Effects of birth date and natal dispersal on faecal glucocorticoid concentrations in juvenile Common hamsters

Highlights

► Birth date and timing of dispersal affected cortisol secretion in juvenile Common hamsters. ► Late-born juveniles had elevated faecal cortisol metabolite (FCM) concentrations shortly after weaning. ► Juveniles that immigrated late in the season had higher FCM levels than early immigrants and residents. ► Elevated FCM levels in late immigrants were independent of birth date. ► Late-borns and late immigrants had less time to prepare for hibernation.

Abstract

In seasonally breeding animals, timing of reproduction in females can influence offspring development and survival. Temporal and energetic constraints are often more pronounced in juveniles born late in the season, and could activate the stress axis. Common hamsters (Cricetus cricetus) are hibernating rodents, and adult females produce up to three litters during the active season. Birth dates range from May to September, and shortly after natal emergence pups are weaned and start to disperse. In this study, we used faecal cortisol metabolites (FCM) as a non-invasive measure of recent glucocorticoid exposure. We compared FCM levels between early- (June/July) and late- (August/September) born juvenile Common hamsters during their first weeks after natal emergence. We further compared FCM levels in juveniles born in the study area and individuals that immigrated either early or late in the season. Additionally, we investigated potential effects of human disturbances on cortisol secretion patterns. FCM levels in late-born juveniles increased during the first weeks post-emergence and were significantly higher than those in early-born individuals during the post-weaning period. Late-immigrating juveniles had significantly higher FCM levels than early immigrants and residents during the same time period. Individuals that inhabited areas frequently used by humans had higher FCM levels than those in low-impact areas. These results indicate that the seasonal timing of birth and dispersal affect cortisol secretion patterns in juvenile Common hamsters. As all juveniles immerged into their hibernacula during early October, we assume that late-born and late-immigrated individuals have less time to prepare for hibernation. This could elevate energetic demands and increase exposure to predators and/or humans during food caching. These factors might lead to increased adrenal activity and thus, elevated FCM concentrations in these individuals. Potential consequences of these time constraints and associated physiological effects could be reflected in the lower overwinter survival rates of juveniles born late in the season.

1. Introduction

In seasonally breeding mammals the timing of reproduction can be crucial for the development [4,23,38,39], sexual maturation [2,23], survival [4,22,41] and lifetime reproductive success [10,41] of juveniles. For example, in the great guinea pig (Cavia magna), juveniles born early in the season grew faster and matured earlier than late-born individuals [23], and in European rabbits (Oryctolagus cuniculus), survival to maturity and lifetime reproductive success was higher in early- than late-born individuals [41]. Juveniles born early in the season have more time to grow and prepare for the forthcoming winter, resulting in higher survival rates compared with juveniles born later in the season [22,38]. Late-born offspring either result from second or third litters or from mothers that started to reproduce later in the season. In the first case, previous reproductive effort may shorten maternal investment for later-born offspring [40]; in the second case, mothers are often young females or individuals in poor condition [1]. Thus, such offspring may face not only higher temporal constraints compared to early-born pups, but also reduced maternal care. This could result in conditional deficits, which in turn have been shown to affect survival rates in several species (e.g. [9,25,31,33,38]).

Common hamsters (Cricetus cricetus) are hibernating rodents with an extended breeding period from April until August [13]. Thus, in contrast to many other hibernators, they produce up to three litters per season [12,34]. In our study area, birth dates ranged from May to September, with litter sizes between one and nine pups (mean ± SD: 4.3 ± 2.1; [12,13]). The altricial young are born after approximately 20 days of gestation [11], and at an age of about 3 weeks they leave the natal burrow for the first time [11,20]. Shortly thereafter, juveniles are weaned and start to disperse [20,47]. Although juveniles are able to reproduce in their year of birth [34,50], this is rather exceptional [3,47], and in our study area only three juvenile females managed to raise a litter during a 9-year observation period (Franceschini et al., unpublished data). The hibernation period starts in late August or early September, with adult males entering their hibernacula before the females [13,24]. Juvenile hamsters terminate above-ground activity during October (Franceschini, unpublished data).

Common hamsters do not depend solely on body fat reserves for hibernation, but also build up food caches during the active season, which can be used during winter [16,32,34]. Thus, young hamsters have to grow, find or construct a burrow, and prepare for the forthcoming winter by accumulating body fat and food stores as energy reserves. To accomplish these preparations, juveniles born late in the season can either delay hibernation onset or complete preparation for the winter in a shorter time span than early-born individuals. In the first case, individuals potentially face unfavourable environmental conditions such as low food availability and ambient temperatures towards the end of the year. The latter option, however, is probably associated with pronounced temporal and energetic constraints. This is supported by the observation that late-born juveniles spend more time per day foraging above-ground during the prehibernation period (September/October) compared to those originating from early litters during the same time of year [48,49]. This extended daily activity of late-born individuals could further increase exposure to predators while searching for suitable food items.

In addition, dispersal was documented in juvenile hamsters in both sexes throughout the active season (Franceschini et al., unpublished data). In arctic ground squirrels, dispersal was associated with increased predation risk [8] and high energetic demands [7]. This could also apply to Common hamsters, although studies focusing on dispersal are lacking for this species. It still can be assumed that individuals immigrating into an area late in the season are confronted with similar challenges as late-born juveniles because they have to more quickly establish a burrow and cache food for the winter.

Free-living animals frequently face unpredictable and/or uncontrollable environmental, physical and social challenges. Such stimuli are stressors that provoke physiological and behavioural responses to maintain or regain homoeostasis [27,43]. The activation of the hypothalamus–pituitary–adrenal (HPA) axis is such a response, resulting in glucocorticoid (GC) secretion by the adrenal cortex. GCs can act as physiological mediators helping the organism to cope with stressors [43]. Among many others, an immediate effect of GC secretion is energy mobilization (i.e. glucose) from storage sites to exercising muscles, for example by stimulating hepatic gluconeogenesis [21,42,45]. These adaptive acute responses, however, can become adverse or even pathological under long, repeated, or more intense exposure to stressors [43]. Prolonged GC production can impact individual fitness and/or survival by inhibiting growth, causing infertility, or suppressing immune response [5,21,26,30,45]. Blood GC concentration is widely used as an indicator of stress response. In recent years, non-invasive methods applying faecal glucocorticoid metabolite analyses have been established to avoid handling-induced effects on the activation of the HPA axis and to facilitate sample collection in the field (reviewed in [29,35]).

In Common hamsters, cortisol secretion patterns have been investigated in adults [14], but information on juveniles is minimal. In adults, high GC levels are associated with reproductive activity: cortisol secretion increased with intrasexual aggression in males and sexual interactions in females [14]. In juveniles, time of birth, natal dispersal, predation pressure, or social conflicts could affect adrenal activity. In this study, we documented cortisol secretion patterns in juvenile Common hamsters by analysing concentrations of faecal cortisol metabolites (FCM). To examine developmental effects on cortisol secretion patterns, we analysed FCM concentrations in juveniles during their first weeks after natal emergence. We investigated potential influences of dispersal on cortisol secretion patterns by assessing FCM levels in resident and immigrated individuals. To further examine possible seasonal effects on adrenal activity, we compared FCM concentrations in juveniles that were born or immigrated either early (June/July) or late (August/September) in the season. Potential effects of social conflicts were investigated by analysing the frequency of sexual, aggressive, and socio-positive interactions in juveniles during the active season. Finally, as our study was carried out in an urban habitat, the presence of humans was considered as a potential stressor affecting adrenal activity. We predict that (1) late-born juveniles have to prepare for winter in a shorter time than early-born individuals and therefore will spend more time foraging above-ground and exhibit higher FCM levels and (2) that late immigrants will face similar time constraints as late-born hamsters and will therefore also show increased FCM levels. Furthermore we controlled for potential effects of disturbance by humans on FCM concentrations.

2. Methods

2.1. Field techniques

The study was conducted in an urban area in southern Vienna, Austria (48°10′26” N, 16°20′39” E). We investigated a population of free-ranging Common hamsters inhabiting the green areas (3.9 ha) surrounding apartment complexes. Adult and juvenile hamsters were captured 5 days/week from March to October 2005. Trapping was carried out during the hamsters’ daily activity periods in the morning (starting with sunrise) and evening hours (until sunset). In our study area, Common hamsters were not active above ground during the night [46]. In juveniles, data collection started in June, when the first juveniles left the natal burrow. Hamsters were captured using Tomahawk live traps baited with peanut butter. The traps were positioned in front of the burrows and checked in 20-min intervals. Hamsters were released from the traps into black, cone-shaped cotton sacks laterally equipped with Velcro fasteners, which enabled an investigation without anaesthesia. Parameters recorded at each capture were date, time, ambient temperature, location (position in a 4 × 4 m grid), sex, age (adult or juvenile), reproductive status (testes width in males, closed/opened vagina in females), and body mass (±1 g). For permanent identification, we marked juveniles on the lower leg by tattoos (animal-applicable tattoo dye) in individual patterns using a sterile needle. For distance recognition, we fur-marked the dorsal region in different patterns using commercial hair dye. We collected faecal samples at every spontaneous defaecation during the investigation and stored them at −20 °C until analysis. Each investigation lasted for about 5–10 min, and the hamsters were then released in front of their burrows, guaranteeing immediate orientation in familiar surroundings. The procedure did not seem to negatively affect the animals: we found no indications for trap avoidance or changes in behaviour, body mass and survival (Franceschini-Zink, unpublished data; Siutz, personal observation). All manipulations were approved by the Austrian Ministry, City of Vienna (MA22-2605/02), and the Ethical Committee for Animal Welfare (GZ 68.210/12-BrGT/2003). To ensure that all juveniles in the study area were detected at emergence, we captured adult females in regular intervals and checked for gestation (indicated by cumulative body mass gain). Indicators for parturition were rapid body mass loss and increasing teat size, partly with milk remains [12]. Breeding burrows of pregnant and lactating females were frequently checked. Based on the day of parturition, we pre-calculated litter emergence date for each female (assuming pups stayed in the burrow for 20 d until natal emergence). This enabled us to assign emerging juveniles to a particular mother. Litter size was determined by counting all juveniles emerging from a female’s breeding burrow. Each pup was individually marked to exclude double counts, and we continued observing the breeding burrows for several days after natal emergence to ensure that no unmarked juveniles were left.

Early-born individuals emerged from the natal burrow between 1 June and 7 July, late-born juveniles between 29 July and 20 September. Almost all individuals classified as late-born juveniles could be assigned to second litters. Only three individuals sampled originated from first litters that were produced late in the season. We performed all analyses with and without those three individuals, respectively, but found no difference in the models. We therefore included them in the analyses. No adult female produced a third litter and no juvenile female reproduced during the study period.

2.2. Behaviour

Behavioural observations were carried out 5 days/week, 2–3 h/day from March to October (from June to October in juveniles). We recorded social interactions using event-sampling techniques. We chose specific observation points to overlook the different parts of the study site. After a 1-h session we switched the observation point. Interactions were classified as aggressive (fights and chases), sexual (copulations and mate chases) [50], and socio-positive (grooming and nose-to-nose contacts). We further recorded initiator and recipient. Throughout the study period, we never observed received or initiated sexual interactions in juvenile hamsters and only one socio-positive interaction was observed (two juveniles, no litter mates). Hence, behavioural analyses were restricted to aggressive interactions.

We calculated the frequency of aggressive interactions/h. To control for potential recipients of interactions, we exclusively analysed observation sessions with at least two individuals present within a distance of ⩽16 m because previous observations demonstrated that hamsters at larger distances did not interact (Franceschini-Zink, unpublished data; Siutz, personal observation).

2.3. Phase definitions

2.3.1. Adult breeding and post-breeding period

Previous studies showed that aggression among adults occurred almost exclusively during the breeding period [14]. As particularly adult males are quite aggressive during breeding, juveniles might be more often displaced or even attacked by adults when foraging near a female burrow. Although aggression was rarely directed towards juveniles, high conflict rates among adults might affect cortisol secretion in juvenile hamsters. We compared FCM levels in juveniles during the adult breeding and post-breeding period. We defined breeding and post-breeding periods in adult hamsters based on testes development in males and vulval development and teat size in females [14]. The breeding period lasted until weaning of the last litter in females, coincident with testes regression in males. The mean date of weaning the last litter was 15 August. Subsequently, the post-breeding period started, lasting until all individuals had terminated above-ground activity (21 October).

2.3.2. Post-emergence intervals

Based on the natal emergence date (i.e. when juveniles left the natal burrow for the first time), we defined three post-emergence intervals for each individual. Pre-weaning (indicated by mother’s still enlarged teat size; corresponding to the first week post-emergence), weaning (decreasing teat size in mothers; weeks 2–4 post-emergence) and post-weaning (regressed teat size in mothers; juveniles started to disperse; weeks 5–7 post-emergence). Due to natal dispersal, the sample size decreased during post-emergence; 7 weeks after emergence, the sample size became too small for statistical analyses. We only used data of juveniles that were born in the study area for comparisons between post-emergence intervals.

2.3.3. Dispersal status

We frequently captured unmarked juveniles of post-weaning age (body mass > 158 g). Since the litters of all resident adult females had already been marked during weaning, older, unmarked individuals were classified as immigrants (about 33% of all juveniles trapped). For FCM analyses, we distinguished between individuals that were born in the study area (residents) and those that immigrated during the season (immigrants). Furthermore, we classified early and late immigrants based on their immigration date (early immigrants: prior to 26 July, corresponding to the adult breeding period; late immigrants: after 17 August, corresponding to the adult post-breeding period; all individuals of which faecal samples were available could be assigned to either period).

2.4. Human impact

Finally, we controlled for potential effects of anthropogenic disturbances on FCM concentrations. The study site was subdivided into two areas that differed in human impact (frequency of passers-by). The first area was a large grass field with one adjacent apartment building, and humans only rarely entered the grass field. This area was classified as “low impact”. The second area consisted of five small lawns surrounded by several apartment buildings, a high frequency of passers-by, and children frequently played on the lawns. This area was classified as “high impact”. Although the areas were separated by buildings, asphalted walkways, concrete walls, and dense shrubbery, hamsters were able to move between them. For FCM analyses, we differentiated between individuals that inhabited the areas with low and high human impact.

2.5. Faecal cortisol metabolites

We dehumidified, crushed and homogenised the faecal samples and suspended 0.2 g of each sample in 4.0 ml of 80% methanol. After centrifugation (3600 rpm, 15 min), we diluted 10 μl of the supernatant 1:10. FCM were determined using an 11-oxoetiocholanolone enzyme immunoassay (EIA), which utilises a group-specific antibody measuring faecal 11,17-dioxoandrostanes, a group of cortisol metabolites (assay detection limit: 2–500 pg; for details see [28]). Intra- and inter-assay coefficients of variance were 10.5% and 9%, respectively. Species validation of the hormone assay was performed previously by Franceschini et al. [14]. FCM concentrations in captive Common hamsters exposed to a stressor were analysed. Levels peaked after 13.7 h (±7.6) [14]. For the analyses of FCM levels, individual means per phase were used.

2.6. Statistics

Statistical analysis was performed in R 2.9.2 [37]. We computed two linear models to test the effects of time of birth and dispersal status, respectively, on juvenile FCM levels. Model selection was based on minimising Akaike’s information criterion (AIC) corrected for small sample sizes (AICc; [19]). In the first (time-of-birth) model, post-emergence intervals (pre-weaning, weaning, post-weaning), and sex were included as predictor variables. In the dispersal model, period (adult breeding or post-breeding), sex, and human impact were used. Although initially included as predictor variables, the parameters body mass, litter size, number of litter (i.e. originating from a female’s first or second), and ambient temperatures were eliminated in both models due to AICc reductions. This procedure also led to elimination of human impact as a predictor variable in the time-of-birth model. Normality of model residuals was tested using Shapiro–Wilk tests. Models were tested for homoscedasticity using Levene-tests and were additionally controlled visually by plotting residuals vs. fitted values. ANOVAs from linear models were computed using marginal (Type III) sums of squares. Post-hoc comparisons were performed using Tukey’s HSD tests. Results are shown as means ± SE.

3. Results

Juvenile Common hamsters were only rarely involved in aggressive interactions (0.09 conflicts/h in relation to the total number of observation hours). We only observed five fights and six chases involving juveniles. Aggression was received mainly from adult females (55%, n = 6), but females never attacked their own offspring. We only twice (18%) observed an adult male chasing a juvenile. Aggression initiated by juveniles (27%, n = 3) was exclusively directed against other juveniles. Conflict behaviour occurred during both the adult breeding (64%) and post-breeding period (36%). Juvenile FCM levels did not differ significantly between the adult breeding and post-breeding period. Because of the low frequency of observed aggressive interactions, conflict behaviour was not included in statistical analyses.

We analysed cortisol secretion patterns in juvenile hamsters during their first weeks after natal emergence and compared early- and late-born individuals. Neither time of birth, post-emergence interval, nor sex had main effects on FCM levels. We did, however, find a significant interaction between time of birth and post-emergence intervals (Table 1). Post-hoc comparisons showed that in early-born juveniles, FCM levels remained relatively constant during the three post-emergence intervals, whereas in late-born individuals FCM levels significantly increased from the weaning to the post-weaning phase (Fig. 1 and Table 2). Early- and late-born juveniles showed similar FCM concentrations during pre-weaning and weaning. However, post-weaning FCM levels of late-born individuals were significantly higher than those of early-born ones (Fig. 1 and Table 2). To test for seasonal effects, we additionally analysed FCM levels in early-born juveniles during the late-born juveniles’ post-weaning period. During this period, FCM concentrations were significantly lower in early-born individuals compared to late-born ones (means ± SE, early-born: 523 ± 79 ng/g, n = 13; late-born: 1073 ± 291 ng/g, n = 5; Student’s t-test, p = 0.03). Immergence into the hibernaculum occurred at similar dates in both groups (early-born: 4 October ± 4.2 d, n = 9; late-born: 3 October ± 3.9 d, n = 11; Student’s t-test: p = 0.77).

Table 1.

ANOVA (Type III tests) table for effects of time of birth (early or late), post-emergence intervals (pre-weaning, weaning, or post-weaning), and sex on FCM-levels (response variable) in juvenile hamsters. For interaction details see Table 2.

Predictor variables	df	F values	p values
Time of birth	1	0.11	0.739
Post-emergence interval	2	3.17	0.052
Sex	1	0.12	0.736
Time of birth × post-emergence interval	2	9.87	<0.001
Time of birth × sex	1	0.93	0.342
Post-emergence interval × sex	2	2.51	0.093

Fig. 1.

FCM concentrations (means ± SE) in early- and late-born juvenile Common hamsters during the pre-weaning, weaning, and post-weaning period. Early-born individuals emerged from the natal burrow between 1 June and 7 July, late-born juveniles between 29 July and 20 September. Groups with the same letter do not differ significantly (for p-values see Tables 1 and 2). Sample sizes: pre-weaning, early-born = 13, late-born = 11; weaning, early-born = 13, late-born = 6; post-weaning, early-born = 5, late-born = 5.

Table 2.

Post-hoc comparisons (Tukey’s HSD test) of FCM-levels in early- and late-born juveniles during their first weeks post-emergence. Late-born individuals during the post-weaning interval had significantly higher levels than all other groups. Only significant group comparisons are shown (for all other comparisons, e.g. early-born juveniles during pre-weaning vs. early-born ones during weaning, p > 0.6). Numbers in parenthesis indicate sample size. Diff…differences between pairs, lwr./upr… lower/upper 95%-confidence intervals.

Group	Diff.	Lwr.	Upr.	p values
Late-born post-weaning (5) vs. early-born pre-weaning (13)	535.9	112.1	959.7	0.006
Late-born post-weaning (5) vs. early-born weaning (13)	611.6	187.8	1035.4	0.001
Late-born post-weaning (5) vs. early-born post-weaning (5)	757.7	248.4	1267.1	0.001
late-born post-weaning (5) vs. late-born pre-weaning (11)	652.6	218.2	1086.9	0.001
Late-born post-weaning (5) vs. late-born weaning (6)	574.9	87.2	1062.5	0.013

Analyses of potential effects of dispersal on FCM concentrations revealed significant main effects of status (resident or immigrant), period (early or late in the season), sex, and human impact. Additionally, significant interactions were found between status and period and between status and sex (Table 3). Juveniles that immigrated into our study site late in the season had significantly higher FCM levels than early immigrants (Fig. 2 and Table 4a). Moreover, late immigrants had significantly higher FCM concentrations than resident juveniles during both phases (Fig. 2 and Table 4a). Sex differences revealed that immigrated males had significantly higher FCM levels than resident males. In female juveniles, residents and immigrants had similar FCM levels. No significant differences were found between the sexes, neither in residents nor immigrants (Table 4b). Human impact affected cortisol secretion patterns (Table 3): individuals in high-impact areas had significantly higher FCM levels than juveniles in low-impact areas (means ± SE, high impact: 738 ± 111 ng/g, n = 15; low impact: 493 ± 25 ng/g, n = 70). We found no differences between the sexes (Student’s t-test, males vs. females, high-impact area: p > 0.99, n(m/f) = 6/9; low-impact area: p = 0.99, n(m/f) = 40/30), and the percentage of early/late born and immigrated individuals was similar in both areas (early-born: high- vs. low-impact: 47% vs. 47%, late-born: 20% vs. 19%, early immigrants 20% vs. 17%, late immigrants 13% vs. 17%). To test for potential seasonal and/or time-of-birth effects, we additionally compared FCM levels of early- and late-born juveniles between the high- and low-impact area. We found higher FCM concentrations in early-born individuals (but not in late-born ones) inhabiting the high-impact area (Student’s t-test, early-born juveniles, high- vs. low-impact area: p = 0.017, n(high/low) = 5/33; means ± SE, high-impact: 710 ± 109 ng/g, low-impact: 464 ± 35 ng/g; late-born juveniles, high- vs. low-impact area: p = 0.14, n(high/low) = 5/13; means ± SE, high-impact: 705 ± 138 ng/g, low-impact: 504 ± 61 ng/g). Sample sizes in the other groups were too small for statistical comparisons.

Table 3.

ANOVA (Type III tests) table for effects of status (resident or immigrant), period (early or late in the season), sex, and area (high or low human impact) on FCM-levels (response variable) in juvenile hamsters. For details on interactions see Table 4.

Predictor variables	df	F values	p values
Status	1	6.6	0.012
Period	1	17.51	<0.001
Sex	1	7.23	0.009
Area	1	17.18	<0.001
Status × period	1	7.41	0.008
Status × sex	1	8.71	0.004

Fig. 2.

FCM levels (means ± SE) in resident (born in the study area) and immigrated juvenile hamsters. Individuals that immigrated early (before 26 July) and late in the season (after 17 August) are compared. Groups with the same letter do not differ significantly (for p-values see Tables 3 and 4). Sample sizes: breeding, residents = 28, immigrants = 15; post-breeding, residents = 41, immigrants = 14.

Table 4.

Post-hoc comparisons (Tukey’s HSD test) of (a) FCM-levels in resident and immigrated juveniles (status) early and late in the season (period). (b) Comparisons of FCM-levels between male and female residents and immigrants. Differences were significant only between male residents and immigrants, with the latter showing higher levels (for all other comparisons, e.g., female residents – female immigrants, p > 0.1). Numbers in parenthesis indicate sample size. Diff… differences between pairs, lwr./upr… lower/upper 95%-confidence intervals.

	Group	Diff.	Lwr.	Upr.	p values
(a)	Late immigrants (14) vs. early immigrants (15)	285.7	52.3	519.1	0.01
	Late immigrants (14) vs. early residents (28)	236.6	31.0	442.1	0.017
	Late immigrants (14) vs. late residents (41)	−195.5	−389.9	−1.2	0.048
	Early residents (28) vs. late residents (41)	41.0	−113.0	195.0	0.898
	Early residents (28) vs. early immigrants (15)	49.1	−151.8	250.1	0.919
	Late residents (41) vs. early immigrants (15)	90.1	−99.4	279.6	0.6
(b)	Male residents (35) vs. male immigrants (18)	−191.5	-371.2	-11.8	0.032

4. Discussion

This study examined potential developmental and seasonal effects on adrenal activity of juvenile Common hamsters by analysing concentrations of faecal cortisol metabolites (FCM) in early- and late-born individuals during their first weeks after natal emergence.

Time of birth apparently affected FCM levels: late-born pups showed elevated values during post-weaning. During this time the juveniles had to find or construct a burrow and prepare for the forthcoming winter, requiring body fat reserves and sufficient food stores. Both early- and late-born juveniles immerge into their hibernacula in early October. The latter therefore have considerably less time to prepare for winter than the former. Accordingly, late-born individuals have been shown to invest more time per day in foraging and food caching above-ground than early-born juveniles [48,49]. Late-born individuals therefore might experience higher temporal and energetic constraints.

Glucocorticoid (GC) release is an important feature of energy mobilization, and GC levels can peak during energetically costly periods [42]. Elevated energy demands affected GC secretion in golden hamsters [6] and house mice [15]: wheel-running activity was related to increased GC secretion in both species. Thus, increased energetic costs due to intense foraging and food-caching in late-born hamsters could explain the elevated FCM levels.

Caching storable food items during the prehibernation period is probably crucial for over-winter survival, particularly in juvenile hamsters. In our study area, individuals were observed to selectively cache food items that vary in their seasonal availability [18]. Selective caching is assumed to be time and energy consuming because these food items have to be searched for, requiring moving longer distances compared to the first weeks post-emergence when the animals feed on grass, clover or dandelion close to the burrow [18].

Factors other than energetic demands could also contribute to the increased FCM levels of post-weaning late-born juveniles. These include increased predation risk associated with increased foraging and above-ground activity, social conflict, greater exposure to low temperatures, and prehibernatory hyperphagia. However, none of these factors appear to be prominent in our data. At our study site, we frequently observed kestrels (Falco tinnunculus), occasionally feral cats, and very rarely martens (Martes foina) and foxes (Vulpes vulpes). Juvenile hamsters are favoured prey for kestrels [36]. Based on casual recording of predator sightings, we did not observe any pronounced peaks in the number of predator sightings (Franceschini et al., unpublished data). On the basis of this limited data on predation, we conclude that it is unlikely that increased predation pressure caused the elevated FCM levels in post-weaning late-born juveniles. Another external factor changing with season is ambient temperature. Late-born individuals might face colder temperatures, activating the HPA-axis (e.g. [17,44]). However, ambient temperature as a factor in the initial model showed no significant effect. Although conflict behaviour affects adrenal activity in adult male Common hamsters [14], aggression in juveniles was very rare and we found no evidence for increased conflict rates in late-born pups. Accordingly, increased adrenal activity is unlikely to be caused by social stress. Finally, some hibernators that rely on fat stores during hibernation have elevated GC levels associated with fat deposition [21,42]. This could explain the elevated FCM levels we observed, if late-born juveniles need to increase fat stores more rapidly than early-born juveniles. However, neither early- nor late-born animals gained body mass rapidly before winter (Siutz et al., unpublished data), making this an unlikely explanation for the increase in late-born FCM levels.

Most late-born juveniles originated from females’ second litters. This calls for considering maternal effects on FCM levels in late-born offspring. These effects could occur via higher maternal GC secretion during gestation and/or lactation of late litters, or via less maternal investment. Previous studies showed that FCM levels in adult female Common hamsters remained low during gestation and lactation [14] and did not differ between first and second litters (Franceschini et al., unpublished data). Moreover, lactation duration did not differ between early- and late-born offspring (Siutz et al., unpublished data). Thus, body mass at weaning was similar in early- and late-born pups, indicating that the latter received sufficient maternal care (Siutz et al., unpublished data).

The fact that early-born juveniles still had significantly lower FCM levels than late-born ones during the late-born juveniles’ post-weaning period indicates that the differences in FCM concentrations do not primarily reflect a seasonal change (from summer to fall).

Considering that none of the factors mentioned above could serve as an explanation, we assume that increased energetic demands led to elevated FCM levels in late-born juveniles and late immigrants.

At our study site, two areas with different human impact (frequency of passers-by or playing children) were clearly distinguishable. We suggest that frequent anthropogenic disturbance acts as a stressor, as reported in other hibernating rodents such as European ground squirrels (Spermophilus citellus), (Brenner et al., unpublished data). Juveniles inhabiting high-impact areas had higher FCM levels than those in low-impact areas. The proportions of early- and late-born as well as resident and immigrated individuals did not differ between the areas. As FCM levels were elevated in late-born juveniles in both high- and low-impact areas, exposure to humans cannot have caused the post-weaning differences between the groups. Human impact had no effect on residents and only a weak effect on immigrants. A reason for this could be that resident individuals in the highly frequented areas were more habituated to this situation than immigrants.

Increased GC secretion could compromise immune function and increase mortality rates [5,21,26,45]. Unfortunately, our sample size shortly before immergence into hibernation was too small to test for potential negative effects of elevated FCM levels on over-winter survival. Preliminary results, however, showed that prehibernation body mass and body fat proportion was significantly lower in late- compared to early-born juveniles (Siutz et al., unpublished data). This indicates that most late-born hamsters could not compensate the time deficit. Accordingly, the overwinter mortality rate of late-born individuals was 80% (24 of 30 individuals) vs. 52% (12 of 23 individuals) for early-born juveniles.

Finally, we compared cortisol secretion patterns between juveniles born in the study area and individuals that immigrated either early or late in the season. Dispersal per se did not seem to affect cortisol secretion patterns: FCM levels were the same in early immigrants and residents. The timing of dispersal, however, did play a role: late immigrators had significantly higher FCM levels than both early immigrants and individuals born on site. Since late-immigrated and late-born juveniles may face a similar situation at the end of the season, the above-mentioned factors might also apply to late immigrants: they had less time to find a suitable hibernaculum and cache food. Note that late immigrators were not born late in the season: body mass comparisons indicated that late immigration did not involve younger individuals. Effects related to birth date can therefore be excluded. Therefore, these results strengthen the assumption that the observed increase in FCM is primarily related to the shorter time available to prepare for hibernation.

In conclusion, our findings indicate that the seasonal timing of birth and dispersal affect cortisol secretion patterns in juvenile Common hamsters. We assume that late-born and late-immigrated individuals experience higher temporal constraints in preparing for hibernation. This could elevate energetic demands, increase exposure to predators and/or humans, and elevate FCM concentrations. The data set, however, does not allow differentiation between acute or chronic activation of the HPA axis. Future experimental studies could focus on determining the effects of different factors. More knowledge about the effects of potential stressors could improve reintroduction and agriculture management projects in this endangered species.