RESEARCH ARTICLE

Precocial Bird Mothers Shape Sex Differences in the Behavior of Their Chicks FLORENT PITTET, CÉCILIA HOUDELIER, AND SOPHIE LUMINEAU*

UMR CNRS 6552 “Ethologie Animale et Humaine”, Université de Rennes I, Rennes, France

ABSTRACT

J. Exp. Zool. 321A:265–275, 2014

Compared to mammals and altricial birds, evaluations of differences related to precocial birds' sex have often been limited to sexual behavior. Nevertheless, the extensive use of precocial bird models for investigating behavioral development issues requires in depth knowledge concerning the emergence of sex differences. Here, we evaluated behavioral differences between Japanese quail chicks in relation to their sex. We know that maternal influences are strong and early social environment shapes behavioral development in this species. Therefore, we investigated the emergence of sex differences in two very different early social situations to evaluate the impact of precocial bird mothers on sex differences. We assessed behavioral differences related to sex of (1) non‐brooded chicks and of (2) brooded chicks, using various ethological tests to evaluate emotional reactivity and social motivation differences related to sex. Our results show that both non‐brooded and brooded chicks present behavioral differences related to sex. They differed greatly according to chicks' early experience. Sex‐related differences between maternally deprived (non‐brooded) chicks concerned mainly fearfulness, whereas differences between male and female brooded chicks concerned mainly their reactions to social isolation. We hypothesize that mothers attenuate sex differences related to fearfulness by being a model for responses to fear‐eliciting situations and by providing a similar secure basis to both males and females. We finally propose that mothers induce differences in chicks' sociality by providing asymmetrical care toward males and females. J. Exp. Zool. 321A:265–275, 2014. © 2014 Wiley Periodicals, Inc. How to cite this article: Pittet F, Houdelier C, Lumineau S. 2014. Precocial bird mothers shape sex differences in the behavior of their chicks. J. Exp. Zool. 321A:265–275.

Behavioral ontogeny is a complex process involving constant interaction between an individual's genetic background and physical and social characteristics of its environment (Hinde, '75; Gottlieb, '91). Behavioral differences related to sex are illustrations of the powerful genetic component that induces divergences between males' and females' traits. Sex‐typical reproductive behavior of a large range of species, including mammals (McPherson and Chenoweth, 2012), birds (Adkins‐Regan, 2009), and fishes (Munakata and Kobayashi, 2010), remains the most studied trait. Probably because of the increasing interest in individual differences in behavior (Réale et al., 2007), the study of sex differences has broadened to include behavioral characteristics, notably traits related to fearfulness and social behavior that are influenced by sex in a large range of species. This aspect has been explored particularly in mammals, revealing various cases of sex‐related differences in both fearfulness (Vandenheede and Bouissou, '93; Haller et al., '99) and social characteristics

(Nelson, '95; Haller et al., '99; Pellis, 2002; Honess and Marin, 2006; Hemelrijk et al., 2008; Ward et al., 2008). Recent studies of the sexual dimorphism of altricial birds' temperament reveal differences between males and females related to exploration and neophobia (Schuett and Dall, 2009; Mainwaring

Conflicts of interest: None.
Correspondence to: Sophie Lumineau, UMR CNRS 6552 “Ethologie Animale et Humaine”, Université de Rennes I, Bâtiment 25, Campus de Beaulieu 263 Avenue du Général Leclerc CS74205, 35042 Rennes, France. E‐mail: [email protected] Received 19 July 2013; Revised 18 February 2014; Accepted 18 February 2014 DOI: 10.1002/jez.1858 Published online 10 March 2014 in Wiley Online Library (wileyonlinelibrary.com).

© 2014 WILEY PERIODICALS, INC.

266 et al., 2011; Ensminger and Westneat, 2012; Mainwaring and Hartley, 2013). Precocial birds, particularly gallinaceans, are choice models for investigating genetic (Agnvall et al., 2012), epigenetic (Goerlich et al., 2012), prenatal (Guibert et al., 2013), and postnatal (Shimmura et al., 2010; Pittet et al., 2012) components of behavioral ontogeny. The increasing interest for these species' behavioral development requires information concerning the ontogeny of sex differences. Surprisingly, this issue remains particularly underexplored in these models as sex differences have been evidenced only in domestic chicks' fearfulness (Jones, '80) and sociality (Vallortigara et al., '90). Currently, Japanese quail is the precocial bird model the most studied for behavioral development (Hegyi and Schwabl, 2010; Casey and Sleigh, in press; De Margerie et al., 2013; Houdelier et al., 2013). However, two papers report inconclusive empirical results concerning the development of emotional and social sex‐related differences of this model. The first study of quail chicks' emotional and social behavior by Mills and Faure ('86) did not evidence differences related to sex. The second study (Launay et al., '93) reported that males were more motivated than females to reach a goalbox containing unfamiliar conspecifics, but these subjects were from strains divergently selected to present enhanced emotional or social responses. Moreover, the fact that the rearing conditions in the two above‐mentioned studies involved large flocks mixing chicks of both sexes could have promoted allelomimesis (Mills and Faure, '86; Odén et al., 2005) and thus thwarted identification of behavioral dimorphism. Differences in behavior linked to sex are considered to be related to differences between the roles played by adult males and females, notably during reproduction. Physical and behavioral dimorphisms are known to be less marked in species whose males and females present similar parental investments and repertoires (Chau et al., 2008). Japanese quail females, as most precocial birds, are the only caregiver; thus males and females are exposed to different selection pressures that should induce important differences related to sex. The present work investigated the emergence of sex‐related behavioral differences during the first weeks of a chick's life under conditions preventing the above‐mentioned allelomimesis effect, as quail were reared in small groups and the influence of their cagemate's sex was evaluated. In spite of their strong genetic basis, sex differences are likely to be affected by environmental factors, particularly early social factors. Caregivers, the main constituents of early social environment, can influence the expression of behavioral differences related to sex through several well‐identified mechanisms. First, caregivers can interact differently with males and females. Asymmetrical care provision by several mammals includes grooming (Rosenzweig et al., '98), inspection of genital regions (Wallen, 2005) or responsiveness to vocal solicitations (Tomaszycki et al., 2001). Similarly, males and females of altricial birds, whose chicks depend heavily on care provided by parents, can also J. Exp. Zool.

PITTET ET AL. present asymmetrical investments, notably by providing food differently to males and females (Ridley and Huyvaert, 2007; Nam et al., 2011). Moreover, divergences in the set‐up and the organization of the neural circuitry during early development can induce males' and females' attention toward similar maternal stimuli to present differences. For instance, as young chimpanzee females spend more time watching their mothers fish for termites (while males spend more time playing) they acquire termite fishing skills earlier (Lonsdorf et al., 2004). Neural dimorphism can also induce differences between males' and females' sensitivity toward a same social event. This trait is illustrated by the sex‐typical influences of mice's early weaning (Livia Terranova and Laviola, '95). Influences of precocial birds' maternal care on the emergence of sex differences remain unknown although their non‐genetic maternal influences have been extensively studied. The easiness of adoption procedures (Richard‐Yris, '94), their uniparental caregiving (Orcutt and Orcutt, '76) and the absence of lactation enable rigorous estimation of maternal care consequences on the behavioral development of chicks. By comparing behavioral characteristics of Japanese quail chicks reared in complete maternal deprivation and those of chicks adopted by foster mothers, several authors illustrated the fundamental role of mothers on behavioral ontogeny. They have evidenced maternal influence on a range of behaviors (Bertin and Richard‐Yris, 2005; De Margerie et al., 2013; Pittet et al., in press), including fearfulness (Bertin and Richard‐Yris, 2004, 2005; Richard‐Yris et al., 2005) and sociality (Bertin and Richard‐Yris, 2005; Formanek et al., 2008). Consequently, we hypothesized that quail mothers influenced the emergence of sex differences. The present study evaluates behavioral differences between male and female Japanese quail chicks in two different situations: either brooded by foster mothers or reared without a maternal female (maternal deprivation). The results for each situation are discussed separately and then compared.

MATERIALS AND METHODS Ethic Statement All animal work was approved by the departmental direction of veterinary services (Ille et Vilaine, Permit number 005283) in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). Our breeding procedure and tests were approved by the regional ethics committee (agreement number: R‐2011‐SLU‐02). Animals and Housing Conditions During the whole experiment, temperature was 20  1°C and light conditions were 14:10 hr light/dark. Food provided ad libitum was a high‐protein cereal diet in the form of a pellet mix for chicks and granulates for adult females (VEGAM, Cesson‐ Sévigné, France).

QUAIL CHICK'S SEX DIFFERENCES Twenty‐two adult female Japanese quail and 100 chicks from a broiler line (from an industrial farm “Les cailles de Chanteloup,” Corps‐Nuds, France) were used. Adult females arrived at the laboratory when they were 5 weeks old. They were immediately individualized by a number ring on their wing and placed individually in wire‐mesh cages (51  40  35 cm3) with opaque lateral walls (preventing visual but not auditory communication) and equipped with a drinker and a feeder. All these cages were in the same breeding room. No other adult birds were placed in this room and no vocalizations from other rooms were audible. Females remained in these cages for 3 weeks before adoption was induced so they could habituate to these environmental conditions. Chicks were obtained a few hours after they had hatched and were individualized by a colored ring around their leg. They spent all their first day, from their arrival until sensitization of mothers, in plastic cages (98  35  42 cm3) with food and water provided ad libitum and equipped with warming lamps (38  1°C). Preparation of Sets The chicks were divided into two groups: a group of brooded chicks (hereafter B) and a group of non‐brooded chicks (NB). Each experimental group included 22 sets of 2 chicks, and the 12 additional chicks remained in the plastic cages. Each set of chicks was placed in a wire‐mesh cage, in the same room. A warming lamp (38  1°C) was placed in the corner of each non‐brooded chicks' cage. The warming lamps were bell‐shaped to limit dispersal of heat. They provided heat consistently and continuously and chicks adjusted their warming by spending more or less time under the warming lamp. The brooded chicks were adopted by adult females following the induction method described below. Cages were placed on three horizontal racks so as to have brooded and non‐brooded cages equally distributed vertically and horizontally. Given the large space between meshes and the small size of newly hatched chicks, fine‐mesh plastic netting was placed on the floor and on the door (7 cm high) before the arrival of chicks. We followed the induction procedure described by Richard‐Yris ('94). Briefly, the day chicks hatched, at the beginning of the dark phase, each set of brooded chicks was placed gently underneath an adult female shut up an hour earlier in a plastic nest box (16  16  16 cm3) with a removable wall and they were then all locked‐up for the night. During this night, chicks' vocalizations and their physical solicitations induced complete expression of maternal behavior by the following morning. Simultaneously, each set of non‐brooded chicks was placed, without a female, in similar nest boxes and locked‐up for the night. The following morning, all the nest boxes were opened (the detachable wall was removed) and once the brood was outside, the nest boxes were removed from the cages. We monitored the first interactions between mothers and brooded chicks and excluded the females that did not warm the chicks during the first hours following the opening of the cages. Thus two of 22 adult females were excluded. They remained in their cages and their chicks were placed in the plastic

267 cages with the additional chicks. Four brooded chicks showed signs of hypothermia (trembling, closed eyes) during this first day of brooding. They were too weak to solicit maternal warming and thus were also removed to prevent mortality, and placed with the additional chicks under heaters where they recovered swiftly. They were replaced by additional chicks, identified by a color ring around a leg. The replacement chicks (1 male and 3 females) were immediately fostered by the maternal females, but were not tested as they did not spend the whole mothering period with their mother. Replacement of chicks is efficient to maintain similar brood sizes in all cages (Pittet et al., 2012). During the 11 days that brooding lasts under natural conditions, we monitored interactions between mothers and chicks. Two non‐brooded chicks from two different cages died after the first day of brooding. We decided not to replace them at this stage. Their cagemates were placed with additional chicks and were not tested, reducing the number of cages containing non‐brooded chicks to 20. On the 11th day, the adult females and the warming lamps were removed and the chicks were left in their cages for 11 more days for their behavioral characteristics to be evaluated as described below. Chicks were weighed when they were 9 days old and 22 days old. Their sex was determined visually when they were 22 days old (sexual dimorphism of throat feathers). Behavioral Measurements The reactions of NB and B to separation from respectively cagemate only or cagemate and mother were evaluated on post‐ hatch day 9 (separation test). After B were separated from their mothers, ethological tests assessed B's and NB's fearfulness and social motivation. Tonic immobility tests and human observer tests evaluated respectively inherent fearfulness and fearfulness toward humans; runway tests evaluated social motivation, and open‐field and emergence tests evaluated both reactions to a novel environment and to social isolation. Emotional Reactivity Tests Tonic Immobility Test. This test followed the protocol described by Jones ('86). Tonic immobility is a reflex response to a fear‐ inducing stimulus; the duration of this response is positively correlated with fearfulness (Mills and Faure, '91). Each chick, when 14 days old, was removed from its cage and placed on its back in a U‐shaped wooden cradle and held in this position for 10 sec prior to release. The experimenter, placed out of the sight of the subject, noted the duration of immobility after manual release, with a maximum of 300 sec. This test was replicated when the chicks were 22 days old to assess the development of their emotional reactivity over this period. Human Observer Test. Chicks were tested in their home cage when 20 days old. The experimenter, using instantaneous scan sampling, passed in front of each home cage every 8 min, recording a total of 32 scans/cage. Each time he passed in front of J. Exp. Zool.

268 a cage, he stopped for few seconds and recorded the reactions of the two chicks. The variable used to assess fearfulness expressed toward humans was the number of scans when a subject expressed a fear reaction (including violent fleeing attempts and avoidance). Social Reactivity Tests Separation Test. When chicks were 9 days old, they were removed, one at a time, from their home cage and placed alone in a similar cage for 3 min in another room. To assess the reactions of NB and B chicks to separation respectively from cagemate or from mother and cagemate, latencies to take first step was noted, as well as numbers of steps and distress calls. Chicks' general activity in this familiar environment is considered to reflect their reaction to separation from cagemate and the strength of filial bond in the case of brooded chicks (Cate, '89; Pittet et al., in press). Runway Test. The apparatus is a 100‐cm‐long wire‐netting tunnel. Eighteen‐ or 19‐day old chicks were transported in a wooden box (18  18  18 cm3) that was then placed at the entrance of the tunnel. A cage (41  24  30 cm3) with three unfamiliar chicks of the same age (non‐brooded additional chicks) was used as a social stimulus and placed at the other end of the tunnel. The closest, “1 chick long (4 cm),” zone to the social stimulus was named proximal zone or zone P. One minute after the transport box had been put in place, it was opened and once the animal was in the tunnel, the box door was closed and, for 5 min, the experimenter noted the time chicks spent in zone P. This variable is related to social attraction to unfamiliar conspecifics (Formanek et al., 2008). Emotional Reactivity and Social Reactivity Tests Aiming to evaluate emotional reactivity and exploratory behavior, emergence and open‐field tests expose subjects to both a novel environment and social isolation. Exploratory data (latency to emerge into a novel environment, number of steps taken, or locomotor activity) yielded by these tests evaluated fearfulness and social behavior (frequency of distress calls, known to be related to social motivation; Forkman et al., 2007). Open‐Field Test. Chicks were placed individually, in the dark, in the center of a wire‐netting arena (120 cm diameter  70 cm high) with a linoleum floor, in the dark. Then the light was switched on, and, hidden behind a one‐way mirror, the experimenter noted, for 5 min, the number of steps taken by chicks as well as the number of distress calls. Chicks were tested when they were 15 and 16 days old. General activity is negatively correlated with emotional reactivity and frequency of distress calls is positively correlated with social motivation (Bertin and Richard‐Yris, 2005). Emergence Test. Chicks, when they were 17 days old, were removed from their home cage and transported in the dark, in a wooden box (18  18  18 cm3) to the experimental room. This box was then placed on one side of the apparatus: a large and well‐ J. Exp. Zool.

PITTET ET AL. lighted wooden box (62  60  33 cm3) with a floor covered with wood‐shavings and an observation window. After the transport box had been placed in the apparatus, it was kept closed for 1 min. Then, the door was left opened for 3 min. The latency to emerge from the box (head and both legs out) was recorded. This variable is a good estimate of fearfulness of a novel environment (Archer, '76; Jones et al., '91; Bertin and Richard‐Yris, 2005). Once the subject was in the test cage, the transport box was closed and the chick was observed for 3 min and the observer noted the frequency of locomotion (walk and run) as well as the frequency of social vocalizations (distress calls). Statistical Analyses After gender determination, we finally had data for 20 non‐brooded males: 12 developed in same‐sex pairs (SS) and 8 developed in mixed‐sex pairs (MS) and for 20 non‐brooded females: 12 SS and 8 MS. Analyses of the brooded set data were limited to the chicks that spent the whole mothering period with their mother. After gender determination, we finally had 18 (7 from same‐sex pairs and 11 from mixed‐sex pairs) brooded males and 18 (9 from same‐sex pairs and 9 from mixed‐sex pairs) brooded females. Behavioral data were analyzed using ANOVAs to detect sex differences, pair (same sex vs. mixed sex) effects and interactions between sex and pair effects, for both non‐brooded and brooded chicks. Weights and tonic immobility durations were measured twice and the effects of repetition were estimated by ANOVAs on repeated data. Normality of residuals was checked and some behavioral data had to be Box–Cox (Box and Cox, '64) transformed to meet the assumptions of normality (tonic immobility durations and behavioral latencies). Bonferroni tests compared pairs. Analyses were performed using XLSTAT 2010 (Addinsoft SARL, Paris, France).

RESULTS Body Weight Age, as expected, influenced weights of both NB and B chicks. We could evidence no sex effect on weights of NB chicks: weights of NB males and females did not differ significantly either when they were 9 or when they were 21 days old. On the contrary, B females were heavier than B males (Fig. 1). An ANOVA revealed a significant interaction between age and sex for B chicks. Bonferroni post‐hoc tests revealed that weights of 9‐day‐old B males and females did not differ but 21‐day‐old B females were heavier than B males (Fig. 1). An ANOVA could not evidence any significant pair effects, interactions between pair and age and interactions between sex, pair and age for either NB or B chicks (P > 0.05). Emotional Reactivity Tonic immobility durations increased with chicks' age for both NB (14 days old: 57.20  6.56 sec, 22 days old: 75.10  8.16 sec;

QUAIL CHICK'S SEX DIFFERENCES

269 from mixed‐sex groups as females' tonic immobility durations were significantly influenced by the sex of the cagemate whereas males' tonic immobility durations were not (Fig. 2). This interaction was not significant for B chicks (Fig. 2). We found no significant interactions between sex and age, pair and age and sex, pair and age for either group (P > 0.05). The human observer test revealed that the amount of fear behavior in the presence of humans differed between NB males and females. NB males showed a higher proportion of fear behaviors than did NB females (males: 46.9  4.1%, females: 32.0  4.4%; F1,38 ¼ 5.956, P ¼ 0.02). This difference related to sex was not significant for B chicks (males: 27.8  3.9%, females: 33.0  4.8%; F1,36 ¼ 0.414, P ¼ 0.5). Pair composition effect and interaction between sex and pair composition were significant neither for NB nor for B chicks (P > 0.05). Latencies to emerge from the box in the emergence test did not differ significantly between males and females either for NB chicks (NB males:

Figure 1. Weights (mean  SEM) of non‐brooded and brooded chicks in relation to age and sex. Results of the ANOVA evaluating effects of interaction between age and sex are detailed above each set of histograms. Bonferroni test results are shown as letters above columns. Means not sharing the same letter are significantly different.

F1,38 ¼ 4.32, P ¼ 0.04) and B chicks (14 days old: 26.03  3.36 sec, 22 days old: 45.53  5.20 sec; F1,36 ¼ 440.64, P ¼ 0.0001). Sex influenced NB chicks' tonic immobility durations, as NB females' tonic immobility lasted longer than that of NB males' (NB males: 53.05  5.91 sec, NB females: 79.25  8.35 sec; F1,38 ¼ 5.055, P ¼ 0.03). This sex‐related difference was not found for B chicks (B males: 38.17  4.65 sec, B females: 36.39  4.90 sec; F1,36 ¼ 0.327, P ¼ 0.57). No significant pair‐type effect could be evidenced either for NB or for B chicks (P > 0.05), but an ANOVA revealed an interaction between pair‐type and sex for NB chicks (Fig. 2). Post hoc comparisons revealed a significant difference between NB males and females from same‐sex groups but not

Figure 2. Tonic immobility durations (mean  SEM) of non‐ brooded and brooded chicks in relation to sex and pair‐type. Results of the ANOVA evaluating effects of interaction between sex and pair‐type are detailed above each set of histograms. Bonferroni test results are shown as letters above columns. Means not sharing the same letter are significantly different. J. Exp. Zool.

J. Exp. Zool.

45.06  7.31

Runway test Emergence test Open‐field test

Significant effects revealed by an ANOVA appear next to the first means. ANOVA:
P < 0.05,

P < 0.01.

25.28  4.81 76.45  11.36 73.3  10.13

86.25  12.75

67.29  9.09

58.28  8.44



39.15  8.20

8.13  3.31 1.50  0.67 21.30  5.55 26.95  6.26

32.19  7.55

18.75  4.54

18.33  6.47


11.35  5.80

179.67  32.48 194.50  20.73 179.58  27.62 196.24  24.42 159.88  24.26 160.56  24.90 159.35  25.05

160.07  24.95

122.00  14.59 95.98  12.08 99.83  14.41 68.17  11.85 90.20  14.65 82.35  13.05

113.44  14.40


115.26  12.36

136.81  22.04 178.75  21.28 107.72  15.53 104.70  21.77

92.46  20.24

Same sex

99.40  19.21

116.44  19.26

212.50  20.74



13.95  3.37

Mixed sex Female

11.67  3.28

Male

10.56  2.84 16.71  4.53

Same sex Mixed sex Female

11.60  3.33

Parameter

Male

Separation test

Test

Step latency (sec) Step (number) Distress calls (number) Time spent zone P (sec) Distress calls (number) Distress calls (number)

23.50  7.35

18.81  7.85

Brooded Non‐brooded

Sociality See Table 1 for social variable data. NB chicks' locomotor behavior (including latency to take first step and number of steps) was not affected by sex (latency of first step: F1,38 ¼ 2.174; number of steps: F1,38 ¼ 0.024), pair (latency of first step: F1,38 ¼ 0.142; number of steps: F1,38 ¼ 2.171) or interaction between these two factors (latency of first step: F3,36 ¼ 0.017; number of steps: F3,36 ¼ 0.056) in the separation test. Comparatively, B males took more steps than did B females (F1,36 ¼ 14.99), but they did not take their first step earlier (F1,36 ¼ 0.025). Numbers of steps taken by B chicks were not affected by other factors. During this separation test, NB and B chicks' vocal behavior was not affected by sex (NB number of distress calls: F1,38 ¼ 0.335; B number of distress calls: F1,36 ¼ 1.216) or interaction between sex and pair (NB: F3,36 ¼ 0.681; B: F3,34 ¼ 1.104), but numbers of distress calls emitted by NB chicks were affected by pair‐ type. NB chicks that developed in mixed‐sex pairs emitted more calls than did NB chicks that developed in same‐sex pairs (F1,38 ¼ 5.700). No such pair effect could be evidenced for B chicks (F1,36 ¼ 2.298). Both NB and B chicks spent a large proportion of their time in the social area of the runway test. An ANOVA revealed no

Table 1. Behavioral responses (mean  SEM) related to sociality of non‐brooded and brooded chicks in relation to sex and pair‐type.

28.1  9.81 sec, NB females: 22.95  9.17 sec; F1,38 ¼ 0.11, P ¼ 0.7) or for B chicks (B males: 32.00  10.70 sec, B females: 51.33  13.63 sec; F1,36 ¼ 1.22, P ¼ 0.3). Once they had emerged from the box, NB chicks' locomotor behavior was not influenced by sex (number of locomotor acts: NB males: 4.80  0.56, NB females: 5.6  0.69; F1,38 ¼ 0.73, P ¼ 0.40), but B males expressed more locomotor acts than did B females (B males: 6.28  0.60, B females: 4.01  0.60; F1,36 ¼ 6.92, P ¼ 0.01). An ANOVA could evidence no significant pair effect or interaction between pair and sex for either group on latency to emerge or frequency of locomotory acts (P > 0.05). NB males took significantly longer than females to take their first steps in the open‐field arena (NB males: 19.90  5.22 sec, NB females: 7.60  3.12 sec; F1,38 ¼ 10.76, P ¼ 0.002). On the other hand, latencies to start moving did not differ significantly between B males and females (B males: 6.72  1.60 sec, B females: 24.01  16.50 sec; F1,36 ¼ 0.243, P ¼ 0.6). Pair‐type did not affect NB's latencies to take first step (NB mixed sex: 17.06  5.66 sec, NB same sex: 11.54  3.71 sec; F1,38 ¼ 2.23, P ¼ 0.14), but B chicks reared in mixed‐sex pairs took longer than chicks reared in same‐sex pairs to start moving (B mixed sex: 24.55  14.75 sec, B same sex: 3.94  1.09 sec; F1,36 ¼ 7.247, P ¼ 0.011). Neither NB chicks' nor B chicks' latencies were influenced by interaction between sex and pair‐type (P > 0.05). The numbers of steps taken by both NB and B chicks were not related to sex, pair or interaction between pair and sex (P > 0.05).

PITTET ET AL.

7.56  2.12

270

QUAIL CHICK'S SEX DIFFERENCES influence of sex (NB: F1,3 ¼ 0.007; B: F1,36 ¼ 0.159), pair (NB: F1,38 ¼ 0.001; B: F1,36 ¼ 0.117) or interactions between sex and pair (NB: F3,36 ¼ 0.354; B: F3,34 ¼ 0.028). Numbers of social vocalizations emitted by NB chicks were not affected by sex, pair or interaction in the emergence (sex: F1,38 ¼ 0.332; pair: F1,38 ¼ 2.542; sex X pair: F3,36 ¼ 0.223) and open‐field tests (sex: F1,38 ¼ 0.311; pair: F1,38 ¼ 1.592; sex X pair: F3,36 ¼ 3.024). On the contrary, B males emitted more distress calls than did females in both the emergence (F1,36 ¼ 5.868) and open‐ field (F1,36 ¼ 11.935) tests. Numbers of calls were not affected by B chicks' pair‐type (emergence test: F1,36 ¼ 0.043; open‐field test: F1,36 ¼ 0.971) or interaction between pair‐type and sex (emergence test: F3,34 ¼ 0.126; open‐field test: F3,34 ¼ 0.108) in these two tests.

DISCUSSION Our aim was to evaluate Japanese quail chicks' sex‐related emotional and social differences in two early‐life social situations: experimental or farm breeding (i.e., mother replaced by a heater) and natural breeding (with a mother). Our results show that behavioral dimorphism related to sex can be evidenced in non‐ brooded as well as brooded chicks, but that its form differs according to chicks' early social rearing conditions. Behavioral Dimorphism of Non‐Brooded and of Brooded Chicks First, our results show that several behavioral traits of both non‐ brooded and brooded chicks differed significantly between males and females. Neither growth nor reactions to social isolation of non‐brooded chicks differed between males and females, but fearfulness did. Non‐brooded males took longer to start moving in an unfamiliar environment and expressed more fear reactions in the presence of humans. Nevertheless, females showed higher inherent fearfulness and their tonic immobility durations were longer. The inconsistency between the results obtained in the different tests is coherent as fear is known to be a multidimensional trait, and tonic immobility durations have been shown to be independent of behavioral expressions evaluated in less stressful procedures (Mignon‐Grasteau et al., 2003; Saint‐Dizier et al., 2008). Contrary to other traits related to non‐brooded chicks' fearfulness, the sex‐ related tonic immobility duration differences depend on the sex of cagemate. Females', but not males', tonic immobility durations were significantly affected by pair‐type. More precisely, the tonic immobility durations of females housed in same‐sex pairs were longer than those of females housed with a male and longer than males' tonic immobility durations whatever their pair‐type. This result indicates that that the presence of a male in a female's social environment reduces their inherent fearfulness. This positive influence of familiar social partners on inherent fearfulness could be considered a case of social buffering (see Kikusui et al., 2006, for review). A closely related species reacted similarly: duration of domestic hens' tonic immobility is reduced when a male is present

271 in the flock (Odén et al., 2005); emotional reactivity of males is higher and their presence has a positive influence on females' emotional reactivity (Launay et al., '93). The social motivation of non‐brooded female chicks was higher than that of males (Vallortigara et al., '90). All these results concerning domestic hens' behavioral dimorphism were associated with the social organization of this species, in which a dominant male plays a guarding role over small groups of females. Authors proposed that the guarding role of males required a higher emotional reactivity while the presence of several females in social groups enhanced their gregarious behavior (Vallortigara et al., '90). The social system of Japanese quail remains largely unknown and, as suggested by Launay et al. ('93), social motivation could have a different biological significance for quail than for other domestic fowl. Nevertheless, our present results showing a higher emotional reactivity of male quail and their positive influence on females' inherent fearfulness suggest a relationship implying a similar guarding role of males. Comparatively, brooded chicks' growth and reactions to social isolation differed between males and females, but not their fearfulness. Weight gains between 9 and 22 days differed between brooded males and females. Actually female Japanese quail reared under farm conditions (i.e., non‐brooded) are heavier than males when adult (Mills et al., '97). Our results showed that this difference is evident for 22‐day‐old chicks when they are reared with a maternal female. We evidenced sex differences concerning brooded chicks' social characteristics. Although times spent near unfamiliar chicks in the runway test did not differ significantly between males and females, males took more steps when isolated in the separation test and reacted vocally consistently more frequently than females when isolated in an unfamiliar environment. In both the emergence and the open‐field tests, males emitted more distress calls when separated from their cagemate, a trait known to indicate need to restore social links (Forkman et al., 2007). Once more, the inconsistency between the results for the runway and social isolation tests highlight the multidimensional aspect of sociality, suggesting that males' reactions to social isolation are stronger than those of females, but that motivation to approach unfamiliar conspecifics does not differ between males and females. These two dimensions of sociality are known to be independent (Schweitzer et al., 2010). Our results revealing the existence of sex‐related behavioral differences among Japanese quail chicks contradicts a previous report. Mills and Faure ('86) reported a lack of dimorphism concerning non‐brooded chicks' tonic immobility and responses to a novel environment. Conversely, when we evaluated similar traits by similar tests, we evidenced sex differences concerning latencies to move into an open‐field and tonic immobility durations. Although Mills and Faure ('86) reported no significant differences between 14‐day‐old males' and females' tonic immobility durations, the mean data they presented seem to indicate that females' immobilizations tended to last longer J. Exp. Zool.

272 (males: 48.8  6.4 sec, females: 73.0  12.7 sec) thus supporting our results. Actually, the slighter differences between males' and females' behavioral data reported in this previous study might be explained by their breeding conditions. In particular, the use of mixed‐sex groups of 20 individuals could have promoted allelomimesis. Our results evidence the fact that sex composition in breeding cages can influence chicks' behavior and thus induce the expression of behavioral dimorphism. A difference between non‐brooded males' and females' tonic immobility durations could be evidenced only by comparing males and females from same‐sex groups. This result, agreeing with previous reports (Odén et al., 2005), confirms that behavioral dimorphism can be reduced by rearing males and females together. Although pair‐type did not affect the expression of sex differences, it did influence other traits, but then similarly for males and females. Our results show that mixing sexes enhanced non‐brooded chicks' vocal reactions to separation and delayed the first step taken by brooded males in the open‐field. These traits (sociality of non‐brooded chicks and fearfulness of brooded chicks) are not sexually dimorphic. Our results suggest that mixed‐sex pairs probably establish a bond differing qualitatively from bonds established between same‐sex chicks, inducing a more important social or emotional reaction when individuals were separated. Influence of Mothers on the Emergence of Sex Differences The fact that behavioral sex differences of brooded chicks differ from those of non‐brooded chicks highlights the influence of mothers on the expression of behavioral dimorphism. First, we could evidence no sex differences concerning brooded chicks' fear reactions, whereas non‐brooded chicks' fear reactions differed in relation to sex. We hypothesize that the mother was used as a model by both males and females to learn how to react to a fear‐inducing stimulus. Young mammals can learn fear responses socially by observing adult models (Cook and Mineka, '89; Gerull and Rapee, 2002), and Japanese quail chicks can, similarly, use their mother as a source of information about danger in their environment. For instance, Bertin & Richard‐Yris (2004) showed that chicks inherited socially the level of their mothers' habituation to humans, a trait that could be involved here given the behavioral dimorphism of non‐brooded, but not of brooded, chicks' reactions to humans. Therefore, we suggest that the presence of a similar model of reaction to fear could have limited differences between brooded males and females concerning fearfulness. Moreover, mothers are primal social partners providing a secure basis during early development. The quality of this secure basis influences the development of fearfulness (Fairbanks and McGuire, '93) providing a similar secure basis for both males and females. This could have limited the development of early behavioral differences in fearfulness. In addition, we found that brooded females' tonic immobility durations were not influenced by the presence of a male cagemate, contrary to those of non‐brooded chicks. This suggests that the J. Exp. Zool.

PITTET ET AL. secure basis provided by a mother during the first days of development reduces the influence of other social partners, including the social buffering effect of males. Two non‐mutually exclusive hypotheses could explain the facts that mothers' presence is able to decrease behavioral dimorphism concerning fearfulness, and enhances sex differences concerning early growth and sociality. First, speed of emancipation could differ between males and females, as suggested by the more pronounced reactions of males to a separation from their mothers. This suggests that males were less emancipated at the end of the breeding period (a hypothesis consistent with the above‐ mentioned potential differences in their developmental calendar) and thus they may have kept closer to their mother during the last days of the brooding period. This strategy is energetically costly as maternal rejection increases during this period (Pittet et al., 2013, 2014). Simultaneously the more emancipated females probably spent more time foraging and this could explain why weight differences appear so early. Similarly, we propose that this difference in emancipation is involved in the emergence of sex differences concerning brooded chicks' sociality. Even when emancipation occurs at an age when the bond between mothers and chicks disrupts naturally (Orcutt and Orcutt, '76), separation from their mother remains a brutal event with important physiological and psychological consequences (Caldji et al., 2000). Considering that emancipation levels differ between males and females when separation occurred, this event could have sex‐related consequences on chicks' later behavior, as for mammals. For instance, precocious weaning induces female, but not male, mice to behave more solitarily (Livia Terranova and Laviola, '95). Our results suggest that, after separation from their mother, females' social bonds with cagemates are weaker than males' bonds. Another explanation could be that mothers' maternal investment in chicks differed between their male and female offspring. Altricial bird caregivers can make asymmetrical efforts related to offspring sex, particularly when providing food (Ridley and Huyvaert, 2007; Nam et al., 2011). Comparatively, the role of mothers of chicks of highly precocial terrestrial species like Japanese quail, that forage on their own, is limited to the transmission of food selectivity (Wauters and Richard‐Yris, 2002; Clarke, 2010). Nevertheless, other maternal care behaviors could be expressed asymmetrically toward males and females. More particularly, we suggest that warming (brooding) of males and females may have been asymmetrical because this behavior can affect both weight gains during brooding and sociality. Warming conditions during development are known to affect quail chicks' weight gains (Krijgsveld et al., 2003): mothers who spend less time warming produce lighter chicks (Pittet et al., 2012). In parallel, mothering styles of Japanese quail have been described recently and one of the two dimensions of care, rejection, is defined mainly by time spent warming chicks (Pittet et al., 2014). Interestingly, the social motivation of chicks that have been warmed less was higher

QUAIL CHICK'S SEX DIFFERENCES after separation from their mothers. We hypothesize that females were warmed more than males, inducing greater weight gains and lower social motivation. This asymmetrical care could be enhanced by sex‐related differences of offspring solicitations. Differences in solicitations are known to enhance differences in care provided to altricial birds' chicks in relation to their sex. For instance, male red‐winged black birds (Agelaius phoeniceus) beg more than do females and consequently they receive more food from their parents (Teather, '92). These solicitation sex‐related differences could induce asymmetrical care provision, as warming is expressed by females in response to chicks' vocal and tactile solicitations and chicks' behavior differs between males and females in the absence of any maternal influence.

CONCLUSION Our results concerning both brooded and non‐brooded chicks indicate that early social experience influences behavioral dimorphism. Mothers seem able, on the one hand, to attenuate chicks' sexual differences related to reactions to fearful situations and, on the other hand, to enhance the emergence of differences related to social reactivity. More broadly, we highlighted that in spite of inherent predispositions, the expression of behavioral differences related to precocial birds' sex can be shaped by their early social environment, as for mammals (Wallen, '96).

ACKNOWLEDGMENTS We thank C. Petton for his help in rearing and maintaining the animals used in this study. We are grateful to Dr. Ann Cloarec for improving the writing and to the referees for their helpful comments on the manuscript.

LITERATURE CITED Adkins‐Regan E. 2009. Hormones and sexual differentiation of avian social behavior. Dev Neurosci 31:342–350. Agnvall B, Jöngren M, Strandberg E, Jensen P. 2012. Heritability and genetic correlations of fear‐related behaviour in Red Junglefowl— possible implications for early domestication. PLoS ONE 7:e35162. Archer J. 1976. The organization of aggression and fear in vertebrates. In: Bateson PPG, Klopfer PH, editors. Perspectives in ethology. New York: Plenium Publishing Corporation. p 231–298. Bertin A, Richard‐Yris M‐A. 2004. Mothers' fear of human affects the emotional reactivity of young in domestic Japanese quail. Appl Anim Behav Sci 89:215–231. Bertin A, Richard‐Yris M‐A. 2005. Mothering during early development influences subsequent emotional and social behaviour in Japanese quail. J Exp Zool A Comp Exp Biol 303:792–801. Box GEB, Cox DR. 1964. An analysis of transformations. J R Stat Soc Ser B 26:211–252. Caldji C, Francis D, Sharma S, Plotsky PM, Meaney MJ. 2000. The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty‐induced fearfulness in the rat. Neuropsychopharmacology 22:219–229.

273 Casey MB, Sleigh MJ. in press. Prenatal visual experience induces postnatal motor laterality in Japanese quail chicks (Coturnix coturnix japonica). Dev Psychobiol. Available online ahead of print: DOI: 10.1002/dev.21116 Cate CT. 1989. Stimulus movement, hen behaviour and filial imprinting in Japanese quail (Coturnix coturnix japonica). Ethology 82:287– 306. Chau MJ, Stone AI, Mendoza SP, Bales KL. 2008. Is play behavior sexually dimorphic in monogamous species? Ethology 114:989– 998. Clarke JA. 2010. White‐tailed ptarmigan food calls enhance chick diet choice: learning nutritional wisdom? Anim Behav 79:25–30. Cook M, Mineka S. 1989. Observational conditioning of fear to fear‐ relevant versus fear‐irrelevant stimuli in rhesus monkeys. J Abnorm Psychol 98:448–459. De Margerie E, Peris A, Pittet F, et al. 2013. Effect of mothering on the spatial exploratory behavior of quail chicks. Dev Psychobiol 55:256– 264. Ensminger AL, Westneat DF. 2012. Individual and sex differences in habituation and neophobia in house sparrows (Passer domesticus). Ethology 118:1085–1095. Fairbanks LA, McGuire MT. 1993. Maternal protectiveness and response to the unfamiliar in vervet monkeys. Am J Primatol 30:119–129. Forkman B, Boissy A, Meunier‐Salaün M‐C, Canali E, Jones RB. 2007. A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiol Behav 92:340–374. Formanek L, Houdelier C, Lumineau S, Bertin A, Richard‐Yris M‐A. 2008. Maternal epigenetic transmission of social motivation in birds. Ethology 114:817–826. Gerull FC, Rapee RM. 2002. Mother knows best: effects of maternal modelling on the acquisition of fear and avoidance behaviour in toddlers. Behav Res Ther 40:279–287. Goerlich VC, Nätt D, Elfwing M, Macdonald B, Jensen P. 2012. Transgenerational effects of early experience on behavioral, hormonal and gene expression responses to acute stress in the precocial chicken. Horm Behav 61:711–718. Gottlieb G. 1991. Experiential canalization of behavioral development theory. Dev Psychol 27:4–13. Guibert F, Lumineau S, Kotrschal K, et al. 2013. Trans‐generational effects of prenatal stress in quail. Proc R Soc B Biol Sci 280:20122368. Haller J, Fuchs E, Halász J, Makara GB. 1999. Defeat is a major stressor in males while social instability is stressful mainly in females: towards the development of a social stress model in female rats. Brain Res Bull 50:33–39. Hegyi G, Schwabl H. 2010. Do different yolk androgens exert similar effects on the morphology or behaviour of Japanese quail hatchlings Coturnix japonica? J Avian Biol 41:258–265. Hemelrijk CK, Wantia J, Isler K. 2008. Female dominance over males in primates: self‐organisation and sexual dimorphism. PLoS ONE 3: e2678. J. Exp. Zool.

274 Hinde R. 1975. Le comportement animal. Paris: Presses Universitaires de France. Honess PE, Marin CM. 2006. Behavioural and physiological aspects of stress and aggression in nonhuman primates. Neurosci Biobehav Rev 30:390–412. Houdelier C, Pittet F, Guibert F, de Margerie E, Lumineau S. 2013. Non‐ genetic inheritance in birds: transmission of behaviour from mother to offspring. Non‐Genet Inherit 1:62–68. Jones RB. 1980. Responses of male and female domestic chicks to a startling stimulus and the effects of a tranquilliser. Behav Processes 5:161–172. Jones B. 1986. The tonic immobility reaction of the domestic fowl: a review. World Poult Sci J 42:82–97. Jones RB, Mills AD, Faure JM. 1991. Genetic and experiential manipulation of fear‐related behavior in Japanese quail chicks (Coturnix coturnix japonica). J Comp Psychol 105:15–24. Kikusui T, Winslow JT, Mori Y. 2006. Social buffering: relief from stress and anxiety. Philos Trans R Soc Lond B Biol Sci 361:2215–2228. Krijgsveld KL, Visser GH, Daan S. 2003. Foraging behavior and physiological changes in precocial quail chicks in response to low temperatures. Physiol Behav 79:311–319. Launay F, Mills AD, Faure JM. 1993. Effects of test age, line and sex on tonic immobility responses and social reinstatement behaviour in Japanese quail Coturnix japonica. Behav Processes 29:1–16. Livia Terranova M, Laviola G. 1995. Individual differences in mouse behavioural development: effects of precocious weaning and ongoing sexual segregation. Anim Behav 50:1261–1271. Lonsdorf EV, Eberly LE, Pusey AE. 2004. Sex differences in learning in chimpanzees. Nature 428:715–716. Mainwaring MC, Hartley IR. 2013. Hatching asynchrony and offspring sex influence the subsequent exploratory behaviour of zebra finches. Anim Behav 85:77–81. Mainwaring MC, Beal JL, Hartley IR. 2011. Zebra finches are bolder in an asocial, rather than social, context. Behav Processes 87: 171–175. McPherson FJ, Chenoweth PJ. 2012. Mammalian sexual dimorphism. Anim Reprod Sci 131:109–122. Mignon‐Grasteau S, Roussot O, Delaby C, et al. 2003. Factorial correspondence analysis of fear‐related behaviour traits in Japanese quail. Behav Processes 61:69–75. Mills A, Faure J. 1986. Apparent absence of sex‐differences in the behavior of Japanese‐quail chicks in 4 behavioral‐tests. IRCS Med Sci Biochem 14:844–845. Mills AD, Faure JM. 1991. Divergent selection for duration of tonic immobility and social reinstatement behavior in Japanese quail (Coturnix coturnix japonica) chicks. J Comp Psychol 105: 25–38. Mills AD, Crawford LL, Domjan M, Faure JM. 1997. The behavior of the Japanese or domestic quail Coturnix japonica. Neurosci Biobehav Rev 21:261–281. Munakata A, Kobayashi M. 2010. Endocrine control of sexual behavior in teleost fish. Gen Comp Endocrinol 165:456–468. J. Exp. Zool.

PITTET ET AL. Nam K‐B, Meade J, Hatchwell BJ. 2011. Do parents and helpers adjust their provisioning effort in relation to nestling sex in a cooperatively breeding bird? Anim Behav 82:303–309. Nelson R. 1995. Aggression and social behavior. In: Nelson JR, editor. An introduction to behavioural endocrinology. Sunderland: Sinaeur Associates, Inc. p 445–484. Odén K, Gunnarsson S, Berg C, Algers B. 2005. Effects of sex composition on fear measured as tonic immobility and vigilance behaviour in large flocks of laying hens. Appl Anim Behav Sci 95:89– 102. Orcutt F, Orcutt A. 1976. Nesting and parental behavior in domestic common quail. Auk 93:135–141. Pellis SM. 2002. Sex differences in play fighting revisited: traditional and nontraditional mechanisms of sexual differentiation in rats. Arch Sex Behav 31:17–26. Pittet F, Coignard M, Houdelier C, Richard‐Yris M‐A, Lumineau S. 2012. Age affects the expression of maternal care and subsequent behavioural development of offspring in a precocial bird. PLoS ONE 7:e36835. Pittet F, Coignard M, Houdelier C, Richard‐Yris M‐A, Lumineau S. 2013. Effects of maternal experience on fearfulness and maternal behaviour in a precocial bird. Anim Behav 85:797–805. Pittet F, Houdelier C, de Margerie E, et al. 2014. Maternal styles in a precocial bird. Anim Behav 87:31–37. Pittet F, Le Bot O, Houdelier C, Richard‐Yris M‐A, Lumineau S. in press. Motherless quail mothers display impaired maternal behavior and produce more fearful and less socially motivated offspring. Dev Psychobiol. Available online ahead of print: DOI: 10.1002/dev.21129 Réale D, Reader SM, Sol D, McDougall PT, Dingemanse NJ. 2007. Integrating animal temperament within ecology and evolution. Biol Rev 82:291–318. Richard‐Yris M. 1994. Comportement parental chez les gallinaces: importance du facteur émotivité dans la vitesse d'émergence des réponses parentales. Apports du modèle caille Japonaise. In: Picard M, Porter RH, et Signoret P, editors. Comportement et bien‐être animal. INRA edition. Paris: INRA. p 61–77. Richard‐Yris M‐A, Michel N, Bertin A. 2005. Nongenomic inheritance of emotional reactivity in Japanese quail. Dev Psychobiol 46:1–12. Ridley AR, Huyvaert KP. 2007. Sex‐biased preferential care in the cooperatively breeding Arabian babbler. J Evol Biol 20:1271–1276. Rosenzweig MR, Leiman AL, Breedlove SM. 1998. Le Sexe. In: Psychobiologie. Bruxelles: DeBoeck Université. p 431–443. Saint‐Dizier H, Leterrier C, Lévy F, Richard S. 2008. Selection for tonic immobility duration does not affect the response to novelty in quail. Appl Anim Behav Sci 112:297–306. Schuett W, Dall SRX. 2009. Sex differences, social context and personality in zebra finches, Taeniopygia guttata. Anim Behav 77:1041–1050. Schweitzer C, Houdelier C, Lumineau S, Lévy F, Arnould C. 2010. Social motivation does not go hand in hand with social bonding between two familiar Japanese quail chicks, Coturnix japonica. Anim Behav 79:571–578.

QUAIL CHICK'S SEX DIFFERENCES Shimmura T, Kamimura E, Azuma T, et al. 2010. Effect of broody hens on behaviour of chicks. Appl Anim Behav Sci 126:125–133. Teather KL. 1992. An experimental study of competition for food between male and female nestlings of the red‐winged blackbird. Behav Ecol Sociobiol 31:81–87. Tomaszycki ML, Davis JE, Gouzoules H, Wallen K. 2001. Sex differences in infant rhesus macaque separation‐rejection vocalizations and effects of prenatal androgens. Horm Behav 39: 267–276. Vallortigara G, Cailotto M, Zanforlin M. 1990. Sex differences in social reinstatement motivation of the domestic chick (Gallus gallus) revealed by runway tests with social and nonsocial reinforcement. J Comp Psychol 104:361–367.

275 Vandenheede M, Bouissou MF. 1993. Sex differences in fear reactions in sheep. Appl Anim Behav Sci 37:39–55. Wallen K. 1996. Nature needs nurture: the interaction of hormonal and social influences on the development of behavioral sex differences in rhesus monkeys. Horm Behav 30:364–378. Wallen K. 2005. Hormonal influences on sexually differentiated behavior in nonhuman primates. Front Neuroendocrinol 26:7–26. Ward C, Bauer EB, Smuts BB. 2008. Partner preferences and asymmetries in social play among domestic dog, Canis lupus familiaris, littermates. Anim Behav 76:1187–1199. Wauters AM, Richard‐Yris MA. 2002. Mutual influence of the maternal hen's food calling and feeding behavior on the behavior of her chicks. Dev Psychobiol 41:25–36.

J. Exp. Zool.