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Within-litter differences in personality and physiology relate to size differences among siblings in cavies
Physiology & Behavior 145 (2015) 22–28
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Within-litter differences in personality and physiology relate to size differences among siblings in cavies A. Guenther ⁎, F. Trillmich Department of Animal Behavior, University of Bielefeld, Bielefeld, Germany
H I G H L I G H T S • • • •
Individual development of siblings depends on relative weight within the litter Siblings already differ in personality three days after birth Relative size contributes to long-term differences in physiology and behaviour Cortisol and RMR, but not growth rate correlate with personality traits
a r t i c l e
i n f o
Article history: Received 5 December 2014 Received in revised form 24 February 2015 Accepted 19 March 2015 Available online 20 March 2015 Keywords: Personality Early development Siblings Individual differences Cortisol Metabolic rate
a b s t r a c t Many aspects of an animal's early life potentially contribute to long-term individual differences in physiology and behaviour. From several studies on birds and mammals it is known that the early family environment is one of the most prominent factors influencing early development. Most of these studies were conducted on highly altricial species. Here we asked whether in the highly precocial cavy (Cavia aperea) the size rank within a litter, i.e. whether an individual is born as the heaviest, the lightest or an intermediate sibling, affects personality traits directly after birth and after independence. Furthermore, we investigated whether individual states (early growth, baseline cortisol and resting metabolic rate) differ between siblings of different size ranks and assessed their relation to personality traits. Siblings of the same litter differed in personality traits as early as three days after birth. Pups born heaviest in the litter were more explorative and in general more risk-prone than their smaller siblings. Physiological state variables were tightly correlated with personality traits and also influenced by the size rank within litter, suggesting that the size relative to littermates constitutes an important factor in shaping an individual's developmental trajectory. Our data add valuable information on how personalities are shaped during early phases of life and indicate the stability of developmentally influenced behavioural and physiological traits. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Despite burgeoning interest in consistent individual variation in suits of behaviours, termed animal personality, the ontogeny of personality types and consistency of personality traits is still scarcely understood [1]. Initial individual differences in states that affect the trade-off between the costs and benefits of behavioural actions are hypothesised to contribute to the development of differences in personality type [2]. In this context ‘state’ might include various aspects of an animal's physiology and morphology [3], such as body mass [4], energy reserves [5], metabolic rate or productivity [6] as well as its interplay with the environment.
⁎ Corresponding author. E-mail address: [email protected] (A. Guenther).
http://dx.doi.org/10.1016/j.physbeh.2015.03.026 0031-9384/© 2015 Elsevier Inc. All rights reserved.
The early social environment an individual experiences is one of the most prominent factors known to influence development. It may have profound immediate and long-term consequences on physiology, behaviour and even fitness [7–9]. Particularly maternal and sibling effects, which can strongly influence early growth and survival, may be important in shaping personality development [10,11]. From mammalian offspring it is known that siblings can influence each other's development as well as the mother's physiology while still in utero [12,13]. Competition for limited resources is considered an important mechanism shaping developmental differences among siblings. Individuals that are relatively (compared to their siblings) large at birth typically have an advantage in competition with smaller siblings that is maintained during development [14]. It has been shown in rats that the relative within-litter body mass was associated with differences in circulating levels of the stress-related hormone corticosterone with heavier pups having lower corticosterone levels [15]. A similar
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It is known that behaviour and physiology in cavies change to some degree according to season, hence, all animals were kept in climate chambers at 20 °C ± 2 °C throughout the experimental period. Light conditions were changed to simulate spring photoperiod. Four weeks prior to the beginning of experiments, the light:dark cycle was set to 12:12 with only artificial light available. Within one week the light:dark rhythm was changed to 10:14 L:D. Thereafter, one male was added to each enclosure and left with the female for three weeks to ensure successful mating. Simulation of the photoperiodic treatment started nine days after adding the males and was achieved by increasing the daily light period by 15 min every ninth day. The juveniles (39 in 18 litters (2 females were not pregnant); 21 females, 18 males, litter size ranging between 1 and 3) were born when the light period lasted between 11.75 and 12.25 h per day. Enclosures were checked daily for newborn pups. Birthdates, litter size, mass and sexes of pups were noted on the day of parturition and each pup was marked individually by a haircut. Based on their weight, the pups were assigned a relative size rank within litter, giving the heaviest pup size rank 1, the second heaviest size rank 2 and so on. The juveniles were weighed again 24 days later to assess the growth rate until weaning. The photoperiodic treatment continued until all tests were conducted with all juveniles. Juveniles were weaned at an age of 21 days by removing the mother but leaving the pups of one litter together until the end of the study.
phenomenon exists in guinea pigs, where pups of larger litters, which are usually smaller, have higher circulating levels of cortisol. Increased aggressive interactions among siblings were observed in correlation with elevated cortisol levels [16]. In humans differences between siblings are known to extend to social and risk-taking behaviour [17]. Some evidence from non-human animals also suggests that interactions between siblings of the same litter may cause potentially long-term effects on physiological and behavioural development. Litter size was associated with differences in emotionality in adult rats [10] and rabbits [8]. In rats, smaller siblings are less bold than their heavier littermates after weaning [11]. In addition, litter sex-ratio may influence personality development prenatally [18,13] and postnatally [19,20]. Sexes might generally develop a different personality type, as for example shown for the spiny mice [21]. Significant differences in affective behaviour between sexes were reported for rodents [22] and we also found differences in exploration behaviour related to sex in cavies [23]. These differences were not immediately apparent in juvenile cavies but developed after maturation. Most likely, gonadal hormones underlie these sex differences. In male rodents, exogenous testosterone was shown to reduce anxiety-like behaviours [24], and correspondingly enhance exploration. We present data on the personality development and physiological traits of litter siblings in wild cavies (Cavia aperea). Cavies have a particular long pregnancy (~60 days) and are highly precocial, resulting in a large scope for prenatal influences on development [25]. Compared to the long pregnancy, young become independent shortly after birth (~ 21 days) [26]. Overall life expectancy in the closely related black backed cavy (Cavia magna) lies between 2.4 and 16 months under natural conditions [27]. The results of an earlier study indicated that individuals born in the same litter differ in birth weight and in their behaviour shortly after weaning [23]. When born into spring conditions, the biggest pup of a litter tended to be more bold and explorative shortly after weaning. The sample size, however, was quite low and we did not investigate possible links to physiological state variables. In this study we tested the hypothesis that size rank within a litter significantly influences behavioural and physiological traits. We study, if cavies express different personality types according to their size rank as early as three days after birth, indicating that personality is influenced by the mother or interaction with siblings in utero prior to birth. We further ask whether these personality differences remain stable until after weaning. In mice and voles a positive relationship between growth, food intake and personality traits were documented [28,29] and interpreted as individual differences in life-history trade-offs [6]. Following this hypothesis we expect that personality differences correlate with differences in physiological states such as growth rate and metabolic rate.
Pups were first tested when three to four days old. To this end, each pup was gently removed from the cage and placed on the open hand of the observer. The hand was then placed in the cage in such a way that the juvenile did not directly face its mother or siblings and the latency to leave the hand was measured (Hand-escape test). The latency (from the moment the hand was touching the floor of the enclosure to the moment when the pup had left the hand) was timed. If it had not left the hand after 60 s, the juvenile was placed back next to its mother. Overall, the procedure did not last more than 2 min to minimize stress to the juveniles in this early phase of life. The test was repeated, when the juveniles were eight to nine days old to assess the repeatability of the Hand-escape latency. Another test was conducted at the age of seven days and again at 12–14 days to measure activity levels, sometimes also referred to as docility of juveniles. Again, the test was short to avoid stressing the juveniles. The animal was removed from the home cage and the time it spent struggling when held on its back in the hand was measured for 30 s before the animal was placed back in the home enclosure (Struggle test).
2. Methods
2.3. Experimental procedure — behavioural tests after weaning (see Fig. 1)
2.1. Subjects and experimental procedure
At an age of 21–30 days, all pups were tested for voluntary exploration behaviour (Long Field test), for forced exploration behaviour in an Open Field test and for boldness in a Novel Object test. All tests were conducted between 9:00 and 12:00 in the morning or 14:00 and
To assess behavioural differences among siblings, 20 adult multiparous female cavies (C. aperea) and 20 adult males derived from the breeding stock of the University of Bielefeld were bred. The animals were descendants from wild cavies caught in Uruguay in 2005 and from Argentinean lineages kept in Bielefeld since 1981. No marked differences in morphology, physiology or behaviour were observed in individuals from different origins and they were bred randomly for several generations. Earlier studies confirmed that wild cavies kept and reared in captivity for up to thirty generations do not differ in behaviour and physiology from wild caught individuals [30]. The females were housed singly in standard enclosures of 0.8 m2 with food and water available ad libitum. Hay, pellet food (Firma Höveler, Germany) and water were available at all times, and fresh carrots or paprika was given every other day. Additionally, vitamin C (1 g/l) was added to the drinking water once a week.
2.2. Experimental procedure — behavioural tests shortly after birth (see Fig. 1)
Fig. 1. Timeline to investigate the behavioural and physiological type of juvenile cavies. Weaning weight was always measured at day 25 and the behavioural and physiological tests were conducted between 21 and 30 days of age, in random order.
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17:00 in the afternoon in random order. Between two consecutive tests, animals were given at least a 1 day break (maximum 5 days). The Long Field test lasted 24 h in total and was conducted in a different room but with conspecifics present to reduce stress due to social isolation. These animals were not directly visible to the focal animal. The setup consisted of a standard housing enclosure and an attached 5 m long corridor separated by a small trap-door. After 21 h habituation to this standard enclosure, the trap-door was opened (at 9:00 ± 10 min). The animal was given 3 h to explore the corridor. Right behind the trap-door, a light sensor recorded how often the animal passed into the corridor and back to its home enclosure. Food and water were available ad libitum throughout the whole test. To test for boldness, the Novel Object test was conducted in the home enclosure of the animal and lasted for 1 h. During this time the siblings were removed from the home enclosure to test the focal animal alone. A 4 cm high green egg cup was placed approximately 20 cm in front of the shelter, where the pup was hiding, and the number of contacts with the novel object within 15 min after the first touching of the object were counted. Siblings were tested with at least one day in between in case the removal from the home enclosure caused stress. Forced exploration behaviour was tested in a 1 m2 open field indicating the fearlessness of the animal. The arena was lit from directly above. The animal was placed in the middle of the arena beneath a quadratic transparent shelter with four exits to all sides. A video camera placed above the set-up recorded the animal's movements for 20 min in total. For the first 10 min the animal was allowed to remain beneath the shelter if it did not want to explore, but the shelter was remotely raised out of the arena after 10 min. The distance the animal ran was recorded from the video using the programme Labimals (Labimals: analysis of laboratory animals 2008 copyright: H. Bohle & E.T. Krause, Bielefeld, Germany). 2.4. Experimental procedure — individual differences in physiological states (see Fig. 1)
approved for keeping and breeding cavies by the local government authority responsible for health, veterinary and food monitoring (Gesundheits-, Veterinär- und Lebensmittelüberwachungsamt) under the licence number 530.42 16 30-1. No mortality occurred throughout the study. At the end of experiments all animals were used for other experiments or kept under stock conditions. 2.6. Statistical analyses All data were analysed using the free software R (version 2.14.1; [34]). Variables of all tests were analysed using univariate mixed effects models. Data derived from the Novel Object test and the Long Field test were count-data and their distribution resembled a Poissondistribution. For their analyses, we used mixed models with a Poisson distribution. Initially, the size rank in litter (3 level factor), the litter size (continuous variable) and sex were included as fixed effects as well as their two-way interactions. Non-significant effects were excluded step-wise. Mother ID was included as a random effect allowing random intercept but not random slopes. To analyse the other behavioural data and growth rate, mixed effect models with the same fixed and random effects structure were used but normal error distributions were fitted. Residuals of the models were checked visually for distribution and variance homogeneity by using Q–Q plots. To estimate correlations between the different behaviours, we extracted the best linear unbiased predictors (BLUPs) from the minimum adequate models. As the distribution of the BLUPs differed between behavioural traits, we used Spearman rank correlations. To account for multiple testing (we calculated 28 correlations), we applied false discovery rate (FDR) adjustment [35]. For analysis of repeatability for the hand escape latency and the struggle activity, we also applied a mixed model design following [36] using 1000 permutations to calculate the significance of the estimated repeatability and 1000 bootstrappings to assess the 95% confidence interval. Because the sample size would have dropped considerably, had we calculated the repeatability for each size rank separately, we did not calculate conditional repeatabilities despite the indicated differences between siblings.
To determine individual differences in states, we measured the growth rate until shortly after weaning (25 days), the baseline plasma cortisol level shortly after weaning and the O2-consumption (ml/min ∗ kg− 1). The absolute growth rate was determined as the weight at day 25 minus the birthweight, divided by 25. The specific growth rate was determined as the growth rate per day and gramme birth weight. Plasma cortisol concentrations were measured at noon (12:00 h ± 10 min) by puncturing the marginal ear vein and collecting about 70 μl blood within 3 min after catching the animal to prevent an increase in blood cortisol concentration [30,31]. Plasma was separated by centrifuging the blood sample for 4 min with 13,000 rpm and then deep frozen at −20 °C. Cortisol concentrations were analysed in duplicate at the department of Behavioural Biology at Münster University by radioimmunoassay without chromatography using specific antibodies against cortisol as described previously [32,33]. O2-consumption was measured by open-flow respirometry for 3.5 h under a continuous flow of outside air of about 80 l/h (Mass Flow Meter FM-360, Tylan Corp., Torrance, CA, U.S.A.). External air was pumped through two visually isolated metabolic chambers. From there it went into two successive coolers (M&C Cooler, Ratingen, Germany) followed by drierite scrubbers (Drierite, Fluka, Steinheim, Germany) for drying. A subsample of this air, measured against outside air, flowed at 600 ml/min through an O2 analyser (Oxzilla FC, Sable Systems, Henderson, NV, U.S.A.). Animals were measured between 09:00 and 18:00 h under low light conditions at 20 ± 1 °C.
In total, 39 pups were born, 18 pups in size rank 1, 14 pups in size rank 2, and 7 pups in size rank 3. To test whether siblings of the same litter differ in behaviour already shortly after birth, we tested pups when they were 3–4 days old and again when 7–8 days old. In both tests, we found differences between siblings of different size ranks. Heaviest pups had the longest latencies to leave the hand while pups of size ranks two and three left significantly faster (size ranks 1–2: t(16) = − 2.5; p = 0.03; size ranks 1–3: t(16) = − 2.8; p = 0.013, see Fig. 2a). There was no difference between pups of size ranks two and three. Pups of size ranks one and two did not differ in struggling activity, while pups of size rank three struggled significantly more than their heavier siblings (size ranks 1–3: t(18) = 2.88; p = 0.01; size ranks 2–3: t(18) = 2.82; p = 0.014, see Fig. 2b). In none of the tests sex or litter size did affect the behaviour of pups. Both tests were repeated after five to six days to assess repeatability. The latency (in sec) to leave the hand of the observer was highly repeatable (R = 0.61 and a CI of 0.32–0.84, p = 0.001). The time spent struggling was not significantly repeatable, however, there was a trend perhaps suggesting some repeatability (R = 0.25, CI = 0–0.52, p = 0.08).
2.5. Ethical note
3.2. Personality after weaning
All experimental procedures were carried out in accordance with German animal protection law and animal facilities are regularly
To test whether differences between siblings persisted until independence of juveniles, we tested all individuals shortly after weaning
3. Results 3.1. Personality shortly after birth
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Fig. 2. Pups of different size ranks differ in personality traits shortly after birth. Panel a shows differences in the latency to leave the hand at an age of three to four days, and panel b shows the time spent struggling at an age of seven days. Shown are the estimated values derived from the mixed models on the behavioural traits ± Standard Error. * indicates significant differences between levels.
(n = 39; 21 females, 18 males). The heaviest pups within litters covered more distance in an open field and hence are considered more fearless than lighter pups (size ranks 1–2: t(15) = − 3.08; p = 0.008; size ranks 1–3: t(15) = −2.5; p = 0.025; see Fig. 3a). There was no difference between pups of size ranks two and three. The same pattern was found for boldness in the Novel Object test (Fig. 3b). Heavy pups interacted significantly more often with a novel object than lighter pups (size ranks 1–2: z = − 3.83; p b 0.00; size ranks 1–3: z = − 3.15, p = 0.002). Again, pups of size ranks N1 did not differ from each other. Pups of different size ranks also differed in their voluntary exploration behaviour as measured in the Long Field test. Heavy pups were the most explorative while lighter pups were less explorative, but in this test size-ranks 2 and 3 also differed from each other (size ranks 1–2: z = − 4.19, p b 0.001; size ranks 1–3: z = − 8.14, p b 0.001; size ranks 2–3: z = − 4.3; p b 0.001; see Fig. 3c). Again we did not find indications for sex differences or effects of the litter size.
3.3. Individual differences in physiological state Absolute growth rate from birth to weaning was measured as average daily weight gain between birth and day 25 (n = 39). It turned out to be equal between siblings of different size ranks (p N 0.2 for all pair-wise comparisons between size ranks; see Fig. 4a). Specific growth rate however, differed significantly between pups of size ranks 1 and 3 (growth size rank 1: 0.7 ± 0.06 g/day ∗ g birthweight−1, growth size rank 3: 0.88 ± 0.07 g/day ∗ g birthweight− 1; t17 = 2.27; p = 0.03) and tended to differ between pups of size ranks 1 and 2 (growth size rank 2: 0.8 ± 0.05 g/day ∗ g birthweight− 1; t17 = 1.86; p = 0.08) being higher in the smaller pups.
Cortisol levels differed significantly between pups of different size ranks (see Fig. 4b). Pups of size rank three showed significantly higher plasma cortisol levels than did their heavier siblings (t(12) = 2.8; p = 0.016). There was no significant difference between pups of size ranks one and two. There was no difference in specific O2-consumption between siblings of different size ranks (see Fig. 4c). Male and female pups did not differ in any of the behavioural and the physiological traits measured. To assess which of the physiological traits are related to personality traits in the cavy, we used pair-wise Spearman rank correlations (see Table 1). In addition, we also correlated the behavioural traits with each other, including the hand-escape latency (measured on day 3) and the struggling behaviour (measured on day 7). 4. Discussion Our results provide new insights into how behavioural and physiological personality traits are shaped by the prenatal and early postnatal social environment and their developmental maintenance. They enhance the knowledge about the maintenance of inter-individual variation in personality types within populations. We detected different personality types in pups of the same litter related to their relative size within the litter. These differences were related to differences in physiological state variables such as plasma cortisol but not to the individual growth rate. 4.1. Differences in personality type and state Pups born as the heaviest in the litter were the most fearless, bold and explorative around weaning. Smaller siblings were less prone to
Fig. 3. Personality types differ between juveniles of different size ranks. Panel a represents fearlessness, measured as the distance moved in an open field. Panel b represents how often individuals touched a novel object within 15 min after the first touch. Panel c represents exploration, measured as the number of activations of a light sensor when entering an unknown environment. Shown are the estimated values derived from the mixed models on the behavioural traits ± Standard Error. * indicates significant differences between levels.
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Fig. 4. Although pups of different size ranks differ in birth weight, the growth rate (a) until weaning does not differ. Pups of different size ranks differ in their plasma cortisol level shortly after weaning (b), but not in their O2-consumption (c). Shown are the estimated values derived from the mixed models on the physiological traits ± Standard Error. * indicates significant differences between levels.
explore an unknown corridor but did not differ from each other in fearlessness or boldness. Comparable effects were found in rats, where smaller pups were less bold than their bigger siblings shortly after weaning [11] and these results are consistent with findings of earlier studies in cavies [23,37]. These studies demonstrated the temporal consistency of all three personality traits from the juvenile stage until adulthood, suggesting that the traits we measured here are indeed personality traits. In accordance with the results of this study, earlier studies found no significant correlations between the personality traits in juveniles, while the number of touches of a novel object was correlated with the number of trips in the Long Field after maturation [37]. Differences in personality types were partly reflected in physiological variables, suggesting an influence of or at least a close connection between an individual's state and the development of its personality type. While metabolic and growth rate during the first 24 days did not differ between pups of different size ranks, pups differed in plasma cortisol concentration around weaning. The growth rate was not correlated to any of the observed behavioural or physiological variables, suggesting that growth proceeded independent of the other traits measured. This result is unexpected as growth rate was shown to be linked to various personality traits not only in other mammals but also in fish, birds and even arthropods [6]. In house mice and voles, for example, early growth was positively linked to activity [28,29,38]. Just as the majority of all studies, the abovementioned studies investigated the relationship between growth and personality in altricial species. There might, however, be a functional difference between altricial and precocial species with respect to the importance of early growth. One study conducted with precocial bighorn ewe also found no relationship between growth and personality [39]. Precocial animals are far shorter and substantially less dependent on milk intake and have lower relative growth rates than altricial young, because they are born much further developed and grow at lower rates after birth. Young of cavies eat solid food almost from the first day of life and can be weaned at 5 days of age with no increase in mortality [40]. Therefore, potential differences in growth rates after birth are harder to detect than in altricial species. Alternatively, early growth rate may be of minor importance for later behavioural and physiological development when young are able to regulate food intake and hence growth this early in life.
In addition to the early growth rate, we investigated resting metabolic rate and baseline cortisol level as both have been proposed as possible mediators of personality differences between individuals and both may be regulated by maternal effects. The smallest pups within a litter had the highest baseline cortisol levels and at the same time covered the least distance in an open field and were the fastest to leave the hand of an observer. These two behavioural traits were also correlated with each other and with cortisol levels. A linkage between cortisol and personality traits seems to exist in a wide range of species. In adult alpine marmots for example, different coping styles were associated with different baseline cortisol levels [41]. In squirrels, baseline cortisol increased with docility [42] and in dairy calves, a positive correlation between plasma cortisol levels and the latency to touch a novel object was found [43]. In male rhesus macaques, high-excitable animals had lower basal cortisol concentrations [44]. All these results point in the direction, that a relatively high baseline cortisol level is characteristic of shy or nervous animals. In the current study, we present the first evidence that this relationship even exists very early after birth (as soon as three days of age) suggesting that cortisol is a key-factor driving the development of personality during life. Cortisol was also tightly correlated to oxygen consumption. Individuals with a relatively high cortisol had low oxygen consumption and oxygen consumption itself was correlated with fearlessness and the hand escape latency. However, there was no statistical difference in oxygen consumption between siblings of different size ranks. These results reflect earlier results [36], where we controlled for the effects of size rank within litter in the resting metabolic rate. In juveniles, the effect of size rank was far from being significant while in mature animals, we found a significant effect. Recent evidence suggests that maternal effects can exert a substantial influence on offspring metabolic rate and indicate the transfer of hormones from mother to embryo as a possible mechanism [45]. It is known from oviparous species that concentrations of egg hormones can vary considerably among- and within-clutches and can affect offspring phenotypes [46]. Female three-spined sticklebacks produce eggs with higher cortisol content and higher oxygen consumption shortly after fertilisation when exposed to predation threats [47]. Likewise, elevation of cortisol in
Table 1 Correlations between behavioural and physiological traits. Fitted values derived from the mixed models are used for correlations. Given are Spearman's rho and the significance level in brackets. Significant correlations are bold. Trait
Hand escape
Struggle
Boldness
Exploration
Fearlessness
Growth rate
Cortisol
Struggle Boldness Exploration Fearlessness Growth rate Cortisol O2 consumption
−0.64 (b0.001) 0.19 (0.3) 0.12 (0.5) 0.5 (0.005) 0.11 (0.55) −0.36 (0.06) −0.92 (b0.001)
0.03 (0.85) 0.09 (0.6) −0.3 (0.09) 0.01 (0.93) −0.23 (0.22) 0.19 (0.3)
0.33 (0.07) 0.28 (0.1) 0.05 (0.95) −0.27 (0.13) −0.28 (0.12)
0.14 (0.44) 0.14 (0.40) 0.26 (0.2) −0.24 (0.17)
−0.09 (0.60) 0.37 (0.04) −0.58 (b0.001)
−0.05 (0.81) −0.16 (0.36)
−0.49 (0.008)
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brown trout eggs increased embryonic RMR [48]. There is also evidence showing that maternal effects on RMR are not restricted to hormonal pathways. Eggs of the clownfish laid on the periphery of a clutch had a 33% lower rate of oxygen consumption than did embryos from the clutch interior [49]. Although we did not find a significant difference in metabolic rate between pups of different size ranks, RMR was tightly correlated with fearlessness and the latency to leave the hand suggesting that it might also be a key-factor in establishing individual differences in personality.
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Acknowledgements We would like to thank the team of the Department of Behavioural Biology of the University of Münster for running the cortisol analyses and Mona Dersen for much appreciated help with the experiments. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR 1232, TR105/25-1).
References 4.2. Possible contribution of the prenatal environment Maternal or parental effects refer to the influence of the parental phenotype on the offspring phenotype independently of the offspring's genotype [50]. These effects can exert influences on offspring either preor postnatally and therefore strongly affect personality development. The fact that we find personality differences in three day old juveniles in the present study implies a strong influence of prenatal maternal effects although initial differences between pups may be enhanced postnatally due to interactions between siblings or mother–offspring interactions. From the closely related guinea pig it is known that aggressive interactions between pups of the same litter occur more often in larger litters where pups have to compete for access to maternal teats, although aggressive interactions were mainly restricted to scramble competition [16]. Prenatal maternal effects in guinea pigs and cavies are well known [51] and have been shown to influence offspring behaviour and physiology until adulthood [52,53]. It is also known that individuals receive different prenatal supplies depending on their position in utero resulting in size differences between pups even several weeks before birth [54,55]. Birth weight depended on the number of pups per uterine horn, but was in general higher for pups in more cervical positions [54]. Consequently, the differences in physiology and behaviour in pups of different size ranks may simply be a result of constraints due to differential maternal provisioning. However, several earlier results render this an unlikely explanation. Juveniles born into a photoperiod simulating autumn conditions show the opposite pattern of behaviour and cortisol with respect to size rank in litter. When born into autumn photoperiod, baseline cortisol values are higher in bigger pups than in smaller ones. Accordingly, bigger pups cover less distance in an open field and are less explorative than their smaller siblings [23]. Together, these results suggest that mothers programme their offspring in an adaptive way. By programming the pups of a litter with different physiological and personality types, mothers could follow a bet-hedging strategy to ensure that at least one of her offspring matches the environmental conditions. Giving the largest in the litter a particularly good chance to survive would appear to make it a kind of ‘core’ offspring as has been described for bird clutches [56]. Producing additional small offspring with somewhat lower survival chances may provide additional benefit, if environmental conditions prove unexpectedly plentiful. However, this hypothesis needs further testing.
5. Conclusion Our data demonstrate that ontogenies within the same litter may be diverse depending on starting conditions as has been shown before for sequential siblings in humans [17,57]. This indicates early niche construction within a litter, which is expected to lead to differential neurophysiological effects that will most likely carry over into the expression of the adult behavioural phenotype. Similar diversification has been shown for altricial rodents [11,58] and it remains to be seen in future comparative studies whether the developmental stage, altricial versus precocial, at birth has systematic consequences for personality development and the extent and timing of changes in its structure.
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Within-litter differences in personality and physiology relate to size differences among siblings in cavies A. Guenther ⁎, F. Trillmich Department of Animal Behavior, University of Bielefeld, Bielefeld, Germany
H I G H L I G H T S • • • •
Individual development of siblings depends on relative weight within the litter Siblings already differ in personality three days after birth Relative size contributes to long-term differences in physiology and behaviour Cortisol and RMR, but not growth rate correlate with personality traits
a r t i c l e
i n f o
Article history: Received 5 December 2014 Received in revised form 24 February 2015 Accepted 19 March 2015 Available online 20 March 2015 Keywords: Personality Early development Siblings Individual differences Cortisol Metabolic rate
a b s t r a c t Many aspects of an animal's early life potentially contribute to long-term individual differences in physiology and behaviour. From several studies on birds and mammals it is known that the early family environment is one of the most prominent factors influencing early development. Most of these studies were conducted on highly altricial species. Here we asked whether in the highly precocial cavy (Cavia aperea) the size rank within a litter, i.e. whether an individual is born as the heaviest, the lightest or an intermediate sibling, affects personality traits directly after birth and after independence. Furthermore, we investigated whether individual states (early growth, baseline cortisol and resting metabolic rate) differ between siblings of different size ranks and assessed their relation to personality traits. Siblings of the same litter differed in personality traits as early as three days after birth. Pups born heaviest in the litter were more explorative and in general more risk-prone than their smaller siblings. Physiological state variables were tightly correlated with personality traits and also influenced by the size rank within litter, suggesting that the size relative to littermates constitutes an important factor in shaping an individual's developmental trajectory. Our data add valuable information on how personalities are shaped during early phases of life and indicate the stability of developmentally influenced behavioural and physiological traits. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Despite burgeoning interest in consistent individual variation in suits of behaviours, termed animal personality, the ontogeny of personality types and consistency of personality traits is still scarcely understood [1]. Initial individual differences in states that affect the trade-off between the costs and benefits of behavioural actions are hypothesised to contribute to the development of differences in personality type [2]. In this context ‘state’ might include various aspects of an animal's physiology and morphology [3], such as body mass [4], energy reserves [5], metabolic rate or productivity [6] as well as its interplay with the environment.
⁎ Corresponding author. E-mail address: [email protected] (A. Guenther).
http://dx.doi.org/10.1016/j.physbeh.2015.03.026 0031-9384/© 2015 Elsevier Inc. All rights reserved.
The early social environment an individual experiences is one of the most prominent factors known to influence development. It may have profound immediate and long-term consequences on physiology, behaviour and even fitness [7–9]. Particularly maternal and sibling effects, which can strongly influence early growth and survival, may be important in shaping personality development [10,11]. From mammalian offspring it is known that siblings can influence each other's development as well as the mother's physiology while still in utero [12,13]. Competition for limited resources is considered an important mechanism shaping developmental differences among siblings. Individuals that are relatively (compared to their siblings) large at birth typically have an advantage in competition with smaller siblings that is maintained during development [14]. It has been shown in rats that the relative within-litter body mass was associated with differences in circulating levels of the stress-related hormone corticosterone with heavier pups having lower corticosterone levels [15]. A similar
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It is known that behaviour and physiology in cavies change to some degree according to season, hence, all animals were kept in climate chambers at 20 °C ± 2 °C throughout the experimental period. Light conditions were changed to simulate spring photoperiod. Four weeks prior to the beginning of experiments, the light:dark cycle was set to 12:12 with only artificial light available. Within one week the light:dark rhythm was changed to 10:14 L:D. Thereafter, one male was added to each enclosure and left with the female for three weeks to ensure successful mating. Simulation of the photoperiodic treatment started nine days after adding the males and was achieved by increasing the daily light period by 15 min every ninth day. The juveniles (39 in 18 litters (2 females were not pregnant); 21 females, 18 males, litter size ranging between 1 and 3) were born when the light period lasted between 11.75 and 12.25 h per day. Enclosures were checked daily for newborn pups. Birthdates, litter size, mass and sexes of pups were noted on the day of parturition and each pup was marked individually by a haircut. Based on their weight, the pups were assigned a relative size rank within litter, giving the heaviest pup size rank 1, the second heaviest size rank 2 and so on. The juveniles were weighed again 24 days later to assess the growth rate until weaning. The photoperiodic treatment continued until all tests were conducted with all juveniles. Juveniles were weaned at an age of 21 days by removing the mother but leaving the pups of one litter together until the end of the study.
phenomenon exists in guinea pigs, where pups of larger litters, which are usually smaller, have higher circulating levels of cortisol. Increased aggressive interactions among siblings were observed in correlation with elevated cortisol levels [16]. In humans differences between siblings are known to extend to social and risk-taking behaviour [17]. Some evidence from non-human animals also suggests that interactions between siblings of the same litter may cause potentially long-term effects on physiological and behavioural development. Litter size was associated with differences in emotionality in adult rats [10] and rabbits [8]. In rats, smaller siblings are less bold than their heavier littermates after weaning [11]. In addition, litter sex-ratio may influence personality development prenatally [18,13] and postnatally [19,20]. Sexes might generally develop a different personality type, as for example shown for the spiny mice [21]. Significant differences in affective behaviour between sexes were reported for rodents [22] and we also found differences in exploration behaviour related to sex in cavies [23]. These differences were not immediately apparent in juvenile cavies but developed after maturation. Most likely, gonadal hormones underlie these sex differences. In male rodents, exogenous testosterone was shown to reduce anxiety-like behaviours [24], and correspondingly enhance exploration. We present data on the personality development and physiological traits of litter siblings in wild cavies (Cavia aperea). Cavies have a particular long pregnancy (~60 days) and are highly precocial, resulting in a large scope for prenatal influences on development [25]. Compared to the long pregnancy, young become independent shortly after birth (~ 21 days) [26]. Overall life expectancy in the closely related black backed cavy (Cavia magna) lies between 2.4 and 16 months under natural conditions [27]. The results of an earlier study indicated that individuals born in the same litter differ in birth weight and in their behaviour shortly after weaning [23]. When born into spring conditions, the biggest pup of a litter tended to be more bold and explorative shortly after weaning. The sample size, however, was quite low and we did not investigate possible links to physiological state variables. In this study we tested the hypothesis that size rank within a litter significantly influences behavioural and physiological traits. We study, if cavies express different personality types according to their size rank as early as three days after birth, indicating that personality is influenced by the mother or interaction with siblings in utero prior to birth. We further ask whether these personality differences remain stable until after weaning. In mice and voles a positive relationship between growth, food intake and personality traits were documented [28,29] and interpreted as individual differences in life-history trade-offs [6]. Following this hypothesis we expect that personality differences correlate with differences in physiological states such as growth rate and metabolic rate.
Pups were first tested when three to four days old. To this end, each pup was gently removed from the cage and placed on the open hand of the observer. The hand was then placed in the cage in such a way that the juvenile did not directly face its mother or siblings and the latency to leave the hand was measured (Hand-escape test). The latency (from the moment the hand was touching the floor of the enclosure to the moment when the pup had left the hand) was timed. If it had not left the hand after 60 s, the juvenile was placed back next to its mother. Overall, the procedure did not last more than 2 min to minimize stress to the juveniles in this early phase of life. The test was repeated, when the juveniles were eight to nine days old to assess the repeatability of the Hand-escape latency. Another test was conducted at the age of seven days and again at 12–14 days to measure activity levels, sometimes also referred to as docility of juveniles. Again, the test was short to avoid stressing the juveniles. The animal was removed from the home cage and the time it spent struggling when held on its back in the hand was measured for 30 s before the animal was placed back in the home enclosure (Struggle test).
2. Methods
2.3. Experimental procedure — behavioural tests after weaning (see Fig. 1)
2.1. Subjects and experimental procedure
At an age of 21–30 days, all pups were tested for voluntary exploration behaviour (Long Field test), for forced exploration behaviour in an Open Field test and for boldness in a Novel Object test. All tests were conducted between 9:00 and 12:00 in the morning or 14:00 and
To assess behavioural differences among siblings, 20 adult multiparous female cavies (C. aperea) and 20 adult males derived from the breeding stock of the University of Bielefeld were bred. The animals were descendants from wild cavies caught in Uruguay in 2005 and from Argentinean lineages kept in Bielefeld since 1981. No marked differences in morphology, physiology or behaviour were observed in individuals from different origins and they were bred randomly for several generations. Earlier studies confirmed that wild cavies kept and reared in captivity for up to thirty generations do not differ in behaviour and physiology from wild caught individuals [30]. The females were housed singly in standard enclosures of 0.8 m2 with food and water available ad libitum. Hay, pellet food (Firma Höveler, Germany) and water were available at all times, and fresh carrots or paprika was given every other day. Additionally, vitamin C (1 g/l) was added to the drinking water once a week.
2.2. Experimental procedure — behavioural tests shortly after birth (see Fig. 1)
Fig. 1. Timeline to investigate the behavioural and physiological type of juvenile cavies. Weaning weight was always measured at day 25 and the behavioural and physiological tests were conducted between 21 and 30 days of age, in random order.
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17:00 in the afternoon in random order. Between two consecutive tests, animals were given at least a 1 day break (maximum 5 days). The Long Field test lasted 24 h in total and was conducted in a different room but with conspecifics present to reduce stress due to social isolation. These animals were not directly visible to the focal animal. The setup consisted of a standard housing enclosure and an attached 5 m long corridor separated by a small trap-door. After 21 h habituation to this standard enclosure, the trap-door was opened (at 9:00 ± 10 min). The animal was given 3 h to explore the corridor. Right behind the trap-door, a light sensor recorded how often the animal passed into the corridor and back to its home enclosure. Food and water were available ad libitum throughout the whole test. To test for boldness, the Novel Object test was conducted in the home enclosure of the animal and lasted for 1 h. During this time the siblings were removed from the home enclosure to test the focal animal alone. A 4 cm high green egg cup was placed approximately 20 cm in front of the shelter, where the pup was hiding, and the number of contacts with the novel object within 15 min after the first touching of the object were counted. Siblings were tested with at least one day in between in case the removal from the home enclosure caused stress. Forced exploration behaviour was tested in a 1 m2 open field indicating the fearlessness of the animal. The arena was lit from directly above. The animal was placed in the middle of the arena beneath a quadratic transparent shelter with four exits to all sides. A video camera placed above the set-up recorded the animal's movements for 20 min in total. For the first 10 min the animal was allowed to remain beneath the shelter if it did not want to explore, but the shelter was remotely raised out of the arena after 10 min. The distance the animal ran was recorded from the video using the programme Labimals (Labimals: analysis of laboratory animals 2008 copyright: H. Bohle & E.T. Krause, Bielefeld, Germany). 2.4. Experimental procedure — individual differences in physiological states (see Fig. 1)
approved for keeping and breeding cavies by the local government authority responsible for health, veterinary and food monitoring (Gesundheits-, Veterinär- und Lebensmittelüberwachungsamt) under the licence number 530.42 16 30-1. No mortality occurred throughout the study. At the end of experiments all animals were used for other experiments or kept under stock conditions. 2.6. Statistical analyses All data were analysed using the free software R (version 2.14.1; [34]). Variables of all tests were analysed using univariate mixed effects models. Data derived from the Novel Object test and the Long Field test were count-data and their distribution resembled a Poissondistribution. For their analyses, we used mixed models with a Poisson distribution. Initially, the size rank in litter (3 level factor), the litter size (continuous variable) and sex were included as fixed effects as well as their two-way interactions. Non-significant effects were excluded step-wise. Mother ID was included as a random effect allowing random intercept but not random slopes. To analyse the other behavioural data and growth rate, mixed effect models with the same fixed and random effects structure were used but normal error distributions were fitted. Residuals of the models were checked visually for distribution and variance homogeneity by using Q–Q plots. To estimate correlations between the different behaviours, we extracted the best linear unbiased predictors (BLUPs) from the minimum adequate models. As the distribution of the BLUPs differed between behavioural traits, we used Spearman rank correlations. To account for multiple testing (we calculated 28 correlations), we applied false discovery rate (FDR) adjustment [35]. For analysis of repeatability for the hand escape latency and the struggle activity, we also applied a mixed model design following [36] using 1000 permutations to calculate the significance of the estimated repeatability and 1000 bootstrappings to assess the 95% confidence interval. Because the sample size would have dropped considerably, had we calculated the repeatability for each size rank separately, we did not calculate conditional repeatabilities despite the indicated differences between siblings.
To determine individual differences in states, we measured the growth rate until shortly after weaning (25 days), the baseline plasma cortisol level shortly after weaning and the O2-consumption (ml/min ∗ kg− 1). The absolute growth rate was determined as the weight at day 25 minus the birthweight, divided by 25. The specific growth rate was determined as the growth rate per day and gramme birth weight. Plasma cortisol concentrations were measured at noon (12:00 h ± 10 min) by puncturing the marginal ear vein and collecting about 70 μl blood within 3 min after catching the animal to prevent an increase in blood cortisol concentration [30,31]. Plasma was separated by centrifuging the blood sample for 4 min with 13,000 rpm and then deep frozen at −20 °C. Cortisol concentrations were analysed in duplicate at the department of Behavioural Biology at Münster University by radioimmunoassay without chromatography using specific antibodies against cortisol as described previously [32,33]. O2-consumption was measured by open-flow respirometry for 3.5 h under a continuous flow of outside air of about 80 l/h (Mass Flow Meter FM-360, Tylan Corp., Torrance, CA, U.S.A.). External air was pumped through two visually isolated metabolic chambers. From there it went into two successive coolers (M&C Cooler, Ratingen, Germany) followed by drierite scrubbers (Drierite, Fluka, Steinheim, Germany) for drying. A subsample of this air, measured against outside air, flowed at 600 ml/min through an O2 analyser (Oxzilla FC, Sable Systems, Henderson, NV, U.S.A.). Animals were measured between 09:00 and 18:00 h under low light conditions at 20 ± 1 °C.
In total, 39 pups were born, 18 pups in size rank 1, 14 pups in size rank 2, and 7 pups in size rank 3. To test whether siblings of the same litter differ in behaviour already shortly after birth, we tested pups when they were 3–4 days old and again when 7–8 days old. In both tests, we found differences between siblings of different size ranks. Heaviest pups had the longest latencies to leave the hand while pups of size ranks two and three left significantly faster (size ranks 1–2: t(16) = − 2.5; p = 0.03; size ranks 1–3: t(16) = − 2.8; p = 0.013, see Fig. 2a). There was no difference between pups of size ranks two and three. Pups of size ranks one and two did not differ in struggling activity, while pups of size rank three struggled significantly more than their heavier siblings (size ranks 1–3: t(18) = 2.88; p = 0.01; size ranks 2–3: t(18) = 2.82; p = 0.014, see Fig. 2b). In none of the tests sex or litter size did affect the behaviour of pups. Both tests were repeated after five to six days to assess repeatability. The latency (in sec) to leave the hand of the observer was highly repeatable (R = 0.61 and a CI of 0.32–0.84, p = 0.001). The time spent struggling was not significantly repeatable, however, there was a trend perhaps suggesting some repeatability (R = 0.25, CI = 0–0.52, p = 0.08).
2.5. Ethical note
3.2. Personality after weaning
All experimental procedures were carried out in accordance with German animal protection law and animal facilities are regularly
To test whether differences between siblings persisted until independence of juveniles, we tested all individuals shortly after weaning
3. Results 3.1. Personality shortly after birth
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Fig. 2. Pups of different size ranks differ in personality traits shortly after birth. Panel a shows differences in the latency to leave the hand at an age of three to four days, and panel b shows the time spent struggling at an age of seven days. Shown are the estimated values derived from the mixed models on the behavioural traits ± Standard Error. * indicates significant differences between levels.
(n = 39; 21 females, 18 males). The heaviest pups within litters covered more distance in an open field and hence are considered more fearless than lighter pups (size ranks 1–2: t(15) = − 3.08; p = 0.008; size ranks 1–3: t(15) = −2.5; p = 0.025; see Fig. 3a). There was no difference between pups of size ranks two and three. The same pattern was found for boldness in the Novel Object test (Fig. 3b). Heavy pups interacted significantly more often with a novel object than lighter pups (size ranks 1–2: z = − 3.83; p b 0.00; size ranks 1–3: z = − 3.15, p = 0.002). Again, pups of size ranks N1 did not differ from each other. Pups of different size ranks also differed in their voluntary exploration behaviour as measured in the Long Field test. Heavy pups were the most explorative while lighter pups were less explorative, but in this test size-ranks 2 and 3 also differed from each other (size ranks 1–2: z = − 4.19, p b 0.001; size ranks 1–3: z = − 8.14, p b 0.001; size ranks 2–3: z = − 4.3; p b 0.001; see Fig. 3c). Again we did not find indications for sex differences or effects of the litter size.
3.3. Individual differences in physiological state Absolute growth rate from birth to weaning was measured as average daily weight gain between birth and day 25 (n = 39). It turned out to be equal between siblings of different size ranks (p N 0.2 for all pair-wise comparisons between size ranks; see Fig. 4a). Specific growth rate however, differed significantly between pups of size ranks 1 and 3 (growth size rank 1: 0.7 ± 0.06 g/day ∗ g birthweight−1, growth size rank 3: 0.88 ± 0.07 g/day ∗ g birthweight− 1; t17 = 2.27; p = 0.03) and tended to differ between pups of size ranks 1 and 2 (growth size rank 2: 0.8 ± 0.05 g/day ∗ g birthweight− 1; t17 = 1.86; p = 0.08) being higher in the smaller pups.
Cortisol levels differed significantly between pups of different size ranks (see Fig. 4b). Pups of size rank three showed significantly higher plasma cortisol levels than did their heavier siblings (t(12) = 2.8; p = 0.016). There was no significant difference between pups of size ranks one and two. There was no difference in specific O2-consumption between siblings of different size ranks (see Fig. 4c). Male and female pups did not differ in any of the behavioural and the physiological traits measured. To assess which of the physiological traits are related to personality traits in the cavy, we used pair-wise Spearman rank correlations (see Table 1). In addition, we also correlated the behavioural traits with each other, including the hand-escape latency (measured on day 3) and the struggling behaviour (measured on day 7). 4. Discussion Our results provide new insights into how behavioural and physiological personality traits are shaped by the prenatal and early postnatal social environment and their developmental maintenance. They enhance the knowledge about the maintenance of inter-individual variation in personality types within populations. We detected different personality types in pups of the same litter related to their relative size within the litter. These differences were related to differences in physiological state variables such as plasma cortisol but not to the individual growth rate. 4.1. Differences in personality type and state Pups born as the heaviest in the litter were the most fearless, bold and explorative around weaning. Smaller siblings were less prone to
Fig. 3. Personality types differ between juveniles of different size ranks. Panel a represents fearlessness, measured as the distance moved in an open field. Panel b represents how often individuals touched a novel object within 15 min after the first touch. Panel c represents exploration, measured as the number of activations of a light sensor when entering an unknown environment. Shown are the estimated values derived from the mixed models on the behavioural traits ± Standard Error. * indicates significant differences between levels.
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Fig. 4. Although pups of different size ranks differ in birth weight, the growth rate (a) until weaning does not differ. Pups of different size ranks differ in their plasma cortisol level shortly after weaning (b), but not in their O2-consumption (c). Shown are the estimated values derived from the mixed models on the physiological traits ± Standard Error. * indicates significant differences between levels.
explore an unknown corridor but did not differ from each other in fearlessness or boldness. Comparable effects were found in rats, where smaller pups were less bold than their bigger siblings shortly after weaning [11] and these results are consistent with findings of earlier studies in cavies [23,37]. These studies demonstrated the temporal consistency of all three personality traits from the juvenile stage until adulthood, suggesting that the traits we measured here are indeed personality traits. In accordance with the results of this study, earlier studies found no significant correlations between the personality traits in juveniles, while the number of touches of a novel object was correlated with the number of trips in the Long Field after maturation [37]. Differences in personality types were partly reflected in physiological variables, suggesting an influence of or at least a close connection between an individual's state and the development of its personality type. While metabolic and growth rate during the first 24 days did not differ between pups of different size ranks, pups differed in plasma cortisol concentration around weaning. The growth rate was not correlated to any of the observed behavioural or physiological variables, suggesting that growth proceeded independent of the other traits measured. This result is unexpected as growth rate was shown to be linked to various personality traits not only in other mammals but also in fish, birds and even arthropods [6]. In house mice and voles, for example, early growth was positively linked to activity [28,29,38]. Just as the majority of all studies, the abovementioned studies investigated the relationship between growth and personality in altricial species. There might, however, be a functional difference between altricial and precocial species with respect to the importance of early growth. One study conducted with precocial bighorn ewe also found no relationship between growth and personality [39]. Precocial animals are far shorter and substantially less dependent on milk intake and have lower relative growth rates than altricial young, because they are born much further developed and grow at lower rates after birth. Young of cavies eat solid food almost from the first day of life and can be weaned at 5 days of age with no increase in mortality [40]. Therefore, potential differences in growth rates after birth are harder to detect than in altricial species. Alternatively, early growth rate may be of minor importance for later behavioural and physiological development when young are able to regulate food intake and hence growth this early in life.
In addition to the early growth rate, we investigated resting metabolic rate and baseline cortisol level as both have been proposed as possible mediators of personality differences between individuals and both may be regulated by maternal effects. The smallest pups within a litter had the highest baseline cortisol levels and at the same time covered the least distance in an open field and were the fastest to leave the hand of an observer. These two behavioural traits were also correlated with each other and with cortisol levels. A linkage between cortisol and personality traits seems to exist in a wide range of species. In adult alpine marmots for example, different coping styles were associated with different baseline cortisol levels [41]. In squirrels, baseline cortisol increased with docility [42] and in dairy calves, a positive correlation between plasma cortisol levels and the latency to touch a novel object was found [43]. In male rhesus macaques, high-excitable animals had lower basal cortisol concentrations [44]. All these results point in the direction, that a relatively high baseline cortisol level is characteristic of shy or nervous animals. In the current study, we present the first evidence that this relationship even exists very early after birth (as soon as three days of age) suggesting that cortisol is a key-factor driving the development of personality during life. Cortisol was also tightly correlated to oxygen consumption. Individuals with a relatively high cortisol had low oxygen consumption and oxygen consumption itself was correlated with fearlessness and the hand escape latency. However, there was no statistical difference in oxygen consumption between siblings of different size ranks. These results reflect earlier results [36], where we controlled for the effects of size rank within litter in the resting metabolic rate. In juveniles, the effect of size rank was far from being significant while in mature animals, we found a significant effect. Recent evidence suggests that maternal effects can exert a substantial influence on offspring metabolic rate and indicate the transfer of hormones from mother to embryo as a possible mechanism [45]. It is known from oviparous species that concentrations of egg hormones can vary considerably among- and within-clutches and can affect offspring phenotypes [46]. Female three-spined sticklebacks produce eggs with higher cortisol content and higher oxygen consumption shortly after fertilisation when exposed to predation threats [47]. Likewise, elevation of cortisol in
Table 1 Correlations between behavioural and physiological traits. Fitted values derived from the mixed models are used for correlations. Given are Spearman's rho and the significance level in brackets. Significant correlations are bold. Trait
Hand escape
Struggle
Boldness
Exploration
Fearlessness
Growth rate
Cortisol
Struggle Boldness Exploration Fearlessness Growth rate Cortisol O2 consumption
−0.64 (b0.001) 0.19 (0.3) 0.12 (0.5) 0.5 (0.005) 0.11 (0.55) −0.36 (0.06) −0.92 (b0.001)
0.03 (0.85) 0.09 (0.6) −0.3 (0.09) 0.01 (0.93) −0.23 (0.22) 0.19 (0.3)
0.33 (0.07) 0.28 (0.1) 0.05 (0.95) −0.27 (0.13) −0.28 (0.12)
0.14 (0.44) 0.14 (0.40) 0.26 (0.2) −0.24 (0.17)
−0.09 (0.60) 0.37 (0.04) −0.58 (b0.001)
−0.05 (0.81) −0.16 (0.36)
−0.49 (0.008)
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brown trout eggs increased embryonic RMR [48]. There is also evidence showing that maternal effects on RMR are not restricted to hormonal pathways. Eggs of the clownfish laid on the periphery of a clutch had a 33% lower rate of oxygen consumption than did embryos from the clutch interior [49]. Although we did not find a significant difference in metabolic rate between pups of different size ranks, RMR was tightly correlated with fearlessness and the latency to leave the hand suggesting that it might also be a key-factor in establishing individual differences in personality.
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Acknowledgements We would like to thank the team of the Department of Behavioural Biology of the University of Münster for running the cortisol analyses and Mona Dersen for much appreciated help with the experiments. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR 1232, TR105/25-1).
References 4.2. Possible contribution of the prenatal environment Maternal or parental effects refer to the influence of the parental phenotype on the offspring phenotype independently of the offspring's genotype [50]. These effects can exert influences on offspring either preor postnatally and therefore strongly affect personality development. The fact that we find personality differences in three day old juveniles in the present study implies a strong influence of prenatal maternal effects although initial differences between pups may be enhanced postnatally due to interactions between siblings or mother–offspring interactions. From the closely related guinea pig it is known that aggressive interactions between pups of the same litter occur more often in larger litters where pups have to compete for access to maternal teats, although aggressive interactions were mainly restricted to scramble competition [16]. Prenatal maternal effects in guinea pigs and cavies are well known [51] and have been shown to influence offspring behaviour and physiology until adulthood [52,53]. It is also known that individuals receive different prenatal supplies depending on their position in utero resulting in size differences between pups even several weeks before birth [54,55]. Birth weight depended on the number of pups per uterine horn, but was in general higher for pups in more cervical positions [54]. Consequently, the differences in physiology and behaviour in pups of different size ranks may simply be a result of constraints due to differential maternal provisioning. However, several earlier results render this an unlikely explanation. Juveniles born into a photoperiod simulating autumn conditions show the opposite pattern of behaviour and cortisol with respect to size rank in litter. When born into autumn photoperiod, baseline cortisol values are higher in bigger pups than in smaller ones. Accordingly, bigger pups cover less distance in an open field and are less explorative than their smaller siblings [23]. Together, these results suggest that mothers programme their offspring in an adaptive way. By programming the pups of a litter with different physiological and personality types, mothers could follow a bet-hedging strategy to ensure that at least one of her offspring matches the environmental conditions. Giving the largest in the litter a particularly good chance to survive would appear to make it a kind of ‘core’ offspring as has been described for bird clutches [56]. Producing additional small offspring with somewhat lower survival chances may provide additional benefit, if environmental conditions prove unexpectedly plentiful. However, this hypothesis needs further testing.
5. Conclusion Our data demonstrate that ontogenies within the same litter may be diverse depending on starting conditions as has been shown before for sequential siblings in humans [17,57]. This indicates early niche construction within a litter, which is expected to lead to differential neurophysiological effects that will most likely carry over into the expression of the adult behavioural phenotype. Similar diversification has been shown for altricial rodents [11,58] and it remains to be seen in future comparative studies whether the developmental stage, altricial versus precocial, at birth has systematic consequences for personality development and the extent and timing of changes in its structure.
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