- Email: [email protected]
Development of social familiarity in ewes
Physiology & Behavior 104 (2011) 392–397
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Development of social familiarity in ewes Matthieu Keller ⁎, Fabien Cornilleau, Eric Archer, Frédéric Lévy INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France CNRS, UMR 6175, F-37380 Nouzilly, France Université de Tours, F-37041 Tours, France
a r t i c l e
i n f o
Article history: Received 8 March 2011 Received in revised form 18 April 2011 Accepted 22 April 2011 Keywords: Social recognition Social familiarity Sheep Estrogens Preference test
a b s t r a c t We assessed the development of individual discrimination resulting from direct contact/familiarization in ewes. Unfamiliar ewes were introduced during 6, 24 or 72 h in groups of already familiarized ewes. At the end of this contact period, the development of social recognition with the animal that has been introduced was assessed using two different types of tests: a Y-maze preference test and a delayed paired close encounters test where tested ewes are successively and randomly interacting with the familiar animal and an unfamiliar conspecific. The results of both tests showed that ewes developed a recognition of the familiar animal in comparison to a completely unfamiliar female. However, this preference was evidenced after 24 h of contact when using the paired close encounters test whereas it appears only after 72 h when using the Y-maze test, suggesting that the paired close encounters test is a more sensitive methodology to assess the development of social familiarization. The importance of estrogens, in the formation of social familiarization was also evaluated. To this end, social recognition in the paired close encounters test was compared between ovariectomized animals receiving estrogen implants or not. Despite significant high levels of estradiol in estrogen implanted females, no major differences in recognition appeared between groups, suggesting that in our conditions estrogens do not have major influence on social familiarization. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Familiarization with individuals resulting from direct exposure to their sensory signatures is the primary mechanism mediating social recognition in many vertebrate species. Previous studies have determined that familiarity plays an important role in the development of social recognition in sheep, a species living in large flocks where the ability to recognize conspecifics is highly developed. For example, in the context of mother–young relationship, ewes develop a recognition of their own lamb within a few hours of contact after lambing [1, 2]; in addition, lambs also respond selectively to their mothers and to agemates of their own group after a few days of contact [3, 4]. A very efficient recognition also exists between adult sheep. Ewes are able to discriminate familiar conspecific on the basis of various sensory cues. For example, sheep discriminate between frontal photographs of different breeds of sheep or between male and female [5]. In similar conditions, sheep also learn significantly faster to discriminate between frontal views of familiar conspecific faces compared with unfamiliar ones [6]. This recognition memory is very enduring since sheep can remember up to 50 different conspecific faces over 2 years [7]. Despite the existence of this efficient recognition between adults, its develop⁎ Corresponding author at: Laboratoire de Physiologie de la Reproduction & des Comportements, UMR 6175 INRA/CNRS/Université de Tours, 37380 Nouzilly, France. Tel.: + 33 2 47 42 72 75; fax: + 33 2 47 42 77 43. E-mail address: [email protected] (M. Keller). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.04.030
mental time course has never been explored in details. In this context, the first series of experiments reported in this manuscript is an initial attempt to assess the development of individual discrimination resulting from direct contact/familiarization in ewes. In addition, although results are sometimes inconsistent, it has been previously reported in rodents that gonadal hormones, particularly estrogens, can exert influences on social preference and social recognition [8, 9]. It has been demonstrated that mice lacking the estrogen receptor α (ERα) show impaired social recognition [10]. In agreement with these results, it has been also reported that long-term estrogens treatment improves social recognition in ovariectomized female rats [11, 12]. In sheep, social preferences of ewes in a two choice tests between faces of male and female widely differ depending on whether the animals are in estrus vs anestrus physiological status. While females in anestrus display a strong preference for face of female conspecific, the same animals show a profound shift in their social preference in the estrus condition, with a marked preference for the face of a ram [6]. In the context of sheep offspring recognition, the recognition processes are established during a short-time window characterised by the fact that the ewe is primed by estrogen and progesterone during the late gestation period and experience vaginocervical stimulation as a result of giving birth. Even if a role for other gonadal hormones cannot be discarded, these data suggest an influence of estrogens on the establishment of social preferences. Indeed, if lamb olfactory memory formation can be induced by artificial vaginocervical stimulation in non parturient ewes, this could
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
only occur after ewes have been artificially primed with estrogens [13]. As a whole these results suggest that estrogens could play a main role in social memory formation in sheep. Therefore, in an additional step, we tested whether estrogens can modulate the establishment of social familiarization towards conspecifics (paired close encounters and Y-maze test). 2. Material and methods 2.1. Animals The animals used were Ile-de-France (IDF; n = 114) ewes and a crossbreed between Romanov × Ile-de-France ewes (RIF; n = 47). The animals were aged between 2 to 6 years old. The RIF animals were used as unfamiliar animals because they had never been in contact with IDF ewes due to the fact that breeding procedures for different breeds are conducted separately in our experimental farm. Ewes of both breeds were kept permanently indoors in separate barns and fed with dehydrated lucerne, maize, straw, and a supplement of vitamins and minerals; they had free access to water. The ewes were individually identifiable by the use of ear tags. In each experience, ewes were randomly assigned to experimental groups and were tested only once to avoid possible habituation to the test. All experimental procedures were performed in accordance with local animal regulations (authorization no. A 37073, French Ministry of Agriculture) and with European Council Directive of 24 November 1986 (86/609/EEC). The experiments were performed in spring, during the anestrus season, at the INRA station in Nouzilly, France. To be sure that all animals were in a similar and physiologically constant state, ewes were diagnosed as seasonally anovulatory by showing persistent low concentrations of circulating progesterone (b1 ng/ml) in weekly progesterone assays, indicating the absence of a functional corpus luteum. These assays were routinely performed with a method adapted from Terqui and Thimonier [14]. 2.2. Development of social familiarization: general experimental procedure At the beginning of the experiments, pairs (n = 2) of IDF ewes were introduced into pens (10 m 2). Pens were visually isolated from each other. After a further 10 days familiarization period between these two IDF ewes, an unfamiliar RIF ewe was introduced within each pair of IDF ewes. Therefore, groups of three females were formed, each comprised of two IDF ewes and one RIF ewe. After various durations following the introduction of the RIF ewe (independent groups with 6 h, n = 32; 24 h, n = 32; or 72 h of contact, n = 30), IDF animals were tested for social preference in two types of tests where their behavior was scored when interacting with the RIF familiar ewe and another unfamiliar RIF ewe, belonging to another group of three animals. In these tests, our working hypothesis is that ewes will respond preferentially to their familiar conspecific (the RIF ewe introduced in their pen) in comparison to a unfamiliar animal as this has been already demonstrated in various situations [1, 15]. This type of discriminative interactions is used as a basis for inferring that the preferred animal is recognized (e.g. [16–18]). Social recognition is thus defined operationally as observable differential interactions or selective responsiveness among particular conspecifics. 2.2.1. Testing procedure: paired close encounters test The first testing procedure used is a paired close encounters test which has previously been found to be an effective and simple method to assess discrimination between familiar vs unfamiliar conspecifics in sheep [4, 19]. Each ewe was tested twice: ewe was once paired with a familiar RIF stimulus ewe while during the other situation the ewe was tested with a RIF stimulus ewe with which she had no prior contact (unfamiliar RIF ewe). At the beginning of each test, the tested ewe was removed from its pen and introduced into a cage measuring 2 m × 2 m
393
situated in a separate barn. After 2 min, a stimulus RIF ewe (either the familiar or the unfamiliar ewe) was introduced in the cage. The number of bleats emitted by each ewe during a 5 min period was then recorded with digital counters; other aggressive (head butts) and investigation (sniffings) behaviors were also recorded (sniffing behavior was defined as a movement of orientation of the nostrils towards the anogenital region or the head of a conspecific). At the end of the test, the ewes were immediately returned to their respective pens. Around 1 to 2 h later, the subject ewes were again tested but with a partner of the opposite category, i.e. with an unfamiliar ewe if they had been paired with a familiar partner for the first test and vice versa. Testing order was counter-balanced across all subject ewes, i.e. half of the animals were first tested with a familiar partner then later paired with an unfamiliar ewe, while the remaining half of the animals were tested in the reverse order. Based upon previous experiments performed in lambs using similar procedures [19], it was hypothesized that ewes paired with a familiar partner would emit fewer distress bleats as well as fewer aggressive and olfactory investigation behavior (head butts and sniffings) than when paired with an unfamiliar partner. 2.2.2. Testing procedure: simultaneous preference test in a Y-maze The same subject animals were observed when exposed to two simultaneously present RIF ewes in a Y-maze consisted of a triangular enclosure (10 × 10 × 6 m), delimited by 1 m high, solid-metal barriers [1]. Two individual pens (2 × 1 m) made of fine wire mesh were located at each arm of the base of the enclosure and contained either the familiar or an alien ewe. Opposite these arms, a starting pen (2 × 1 m) served to hold the ewe before releasing her into the testing enclosure. The testing area was divided into three main zones by string: two 1 m-wide contact zones in front of the two stimuli ewe pens and a neutral zone. In each case, the unfamiliar RIF ewe had been housed in a different yard than the tested ewe. The position of familiar and unfamiliar ewes was reversed at each test. The ewe was left in the starting pen (2 × 1 m) for 1 min, giving her the opportunity to see and hear both stimulus ewes before being released. In all cases, both stimulus ewes had bleated before the tested ewe was released. Two experimenters recorded the total time spent in the contact zone near each stimulus ewe during 5 min. The tested ewe was scored as being in a contact zone when its four feet were entirely inside the string boundary. When the 5 min trial ended, the tested ewe was immediately returned to its own pen. The left and right location of the familiar and unfamiliar stimulus ewes was randomly determined for each test. To avoid neophobic reactions during the test, ewes were habituated to the experimental device for 5 min during 3 consecutive days prior the beginning of the experiment [1]. In each group of three animals, both IDF ewes were tested but only as experimental animals; therefore these IDF ewes were used only once in each type of test (paired close encounters and Y-maze test). RIF animals were used as stimulus animal: these animals were only used in a limited number of tests (4 times in each type of test) so that the potential effect of prior experience as a stimulus animal had only minor impact on subject's subsequent behavior. The order of each type of test (paired close encounters and Y-maze test) was counterbalanced with half of the animals being tested in the paired close encounters test first and the other half being tested with the Y-maze test first. 2.3. Influence of estrogens on social familiarization Sexually mature Ile-de-France ewes (n = 20) were ovariectomized at least 1 month before experimentation. One week before the study, half of the animals (n = 10) were implanted s.c. with a 16-cm Silastic capsule containing 17β-estradiol (Sigma, St-Louis, MO, USA) to maintain a high but physiological circulating concentrations of estrogens (peak follicular phase concentration, approx. 10–15 pg/ml, [20]). This
394
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
range of concentration has been chosen to have the largest physiological difference between groups. Implants were removed after the familiarization test, one week later. The other half of the animals was implanted with empty silastic tubing. Given the results showing that the paired close encounters test appeared to be the most sensitive to assess recognition following social familiarization (see the first part of the Results section), we decided to use only this test. In addition, we performed the test after a duration of contact of 24 h because ewes start to exhibit preferential responses with the recently introduced partner in comparison to unfamiliar animals at this timepoint (see Results section). We hypothesized that both the type of test and delay would be appropriate to reveal a facilitatory (i.e. increased preference) or an inhibitory (i.e. reduced preference) effect of estradiol on social familiarization. Just after the end of the test, the plasma concentration of estradiol was measured to check whether the expected levels of estradiol were obtained. After blood sampling, the estradiol concentrations were estimated using the 125I E2 Diasorin RIA kit (Sorin Diagnostic, Antony, France), with a slight modification for the analysis of estradiol concentrations in ovine plasma. Duplicate 200 μl aliquots of plasma samples were extracted in 3 ml of ethyl-acetate/cyclohexane (V/V) mixed for 5 min. After 2 h, the tubes were centrifuged for 15 min and frozen in liquid nitrogen. The solvent layer was decanted, transferred into glass tubes, and evaporated under nitrogen. Standards and samples were reconstituted in 150 μl of 0.1 M PBS (pH 7.4) that contained 1 g/ml BSA and 1 mM EDTA. Samples were incubated in primary antibody (150 μl) for 24 h at 48 C and 125I estradiol (100 μl, 30 000 cpm) was added to each tube for 24 h at 48 C. The secondary antibody (500 μl of precipitating solution) was added, mixed, and incubated at 48 C for 1 h. Subsequently, 2 ml of 0.025 M Tris buffer (pH 7.4) was added, the tubes were centrifuged at 2500 g for 30 min, the supernatant was discarded, and the radioactivity was measured. Following these modifications, the cross-reactivities were found to be less than 0.5% for estrone and estriol and less than 0.1% for ethinylestradiol, progesterone, testosterone, androstenediol, estradiol-3-glucuronide, and estradiol-17-glucuronide. The assay sensitivity was 0.78 pg/ml. At 4.8 pg/ml (nine assays), the intra-assay and inter-assay coefficients of variation were 10.7% and 7.1%, respectively. Recovery was determined by adding known amounts of estradiol to four ovine plasma samples with low endogenous estradiol concentrations. The percentage recovery, calculated as recovered/ expected, was 90.5%. The linearity of the dilutions was tested by assaying four plasma samples with high estradiol levels that were diluted with ovine plasma free of estradiol. The observed/expected ratios were between 93% and 100% at 1:2, 1:4, and 1:8 dilutions.
(identity of the stimulus animal: F1,91 = 44.95, p b 0.001; interaction: F2,91 = 5.32, p = 0.006). Post-hoc analyses revealed that distress bleats and olfactory investigations were significantly higher for unfamiliar than for familiar stimulus ewes after 24 and 72 h of contact but not for the 6 h of contact group. No significant effect or differences were detected for head butts at the various time points. Regarding the Y-maze procedure, a two-way ANOVA with duration of contact (6, 24 or 72 h) as independent factor and the stimulus animal (familiar vs unfamiliar) as related factor indicated a significant effect of the stimulus animal (F1,91 = 9.82, p = 0.002; Fig. 2) as well as a significant interaction (F2,91 = 4.61, p = 0.012). Post-hoc tests revealed a higher and significant difference in the time spent near the familiar in comparison to the unfamiliar ewe after 72 h of contact. 3.2. Influence of estradiol on the development of social familiarization in ewes As illustrated in Fig. 3, plasma assays performed just after the behavioral test and before the removal of estradiol implants show a
2.4. Statistical analysis All data, except plasma estradiol levels, were analyzed using twoway repeated measures analysis of variance (ANOVA). When appropriate, all ANOVAs were followed by Tukey highest signification difference (HSD) post-hoc comparisons adapted for repeated measures anova. Only significant (pb 0.05) effects detected by the ANOVAs are mentioned in detail in the Results section. Estradiol levels were compared by using a t-test. Data were analyzed using the software Statistica (10.0). 3. Results 3.1. Development of social recognition among ewes In the paired close encounters test, a two-way ANOVA for repeated measures with duration of contact (6, 24 or 72 h) as independent factor and the identity of the stimulus ewes (familiar vs unfamiliar) as related factor indicated a significant effect of the identity of the stimulus animal as well as a significant interaction for both the number of distress bleats (identity of the stimulus animal: F1,91 = 19.93, p b 0.001; interaction: F2,91 = 3.22, p = 0.044; Fig. 1) and the number of olfactory investigations
Fig. 1. Number of A) distress bleats, B) olfactory investigations and C) head butts expressed by tested ewes towards a familiar or an unfamiliar partner during the 3-min paired close encounters test. *Familiar different from unfamiliar, p b 0.05.
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
395
Fig. 2. Time spent by tested ewes near a familiar or an unfamiliar ewe during a 5-min preference test performed after 6 h, 24 h or 72 h after the introduction of the familiar ewe in the pen of the tested ewe. *Familiar different from unfamiliar, p b 0.05.
very high and significant level of estradiol in OVX+ E2 animals (9.88 ± 1.70 pg/ml) in comparison to OVX ewes (1.77 ± 0.53 pg/ml; t = −14.38, p b 0.01), thereby demonstrating the efficiency of implants in establishing a high plasmatic estradiol profile. During behavioral interactions in the paired close encounters test, a two-way ANOVA with physiological status (OVX vs OVX + E2) as independent factor and the stimulus animal (familiar vs unfamiliar) as related factor revealed a significant effect of the stimulus ewe only for olfactory investigations, with ewes emitting more olfactory investigations in presence of the unfamiliar RIF ewe than when paired with the familiar RIF ewe (F1,18 = 19.17, p b 0.001) (Fig. 4). This effect was confirmed by post-hoc tests. Other behavioral measures did not differ according treatment or stimulus animal. 4. Discussion 4.1. Development of social familiarity in ewes A first important conclusion emerge from this study: in domestic ewes, the development of social familiarity is established very rapidly after the introduction of a novel and unfamiliar conspecific. Indeed,
Fig. 3. Plasma levels of estradiol in OVX and OVX + E2 groups before and after the subcutaneous deposit of estradiol implants. *OVX different from OVX + E2, p b 0.05.
Fig. 4. Number of A) distress bleats, B) olfactory investigations and C) head butts expressed by OVX or OVX + E2 tested ewes towards a familiar or an unfamiliar partner during a 3-min paired close encounters test. *Familiar different from unfamiliar, p b 0.05.
after one day of contact, ewes that were paired with a RIF partner emitted significantly fewer distress bleats and displayed fewer olfactory investigations than when they were paired with an unfamiliar RIF partner. Such discriminative responsiveness indicates that ewes are capable of recognizing familiar conspecifics following a 1-day contact period. In the simultaneous two-choice test, which was adopted in an attempt to alleviate the problems associated with consecutive tests in which ewes are paired with different categories of partners (familiar vs unfamiliar), the ewes displayed a significant preference only after 3 days of contact. Thus, the simultaneous twochoice test in the Y-maze appears to be a less sensitive method for assessing social recognition than the pair test. Lack of differential responsiveness to the categories of stimulus ewes used in the Y-maze indicates that this discrimination task might have been more difficult. The inconsistent results between the two test methods have already been reported previously [19] and might reflect motivational differences as well as differential availability of salient cues from the stimulus animals. The two ewes that were confined together in a small cage during the pair tests had complete access to an array of phenotypic cues from their partner. In contrast, the possibility of direct contact was limited in the choice test, which could result, to some extent, in reduced access to the stimulus ewes' phenotypic traits. True discrimination between the two stimulus ewes should occur only if the subjects had sufficient exposure to the appropriate signatures of those animals. Thus, animals that did not approach and adequately investigate both stimulus ewes might have been incapable of discriminating between them. The negative effect of restricted exposure to salient phenotypic traits on social recognition would
396
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
presumably be most pronounced when such discrimination involved the perception of subtle cues or differences between individuals. In any event, the different results we obtained with the two test procedures point out the difficulty in identifying appropriate situations for assessing social recognition and argue against attempting to draw clear conclusions from negative results in this area of research [19]. It must be here noted that ewes were kept in small groups (three animals), therefore it is possible that under more natural conditions in larger flocks, with the possibility of segregation into different subgroups, the development of social familiarization may be delayed. In this context, it has been demonstrated that several weeks are needed for two flocks to mix so that individuals are randomly distributed in the new flock, a delay possibly corresponding to the duration of familiarization between the animals of each flock [21]. Definitive conclusions regarding the relative importance of sensory channels for social recognition in ewes cannot be offered from the present experiment. Olfaction is involved in several contexts of social recognition in sheep, including mother–young recognition or recognition between adults as well as between young [1, 2, 22–27]. In our experiments, ewes could rely on their sense of smell to discriminate between conspecifics when they are in close proximity to those animals, especially in the paired close encounters test where olfactory investigations seems to be one of the sensitive behavioral items to assess discriminative interactions. Beside olfaction, visual and auditory cues may also play a role in the recognition of adult conspecifics. For example, sheep are able to discriminate between frontal views of familiar or unfamiliar sheep or between male and female [5, 6, 15]. Auditory cues serve as a basis for recognition between lambs [28] or between the ewe and her lamb [29, 30]. In the present study, ewes emitted more call bleats in the presence of unfamiliar animals suggesting that auditory cues also play a role in social recognition among adult sheep. Whether each of these sensory modalities can be sufficient for familiarity discrimination or a combination of them is involved remains to be explored. Finally, it cannot be determined from the present study whether ewes recognize their conspecifics on an individual basis (true individual recognition) or alternatively distinguish between members of super-ordinate social categories, such as familiar vs unfamiliar; group member vs alien. Either type of social recognition could account for the observed discrimination between members vs unfamiliar ewes. Likewise, the lack of differential responses to stimulus ewes during the simultaneous choice tests in the Y-maze does not necessarily imply that ewes are unable to distinguish between those individuals. 4.2. Lack of modulatory role of estrogens in the development of social familiarity in ewes Estradiol treatment seems not to influence social recognition greatly in the paired close encounters test as no significant differences can be observed between the OVX and OVX + E2 groups. Furthermore, the absence of any estrogens did not prevent social recognition and these results confirm the results obtained in the anovulatory intact animals tested during the anestrus season. The absence of any effect of estrogens on social recognition in sheep is in sharp contrast to rodents where estrogens have been shown to influence the establishment of female social recognition in similar situations. In rats, a pro-estrus facilitation of social recognition memory has been reported when paired with vaginocervical stimulation [31]. Exogenous estrogen administration has been reported to prolong social recognition to 2 h in ovariectomized rats [11] and 24 h in ovariectomized mice [12]. Using a habituation/dishabituation social recognition paradigm, a better recognition memory was found in ovariectomized rats treated with estrogen and progesterone [32]. Studies have also pointed to an important role of estrogen receptor α and to a lower magnitude of estrogen receptor β in the formation of social recognition [8–10].
The lack of effect of estrogens in our study may be dependent upon various factors. The first could be related to the number of animals used. Indeed, while in the first range of experiments, we tested around 30 animals/group, only 10 females/groups were tested in the second part of the study. The number of head butts in OVX + E2 females would potentially become significant with a higher number of animals. The second factor could be related to the test used: while previous studies in rodents have mainly used habituation/dishabituation testing protocols to assess social recognition memory [10, 33–35], the situation used in the present study is somewhat different. As demonstrated in the first experiment, the paired close encounters test seems to be a robust and sensitive situation. By contrast, the Y-maze preference test is less sensitive as more time is needed before differences between groups could emerge; it is therefore conceivable that differential effects of estrogens may affect social recognition memory in the Y-maze preference test. In addition, dose and/or length of hormonal treatment may be another explanatory factor. Dose-dependent effects of estrogens on learning and memory tasks have been reported, sometimes with contradictory results [36–39]. Different hormonal levels may influence the learning strategies involved [40, 41]: exogenous treatments producing high estrogen levels being associated with impaired learning whereas those producing lower ones with its facilitation [42, 43]. Overall, our results do not exclude any potential modulatory effects of estrogens and further experiments using estrogen administration at different doses or in different situations should be conducted before drawing any definitive conclusions.
Acknowledgements We would like to thank all the shepherds of the experimental farm for the care of the animals. We also want to thank Anne-Lise Lainé and Lionel Lardic who performed the hormonal assays. We are also grateful to Dr. Alain Caraty for valuable suggestions with the second experiment included in this manuscript. Finally, the authors wish to thank Dr. Pascal Poindron for discussion regarding experimental protocols included in this manuscript. This work was partly funded by an INRA PHASE grant. Matthieu Keller is a research associate of the French Centre National de la Recherche Scientifique.
References [1] Keller M, Meurisse M, Poindron P, Nowak R, Ferreira G, Shayit M, Lévy F. Maternal experience influences the establishment of visual/auditory, but not olfactory recognition of the newborn lamb by ewes at parturition. Dev Psychobiol 2003;43: 167–76. [2] Poindron P, Lévy F, Keller M. Maternal responsiveness and maternal selectivity in domestic sheep and goats: the two facets of maternal attachment. Dev Psychobiol 2007;49:54–70. [3] Nowak R, Keller M, Val-Laillet D, Lévy F. Perinatal visceral events, neural activation, and development of mother young bonding in sheep. Horm Behav 2007;52:92–8. [4] Porter RH, Désiré L, Bon R, Orgeur P. The role of familiarity in the development of social recognition by lambs. Behaviour 2001;138:207–19. [5] Kendrick KM, Atkins K, Hinton MR, Broad KD, Fabre-Nys C, Keverne EB. Facial and vocal discrimination in sheep. Anim Behav 1995;49:1665–76. [6] Kendrick KM, Atkins K, Hinton MR, Heavens P, Keverne EB. Are faces special for sheep? Evidence from facial and object discrimination learning tests showing effects of inversion and social familiarity. Behav Processes 1996;38:19–35. [7] Kendrick KM, Da Costa AP, Leigh AE, Hinnton MR, Pierce JW. Sheep don't forget a face. Nature 2001;414:165–6. [8] Sànchez-Andrade G, Kendrick KM. The main olfactory system and social learning in mammals. Behav Brain Res 2009;200:323–35. [9] Sànchez-Andrade G, Kendrick KM. Roles of α- and β-estrogen receptors in mouse social recognition memory: effects of gender and the estrous cycle. Horm Behav 2011;59:114–22. [10] Choleris E, Gustafsson JA, Korach KS, Muglia LJ, Pfaff DW, Ogawa S. An estrogendependent four-gene micronet regulating social recognition: a study with oxytocin and estrogen receptor-alpha and -beta knockout mice. Proc Natl Acad Sci USA 2003;100:6192–7. [11] Hlinak Z. Social recognition in ovariectomized and estradiol-treated female rats. Horm Behav 1994;47:350–7.
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397 [12] Tang AC, Nakazawa M, Romeo RD, Reeb BC, Sisti H, McEwen BS. Effects of longterm estrogen replacement on social investigation and social memory in ovariectomized C57BL/6 mice. Horm Behav 2005;47:350–7. [13] Kendrick KM, Keverne EB. Importance of progesterone and estrogen priming for the induction of maternal behavior by vaginocervical stimulation in sheep: effects of maternal experience. Physiol Behav 1991;49:745–50. [14] Terqui M, Thimonier J. New rapid radioimmunologic method for estimation of plasma progesterone. Application to early diagnosis of gestation in the ewe and goat. C R Acad Sci Hebd Seances Acad Sci D 1974;279:1109–12. [15] Ferreira G, Keller M, Saint-Dizier H, Perrin G, Lévy F. Transfer between views of conspecific faces at different ages or in different orientations by sheep. Behav Processes 2004;67:491–9. [16] Beecher MD. From individual to species recognition: theories and mechanisms. Signature systems and kin recognition. Amer Zool 1982;22:477–90. [17] Porter RH, Blaustein AR. Mechanisms and ecological correlates of kin recognition. Sci Prog 1989;73:53–66. [18] Tang-Martinez Z. The mechanisms of kin discrimination and the evolution of kin recognition in vertebrates: a critical re-evaluation. Behav Processes 2001;53:21–40. [19] Ligout S, Porter RH, Bon R. Social discrimination in lambs: persistence and scope. Appl Anim Behav Sci 2002;76:239–48. [20] Evans NP, Dahl GE, Glover BH, Karsch FJ. Central regulation of pulsatile gonadotropinreeasing hormone (GnRH) secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 1994;34:1806–11. [21] Arnold GW, Pahl PJ. Some aspects of social behaviour in domestic sheep. Anim Behav 1974;3:592–600. [22] Baldwin BA, Meese GB. The ability of sheep to distinguish between conspecifics by means of olfaction. Physiol Behav 1977;18:803–8. [23] Blissett MJ, Boland KP, Cottrell DF. Discrimination between odours of fresh oestrous and non-oestrous ewe urine by rams. Appl Anim Behav Sci 1990;25:51–9. [24] Lévy F, Keller M, Poindron P. Olfactory regulation of maternal behavior in mammals. Horm Behav 2004;46:284–302. [25] Ligout S, Keller M, Porter RH. The role of olfactory cues in the discrimination of agemates by lambs. Anim Behav 2004;68:785–92. [26] Lévy F, Keller M. Neurobiology of maternal behavior in sheep. Adv Study Behav 2008;38:399–437. [27] Lévy F, Keller M. Olfactory mediation of maternal behavior in selected mammalian species. Behav Brain Res 2009;200(2):336–45. [28] Ligout S, Sèbe F, Porter RH. Vocal discrimination of kin and non-kin agemates among lambs. Behaviour 2004;141:355–69. [29] Searby A, Jouventin P. Mother–lamb acoustic recognition inn sheep: a frequency coding. Proc Royal Soc Lon 2003;270:1765–71.
397
[30] Sèbe F, Nowak R, Poindron P, Aubin T. Establishment of vocal communication and discrimination between ewes and their lamb in the first two days after parturition. Dev Psychobiol 2007;49:375–86. [31] Larrazolo-Lopez A, Kendrick KM, Aburto-Arciniega M, Arriaga-Avila V, Morimoto S, Frias M, Guevara-Guzman R. Vaginocervical stimulation enhances social recognition memory in rats via oxytocin release in the olfactory bulb. Neuroscience 2008;152:585–93. [32] Spiteri T, Agmo A. Ovarian hormones modulate social recognition in female rats. Physiol Behav 2009;98:247–50. [33] Choleris E, Ogawa S, Kavaliers M, Gustafsson JA, Korach KS, Muglia LJ, Pfaff DW. Involvement of estrogen receptor α, β and oxytocin in social discrimination: a detailed behavioural analysis with knockout female mice. Genes Brain Behav 2006;5:528–39. [34] Gheusi G, Bluthé RM, Goodall G, Dantzer R. Social and individual recogntion in rodents: methodological aspects and neurobiological bases. Behav Processes 1994;33:59–88. [35] Imwalle DB, Scordalakes EM, Rissman EF. Estrogen receptor alpha influences socially motivated behaviors. Horm Behav 2002;42:484–91. [36] Frick KM, Fernandez SM, Bulinski SC. Estrogen replacement improves spatial reference memory and increases hippocampal synaptophysin in aged female mice. Neuroscience 2002;115:547–58. [37] Fugger HN, Cunningham SG, Rissman EF, Foster TC. Sex differences in the activational effect of ERα on spatial learning. Horm Behav 1998;34:163–70. [38] Leuner B, Mendolia-Loffredo S, Shors TJ. High levels of estrogen enhance associative memory formation of ovariectomized females. Psychoneuroendocrinology 2004;29:883–90. [39] Rissanen A, Puolivali J, van Groen T, Riekkinen PJ. In mice tonic estrogen replacement therapy improves non-spatial and spatial memory in a water maze task. Neuroreport 1999;10:1369–72. [40] Korol DL. Role of estrogen in balancing contributions from multiple memory systems. Neurobiol Learn Mem 2004;82:309–23. [41] Zurkovsky L, Brown SL, Boyd S, Fell JA, Korol DL. Estrogen modulates learning in female rats by acting directly at distinct memory systems. Neuroscience 2007;144:26–37. [42] Holmes MM, Wide JK, Galea LA. Low levels of estradiol facilitate, whereas high levels of estradiol impair, working memory performance on the radial arm maze. Behav Neurosci 2002;116:928–34. [43] Wide JK, Hanratty K, Ting J, Galea LA. High level estradiol impairs and low level estradiol facilitates non-spatial working memory. Behav Brain Res 2004;155:45–53.
Contents lists available at ScienceDirect
Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Development of social familiarity in ewes Matthieu Keller ⁎, Fabien Cornilleau, Eric Archer, Frédéric Lévy INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France CNRS, UMR 6175, F-37380 Nouzilly, France Université de Tours, F-37041 Tours, France
a r t i c l e
i n f o
Article history: Received 8 March 2011 Received in revised form 18 April 2011 Accepted 22 April 2011 Keywords: Social recognition Social familiarity Sheep Estrogens Preference test
a b s t r a c t We assessed the development of individual discrimination resulting from direct contact/familiarization in ewes. Unfamiliar ewes were introduced during 6, 24 or 72 h in groups of already familiarized ewes. At the end of this contact period, the development of social recognition with the animal that has been introduced was assessed using two different types of tests: a Y-maze preference test and a delayed paired close encounters test where tested ewes are successively and randomly interacting with the familiar animal and an unfamiliar conspecific. The results of both tests showed that ewes developed a recognition of the familiar animal in comparison to a completely unfamiliar female. However, this preference was evidenced after 24 h of contact when using the paired close encounters test whereas it appears only after 72 h when using the Y-maze test, suggesting that the paired close encounters test is a more sensitive methodology to assess the development of social familiarization. The importance of estrogens, in the formation of social familiarization was also evaluated. To this end, social recognition in the paired close encounters test was compared between ovariectomized animals receiving estrogen implants or not. Despite significant high levels of estradiol in estrogen implanted females, no major differences in recognition appeared between groups, suggesting that in our conditions estrogens do not have major influence on social familiarization. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Familiarization with individuals resulting from direct exposure to their sensory signatures is the primary mechanism mediating social recognition in many vertebrate species. Previous studies have determined that familiarity plays an important role in the development of social recognition in sheep, a species living in large flocks where the ability to recognize conspecifics is highly developed. For example, in the context of mother–young relationship, ewes develop a recognition of their own lamb within a few hours of contact after lambing [1, 2]; in addition, lambs also respond selectively to their mothers and to agemates of their own group after a few days of contact [3, 4]. A very efficient recognition also exists between adult sheep. Ewes are able to discriminate familiar conspecific on the basis of various sensory cues. For example, sheep discriminate between frontal photographs of different breeds of sheep or between male and female [5]. In similar conditions, sheep also learn significantly faster to discriminate between frontal views of familiar conspecific faces compared with unfamiliar ones [6]. This recognition memory is very enduring since sheep can remember up to 50 different conspecific faces over 2 years [7]. Despite the existence of this efficient recognition between adults, its develop⁎ Corresponding author at: Laboratoire de Physiologie de la Reproduction & des Comportements, UMR 6175 INRA/CNRS/Université de Tours, 37380 Nouzilly, France. Tel.: + 33 2 47 42 72 75; fax: + 33 2 47 42 77 43. E-mail address: [email protected] (M. Keller). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.04.030
mental time course has never been explored in details. In this context, the first series of experiments reported in this manuscript is an initial attempt to assess the development of individual discrimination resulting from direct contact/familiarization in ewes. In addition, although results are sometimes inconsistent, it has been previously reported in rodents that gonadal hormones, particularly estrogens, can exert influences on social preference and social recognition [8, 9]. It has been demonstrated that mice lacking the estrogen receptor α (ERα) show impaired social recognition [10]. In agreement with these results, it has been also reported that long-term estrogens treatment improves social recognition in ovariectomized female rats [11, 12]. In sheep, social preferences of ewes in a two choice tests between faces of male and female widely differ depending on whether the animals are in estrus vs anestrus physiological status. While females in anestrus display a strong preference for face of female conspecific, the same animals show a profound shift in their social preference in the estrus condition, with a marked preference for the face of a ram [6]. In the context of sheep offspring recognition, the recognition processes are established during a short-time window characterised by the fact that the ewe is primed by estrogen and progesterone during the late gestation period and experience vaginocervical stimulation as a result of giving birth. Even if a role for other gonadal hormones cannot be discarded, these data suggest an influence of estrogens on the establishment of social preferences. Indeed, if lamb olfactory memory formation can be induced by artificial vaginocervical stimulation in non parturient ewes, this could
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
only occur after ewes have been artificially primed with estrogens [13]. As a whole these results suggest that estrogens could play a main role in social memory formation in sheep. Therefore, in an additional step, we tested whether estrogens can modulate the establishment of social familiarization towards conspecifics (paired close encounters and Y-maze test). 2. Material and methods 2.1. Animals The animals used were Ile-de-France (IDF; n = 114) ewes and a crossbreed between Romanov × Ile-de-France ewes (RIF; n = 47). The animals were aged between 2 to 6 years old. The RIF animals were used as unfamiliar animals because they had never been in contact with IDF ewes due to the fact that breeding procedures for different breeds are conducted separately in our experimental farm. Ewes of both breeds were kept permanently indoors in separate barns and fed with dehydrated lucerne, maize, straw, and a supplement of vitamins and minerals; they had free access to water. The ewes were individually identifiable by the use of ear tags. In each experience, ewes were randomly assigned to experimental groups and were tested only once to avoid possible habituation to the test. All experimental procedures were performed in accordance with local animal regulations (authorization no. A 37073, French Ministry of Agriculture) and with European Council Directive of 24 November 1986 (86/609/EEC). The experiments were performed in spring, during the anestrus season, at the INRA station in Nouzilly, France. To be sure that all animals were in a similar and physiologically constant state, ewes were diagnosed as seasonally anovulatory by showing persistent low concentrations of circulating progesterone (b1 ng/ml) in weekly progesterone assays, indicating the absence of a functional corpus luteum. These assays were routinely performed with a method adapted from Terqui and Thimonier [14]. 2.2. Development of social familiarization: general experimental procedure At the beginning of the experiments, pairs (n = 2) of IDF ewes were introduced into pens (10 m 2). Pens were visually isolated from each other. After a further 10 days familiarization period between these two IDF ewes, an unfamiliar RIF ewe was introduced within each pair of IDF ewes. Therefore, groups of three females were formed, each comprised of two IDF ewes and one RIF ewe. After various durations following the introduction of the RIF ewe (independent groups with 6 h, n = 32; 24 h, n = 32; or 72 h of contact, n = 30), IDF animals were tested for social preference in two types of tests where their behavior was scored when interacting with the RIF familiar ewe and another unfamiliar RIF ewe, belonging to another group of three animals. In these tests, our working hypothesis is that ewes will respond preferentially to their familiar conspecific (the RIF ewe introduced in their pen) in comparison to a unfamiliar animal as this has been already demonstrated in various situations [1, 15]. This type of discriminative interactions is used as a basis for inferring that the preferred animal is recognized (e.g. [16–18]). Social recognition is thus defined operationally as observable differential interactions or selective responsiveness among particular conspecifics. 2.2.1. Testing procedure: paired close encounters test The first testing procedure used is a paired close encounters test which has previously been found to be an effective and simple method to assess discrimination between familiar vs unfamiliar conspecifics in sheep [4, 19]. Each ewe was tested twice: ewe was once paired with a familiar RIF stimulus ewe while during the other situation the ewe was tested with a RIF stimulus ewe with which she had no prior contact (unfamiliar RIF ewe). At the beginning of each test, the tested ewe was removed from its pen and introduced into a cage measuring 2 m × 2 m
393
situated in a separate barn. After 2 min, a stimulus RIF ewe (either the familiar or the unfamiliar ewe) was introduced in the cage. The number of bleats emitted by each ewe during a 5 min period was then recorded with digital counters; other aggressive (head butts) and investigation (sniffings) behaviors were also recorded (sniffing behavior was defined as a movement of orientation of the nostrils towards the anogenital region or the head of a conspecific). At the end of the test, the ewes were immediately returned to their respective pens. Around 1 to 2 h later, the subject ewes were again tested but with a partner of the opposite category, i.e. with an unfamiliar ewe if they had been paired with a familiar partner for the first test and vice versa. Testing order was counter-balanced across all subject ewes, i.e. half of the animals were first tested with a familiar partner then later paired with an unfamiliar ewe, while the remaining half of the animals were tested in the reverse order. Based upon previous experiments performed in lambs using similar procedures [19], it was hypothesized that ewes paired with a familiar partner would emit fewer distress bleats as well as fewer aggressive and olfactory investigation behavior (head butts and sniffings) than when paired with an unfamiliar partner. 2.2.2. Testing procedure: simultaneous preference test in a Y-maze The same subject animals were observed when exposed to two simultaneously present RIF ewes in a Y-maze consisted of a triangular enclosure (10 × 10 × 6 m), delimited by 1 m high, solid-metal barriers [1]. Two individual pens (2 × 1 m) made of fine wire mesh were located at each arm of the base of the enclosure and contained either the familiar or an alien ewe. Opposite these arms, a starting pen (2 × 1 m) served to hold the ewe before releasing her into the testing enclosure. The testing area was divided into three main zones by string: two 1 m-wide contact zones in front of the two stimuli ewe pens and a neutral zone. In each case, the unfamiliar RIF ewe had been housed in a different yard than the tested ewe. The position of familiar and unfamiliar ewes was reversed at each test. The ewe was left in the starting pen (2 × 1 m) for 1 min, giving her the opportunity to see and hear both stimulus ewes before being released. In all cases, both stimulus ewes had bleated before the tested ewe was released. Two experimenters recorded the total time spent in the contact zone near each stimulus ewe during 5 min. The tested ewe was scored as being in a contact zone when its four feet were entirely inside the string boundary. When the 5 min trial ended, the tested ewe was immediately returned to its own pen. The left and right location of the familiar and unfamiliar stimulus ewes was randomly determined for each test. To avoid neophobic reactions during the test, ewes were habituated to the experimental device for 5 min during 3 consecutive days prior the beginning of the experiment [1]. In each group of three animals, both IDF ewes were tested but only as experimental animals; therefore these IDF ewes were used only once in each type of test (paired close encounters and Y-maze test). RIF animals were used as stimulus animal: these animals were only used in a limited number of tests (4 times in each type of test) so that the potential effect of prior experience as a stimulus animal had only minor impact on subject's subsequent behavior. The order of each type of test (paired close encounters and Y-maze test) was counterbalanced with half of the animals being tested in the paired close encounters test first and the other half being tested with the Y-maze test first. 2.3. Influence of estrogens on social familiarization Sexually mature Ile-de-France ewes (n = 20) were ovariectomized at least 1 month before experimentation. One week before the study, half of the animals (n = 10) were implanted s.c. with a 16-cm Silastic capsule containing 17β-estradiol (Sigma, St-Louis, MO, USA) to maintain a high but physiological circulating concentrations of estrogens (peak follicular phase concentration, approx. 10–15 pg/ml, [20]). This
394
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
range of concentration has been chosen to have the largest physiological difference between groups. Implants were removed after the familiarization test, one week later. The other half of the animals was implanted with empty silastic tubing. Given the results showing that the paired close encounters test appeared to be the most sensitive to assess recognition following social familiarization (see the first part of the Results section), we decided to use only this test. In addition, we performed the test after a duration of contact of 24 h because ewes start to exhibit preferential responses with the recently introduced partner in comparison to unfamiliar animals at this timepoint (see Results section). We hypothesized that both the type of test and delay would be appropriate to reveal a facilitatory (i.e. increased preference) or an inhibitory (i.e. reduced preference) effect of estradiol on social familiarization. Just after the end of the test, the plasma concentration of estradiol was measured to check whether the expected levels of estradiol were obtained. After blood sampling, the estradiol concentrations were estimated using the 125I E2 Diasorin RIA kit (Sorin Diagnostic, Antony, France), with a slight modification for the analysis of estradiol concentrations in ovine plasma. Duplicate 200 μl aliquots of plasma samples were extracted in 3 ml of ethyl-acetate/cyclohexane (V/V) mixed for 5 min. After 2 h, the tubes were centrifuged for 15 min and frozen in liquid nitrogen. The solvent layer was decanted, transferred into glass tubes, and evaporated under nitrogen. Standards and samples were reconstituted in 150 μl of 0.1 M PBS (pH 7.4) that contained 1 g/ml BSA and 1 mM EDTA. Samples were incubated in primary antibody (150 μl) for 24 h at 48 C and 125I estradiol (100 μl, 30 000 cpm) was added to each tube for 24 h at 48 C. The secondary antibody (500 μl of precipitating solution) was added, mixed, and incubated at 48 C for 1 h. Subsequently, 2 ml of 0.025 M Tris buffer (pH 7.4) was added, the tubes were centrifuged at 2500 g for 30 min, the supernatant was discarded, and the radioactivity was measured. Following these modifications, the cross-reactivities were found to be less than 0.5% for estrone and estriol and less than 0.1% for ethinylestradiol, progesterone, testosterone, androstenediol, estradiol-3-glucuronide, and estradiol-17-glucuronide. The assay sensitivity was 0.78 pg/ml. At 4.8 pg/ml (nine assays), the intra-assay and inter-assay coefficients of variation were 10.7% and 7.1%, respectively. Recovery was determined by adding known amounts of estradiol to four ovine plasma samples with low endogenous estradiol concentrations. The percentage recovery, calculated as recovered/ expected, was 90.5%. The linearity of the dilutions was tested by assaying four plasma samples with high estradiol levels that were diluted with ovine plasma free of estradiol. The observed/expected ratios were between 93% and 100% at 1:2, 1:4, and 1:8 dilutions.
(identity of the stimulus animal: F1,91 = 44.95, p b 0.001; interaction: F2,91 = 5.32, p = 0.006). Post-hoc analyses revealed that distress bleats and olfactory investigations were significantly higher for unfamiliar than for familiar stimulus ewes after 24 and 72 h of contact but not for the 6 h of contact group. No significant effect or differences were detected for head butts at the various time points. Regarding the Y-maze procedure, a two-way ANOVA with duration of contact (6, 24 or 72 h) as independent factor and the stimulus animal (familiar vs unfamiliar) as related factor indicated a significant effect of the stimulus animal (F1,91 = 9.82, p = 0.002; Fig. 2) as well as a significant interaction (F2,91 = 4.61, p = 0.012). Post-hoc tests revealed a higher and significant difference in the time spent near the familiar in comparison to the unfamiliar ewe after 72 h of contact. 3.2. Influence of estradiol on the development of social familiarization in ewes As illustrated in Fig. 3, plasma assays performed just after the behavioral test and before the removal of estradiol implants show a
2.4. Statistical analysis All data, except plasma estradiol levels, were analyzed using twoway repeated measures analysis of variance (ANOVA). When appropriate, all ANOVAs were followed by Tukey highest signification difference (HSD) post-hoc comparisons adapted for repeated measures anova. Only significant (pb 0.05) effects detected by the ANOVAs are mentioned in detail in the Results section. Estradiol levels were compared by using a t-test. Data were analyzed using the software Statistica (10.0). 3. Results 3.1. Development of social recognition among ewes In the paired close encounters test, a two-way ANOVA for repeated measures with duration of contact (6, 24 or 72 h) as independent factor and the identity of the stimulus ewes (familiar vs unfamiliar) as related factor indicated a significant effect of the identity of the stimulus animal as well as a significant interaction for both the number of distress bleats (identity of the stimulus animal: F1,91 = 19.93, p b 0.001; interaction: F2,91 = 3.22, p = 0.044; Fig. 1) and the number of olfactory investigations
Fig. 1. Number of A) distress bleats, B) olfactory investigations and C) head butts expressed by tested ewes towards a familiar or an unfamiliar partner during the 3-min paired close encounters test. *Familiar different from unfamiliar, p b 0.05.
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
395
Fig. 2. Time spent by tested ewes near a familiar or an unfamiliar ewe during a 5-min preference test performed after 6 h, 24 h or 72 h after the introduction of the familiar ewe in the pen of the tested ewe. *Familiar different from unfamiliar, p b 0.05.
very high and significant level of estradiol in OVX+ E2 animals (9.88 ± 1.70 pg/ml) in comparison to OVX ewes (1.77 ± 0.53 pg/ml; t = −14.38, p b 0.01), thereby demonstrating the efficiency of implants in establishing a high plasmatic estradiol profile. During behavioral interactions in the paired close encounters test, a two-way ANOVA with physiological status (OVX vs OVX + E2) as independent factor and the stimulus animal (familiar vs unfamiliar) as related factor revealed a significant effect of the stimulus ewe only for olfactory investigations, with ewes emitting more olfactory investigations in presence of the unfamiliar RIF ewe than when paired with the familiar RIF ewe (F1,18 = 19.17, p b 0.001) (Fig. 4). This effect was confirmed by post-hoc tests. Other behavioral measures did not differ according treatment or stimulus animal. 4. Discussion 4.1. Development of social familiarity in ewes A first important conclusion emerge from this study: in domestic ewes, the development of social familiarity is established very rapidly after the introduction of a novel and unfamiliar conspecific. Indeed,
Fig. 3. Plasma levels of estradiol in OVX and OVX + E2 groups before and after the subcutaneous deposit of estradiol implants. *OVX different from OVX + E2, p b 0.05.
Fig. 4. Number of A) distress bleats, B) olfactory investigations and C) head butts expressed by OVX or OVX + E2 tested ewes towards a familiar or an unfamiliar partner during a 3-min paired close encounters test. *Familiar different from unfamiliar, p b 0.05.
after one day of contact, ewes that were paired with a RIF partner emitted significantly fewer distress bleats and displayed fewer olfactory investigations than when they were paired with an unfamiliar RIF partner. Such discriminative responsiveness indicates that ewes are capable of recognizing familiar conspecifics following a 1-day contact period. In the simultaneous two-choice test, which was adopted in an attempt to alleviate the problems associated with consecutive tests in which ewes are paired with different categories of partners (familiar vs unfamiliar), the ewes displayed a significant preference only after 3 days of contact. Thus, the simultaneous twochoice test in the Y-maze appears to be a less sensitive method for assessing social recognition than the pair test. Lack of differential responsiveness to the categories of stimulus ewes used in the Y-maze indicates that this discrimination task might have been more difficult. The inconsistent results between the two test methods have already been reported previously [19] and might reflect motivational differences as well as differential availability of salient cues from the stimulus animals. The two ewes that were confined together in a small cage during the pair tests had complete access to an array of phenotypic cues from their partner. In contrast, the possibility of direct contact was limited in the choice test, which could result, to some extent, in reduced access to the stimulus ewes' phenotypic traits. True discrimination between the two stimulus ewes should occur only if the subjects had sufficient exposure to the appropriate signatures of those animals. Thus, animals that did not approach and adequately investigate both stimulus ewes might have been incapable of discriminating between them. The negative effect of restricted exposure to salient phenotypic traits on social recognition would
396
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397
presumably be most pronounced when such discrimination involved the perception of subtle cues or differences between individuals. In any event, the different results we obtained with the two test procedures point out the difficulty in identifying appropriate situations for assessing social recognition and argue against attempting to draw clear conclusions from negative results in this area of research [19]. It must be here noted that ewes were kept in small groups (three animals), therefore it is possible that under more natural conditions in larger flocks, with the possibility of segregation into different subgroups, the development of social familiarization may be delayed. In this context, it has been demonstrated that several weeks are needed for two flocks to mix so that individuals are randomly distributed in the new flock, a delay possibly corresponding to the duration of familiarization between the animals of each flock [21]. Definitive conclusions regarding the relative importance of sensory channels for social recognition in ewes cannot be offered from the present experiment. Olfaction is involved in several contexts of social recognition in sheep, including mother–young recognition or recognition between adults as well as between young [1, 2, 22–27]. In our experiments, ewes could rely on their sense of smell to discriminate between conspecifics when they are in close proximity to those animals, especially in the paired close encounters test where olfactory investigations seems to be one of the sensitive behavioral items to assess discriminative interactions. Beside olfaction, visual and auditory cues may also play a role in the recognition of adult conspecifics. For example, sheep are able to discriminate between frontal views of familiar or unfamiliar sheep or between male and female [5, 6, 15]. Auditory cues serve as a basis for recognition between lambs [28] or between the ewe and her lamb [29, 30]. In the present study, ewes emitted more call bleats in the presence of unfamiliar animals suggesting that auditory cues also play a role in social recognition among adult sheep. Whether each of these sensory modalities can be sufficient for familiarity discrimination or a combination of them is involved remains to be explored. Finally, it cannot be determined from the present study whether ewes recognize their conspecifics on an individual basis (true individual recognition) or alternatively distinguish between members of super-ordinate social categories, such as familiar vs unfamiliar; group member vs alien. Either type of social recognition could account for the observed discrimination between members vs unfamiliar ewes. Likewise, the lack of differential responses to stimulus ewes during the simultaneous choice tests in the Y-maze does not necessarily imply that ewes are unable to distinguish between those individuals. 4.2. Lack of modulatory role of estrogens in the development of social familiarity in ewes Estradiol treatment seems not to influence social recognition greatly in the paired close encounters test as no significant differences can be observed between the OVX and OVX + E2 groups. Furthermore, the absence of any estrogens did not prevent social recognition and these results confirm the results obtained in the anovulatory intact animals tested during the anestrus season. The absence of any effect of estrogens on social recognition in sheep is in sharp contrast to rodents where estrogens have been shown to influence the establishment of female social recognition in similar situations. In rats, a pro-estrus facilitation of social recognition memory has been reported when paired with vaginocervical stimulation [31]. Exogenous estrogen administration has been reported to prolong social recognition to 2 h in ovariectomized rats [11] and 24 h in ovariectomized mice [12]. Using a habituation/dishabituation social recognition paradigm, a better recognition memory was found in ovariectomized rats treated with estrogen and progesterone [32]. Studies have also pointed to an important role of estrogen receptor α and to a lower magnitude of estrogen receptor β in the formation of social recognition [8–10].
The lack of effect of estrogens in our study may be dependent upon various factors. The first could be related to the number of animals used. Indeed, while in the first range of experiments, we tested around 30 animals/group, only 10 females/groups were tested in the second part of the study. The number of head butts in OVX + E2 females would potentially become significant with a higher number of animals. The second factor could be related to the test used: while previous studies in rodents have mainly used habituation/dishabituation testing protocols to assess social recognition memory [10, 33–35], the situation used in the present study is somewhat different. As demonstrated in the first experiment, the paired close encounters test seems to be a robust and sensitive situation. By contrast, the Y-maze preference test is less sensitive as more time is needed before differences between groups could emerge; it is therefore conceivable that differential effects of estrogens may affect social recognition memory in the Y-maze preference test. In addition, dose and/or length of hormonal treatment may be another explanatory factor. Dose-dependent effects of estrogens on learning and memory tasks have been reported, sometimes with contradictory results [36–39]. Different hormonal levels may influence the learning strategies involved [40, 41]: exogenous treatments producing high estrogen levels being associated with impaired learning whereas those producing lower ones with its facilitation [42, 43]. Overall, our results do not exclude any potential modulatory effects of estrogens and further experiments using estrogen administration at different doses or in different situations should be conducted before drawing any definitive conclusions.
Acknowledgements We would like to thank all the shepherds of the experimental farm for the care of the animals. We also want to thank Anne-Lise Lainé and Lionel Lardic who performed the hormonal assays. We are also grateful to Dr. Alain Caraty for valuable suggestions with the second experiment included in this manuscript. Finally, the authors wish to thank Dr. Pascal Poindron for discussion regarding experimental protocols included in this manuscript. This work was partly funded by an INRA PHASE grant. Matthieu Keller is a research associate of the French Centre National de la Recherche Scientifique.
References [1] Keller M, Meurisse M, Poindron P, Nowak R, Ferreira G, Shayit M, Lévy F. Maternal experience influences the establishment of visual/auditory, but not olfactory recognition of the newborn lamb by ewes at parturition. Dev Psychobiol 2003;43: 167–76. [2] Poindron P, Lévy F, Keller M. Maternal responsiveness and maternal selectivity in domestic sheep and goats: the two facets of maternal attachment. Dev Psychobiol 2007;49:54–70. [3] Nowak R, Keller M, Val-Laillet D, Lévy F. Perinatal visceral events, neural activation, and development of mother young bonding in sheep. Horm Behav 2007;52:92–8. [4] Porter RH, Désiré L, Bon R, Orgeur P. The role of familiarity in the development of social recognition by lambs. Behaviour 2001;138:207–19. [5] Kendrick KM, Atkins K, Hinton MR, Broad KD, Fabre-Nys C, Keverne EB. Facial and vocal discrimination in sheep. Anim Behav 1995;49:1665–76. [6] Kendrick KM, Atkins K, Hinton MR, Heavens P, Keverne EB. Are faces special for sheep? Evidence from facial and object discrimination learning tests showing effects of inversion and social familiarity. Behav Processes 1996;38:19–35. [7] Kendrick KM, Da Costa AP, Leigh AE, Hinnton MR, Pierce JW. Sheep don't forget a face. Nature 2001;414:165–6. [8] Sànchez-Andrade G, Kendrick KM. The main olfactory system and social learning in mammals. Behav Brain Res 2009;200:323–35. [9] Sànchez-Andrade G, Kendrick KM. Roles of α- and β-estrogen receptors in mouse social recognition memory: effects of gender and the estrous cycle. Horm Behav 2011;59:114–22. [10] Choleris E, Gustafsson JA, Korach KS, Muglia LJ, Pfaff DW, Ogawa S. An estrogendependent four-gene micronet regulating social recognition: a study with oxytocin and estrogen receptor-alpha and -beta knockout mice. Proc Natl Acad Sci USA 2003;100:6192–7. [11] Hlinak Z. Social recognition in ovariectomized and estradiol-treated female rats. Horm Behav 1994;47:350–7.
M. Keller et al. / Physiology & Behavior 104 (2011) 392–397 [12] Tang AC, Nakazawa M, Romeo RD, Reeb BC, Sisti H, McEwen BS. Effects of longterm estrogen replacement on social investigation and social memory in ovariectomized C57BL/6 mice. Horm Behav 2005;47:350–7. [13] Kendrick KM, Keverne EB. Importance of progesterone and estrogen priming for the induction of maternal behavior by vaginocervical stimulation in sheep: effects of maternal experience. Physiol Behav 1991;49:745–50. [14] Terqui M, Thimonier J. New rapid radioimmunologic method for estimation of plasma progesterone. Application to early diagnosis of gestation in the ewe and goat. C R Acad Sci Hebd Seances Acad Sci D 1974;279:1109–12. [15] Ferreira G, Keller M, Saint-Dizier H, Perrin G, Lévy F. Transfer between views of conspecific faces at different ages or in different orientations by sheep. Behav Processes 2004;67:491–9. [16] Beecher MD. From individual to species recognition: theories and mechanisms. Signature systems and kin recognition. Amer Zool 1982;22:477–90. [17] Porter RH, Blaustein AR. Mechanisms and ecological correlates of kin recognition. Sci Prog 1989;73:53–66. [18] Tang-Martinez Z. The mechanisms of kin discrimination and the evolution of kin recognition in vertebrates: a critical re-evaluation. Behav Processes 2001;53:21–40. [19] Ligout S, Porter RH, Bon R. Social discrimination in lambs: persistence and scope. Appl Anim Behav Sci 2002;76:239–48. [20] Evans NP, Dahl GE, Glover BH, Karsch FJ. Central regulation of pulsatile gonadotropinreeasing hormone (GnRH) secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 1994;34:1806–11. [21] Arnold GW, Pahl PJ. Some aspects of social behaviour in domestic sheep. Anim Behav 1974;3:592–600. [22] Baldwin BA, Meese GB. The ability of sheep to distinguish between conspecifics by means of olfaction. Physiol Behav 1977;18:803–8. [23] Blissett MJ, Boland KP, Cottrell DF. Discrimination between odours of fresh oestrous and non-oestrous ewe urine by rams. Appl Anim Behav Sci 1990;25:51–9. [24] Lévy F, Keller M, Poindron P. Olfactory regulation of maternal behavior in mammals. Horm Behav 2004;46:284–302. [25] Ligout S, Keller M, Porter RH. The role of olfactory cues in the discrimination of agemates by lambs. Anim Behav 2004;68:785–92. [26] Lévy F, Keller M. Neurobiology of maternal behavior in sheep. Adv Study Behav 2008;38:399–437. [27] Lévy F, Keller M. Olfactory mediation of maternal behavior in selected mammalian species. Behav Brain Res 2009;200(2):336–45. [28] Ligout S, Sèbe F, Porter RH. Vocal discrimination of kin and non-kin agemates among lambs. Behaviour 2004;141:355–69. [29] Searby A, Jouventin P. Mother–lamb acoustic recognition inn sheep: a frequency coding. Proc Royal Soc Lon 2003;270:1765–71.
397
[30] Sèbe F, Nowak R, Poindron P, Aubin T. Establishment of vocal communication and discrimination between ewes and their lamb in the first two days after parturition. Dev Psychobiol 2007;49:375–86. [31] Larrazolo-Lopez A, Kendrick KM, Aburto-Arciniega M, Arriaga-Avila V, Morimoto S, Frias M, Guevara-Guzman R. Vaginocervical stimulation enhances social recognition memory in rats via oxytocin release in the olfactory bulb. Neuroscience 2008;152:585–93. [32] Spiteri T, Agmo A. Ovarian hormones modulate social recognition in female rats. Physiol Behav 2009;98:247–50. [33] Choleris E, Ogawa S, Kavaliers M, Gustafsson JA, Korach KS, Muglia LJ, Pfaff DW. Involvement of estrogen receptor α, β and oxytocin in social discrimination: a detailed behavioural analysis with knockout female mice. Genes Brain Behav 2006;5:528–39. [34] Gheusi G, Bluthé RM, Goodall G, Dantzer R. Social and individual recogntion in rodents: methodological aspects and neurobiological bases. Behav Processes 1994;33:59–88. [35] Imwalle DB, Scordalakes EM, Rissman EF. Estrogen receptor alpha influences socially motivated behaviors. Horm Behav 2002;42:484–91. [36] Frick KM, Fernandez SM, Bulinski SC. Estrogen replacement improves spatial reference memory and increases hippocampal synaptophysin in aged female mice. Neuroscience 2002;115:547–58. [37] Fugger HN, Cunningham SG, Rissman EF, Foster TC. Sex differences in the activational effect of ERα on spatial learning. Horm Behav 1998;34:163–70. [38] Leuner B, Mendolia-Loffredo S, Shors TJ. High levels of estrogen enhance associative memory formation of ovariectomized females. Psychoneuroendocrinology 2004;29:883–90. [39] Rissanen A, Puolivali J, van Groen T, Riekkinen PJ. In mice tonic estrogen replacement therapy improves non-spatial and spatial memory in a water maze task. Neuroreport 1999;10:1369–72. [40] Korol DL. Role of estrogen in balancing contributions from multiple memory systems. Neurobiol Learn Mem 2004;82:309–23. [41] Zurkovsky L, Brown SL, Boyd S, Fell JA, Korol DL. Estrogen modulates learning in female rats by acting directly at distinct memory systems. Neuroscience 2007;144:26–37. [42] Holmes MM, Wide JK, Galea LA. Low levels of estradiol facilitate, whereas high levels of estradiol impair, working memory performance on the radial arm maze. Behav Neurosci 2002;116:928–34. [43] Wide JK, Hanratty K, Ting J, Galea LA. High level estradiol impairs and low level estradiol facilitates non-spatial working memory. Behav Brain Res 2004;155:45–53.