ORIGINAL PAPER

P. Sartorelli Æ S. Calderola Æ M. Sala Æ C. CitterioP. Lanfranchi

Seasonal changes in serum metabolites in free-ranging alpine marmots(Marmota marmota)

Accepted: 16 February 2004 / Published online: 23 March 2004� Springer-Verlag 2004

Abstract Circannual changes in serum parameters andbody mass were studied in free-ranging Marmota mar-mota that had been shot in Switzerland (Grisons) formanagement reasons in May, July, and September of1995, 1996, and 1997; and in April 1996. Markers oflipid (triglycerides, cholesterol), protein (total protein,urea-nitrogen) and mineral (calcium, inorganic phos-phate) metabolism were evaluated in 111 haemolysis-free serum samples; the effects of sex, age, reproductivestatus, season and year were tested. Mean body masswas higher in adult males than in adult females in Julyand September, and serum cholesterol concentrationswere lower in adult males in May. Pregnant females hadlower concentrations of total protein than non-pregnantfemales, and triglyceride concentrations were negativelycorrelated with the number of uterine ampullae. Inor-ganic phosphate decreased and total protein increasedwith age. In adults, triglycerides, cholesterol and urea-nitrogen increased mainly from May to July and de-creased between September and May. Total protein,calcium and phosphate did not change throughout theyear. This suggests that, during the active season, in-gested lipids and amino acids were utilised for metabolicneeds or lipid storage, while in winter, lipids were ca-tabolised and protein was spared. Quantitative differ-ences between years, observed for triglycerides and totalprotein during the active season, were probably due todifferent climatic conditions in each year.

Keywords Marmota marmota Æ Blood chemistry ÆLipid Æ Protein Æ Minerals

Introduction

Haematochemical parameters are widely used indomestic and wild animals as indicators of metabolicchanges in patho-physiological conditions, such as ali-mentary changes, pregnancy, lactation, stress andhibernation. The aim of this work was to measuremarkers of lipid (triglycerides, cholesterol), protein (to-tal protein, blood urea-nitrogen) and mineral metabo-lism (calcium, inorganic phosphate) in blood serumduring the active season in free-ranging alpine marmots(Marmota marmota).

Alpine marmots are herbivorous rodents of thefamily Sciuridae that live in the Alps and othermountains in Europe, mostly at altitudes of 1,500–3,000 m. As an adaptation to hard winter conditions,alpine marmots hibernate in family groups in deepburrows for 6 to 7 months each year (Arnold 1988).Marmots leave the burrows in March or April, whensnow is still present and food is often scarce, andcontinue to live of their fat reserves until the snowmelts. At this time, there are additional energetic costsfor reproduction and territorial defence. The highenergy requirement is mainly met from lipolysis, asreported by Hill and Florant (1999) for Marmotaflaviventris in Colorado in April. Alpine marmots loseup to 50% of their body mass (Arnold 1990a, 1990b),mobilising fat reserves that were accumulated duringthe previous short warm season. This cycle of alternateanabolic and catabolic states (Kortner and Heldmaier1995) makes the marmot an interesting species formetabolic studies.

In addition to studying metabolic adaptation, thiswork provides reference values for haematochemicalparameters of alpine marmots; no such data have beenreported for Marmota marmota, although Wilson et al.

P. Sartorelli (&) Æ M. Sala Æ C. Citterio Æ P. LanfranchiDipartimento di Patologia Animale Igiene e Sanita PubblicaVeterinaria, Sezione di Patologia Generale e Parassitologia,Via Celoria 10 20133, Milan, ItalyE-mail: paola.sartorelli@unimi.itTel.: +39-0250318100Fax: +39-0250318095

S. CalderolaResearch Institute for Wildlife Ecology,Veterinary University, Vienna, Austria

Present address: S. CalderolaRegione Piemonte, Osservatorio Regionale sulla Fauna Selvatica,Torino, Italy

J Comp Physiol B (2004) 174: 355–361DOI 10.1007/s00360-004-0421-9

(1992) have published some information on captiveMarmota flaviventris.

Material and methods

Because of damage caused by the marmots to high-value pastures,a culling program has been carried out for several years by theGame and Fish Inspectorate of the Canton Grisons. The study areawas located in the locality of Bivio, 46�28¢N/9�41¢E (Retic Alps,Canton Grisons, Switzerland). The area was a 5-ha pasture at1,950–2,000 m altitude. The climate and vegetation are typical forhigh-alpine habitats. From November until the end of April, thearea is mostly covered by snow. In summer, the lower part is mownand grazed by cattle. Roe deer (Capreolus capreolus) are present inthe study area during the whole year, while red deer (Cervus ela-phus), ibex (Capra ibex) and chamois (Rupicapra rupicapra) arepresent in spring.

Meteorological data were purchased from the Swiss Meteoro-logical Service; since a meteorological station was not present in thestudy area, data from nearby stations were pooled, as suggested bythe Service. We recorded the mean minimum temperature eachmonth from November to April, and the mean temperature, meanradiation and mean rainfall each month from April to Septemberbecause of the influence of these variables on grass growth.

Since 1994, multidisciplinary studies involving several researchgroups have been undertaken on marmots shot by the gamekeep-ers. Information on the marmots’ activity in the study area wasprovided by the gamekeepers of the Game and Fish Inspectorate,who monitored the territory. The culling operations took placewhen marmots emerged from the winter burrows in spring (as soonas possible after the first sighting), when juveniles came out in July,and in September (as late as possible before entry into hibernation).The time of first and last sighting, the presumed length of winterhibernation and the winter mean minimum temperature are shownin Table 1. Since marmots may stay awake in the burrow whenweather is unfavourable, it was impossible to know the actuallength of hibernation. The 185 animals used for the present workwere culled in May, July and September in 1995, 1996 and 1997. In1996 they were also culled in April. The meteorological data for theactive seasons and the number of the serum samples collected andanalysed are shown in Table 2.

Marmots were transferred to a field laboratory immediatelyafter death, then numbered, sexed, weighed (the full mass of theanimals was considered), and assigned to one of three age classes(1=juveniles; 2=yearlings; 3=from the second year onward toadults), according to body mass (Arnold 1990b) and tooth wear(Ratti 1970). Postmortem inspection was performed to identifypregnant females and the number of uterine ampullae. Sincejuveniles first emerge from natal burrows during the first half ofJuly, this age class was not present in the May samples. Bloodsamples, when possible, or coagula were taken from the heart. The

Table 1 Mean minimum temperature during the winter seasons and calculated length of hibernation. Start of hibernation was taken as thetime that marmots were last seen above ground in autumn and end of hibernation when marmots were seen the first time in spring

Winter season Mean minimum temperature (�C) Hibernation

Nov Dec Jan Feb Mar Apr Start End Length (days)

1994–1995 �0.1 �5.8 �11.1 �7.0 �9.1 �2.1 Oct 7 Apr 9 1831995–1996 �5.0 �8.7 �8.0 �11.8 �8.3 �2.4 Oct 10 Mar 16 1581996–1997 �5.0 �7.9 �7.2 �6.9 �3.3 �4.3 Sept 29 Apr 3 186

Table 2 Mean temperature, radiation and rainfall during the active seasons; date of sampling, number of serum samples collected andnumber of haemolysis-free serum samples analysed, divided by age class and sex. For adult females, the number of pregnant (p) and non-pregnant (np) animals is reported

Year Month Meantemperature(�C)

Meanradiation(J/m2)

Meanrainfall(mm)

Samplingday

Serumsamplescollectedn

Serum samples analysed

Totaln

Juveniles Yearlings Adults

M F M F M F

p np

1995 April 1.2 530 131May 5.0 655 111 16th 19 12 3 7 1 1June 7.2 614 108July 12.7 735 114 19th 28 17 1 1 8 7Aug 9.7 499 144Sept 4.3 380 140 27th 2 2 1 1

1996 April 1.2 559 25 15th 19 7 1 3 2 1May 5.0 579 169 6th 19 14 8 2 4June 9.4 77 108July 9.7 668 181 23th 22 9 4 1 3 1Aug 9.3 535 181Sept 4.0 435 410 16th 18 5 2 3

1997 April �0.3 616 60May 5.0 691 91 13th 17 16 6 3 3 4June 7.8 545 194July 9.3 599 156 22th 24 17 4 2 1 4 6Aug 11.2 578 119Sept 8.6 507 29 15th 17 12 2 3 2 1 2 2

356

times of shooting, blood sampling and freezing of blood serumwere recorded and tested for any effect on serum parameters.During the first year of study, blood samples were allowed to clot atroom temperature, and in the second and the third year, at 18 �C ina water bath or an air thermostat. Serum was prepared by centri-fuging blood at 1,000·g for 15 min and was stored at �20 �C fortransport to the laboratory (Milan, Italy), where it was analysedwithin 2 months. Serum samples were assigned to five classes byvisual assessment of their degree of haemolysis: from 0=nohaemolysis to 4=very haemolytic. The following haematochemicalparameters were evaluated with an Auto Analyzer (Abbott VP) andcommercial kits: cholesterol, triglycerides, total protein, urea-nitrogen, calcium, inorganic phosphate (Hospitex Diagnostics,Firenze, Italy).

Statistical analysis was performed using Statistica 5.1 (StatsoftInc., Tulsa, Oklahoma, USA), with the level of significance ofp<0.05. Homogeneity of variance was tested by the Levene test,and adherence to normal distribution by the Kolmogorov-Smirnovtest. Non-normal data were log-transformed. The following cor-relations were evaluated by means of the Spearman rank correla-tion test: (1) haemolysis and time elapsed between shooting andblood sampling; (2) haemolysis and time elapsed between bloodsampling and serum freezing; (3) haemolysis and haematochemicalvalues; (4) haematochemical values and time between shooting andserum freezing; (5) body mass and haematochemical parameters;(6) the different haematochemical parameters.

Since haemolysis was seen to affect some haematochemicalparameters, the values obtained from haemolytic sera (haemoly-sis-classes 2, 3, 4), accounting for 40% of samples, were dis-carded. Therefore, final statistical analysis was performed on 111sera, 15 for age-class 1, 18 for age-class 2 and 78 for age-class 3(Table 2). The non-parametric tests of Kruskall-Wallis and UMann-Whitney were used to test for differences in body mass andhaematochemical parameters between age classes and sexes.Differences between months or years were evaluated by theKruskall-Wallis test only for adults (age-class 3) because fewjuveniles and yearlings were culled.

Results

Meteorological data. The mean minimum temperaturein winter tended to be higher in 1997, but it was stillbelow zero in April (Table 1). In the active season, meantemperature, radiation and rainfall were similar for the 3years (Table 2).

Body mass. All culled animals (n=185) were consid-ered for statistical analysis. Values for juveniles, year-lings and adults are reported in Table 3. Mean values forthe three age-classes differed significantly (p<0.001).Statistical analysis of the effects of sex, season and yearwas performed only on data from adult marmots. Meanbody mass of males was higher than that of females inJuly and September. In both sexes body mass increased(p<0.001) from May to July, with a plateau until Sep-tember without differences among years. The mean de-crease from September to May was approximately 25%for females and 30% for males.

Haematochemical parameters. Haemolysis rate wassignificantly correlated with the time between shootingand blood sampling (r=0.39; p<0.001), and with thetime between shooting and serum freezing (r=0.29;p<0.001). Most haemolytic sera were obtained in 1995,when clotting was slow in the absence of a thermostat. T

able

3Bodymass

(mean±

SD)andnumbersofalljuvenile,

yearlingandadultMarm

ota

marm

ota

(n=

185).Since

values

foradultmalesandfemaleswerestatisticallydifferent

(p<

0.01),they

are

reported

separately.Thebodymass

(mean±

SD)andnumber

ofanim

alswhose

serum

sampleswereanalysed(n=

111)are

given

inparentheses

when

somesamples

havebeenomittedfrom

further

analysis

Year

Month

Juveniles

Yearlings

Adults

Males

Fem

ales

(g)

n(g)

n(g)

n(g)

n

1995

May

01,783±

699(2,133±

899)

5(3)

3,226±

572(3,420±555)

10(7)

3,378±

257(3,300±

0)

4(2)

July

907±

311(950)

5(1)

2,950

14,568±

605(4,500±666)

11(8)

3,950±

546(3,993±

526)

11(7)

September

03,650

10

3,350

11996

April

01,018±

122(1,170)

6(1)

3,267±

235(3,135±117)

8(3)

2,999±

296(3,175±

208)

5(3)

May

00

3,352±

247(3,344±288)

12(8)

3,214±

234(3,258±

222)

7(6)

July

928±

202(940±

268)

8(5)

2,750(nd)

1(0)

4,636±

509(4,900±424)

7(3)

3,433±

7133,400

6(1)

September

1,550±

108

33,625±

177

24,667±

661(5,375±350)

9(2)

4,383±

441(4,650±

328)

4(3)

1997

May

01,892±

236

63,738±

354(3,750±433)

4(3)

3,521±

940

7July

1,069±

282(1,287±

312)

8(4)

2,633±

416

34,250±

465

43,983±

455(3,842±

467)

9(6)

September

1,825±

178

53,033±

202

34,420±

1117(4,075±

389)

5(2)

3,650±

950(3,250±

636)

4(2)

357

Haemolysis was positively correlated with serum con-centrations of total protein (r=0.46; p<0.001) andcalcium (r=0.65; p<0.001). For all haematochemicalparameters, only results from sera of haemolysis-class 0and 1 are reported (n=111). Values for juveniles, year-lings and adults are reported in Tables 4, 5 and 6,respectively.

Effect of sex. There were no significant differences forhaematochemical parameters between sexes for age-classes 1 and 2; for age-class 3, lower levels of cholesterolwere observed in May in adult males (Table 7).

Effect of age. To test for differences in age classes,values for both sexes were pooled. There were no sig-nificant differences between age classes for any haema-tochemical parameters. However, there was a negativecorrelation in July between age and phosphate(r=�0.33; p<0.05), and a positive correlation betweenage and total protein (r=0.32; p<0.05).

Effect of reproductive status. To test for any effect ofpregnancy, values of adult pregnant females culled in

May were compared with those of non-pregnant femalesand males in the same month. In pregnant females, totalprotein concentrations were significantly lower than innon-pregnant females (Table 7) and there was a signifi-cant negative correlation between concentrations of tri-glycerides and the number of uterine ampullae (r=0.51;p<0.05). There were significant differences in cholesterolconcentrations between males and females, but not be-tween pregnant and non-pregnant females.

Effect of year. In age-class 3, concentrations for serumprotein were higher in 1997 than in 1995 and 1996. InSeptember 1997, concentrations of triglycerides werehigher than in previous years.

Effect of season. In age-class 3, triglyceride concen-trations increased significantly from May to July in 1995and 1996, and in 1997, also to September. Mean con-centrations in May were always lower than those inSeptember of the previous year. Cholesterol increasedfrom May to July but then remained unchanged toSeptember. As for triglycerides, lower concentrationswere recorded in May than in the previous September.

Table 5 Hematochemical values (mean±SD) of free-living yearling marmots

Year Month Urea Total protein Cholesterol Triglycerides Calcium Inorganic phosphate n(mmol l–1) (g l–1) (mmol l–1) (mmol l–1) (mmol l–1) (mmol l–1)

1995 May 20.2±13.8 65.0±9.3 4.8±2.8 1.6±0.8 3.8±0.6 5.8±1.8 3July 23.0 69.8 5.6 3.5 3.6 3.8 1September 15.9 63.6 3.0 3.2 2.9 2.9 1

1996 April 12.3 102.9 5.5 1.6 0.2 6.2 11997 May 22.2±4.1 63.3±13.5 4.8±0.7 1.8±0.7 2.8±0.3 2.5±0.8 6

July 18.4±4.5 62.6±4.7 4.7±0.8 1.8±0.2 3.2±0.3 3.4±0.4 3September 33.3±11.4 90.4±8.7 4.9±0.6 4.4±1.7 4.3±0.9 3.4±1.3 3

Table 4 Hematochemical values (mean±SD) of free-living juvenile marmots

Year Month Urea Total protein Cholesterol Triglycerides Calcium Inorganic phosphate n(mmol l–1) (g l–1) (mmol l–1) (mmol l–1) (mmol l–1) (mmol l–1)

1995 July 20.8 40.8 3.4 2.3 5.63 7.3 11996 July 19.2±3.2 40.7±14.4 3.7±1.8 2.3±0.8 3.5±0.5 3.8±0.3 51997 July 25.2±3.9 66.0±17.5 5.7±0.7 1.6±0.8 3.8±1.2 3.7±0.6 4

September 30.3±9.0 72.4±14.2 5.7±0.6 4.3±1.7 3.7±0.8 4.3±2.1 5

Table 6 Hematochemical values (mean±SD) of free-living adult marmots

Year Month Urea Total protein Cholesterol Triglycerides Calcium Inorganic phosphate n(mmol l–1) (g l–1) (mmol l–1) (mmol l–1) (mmol l–1) (mmol l–1)

1995 May 20.9±5.0 58.0±15.1 3.3±0.8 2.2±0.7 3.4±0.5 4.1±1.8 9July 23.7±5.3 61.7±15.5 4.3±1.2 2.4±0.7 3.2±0.5 3.2±1.2 15September 25.7 52.9 3.3 2.65 3.4 3.6 1

1996 April 10.0±4.0 65.6±15.7 2.5±1.6 2.0±0.92 3.1±0.9 4.5±2.5 6May 17.7±5.5 65.2±15.8 2.8±0.9 2.2±0.7 3.5±0.5 4.6±1.6 14July 34.6±14.5 58.2±3.2 4.4±1.0 2.5±1.1 3.2±0.5 3.5±0.4 4September 23.9±9.2 62.1±12.1 4.4±2.1 2.6±0.7 3.2±0.4 2.9±1.0 5

1997 May 28.4±13.3 77.7±24.2 3.3±0.8 1.6±0.6 2.7±0.5 2.5±1.6 10July 25.9±8.1 72.4±9.9 5.4±1.1 2.4±0.8 3.4±0.4 3.5±0.4 10September 28.7±4.3 76.4±8.5 5.1±1.4 4.2±1.7 3.5±0.8 5.4±3.8 4

358

Both cholesterol (r=0.36; p<0.01) and triglyceride(r=32; p<0.01) concentrations were correlated posi-tively with body mass. Total-protein concentration didnot change during the active season. Values recorded inMay were similar to those of the previous September.Urea-nitrogen increased significantly from May to July,then remained unchanged until September. Urea-nitro-gen concentrations were positively correlated with bodymass (r=0.23; p<0.05).

Serum calcium and inorganic phosphate did notchange significantly throughout the year. There werepositive correlations between calcium and total protein(r=0.44; p<0.001) and between calcium and inorganicphosphate (r=0.40; p<0.001).

Discussion

Until this study, reference values for blood parametersfor Marmota marmota were lacking. Some data havebeen reported for yellow-bellied marmots (Marmotaflaviventris) kept under laboratory conditions and fedad libitum with commercial food (Wilson et al. 1992).Thus there were no data published on seasonalchanges in haematochemistry related to environmentalparameters such as food quality or the energyrequirements of free-ranging animals in any species ofMarmota. Studies on blood parameters of wild ani-mals in field conditions must be carefully planned, andrapid processing of blood samples is essential if pre-analytical errors (Lumsden 1998) due to haemolysis ordelay in storage (Alleman 1990) are to be avoided. Inprevious work on culled, free-ranging Capra ibex(Sartorelli et al. 1994; Sartorelli et al. 1997), we ob-tained good-quality serum samples and reported onthe effects of stress and of pre-analytical errors onserum metabolites. On this basis, in the present studywe excluded those serum parameters, such as glucoseand non-esterified fatty acids, that could have beenuseful in the study of energy metabolism in marmotsbut were shown to be greatly affected by stress and byserum storage delay.

The absence of the expected decrease in serum tri-glycerides with increasing time between blood samplingand serum freezing (Sartorelli et al. 1994) could be dueto low room temperature or to low lipase activity duringthe active season, when lipase activity is high in adiposetissue (Wilson et al. 1992).

Effect of sex. The greater body-mass loss and lowercholesterol concentrations after hibernation in malesthan in females were probably due to greater energyexpenditure in the males.

Effect of age. The higher serum concentrations ofphosphate in juveniles were probably a reflection ofbone growth, as reported for other species; also the in-crease in serum protein with age is common in animals,and is generally due to higher globulin concentrations(Kaneko et al. 1997). However, the electrophoreticpattern of Marmota serum protein has not beenreported.

Effect of reproductive status. The lower concentrationsof serum protein in pregnant marmots may be partlyexplained by an increase in plasma volume duringpregnancy, but since other serum metabolites did notproportionally decrease, utilisation of protein by thedeveloping foetus is a more likely explanation. A de-crease in serum-albumin concentration is a commonoccurrence in pregnant animals (Kaneko et al. 1997).Energy was probably partly supplied by triglycerides, asindicated by the negative correlation between triglycer-ide concentration and the number of uterine ampullae.Mating takes place soon after waking from hibernation,when food is still scarce and the major energy source isbody-fat reserves (Arnold 1990a; Hill and Florant 1999).Total reproductive costs are so high in alpine marmotsthat females reproduce on average only every secondyear (Hacklander et al. 2003; Hacklander and Arnold1999).

Effect of season. Concentrations of both triglyceridesand cholesterol increased in adults of both sexes fromMay to July in each of the 3 years examined, concomi-tant with the period of maximum body-mass gain. Fuelmolecules, such as glucose, whose uptake by M. flavi-ventris is maximal in the same period (Tokuyama et al.1991), amino acids, and fatty acids, in particular essen-tial polyunsaturated fatty acids (Florant 1998; Hill andFlorant 2000), are converted by the liver to lipoproteinfor accumulation of energy reserves. Lipoprotein-lipaseactivity in adipose tissue is high in M. flaviventris duringthe active season (Wilson et al. 1992). The increase intriglyceride concentrations in September was unex-pected, since a decrease was reported for M. flaviventris(Wilson et al.1992). Marmots reduce food intake and themass of their small-intestine decreases before hiberna-tion (Hume et al. 2002). The difference between the twostudies could therefore be due to differences in times ofsampling in relation to time of entry into hibernation.During hibernation, the activity of hormone-sensitivelipase in adipose tissue increases (Wilson et al. 1992),and stored lipids are utilised for energy (Arnold 1990a),as indicated by loss of body mass. The decrease in bothcholesterol and triglyceride concentrations betweenSeptember and April/May suggests that mobilised lipidsare oxidised for warming, and that few lipoproteins are

Table 7 Serum concentrations (mean±SD) of cholesterol and totalprotein in free-living adult Marmota marmotain May 1995, 1996and 1997

Cholesterol Total protein n(mmol l–1) (g l–1)

Males 2.7±0.8 65.0±17.7 18Females Pregnant 3.6±0.6 58.3±11.9 9

Non-pregnant 3.7±0.3 86.4±23.8 6

359

formed to meet the energy requirements of peripheraltissues, in contrast to a non-hibernating alpine species,the ibex (Sartorelli et al. 1997).

Reference values for serum triglycerides and choles-terol have not been reported for M. marmota; values fortriglycerides in the present work were higher than thosereported for captive M. flaviventris under laboratoryconditions with commercial food provided ad libitum(Wilson 1992). Total protein levels were in the range re-ported for other rodents (Kaneko et al. 1997). The in-crease in urea-nitrogen concentrations during the activeseason was most likely a result of the ingestion of protein-rich forage and/or endogenous amino-acid catabolism.Also, in hindgut fermenters such as marmots, ammoniaproduced by caecal bacteria is largely absorbed into theportal blood, then converted to urea in the liver (Stevensand Hume 1995). In either case, the lack of change inserum total protein during the active season supports thehypothesis that surplus absorbed amino acids are con-verted to lipids for storage (Tokuyama et al. 1991).

During the cold season, protein seems to be spared,since serum concentrations of total protein remain un-changed and urea-nitrogen levels tend to decrease,indicating reduced catabolism. In hibernating femalebears, which also do not eat, drink or urinate, bloodconcentrations of amino acids, total protein, urea anduric acid remain unchanged throughout winter and leanbody mass remains constant (Nelson 1980). Urea wasassumed to be a precursor for non-essential amino-acidsynthesis in the liver (Hissa et al. 1994). Urea recyclingby this means could be an advantageous adaptationshared by different species, since elevated concentrationsof blood urea in hibernating animals, when urine pro-duction is suppressed, could be toxic for many organs.

Serum calcium was expected to change seasonallybecause the output of non-esterified fatty acids fromadipocytes is accompanied by calcium entry (Luthmanand Holtenius 1972). However, since a stable concen-tration of serum calcium is required for many biologicalprocesses, calcium may have been mobilised from bone.Alpine marmots have been reported to actively searchfor supplemental mineral supplies during the activeseason (Massemin et al. 1996), and high levels of intes-tinal calcium and phosphorus have been reported inwoodchucks (Marmota monax) (Staaland et al. 1995).Mineral homeostasis during hibernation needs to befurther investigated since, to our knowledge, no data areavailable for any species other than marmots. Serumcalcium and protein concentrations are positively cor-related because of the role of protein in calcium trans-port (Kaneko et al. 1997).

Effect of year. That serum triglyceride and total-pro-tein concentrations were higher in September 1997 thanin September 1996 suggests that seasonal conditions weremore favourable in 1997, with greater primary produc-tion extending the active period. This would have al-lowed the animals to stay longer above ground to forage

at a time when, in other years, food intake and metabolicrate were declining in preparation for hibernation (Wardet al. 1981). In 1997, marmots entered hibernation laterthan in 1996, a month after the autumn culling.

In conclusion, our data provide the first referencevalues for free-ranging M. marmota in their alpinehabitat for some haematochemical parameters and theirseasonal variation related to cyclic fat deposition andmobilisation. Individual variation was high, probablydue to differences in energy status during winter hiber-nation and spring reproduction. Nevertheless, our datasuggest a strategy of protein utilisation during the activeseason (summer), while fat is spared, and the oppositeduring winter. Further haematochemical studies onmarked and recaptured animals, together with analysisof differences in food intake over months and years,could reduce errors due to individual variation and al-low more precise evaluation of the haematochemicalchanges. Additionally, the data presented here may beuseful for an integrated evaluation of the health status offree-living alpine marmots.

Acknowledgments The authors are grateful to the management andgamekeepers of Jagd und Fisch Inspectorat of Grisons, Switzerlandfor facilities and collaboration in fieldwork. Particular acknowl-edgment is made to the former Director, Dr Peider Ratti, who wasthe leading spirit of the project, to U. Bruns and F. Frey-Roos forcooperation in sample collection, to Prof. W. Arnold for helpfulcomments on the manuscript, and to an anonymous sponsor whosupported part of the project. We thank the Swiss MeteorologicalService for providing the meteorological data. Procedures followedduring this study conformed to the current laws regulating suchresearch in Switzerland.

References

Alleman AR (1990) The effect of hemolysis and lipemia on serumbiochemical constituents. Vet Med 85:1272–1284

Arnold W (1988) Social thermoregulation during hibernation inalpine marmots (Marmota marmota). J Comp Physiol B158:151–156

Arnold W (1990a) The evolution of marmot sociality: I. why dis-perse late? Behav Ecol Sociobiol 27:229–237

Arnold W (1990b) The evolution of marmot sociality: II. costs andbenefits of joint hibernation. Behav Ecol Sociobiol 27:239–246

Florant GL (1998) Lipid metabolism in hibernators: the impor-tance of essential fatty acids. Am Zool 38:331–340

Hacklander K, Arnold W (1999) Male-caused failure of femalereproduction and its adaptive value in alpine marmots (Mar-mota marmota). Behav Ecol 10:592–597

Hacklander K, Mostl E, Arnold W (2003) Reproductive suppres-sion in female alpine marmots (Marmota marmota). Anim Be-hav 65:1133–1140

Hill VL, Florant GL (1999) Patterns of fatty acid composition infree ranging yellow-bellied marmots (Marmota flaviventris).Can J Zool 77:1494–1503

Hill VL, Florant GL (2000) The effect of a linseed oil diet onhibernation in yellow-bellied marmots (Marmota flaviventris).Physiol Behav 68:431–437

Hissa R, Siekkinen J, Hohtola E, Saarela S, Hakala A, Pudas J(1994) Seasonal patterns in the physiology of the Europeanbrown bear (Ursus arctos arctos) in Finland. Comp BiochemPhysiol 109A:781–791

360

Hume ID, Beiglbock C, Ruf T, Frey-Roos F, Bruns U, Arnold W(2002) Seasonal changes in morphology and function of thegastrointestinal tract of free-living alpine marmots (Marmotamarmota). J Comp Physiol B 172:197–207

Kaneko JJ, Harvey JW, Bruss ML (1997) Clinical biochemistry ofdomestic animals. Academic Press, San Diego

Kortner G, Heldmaier G (1995) Body weight cycles and energybalance in the Alpine marmot (Marmota marmota). PhysiolZool 68:149–163

Lumsden JH (1998) Laboratory data interpretation. In: DavidsonMG, Else RW, Lumdsen JH (eds) Manual of small animalclinical pathology. British Small Animal Veterinary Associa-tion, Kingsley House, Gloucester, pp 27–32

Luthman J, Holtenius P (1972) Norepinephrine induced fatty liverand hypocalcemia in sheep. Acta Vet Scand 13:31–41

Massemin S, Gibault C, Ramousse R, Butet A (1996) Premieresdonnee sur la regime alimentaire de la marmotte alpine (Mar-mota marmota) en France. Mammalia 60:351–361

Nelson RA (1980) Protein and fat metabolism in hibernating bears.Fed Proc 39:2955–2958

Ratti P (1970) Beitrag zur Kenntnis des Alpenmurmeltieres (Mar-mota marmota). Schweiz Arch Tierheilkunde 112:283–295

Sartorelli P, Agnes F, Lanfranchi P (1994) Parametri ematochimicinello stambecco: influenza di fattori fisiologici ed errori

preanalitici. In: Argomenti di Patologia Veterinaria, Fondaz.Iniz. Zooprofilattiche e Zootecniche, Brescia, pp 331–337

Sartorelli P, Agnes F, Lanfranchi P (1997) Pathophysiologicalsignificance of hematochemical parameters of Capra ibex.Hystrix 9:39–44

Staaland H, White RG, Kortner H (1995) Mineral concentrationsin the alimentary tract of northern rodents and lagomorphs.Comp Biochem Physiol 112A:619–627

Stevens CE, Hume ID (1995) Comparative physiology of the ver-tebrate digestive system, 2nd edn. Cambridge University Press,Cambridge

Tokuyama K, Galantino HL, Green R, Florant GL (1991) Sea-sonal glucose uptake in marmots (Marmota flaviventris): therole of pancreatic hormones. Comp Biochem Physiol100A:925–930

Ward JM, Armitage KB (1981) Circannual rhythms of food con-sumption, body mass, and metabolism in yellow-bellied mar-mots. Comp Biochem Physiol 69A:621–626

Wilson B, Deeb S, Florant GL (1992) Seasonal changes in hor-mone-sensitive and lipoprotein lipase mRNA concentrations inmarmot white adipose tissue. Am J Physiol 262:R177–R181

361