recorded s the seson progressed. The onset of torpor expression resulted in stedy decrese in mss; furthermore, the rte of mss decrese ws dependent on the mount of time spent torpid. Thus, despite the vilbility, nd consumption, of food during the hiberntion seson (Humphries et l. 2001), chipmunks still utilize ft stores during torpor bouts or the subsequent interbout rousl periods, s witnessed by the lower RER in the winter (0.74 ± 0.03 compred to 0.84 ± 0.14 in the summer). Once the nimls hd cesed hibernting for the seson, body mss returned to levels similr to those t cpture, consistent with previous studies (Pnusk 1959; Scott nd Fisher 1972). Sesonl differences in T b nd diurnl rhythms T b ptterns were significntly more complex thn chnges in body mss. The first bouts of winter torpor were short, resembling the test-drops tht chrcterise the strt of ll

10 288 J Comp Physiol B (2010) 180: T b MR (W. kg -1 ) C (W C -1 kg -1 ) b b T b Fig. 4 Metbolic rte, T b nd therml conductnce in summer- nd winter-cclimted chipmunks conducted during normothermic periods t 8, 15, 22, nd 29 C. All vlues presented re mens (± SD). Summer dt re represented by blck circles, while winter vlues re indicted in white (n = 9 for ech seson). Different lettering indictes significnt effects of temperture. Indictes sesonl differences hiberntion periods (Strumwsser 1958; Pivorun 1977; Lymn et l. 1982). However, three individuls never progressed pst this stge into the deeper stges of hiberntion. This vrition in torpor expression is typicl of lbortorybsed studies on this species (French 2000; Humphries et l. 2001). Food supplementtion throughout the winter my hve hd n dverse effect on torpor expression, since c b d b Bout Length (h) BMR (W kg h -1 ) T b in Torpor Time in Torpor (%) c b d # Torpor Bouts Time in Torpor (%) Fig. 5 Correltions mong physiologicl vribles mesured in torpid chipmunks. Demonstrtes negtive correltion between verge bout length nd verge T b in torpor (r 2 = 0.81), b depicts the negtive correltion between dily mss chnges nd the proportion of time spent in torpor (r 2 = 0.50), c shows the negtive correltion between bsl metbolic rte during periods of normothermic rousl nd totl torpor bouts exhibited throughout the winter (r 2 = 0.53), nd d illustrtes the influence tht torpor expression hs on normothermic T b itself (r 2 = 0.67). Filled circles re derived from winter chipmunks exhibiting vrition in torpor expression. Open squres show the verge (±SD) from summer, normothermic nimls for comprison incresed hord size negtively ffects torpor expression (French, 2000; Humphries et l. 2003; Lndry-Cuerrier et l. 2008). Furthermore, the food provided (sunflower seed), is high in polyunsturted ftty-cids, which, lthough importnt for hiberntion, cn lso reduce torpor bout length t high dietry concentrtions (Geiser et l. 1997; Munro et l. 2005). To void dietry confounds, future studies should include more nturlized diet over the winter periods. Overll, it ppers tht lthough ll nimls hd the bility to hibernte in the winter, most did not hibernte s deeply s hs been observed in free-rnging individuls (Humphries et l. 2003; Lndry-Cuerrier et l. 2008), remining t torpid T b greter thn 14 C or only going torpid during the night. Similr ptterns hve been observed in the wild, nd it is believed tht, lthough hiberntion benefits the niml in terms of energy svings, it is of greter benefit to the individul to reduce the time spent torpid when energy stores, in the form of stored food, re sufficient (French 1985; 2000; Humphries et l. 2003, b; Lndry-Cuerrier et l. 2008). Indeed, the tight correltion between verge torpor bout durtion nd T b during torpor in this study (Fig. 5), yields Q 10 of 4, suggesting tht the time invested in torpor, lthough prtly explined by torpor T b (French 1982; Thoms nd Geiser 1997; Buck nd Brnes % Mss chnge dy -1 Normothermic T b

11 J Comp Physiol B (2010) 180: ), is more likely driven by the metbolic rte experienced during torpor itself (Geiser nd Kengy 1988), shedding light on the inherent reltionship between metbolism nd torpor use ptterns in hiberntors. It should be noted, however, tht while torpor in Estern chipmunks, t lest in the northern prts of their distribution rnge, is often reduced in the presence of lrge mounts of stored energy, it is rrely bndoned completely (Humphries nd Rodgers 2004; Munro et l. 2005; Lndry-Cuerrier et l. 2008). This indictes some benefit to hiberntion outside of energy conservtion. Indeed, it could lso represent bet-hedging strtegy llowing them to crry unused food stores over to the next hiberntion seson (see Munro et l. 2008) or reduce the inherent costs of normothermic thermoregultion during periods of extreme cold (Lndry-Cuerrier et l. 2008). Torpor expression ws not the only spect of the chipmunk s T b regultion to differ between sesons. Diurnl ptterns in T b were vstly different s well. A cler bimodl dily pttern ws observed during the summer (Fig. 3), consistent with the dily ctivity ptterns of chipmunks (Elliot 1978; Richter 1978; Decoursey et l. 1998). Animls were most often t rest overnight, when the lowest T b ws observed. T b generlly incresed shortly before sunrise, nd the period of gretest ctivity ws observed in the erly morning. Diurnl ptterns in normothermic T b were much less evident, nd nerly bsent in some individuls in the winter. In generl, pek normothermic T b ws observed during the light hours, which would be consistent with the higher levels of ctivity observed in the chipmunks during the dy. Similrly, the lowest normothermic T b vlues from ll individuls were consistently recorded overnight. However, this pttern my hve been influenced by the few individuls tht brely hibernted or the two individuls tht only ever went into torpor overnight nd tht were lwys ctive during the dy. The four hiberntors tht hd the longest torpor bouts showed very little in the wy of diurnl normothermic T b ptterns; their ptterns of entry to nd rousl from torpor lso did not follow ny distinct pttern. This lck of dily T b ptterns in hibernting nimls hs lso been observed in Antolin ground squirrels (Spermophilus xnthoprymnus) (Krt Gür et l. 2009). Other studies monitoring T b in chipmunks hve filed to find sesonl differences in normothermic T b s (Neumnn, 1967; Wng nd Hudson, 1971). In the present study, however, chipmunks hd lower normothermic T b in the winter. Although the difference ws less thn 1 C, the fct tht normothermic winter T b ws consistently lower is significnt. Interestingly, similr drop in normothermic T b, ttributed to reduction in ctivity or in energy expenditure, hs been observed in both dily heterotherms (Christin nd Geiser 2007) s well s hiberntors (Krt Gür et l. 2009)in periods immeditely preceding torpor use. Defending lower normothermic T b could permit slight decreses in energy expenditure. In normothermic mmmls, both hiberntors nd non-hiberntors, dily oscilltions in T b re generlly lrger t lower T (Refinetti nd Menker 1992). This hs been ttributed to lrger drop in T b when thermoregultion is prtilly shutdown during sleep (Gordon 1993), lthough corresponding chnges in dily mximum T b re rre (Refinetti nd Menker 1992). Given the lower normothermic T b observed in winter-cclimted chipmunks, it suggests tht nimls in the present study spent considerble mounts of time resting or sleep during their rousl episodes. T b ws reltively low (\38 C; Fig. 5d) during the normothermic periods of individuls tht hibernted more deeply, possibly becuse the nimls were not ctive or wke during the entirety of these rousls. Indeed, it hs been suggested tht normothermic periods my be times when hiberntors mke up for the loss of sleep tht occurs during torpor (Dn et l. 1991; Lrkin et l. 2002). Although the nimls must hve been feeding, since normothermic periods re the only times in which the chipmunks cn et enough food to sustin themselves during their next hiberntion bout (Humphries et l. 2001), bsed on bsolute T b vlues nd visul inspections of the nimls cges, very little ctivity ws observed during these times compred to similr times of dy in the fll. Sesonlity in metbolic cpbility This is the first study to observe nd quntify the thermoregultory chrcteristics of nimls in their hiberntion seson during their brief periods of normothermi. All of the nimls from the winter phse expressed torpor to some degree coinciding with decrese in normothermic MR. The degree of torpor expression ws negtively correlted to both normothermic BMR nd T b (Fig. 5c, d); trends previously observed t n inter-specific level (McNb 2002; Christin nd Geiser 2007; Cooper nd Geiser 2008). Interestingly, despite mss, MR nd, T b exhibiting sesonl differences, C wet remined constnt between sesons. The winter nimls did not pper to gin ny dditionl fur nd often did not re-grow fur on the re of the stomch, which hd been shved for surgery. This is consistent with reports from the literture tht indicte tht chipmunks generlly only moult once nnully, round the middle of the summer (Yerger 1955). Field studies in which n individul s hir ws clipped in the fll ( common form of identifiction) still mintined the sme mrking in the summer (Snyder 1982; Munro et l. 2008). This would indicte tht chnges in insultion re not prt of the norml dpttions to the cold in this species. Similrly, the presence or bsence of fur does not pper to ffect the expression of hiberntion. Kuffmn et l. (2004) found tht shved

12 290 J Comp Physiol B (2010) 180: ground squirrels continued to hibernte s norml, lthough the costs of rousl nd mintining elevted metbolism during normothermic bouts incresed. They concluded tht lthough insultion during hiberntion cn permit greter energy svings, it is not necessry (Kuffmn et l., 2001; Kuffmn et l., 2004). Therefore, it is not surprising tht the nimls in this study showed no chnge in therml conductnce between sesons. Another common dpttion to the cold tht could hve been employed by the nimls, n incresed in thermogenic cpcity, ws not supported by dt in this study. It is possible tht erly in the winter, when the chipmunks rech their pek mss (Fig. 1), the nimls would hve shown greter response to the cold. Mss gin is common occurrence during cold cclimtion in smll mmmls, ccompnied by n increse in brown dipose tissue, which in turn increses the degree of non-shivering thermogenesis (Heroux et l. 1959). However, becuse neither shivering nor non-shivering thermogenesis were mesured in this study, it is unknown whether or not the increse in mss seen t the strt of the study ws relted to cold cclimtion or to sesonl mss increse in nticiption of hiberntion. The subsequent decrese in mss, however, cn be redily ttributed to reducing ft stores during torpor bouts; the proportion of time spent torpid ws correlted with rte of mss decrese (Fig. 5b). It is interesting to note tht the chipmunks, t lest t the lower T s, did not switch metbolic fuel sources between sesons, s indicted by the comprble RER vlues below therml neutrlity. RER of mesured t those T s indicte the sole usge of ft s substrte for MR (Blxter 1989), consistent with work on other endotherms in the cold (Hrt 1971; Wlsberg et l. 1997). Generl conclusions Although previous studies hve described either the sesonl chnges in chipmunk T b (see Neumnn 1967; Wng nd Hudson 1971; Scott nd Fisher 1972; Pivorun 1976b; Kwmichi 1996; French 2000; Humphries et l. 2001; Munro et l. 2005), or the mplitude of dily T b vrition (Wng nd Hudson 1971; Decoursey et l. 1998), ours is the first study to combine the nlysis of both of these cycles with mesurement of resting nd bsl rtes of metbolism, nd the potentil influence of hiberntion nd winter cclimtion on the normothermic rousl sttes. The chipmunks in our study demonstrted sesonlity in both body mss nd T b regultion chrcteristic of mmmlin heterotherms. While field-studies hve found no correltion between mss t immergence nd torpor expression (Humphries et l. 2003; Lndry-Cuerrier et l. 2008), nimls in our study, in conditions which permitted the collection of body mss dt unvilble from nimls in the wild, showed strong reltionship between fll mss gin nd the degree of torpor expression; the individuls who put on the most mss spent more time in torpor. Torpor expression ws restricted to the months of December through April, nd normothermic T b tht occurred during the periodic rousls ws consistently lower. This sesonlity in T b regultion mnifested itself s slight decrese in MR nd evportive wter loss in the winter, but no chnge in therml conductnce. It ppers tht the physiologicl dpttions necessry for sesonl expression of torpor coincide with lower T b nd MR during the normothermic periods between torpor bouts. The fct tht these levels re correlted with incresed torpor expression strongly suggests tht conferring greter energy svings to the niml during torpor nd normothermi is n essentil feture of winter survivl in chipmunks nd likely mny other hibernting mmmls. Acknowledgments All procedures involving the use of nimls were pproved by the Brock University Animl Cre nd Use Committee (AUPP # ). Collection of the nimls ws uthorized by the Ontrio Ministry of Nturl Resources nd the Algonquin Provincil Prk Superintendent. We would like to thnk the stff nd reserchers of the Wildlife Reserch Sttion (Algonquin Provincil Prk) for logisticl support throughout the summer phse nd the trpping sessions. Suggestions on housing nd husbndry of the nimls were provided by Murry M. Humphries nd winter burrow T dt provided by Dniel Munro nd the Ruiter Vlley Chipmunk Project. Help with cring for the nimls s well s reviewing the mnuscript ws provided by Vivin Cden. Two nonymous reviewers provided helpful comments on n erly drft of the mnuscript. This project ws funded by the Cndin Foundtion for Innovtion, Premier s Reserch Excellence Awrd, nd n NSERC operting grnt to GJT (Grnt No ) nd n Ontrio Grdute Scholrship in Science nd Technology to DLL. References Brnes BM, Buck CL (2000) Hiberntion in the extreme, burrow nd body tempertures, metbolism, nd limits to torpor bout length in rctic ground squirrels. In: Heldmier G, Klingenspor M (eds) Life in the cold: eleventh interntionl hiberntion symposium. Springer, New York, pp Brtholomew GA (1972) Body temperture nd energy metbolism. In: Gordon MS, Brtholomew GA, Grinnell AD, Jorgensen CB, White FN (eds) Animl physiology: principles nd dpttions. Mcmilln, New York, pp Blxter K (1989) Energy metbolism in nimls nd mn. Cmbridge University Press, Cmbridge Buck CL, Brnes BM (2000) Effects of mbient temperture on metbolic rte, respirtory quotient, nd torpor in n rctic hiberntor. 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