Lucie Rigaill

James P Higham

Phyllis C Lee

Amandine Blin

Dr Cécile Garcia
email: cecile.garcia@evolhum.cnrs.fr

Multimodal Sexual Signaling and Mating Behavior in Olive Baboons (Papio anubis)

Keywords: sexual communication, multimodal signals, mating decisions, sexual swelling, ovulation

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Animals communication often depends on multiple signal elements expressed in multiple modalities [START_REF] Candolin | The use of multiple cues in mate choice[END_REF][START_REF] Hebets | Complex signal function: developing a framework of testable hypotheses[END_REF][START_REF] Partan | Multisensory animal communication[END_REF][START_REF] Partan | Multisensory animal communication[END_REF]Marler 1999, 2005]. These signals may provide information on many different characteristics, including social and reproductive status with consequences for fitness and reproductive success; as such they are often under strong sexual selection. Primates are an excellent model for studies of multimodal communication and recent papers have urged more studies along these lines [START_REF] Slocombe | The language void: the need for multimodality in primate communication research[END_REF]. Old World monkeys have developed multiple signals with different hypothesized roles in the signaling of reproductive status, and several of these, such as sexual swellings and copulation calls, have a venerable place in the study of sexual selection. However, few attempts have been made to assess the extent to which primate females simultaneously signal fertility in multiple modalities, and how different signals may combine to influence patterns of male mate choice in group-living species.

In a number of primate species, males tend to mate during the period when ovulation is most likely [START_REF] Daspre | Signals of female reproductive quality and fertility in colony-living baboons (Papio h. anubis) in relation to ensuring paternal investment[END_REF][START_REF] Deschner | Female sexual swelling size, timing of ovulation, and male behavior in wild West African chimpanzees[END_REF][START_REF] Thompson | Male mating interest varies with female fecundity in Pan troglodytes schweinfurthii of Kanyawara, Kibale National Park[END_REF][START_REF] Engelhardt | Assessment of female reproductive status by male longtailed macaques, Macaca fascicularis, under natural conditions[END_REF][START_REF] Garcia | Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)[END_REF][START_REF] Gesquiere | Mechanisms of sexual selection: sexual swellings and estrogen concentrations as fertility indicators and cues for male consort decisions in wild baboons[END_REF][START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF] suggesting that they are able to make mating decisions based on the discrimination of fertility differences within female reproductive cycles. However, the signals and cues available to males for their mating decisions, and the relative reliability of these, are still poorly understood. There are several potential cues or signals involved in female primate ovulatory signaling: female behaviors [START_REF] Bullivant | Women's sexual experience during the menstrual cycle: identification of the sexual phase by noninvasive measurement of luteinizing hormone[END_REF][START_REF] Engelhardt | Female sexual behaviour, but not sex skin swelling, reliably indicates the timing of the fertile phase in wild long-tailed macaques (Macaca fascicularis)[END_REF][START_REF] Haselton | Ovulatory shifts in human female ornamentation: near ovulation, women dress to impress[END_REF][START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF][START_REF] Miller | Ovulatory cycle effects on tip earnings by lap dancers: economic evidence for human estrus?[END_REF][START_REF] Van Belle | Sexual behavior across ovarian cycles in wild black howler monkeys (Alouatta pigra): male mate guarding and female mate choice[END_REF], visual cues (e.g. sexual swelling and redness of face and sexual skin) [START_REF] Bercovitch | Sex skin[END_REF][START_REF] Dubuc | Sexual skin color contains information about the timing of the fertile phase in free-ranging Macaca mulatta[END_REF][START_REF] Gesquiere | Mechanisms of sexual selection: sexual swellings and estrogen concentrations as fertility indicators and cues for male consort decisions in wild baboons[END_REF][START_REF] Girolami | Female perineal swelling and its effects on male sexual arousal: an apparent sexual releaser in the chacma baboon (Papio ursinus)[END_REF]Higham et al. 2008aHigham et al. , 2008b[START_REF] Higham | Color signal information content and the eye of the beholder: a case study in the rhesus macaque[END_REF][START_REF] Higham | Familiarity affects assessment of Rigaill-Page 31 facial signals of female fertility by free-ranging male rhesus macaques[END_REF][START_REF] Manning | Fluctuating asymmetry and body-weight in men and women: implication for sexual selection[END_REF][START_REF] Nunn | The evolution of exaggerated sexual swellings in primates and the graded-signal hypothesis[END_REF][START_REF] Roberts | Female facial attractiveness increases during the fertile phase of the menstrual cycle[END_REF][START_REF] Waitt | Selective attention toward female secondary sexual color in male rhesus macaques[END_REF]], auditory signals (e.g. copulation calls) [Maestripieri et al. 2005;[START_REF] Pfefferle | Estrogen and progestogen correlates of the structure of female copulation calls in semi-freeranging Barbary macaques (Macaca sylvanus)[END_REF][START_REF] Pipitone | Women's voice attractiveness varies across the menstrual cycle[END_REF][START_REF] Pradhan | The evolution of female copulation calls in primates: a review and a new model[END_REF][START_REF] Semple | Information content of female copulation calls in yellow baboons[END_REF], and olfactory cues [Cerda Molina et al. 2006a, 2006b;[START_REF] Clarke | What role do olfactory cues play in chacma baboon mating?[END_REF][START_REF] Havlıcek | Non-advertized does not mean concealed: body odour changes across the human menstrual cycle[END_REF][START_REF] Keverne | Sex attractants in primates[END_REF][START_REF] Matsumoto-Oda | Sniffing behaviors in Mahale chimpanzees[END_REF]Michael andKeverne 1968, 1970;[START_REF] Michael | Influence of olfactory signals on the reproductive behavior of social groups of rhesus monkeys (Macaca mulatta)[END_REF][START_REF] Ober | HLA and mate choice in humans[END_REF][START_REF] Palagi | Sniffing behavior in Lemur catta: seasonality, sex, and rank[END_REF]].

In several primate species, the sexual swelling is one of the most conspicuous cues of fertility that could be used by males to allocate their mating activities [START_REF] Dixson | Primate sexuality: comparative studies of the Prosimians, Monkeys, Apes, and Humans[END_REF]]. Besides swelling, several primate species exhibit color changes in the anogenital area and/or the face in relation to reproductive state [rhesus macaques [START_REF] Dubuc | Sexual skin color contains information about the timing of the fertile phase in free-ranging Macaca mulatta[END_REF][START_REF] Higham | Color signal information content and the eye of the beholder: a case study in the rhesus macaque[END_REF], 2011;mandrills: Setchell et al. 2006], but few studies have investigated the potential simultaneous links between female fertility, mating decisions, and red skin coloration in Catarrhine species. Changes in face coloration, but not in hindquarters, were related to the timing of ovulation in rhesus macaques [START_REF] Dubuc | Sexual skin color contains information about the timing of the fertile phase in free-ranging Macaca mulatta[END_REF][START_REF] Higham | Color signal information content and the eye of the beholder: a case study in the rhesus macaque[END_REF]] and the closely related Japanese macaques [START_REF] Fujita | Interaction between male and female mating strategies and factors affecting reproductive outcome[END_REF]], whereas a study of baboons found no relationship between sexual skin coloration and ovulation [Higham et al. 2008b]. The role of sexual skin coloration in mate preferences and mating decisions remains unclear for most species [START_REF] Gauthier | Reproductive parameters and paracallosal skin color changes in captive female guinea baboons, Papio papio[END_REF][START_REF] Hausfater | Dominance and reproduction in baboons (Papio cynocephalus)[END_REF][START_REF] Strum | Variations in fecundity with age and environment in olive baboons (Papio anubis)[END_REF].

Similarly, whereas olfactory cues are associated with ovulation in a number of mammalian species, few attempts have been made to identify their use as a sexual signal in Catarrhines [START_REF] Heymann | The neglected sense-olfaction in primate behavior, ecology, and evolution[END_REF][START_REF] Stevenson | An initial evaluation of the functions of human olfaction[END_REF]. Several studies have reported sniffing behaviors in a sexual context [START_REF] Clarke | What role do olfactory cues play in chacma baboon mating?[END_REF][START_REF] Engelhardt | Assessment of female reproductive status by male longtailed macaques, Macaca fascicularis, under natural conditions[END_REF][START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF][START_REF] Matsumoto-Oda | Vaginal fatty acids produced by chimpanzees during menstrual cycles[END_REF][START_REF] Campbell | Primate sexuality and reproduction[END_REF][START_REF] Van Belle | Sexual behavior across ovarian cycles in wild black howler monkeys (Alouatta pigra): male mate guarding and female mate choice[END_REF]] and olfactory cues are known to advertise reproductive state, dominance rank, and individual identity in species such as lemurs and mandrills [START_REF] Palagi | Sniffing behavior in Lemur catta: seasonality, sex, and rank[END_REF][START_REF] Setchell | Chemical composition of scent-gland secretions in an Old World monkey (Mandrillus sphinx): influence of sex, male status, and individual identity[END_REF]. Female odors are also suggested to elicit both behavioral and endocrinological responses in male primates [Cerda-Molina et al. 2006a, 2006b;[START_REF] Keverne | Sex-attractant properties of ether extracts of vaginal secretions from rhesus monkeys[END_REF][START_REF] Michael | Influence of olfactory signals on the reproductive behavior of social groups of rhesus monkeys (Macaca mulatta)[END_REF], and studies of macaques [START_REF] Engelhardt | Assessment of female reproductive status by male longtailed macaques, Macaca fascicularis, under natural conditions[END_REF] and baboons [START_REF] Clarke | What role do olfactory cues play in chacma baboon mating?[END_REF][START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF] have suggested that olfactory cues may be critical elements in primate fertility signaling systems.

Earlier studies investigating primate sexual behavior have also yielded a contrasting picture of the extent to which female proceptive behavior may indicate the timing of ovulation to males. Female behavior has been related to the timing of ovulation in some species [e.g. humans: [START_REF] Bullivant | Women's sexual experience during the menstrual cycle: identification of the sexual phase by noninvasive measurement of luteinizing hormone[END_REF]capuchins: Carosi and Visalberghi 2002;long-tailed macaques: Engelhardt et al. 2005], although such relationships may not be universal [e.g. Barbary macaques: [START_REF] Brauch | Female sexual behavior and sexual swelling size as potential cues for males to discern the female fertile phase in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar[END_REF]; Japanese macaques [START_REF] Garcia | Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)[END_REF]olive baboons: Higham et al. 2009]. In primate species with a conspicuous sexual swelling whose size is correlated with the underlying probability of ovulation, such as baboons [START_REF] Daspre | Signals of female reproductive quality and fertility in colony-living baboons (Papio h. anubis) in relation to ensuring paternal investment[END_REF][START_REF] Gesquiere | Mechanisms of sexual selection: sexual swellings and estrogen concentrations as fertility indicators and cues for male consort decisions in wild baboons[END_REF]Higham et al. 2008a], female proceptive behavior can be predicted to be more weakly linked to ovarian cycle stage. However, the importance of this signal in baboon ovulatory signaling has been relatively poorly investigated. In the only relevant study using hormonal profiles and female behaviors, [START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF] showed that proceptive behaviors were unrelated to the timing of ovulation in baboons, although a number of potential indicators of female proceptivity were not measured and analyzed (i.e. female initiations and approaches).

Auditory cues in the context of sexual behavior, such as copulation calls, are relatively well described in some species of Old World monkeys and apes [long-tailed macaques: [START_REF] Engelhardt | Information content of female copulation calls in wild long-tailed macaques (Macaca fascicularis)[END_REF]Guinea baboons: Maestripieri et al. 2005; Barbary macaques: [START_REF] Pfefferle | Estrogen and progestogen correlates of the structure of female copulation calls in semi-freeranging Barbary macaques (Macaca sylvanus)[END_REF]yellow baboons: Semple et al. 2002;chimpanzees: Townsend et al. 2011]. These signals seem to be related to sexual hormones such as estrogen and progesterone levels though studies of almost all species have found that they do not accurately indicate the timing of ovulation [e.g. long-tailed macaques: [START_REF] Engelhardt | Assessment of female reproductive status by male longtailed macaques, Macaca fascicularis, under natural conditions[END_REF]Barbary macaques: Pfefferle et al. 2008; but see crested macaques, [START_REF] Higham | Sexual signaling in the crested macaque and the evolution of primate fertility signals[END_REF], which may be an exception]. Copulation calls provide information on female identities [START_REF] Semple | Individuality and male discrimination of female copulation calls in the yellow baboon[END_REF]Towsend et al. 2011] which could influence female mating success [Maestripieri et al. 2005;[START_REF] Pfefferle | Female Barbary macaque (Macaca sylvanus) copulation calls do not reveal the fertile phase but influence mating outcome[END_REF] or decrease the capacity for male monopolization [START_REF] Engelhardt | Information content of female copulation calls in wild long-tailed macaques (Macaca fascicularis)[END_REF][START_REF] Townsend | Copulation calls in female chimpanzees (Pan troglodytes schweinfurthii) convey identity but do not accurately reflect fertility[END_REF]]. [START_REF] Maestripieri | Primate copulation calls and postcopulatory female choice[END_REF] suggested that copulation calls in baboons may aid postcopulatory female choice by enhancing sperm competition or promoting mate guarding, but as with olfactory cues and sex skin signals, their role in multimodal sexual signaling remains unclear for most species. Among many primates with hierarchically-structured matrilineal social systems, males are also expected to use social parameters as further predictors of female fertility.

They are predicted to discriminate in mating decisions based on fertility differences between females (if fertility changes with reproductive history or social parameters such as age and social rank, for instance), with males biasing their sexual behaviors particularly towards high-ranking and older females [START_REF] Garcia | Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)[END_REF][START_REF] Muller | Male chimpanzees prefer mating with old females[END_REF][START_REF] Proctor | Male chimpanzees' grooming rates vary by female age, parity, and fertility status[END_REF]. Discriminating between high/low rank females or aged/young females may however only be relevant when both such females are simultaneously available. Therefore, information from all other signal modalities should be salient in male mating behavior.

Baboons (Papio anubis) are an excellent species for exploring multimodal ovulatory signaling, since females exhibit a prominent sexual swelling, utter copulation calls during mating and produce vaginal secretions that could potentially offer information concerning the timing of ovulation. However, the simultaneous study of behavioral, visual (i.e. sexual skin size and color), auditory, and olfactory cues as signals of reproductive state in baboons has not been undertaken. This study was structured around four linked aims: 1) To determine the role of each female sexual signal (behavior, swelling, vocalization) in indicating the likely fertile phase; 2) To assess the inter-individual information content of signals by looking for correlates of female attributes such as social rank and age; 3) To relate male response to those signals by investigating changes in male sexual behavior (sexual mounts and olfactory inspections) with respect to the timing of ovulation and to the female characteristics. We seek to clarify whether males are discriminating in mating choices, e.g. whether they are most likely to mate with a given female at a given time (based on the likely fertile phase) and whether they are able to discriminate between females and to choose among several potential mates; 4) Ultimately, to examine whether females signal fertility in multiple modalities, and the extent to which males may utilize these different modalities in their decision-making.

METHODS

This research protocol was reviewed and approved by the appropriate institutional animal care and use committee (Comité Régional d'Ethique sur l'Expérimentation Animale, Marseille, France). All research adhered to the legal requirements of the USA, and the specific principles of the American Society of Primatologists for the ethical treatment of animals.

Subjects and Housing

The subjects were 9 adult female olive baboons ranging in age from 5 to 19 years and housed in semi-free ranging conditions at the CNRS Primatology Station (Rousset-sur-Arc, France). We sampled 3 cycling and multiparous females (mean age = 16.0 ± SD 6.6 years) and 6 cycling and nulliparous females (mean age = 6.2 ± SD 0.8 years).

These females were part of a social group housed in Park 2 (220 m² enclosure with climbing apparatus), consisting of 57 individuals (2 adult males, 11 adult females, 7 adolescent females, 8 subadult males, 21 juveniles and 8 infants). This study was conducted from February 2011 to the end of April 2011. Group composition remained unchanged during the study period and no major changes in the dominance hierarchy were observed. Animals had free access to water and they were fed three times per day with fruits and monkey pellets. Baboons at this study site breed throughout the year [START_REF] Garcia | Impact of social environment on variation in menstrual cycle length in captive female olive baboons (Papio anubis)[END_REF].

Behavioral observations

Data were collected by a single observer (LR) via thirty-minute continuous focal animal sampling [START_REF] Altmann | Observational study of behavior: sampling methods[END_REF]], yielding a total of 156 hours of observation (17.3 hours per female on average) over the 3-month study period. Focal samples were evenly distributed across days and weeks for all females, and were distributed between 9:00 A.M. and 5:00 P.M. Focal subjects were rotated throughout the week, although this depended somewhat on the schedule of image collection. Every effort was made to standardize the amount of time each female was sampled monthly by tabulating the number of samples for each female and making a special effort to sample any female for which data were lacking. During the 30-min samples, all social and sexual behaviors were recorded, along with the direction of behavior between the focal female and any adult male, and the identity of the other individual involved. Ad libitum data were also recorded for dominance hierarchy and mating activity. The total daily observation time was identical between days, and therefore there was no bias related to observation time on a given day in the estimation of the number of mounts and olfactory inspections observed per day.

Female and male dominance ranks were monitored using ad libitum data and were assessed by transcribing submissive (avoidances, retreats) and aggressive behaviors (initiation of attacks, threats, chases) as well as the outcome of dyadic interactions into an agonistic interaction matrix [see methods in [START_REF] Garcia | Dominance and reproductive rates in captive female olive baboons, Papio anubis[END_REF]].

Female dominance rank order was deduced from the matrix of interactions among all females in the group and female ranks were expressed as a percentage of females dominated, with relative ranks ranging from 0 (low rank) to 1 (high rank). For the two adult males, we distinguished the α male, i.e. the dominant male, and the β male.

During focal animal sampling, we recorded social behaviors between focal females and males, e.g. when a female was grooming or being groomed. Concerning sexual behaviors, we recorded behavioral indicators of proceptivity [START_REF] Beach | Sexual attractivity, proceptivity and receptivity in female mammals[END_REF][START_REF] Campbell | Primate sexuality and reproduction[END_REF]]. Proceptivity refers to sexual behaviors displayed by females toward males to indicate their motivation to copulate [START_REF] Van Belle | Sexual behavior across ovarian cycles in wild black howler monkeys (Alouatta pigra): male mate guarding and female mate choice[END_REF]]: these were approaches to males, sexual presentations to males, and copulation calls uttered during copulation (see Table 1 for definitions and distribution). Male sexual behaviors directed toward females were used to assess male responses to females' signals; we recorded approaches by males, holding, ejaculatory and non-ejaculatory mounts, and olfactory (nasal) inspections of the anogenital area (Table 1). Changes in the rates of olfactory inspections by males were also used as a proxy for changes in the intensity of female olfactory signals. We also recorded proximity between males and the focal female at 1min intervals; distances were classified in four categories (contact, less than 1 m, 1 to 2 m and more than 2 m). Olfactory inspections and mounts (ejaculatory and nonejaculatory) were also recorded by ad libitum observations.

Reproductive parameters

During the follicular phase of the menstrual cycle, the olive baboon anogenital area (AGA) increases in turgescence due to estrogenic stimulation [START_REF] Dixson | Primate sexuality: comparative studies of the prosimians, monkeys, apes and Human beings[END_REF]]. Ovulation most commonly occurs during the last few days of maximal tumescence [START_REF] Hendrickx | Observations on the menstrual cycle, optimal mating time and pre-implantation embryos of the baboon, Papio anubis and Papio cynocephalus[END_REF][START_REF] Koyama | Plasma estrogen, progestin, and luteinizing hormone during the normal menstrual cycle in the baboon: role of luteinizing hormone[END_REF], typically two days before the swelling subsides [START_REF] Bercovitch | Female weight and reproductive condition in a population of olive baboons (Papio anubis)[END_REF][START_REF] Daspre | Signals of female reproductive quality and fertility in colony-living baboons (Papio h. anubis) in relation to ensuring paternal investment[END_REF]Higham et al. 2008a;[START_REF] Shaikh | Temporal relationship of hormonal peaks to ovulation and sex skin deturgescence in the baboon[END_REF]. After ovulation, this decrease in swelling size coincides with a postovulatory rise in progesterone and a decrease in estrogen concentrations [START_REF] Stevens | Some reproductive studies in the baboon[END_REF]]. Daily records of morphological changes in the AGA and menstruation were made over 3 months. In addition, each female's reproductive history (parity and mean interbirth interval following surviving offspring) was known from colony records. It was not possible to investigate sexual signals with respect to infant survival because mortality rates are very low in this semi-free ranging population (Garcia, unpubl. data).

AGA characteristics analyzed here were: post-menstrual flat (immediately following menstruation, AGA is flat and uncolored), moderate genital swelling (reddening or partial tumescence of the AGA), large genital swelling (the volume of the swelling is maximal and bright pink), deflating swelling (the onset of detumescence was recognized from the appearance of wrinkles, reduction of visible tumescence and loss of coloration), full detumescence (no tumescence and pale colored).

Cycle length was calculated from the first day of menstruation to the subsequent first day of menstruation. The follicular phase was defined as the interval between the first day of menstruation and the estimated day of ovulation (2 days before detumescence), whereas the luteal phase comprised the period between the day of ovulation and the day before the next menstruation [see also [START_REF] Garcia | Impact of social environment on variation in menstrual cycle length in captive female olive baboons (Papio anubis)[END_REF]. A conceptive cycle was defined as the cycle prior to the onset of red coloration of the paracallosal skin of females, indicating pregnancy [START_REF] Altmann | The pregnancy sign in savannah baboons[END_REF]]. We analyzed a total of 13 menstrual cycles from the nine females, with all but one cycles non-conceptive.

We determined three distinct phases during the menstrual cycle. The periovulatory period, or fertile window, was then considered to be the time block covering the 2 days before detumescence (D0) plus the three preceding days in order to account for the life span of sperm in the female tract [humans, [START_REF] Wilcox | Timing of sexual intercourse in relation to ovulation: effects on the probability of conception, survival of the pregnancy and sex of the baby[END_REF]. The fertile phase was then defined as a period of 5 days (D-1 to D-5) [e.g. [START_REF] Barelli | Sexual swellings in wild white-handed gibbon females (Hylobates lar) indicate the probability of ovulation[END_REF][START_REF] Brauch | Female sexual behavior and sexual swelling size as potential cues for males to discern the female fertile phase in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar[END_REF][START_REF] Engelhardt | Assessment of female reproductive status by male longtailed macaques, Macaca fascicularis, under natural conditions[END_REF][START_REF] Heistermann | Female ovarian cycle phase affects the timing of male sexual activity in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar[END_REF], the period preceding this phase (D-6 to D-10) was taken to represent the prefertile phase (5 days) while the 5 days following the fertile phase were defined as postfertile phase (D0 to D+4). We only used data collected during this 15-day period in analyses.

Assessment of sexual skin size and color

Objective analysis of size and color of the sexual swelling followed Higham et al. [2008b]. We used a Canon EOS 350D camera with a 8 megapixel CMOS censor and an EF 28-135mm f/3.5-5.6 IS USM lens. White balance was set manually with a X-Rite White Balance Card (GretagMacbeth ColorChecker) before every daily data collection.

We collected images in RAW format with the flash disabled and with the shutter speed and aperture size determined automatically by the camera. We conducted image collection between 11:30 AM and 12:30 PM, a period preceding food distribution and characterized by locomotor activities.

To standardize images of size and color of the sexual swelling, we used a 30 x 30 cm grid (with vertical and horizontal lines marked at 1 cm intervals) and a Gretag X-Rite Color Checker (24 colored squares of known and varying reflectance) placed on an easel 5 cm off the ground. Images of these two charts were taken immediately after the sexual swelling image. We placed the charts at the same distance from the camera as the subjects with camera settings being standardized such that the image of the swelling and the corresponding images of the charts were captured on all the same camera settings (same shutter speed and aperture size) [START_REF] Bergman | A simple method for measuring colour in wild animals: validation and use on chest patch colour in geladas (Theropithecus gelada)[END_REF][START_REF] Dubuc | Sexual skin color contains information about the timing of the fertile phase in free-ranging Macaca mulatta[END_REF]Higham et al. 2008b]. This technique allowed images to be standardized such that size and color measurements were comparable across all images. A total of 95 images (mean=10; range=4-12 images) were used for color and size analysis. Females were photographed every day during their fully tumescent phase, and every other day during other phases.

We measured AGA color by integrating the color chart and swelling images into a single image. This image was converted to PNG format using Photoshop CS 5 (version 8) and exported in ColourWorker software (version 1.1 Beta). This software allows reflectance spectra to be extracted from selected parts of images as if they had been measured with a spectrometer [START_REF] Stevens | Studying primate color: towards visual system-dependent methods[END_REF]]. For the required reference spectra, we used samples of red signals from the long-tailed macaque (Macaca fascicularis), red uakari (Cacajao rubicundus), and mandrill (Mandrillus sphinx) taken from http://vision.psychol.cam.ac.uk/spectra/, as well as the human skin spectra with which the program already contains [START_REF] Higham | Color signal information content and the eye of the beholder: a case study in the rhesus macaque[END_REF]]. We assessed color from the whole sexual swelling and excluded anatomical parts like tails and callosities (Figure 1), taking measurements 3 times for each swelling, and using an average. The software estimates reflectance spectra in 5 nm intervals between 380 and 700 nm. We used these reflectance spectra to calculate the quantal catch of each of the three olive baboon receptors in response to the color [e.g. [START_REF] Stevens | Studying primate color: towards visual system-dependent methods[END_REF]]. As peak spectral sensitivities are conserved across Old World monkeys [START_REF] Jacobs | Uniformity of colour vision in Old World monkeys[END_REF], we took Papio papio peak spectral sensitivities from [START_REF] Bowmaker | Photosensitive and photostable pigments in the retinae of Old World monkeys[END_REF][START_REF] Candolin | The use of multiple cues in mate choice[END_REF] and used a rhodopsin template [START_REF] Govardosvskii | In search of the visual pigment template[END_REF]] to estimate full sensitivity curves for each receptor. We then integrated the spectra and sensitivity curves using across a D65 "daylight" illuminance spectra across the whole visible spectrum to obtain our quantal catches for each receptor [START_REF] Stevens | Studying primate color: towards visual system-dependent methods[END_REF]]. As measures for use in our analyses, we then calculated the red-green opponency channel as (LW-MW)/(MW+LW) (to assess how swellings varied chromatically at long-wavelengths), and (MW+LW)/2 as a luminance measure of how light-dark the swelling appeared [START_REF] Higham | Color signal information content and the eye of the beholder: a case study in the rhesus macaque[END_REF][START_REF] Stevens | Studying primate color: towards visual system-dependent methods[END_REF]].

We used a method adapted from Higham et al. [2008b] to measure different dimensions of swellings using Photoshop CS5: the maximal swelling height (A), the maximal swelling width (B) and the maximal labial width (C) (Figure 1). Measures were marked on the swelling's photo and copied on to the grid's photo where they were measured.

Statistical analysis

Data are expressed as mean ± standard error of the mean (SE). We analyzed data using all 13 cycles (one cycle for 5 females and two cycles for 4 females). We averaged data from any 2 cycles per female, so that N=9 for the final analyses. Our sample size is comparable with previous studies of sexual swellings and reproductive behavior [e.g. [START_REF] Emery | Size of sexual swellings reflects ovarian function in chimpanzees (Pan troglodytes)[END_REF][START_REF] Engelhardt | Female sexual behaviour, but not sex skin swelling, reliably indicates the timing of the fertile phase in wild long-tailed macaques (Macaca fascicularis)[END_REF]Higham et al. 2008a[START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF].

Behavioral and swelling data were averaged for each female's three phases of the cycle (prefertile, fertile and postfertile). A mean rate (frequency per hour) was calculated from behavioral data for the three cycle phases. We also calculated the number of nonejaculatory and ejaculatory mounts per day (sum of mounts observed during focal samples and ad libitum sampling) as well as the number of olfactory inspections per day. We log-transformed data and attempted to perform generalized linear models (GLM), but post-hoc tests of the homogeneity of variance in each model showed that data violated model assumptions. We therefore analyzed data using non-parametric statistics and permutation tests. Cycle length was analyzed by Friedman and Wilcoxon exact tests. Because of the size of our sample and the occasional missing value, we conducted permutation tests [START_REF] Mundry | Testing related samples with missing values: a permutation approach[END_REF][START_REF] Mundry | Use of statistical programs for nonparametric tests of small samples often leads to incorrect P values: examples from Animal Behaviour[END_REF]] that prevent for type I errors and incorrect P-values. We generated 10,000 permutations with two-tailed P-values. The permutation analysis tests for an effect of cycle phase on the swelling data (color and size) and other variables of interest (proceptive behaviors, copulation calls, and male sexual behaviors i.e. mounts and olfactory inspections). The analysis allows comparison of behavioral frequencies and swelling data across the three different phases. If results showed significant variation across these phases, we undertook posthoc tests to compare each set of two phases against each other.

To test for a relationship between female signaling or male response and social characteristics (age and social rank), we used Spearman's rank exact correlation test.

Given that age and parity are not independent, we only used age in our analysis.

Correlations were performed for the overall 15-day period covering the prefertile, fertile and postfertile phases. We therefore combined the Spearman's P-values using Fisher's omnibus test to correct for multiple testing [START_REF] Haccou | Statistical analyses of behavioural data[END_REF][START_REF] Hohmann | The relationship between socio-sexual behavior and salivary cortisol in bonobos: tests of the tension regulation hypothesis[END_REF]]. This procedure combines a number of P-values into a single χ²-distributed variable with its degree of freedom equaling twice the number of P-values.

It may appear slightly circular to use detumescence to delineate the cycle phases and then relating swelling size to these phases. However, we used very fine-scale measurements of swelling size, and there is no reason to assume that such measurements will always peak just before detumescence (during our assumed fertile period). As such the swelling size measurements and our fertile phase estimates are independent. All analyses were conducted using SPSS 18.0 for Windows and R statistical software v.2.14.2 (R Development Core Team 2012) and used two-tailed probabilities with a significance level of 0.05.

RESULTS

Menstrual cycle length

The mean duration of the menstrual cycle was 37.77 ± SE 3.33 days (range: 21-65 days). Cycle length did not differ significantly among the nine females (exact Friedman's test: χ 2 =8.00, df=8, P=0.43, N=9) or between successive cycles on the same females (exact Wilcoxon's test: Z=0.00, P=1.00, N=4). The follicular phase had an overall mean duration of 21.00 ± SE 1.68 days (range: 15-38 days) and the luteal phase lasted 16.77 ± SE 2.80 days (range: 4-44 days). Only one (the α female) of the 9 females conceived during the study period (during the second cycle after resumption of cycling following post-partum amenorrhea).

Female age was not associated with menstrual cycle length (exact Spearman's correlation test, rs=-0.08, P=0.080). We found a negative relationship between female relative rank and cycle length, with low-ranking females having longer cycles than dominant ones (rs=-0.78, P=0.002, N=9). These longer cycles were due to longer luteal phases (rs=-0.73, P=0.005, N=8); there were no changes in the duration of follicular phase with respect to rank.

Relation between female sexual signals and the cycle phase

Fine-scale measurements of maximal swelling height (A) varied significantly according to cycle phase, with maximal size during the fertile phase (Permutation test, prefertile vs. fertile: │t│10000=6.79, P=0.017; fertile vs. postfertile: │t│10000=7.08, P=0.049).

Concerning maximal swelling width (B) and maximal labial width (C), the difference was only significant for the comparison between the prefertile and the fertile phases (B: │t│10000=6.67, P=0.022; C: │t│10000=7.31, P=0.008). Swelling height was strongly correlated with labial and swelling widths during the fertile phase (A-C: rs=0.68, P<0.001; A-B: rs=0.69, P<0.001; N=9); whereas labial and swelling widths did not covary (rs=0.26, P=0.18, N=9).

Concerning color, neither swelling luminance nor the red-green opponency channel varied with respect to cycle phase (luminance: │t│10000=10.54, P=0.26; redgreen: │t│10000=0.62, P=0.91).

We recorded 186 events of proceptive behavior (see table 1 for distribution; mean per female=20.67 ± SE 11.40, range=6-41), and 46 events of social grooming during the study period. Females approached more males during the fertile phase (│t│10000=39.24, P=0.040; prefertile vs. fertile: │t│10000=7.00, P=0.044; fertile vs. postfertile │t│10000=8.19, P=0.020; Figure 2) and spent more time in contact with males during the fertile phase (│t│10000=62.38, P=0.013; fertile vs. postfertile: │t│10000=10.72, P=0.002). The rates of female presentations did not vary according to cycle phase (│t│10000=0.57, P=0.95). Grooming performed by females towards males did not vary according to cycle phase (grooming frequency: │t│10000=23.37, P=0.16).

The number of copulation calls emitted by females per hour did not vary according to cycle phase (│t│10000=4.53, P=0.16).

Concerning the relationship between the different female signals, we found that the frequency of female approaches during the fertile phase was strongly related to finescale variation in swelling size, e.g. swelling width (rs=0.77, P=0.015, N=9). None of the other female signals were correlated to each other (all P>0.10, N=9). Moreover, size (A, B and C) and color (luminance and red-green channel) of the swelling did not covary (all P>0.10, N=9).

Female sexual signals in relation to female characteristics

Neither female age nor rank was correlated with swelling color (effect of age: luminance: rs=0.48, P=0.19; red-green: rs=-0.28, P=0.47; N=9; effect of rank: luminance: rs=0.28, P=0.46; red-green: rs=0.28, P=0.46; N=9) or swelling size during the fertile phase (all P>0.10, N=9).

Rates of female approaches and presentations during the postfertile phase differed according to female rank (Fisher's omnibus test: approaches: χ²=12.91, df=6, P=0.044; presentations: χ²=13.65, df=6, P=0.034), with high-ranking females performing more approaches (rs=0.78, P=0.023, N=8) and more presentations (rs=0.85, P=0.005, N=8) toward males than subordinate females did. None of the female proceptive behaviors during the fertile phase were correlated with female age (all P>0.10, N=9). Copulation calls and olfactory cues were not related to female age or rank (fertile phase, all P>0.10, N=9).

Male sexual behaviors with respect to the timing of ovulation and to the female characteristics

The analysis of male sexual responses to female signals was based on a total of 278 observed behavioral interactions (see table 1 for distribution; mean per individual=30.89 ± SE 17.85,. Rates of male approaches and holding behaviors varied significantly according to cycle phase (approaches: │t│10000=102.97, P<0.001; holding: │t│10000=73.07, P<0.001), with higher rates during the fertile phase (approaches: prefertile vs. fertile: │t│10000=11.28, P=0.002; fertile vs. postfertile: │t│10000=13.31, P<0.001, Figure 3; holding: fertile vs. postfertile: │t│10000=11.82, P<0.001). Male olfactory inspections tended to be higher during the fertile and postfertile phases (number of olfactory inspections received by females per day: prefertile vs. fertile: │t│10000=6.00, P=0.059; prefertile vs. postfertile: │t│10000=6.25, P=0.063, Figure 4).

We also recorded 88 events of social grooming. We found that grooming frequency varied according to cycle phase (│t│10000=36.31, P=0.034) and was highest during the fertile phase (fertile vs. postfertile: │t│10000=8.31, P=0.012).

We recorded a total of 76 mounts performed by adult males during the study period (i.e. during focal observations and ad libitum sampling); 90.79% were ejaculatory mounts. The number of ejaculatory mounts performed by males per day tended to vary between the fertile and postfertile phases (│t│10000=3.30, P=0.061), whereas the number of non-ejaculatory mounts per day did not vary according to cycle phase (│t│10000=5.68, P=0.28).

The number of ejaculatory mounts performed by adult males and received by a female per day during the fertile phase tended to be positively associated with her rank, with high-ranking females tending to receive more ejaculatory mounts (Fisher's omnibus test: χ²=16.77, df=6, P=0.01; rs=0.63, P=0.067, N=9). None of the other male behaviors were related to female age or rank (fertile phase, all P>0.10, N=9).

The number of ejaculatory mounts received by females per day during the fertile phase was not correlated with the size or the color of the sexual swelling (A: rs=-0.26; B: rs=0.07; C: rs=0.47; luminance: rs=0.58; red-green: rs=-0.60; all P>0.10, N=9). We found a negative relationship between the number of male olfactory inspections, and swelling height (rs=-0.76, P=0.017; N=9).

DISCUSSION

Male baboons in our study can reliably discriminate intra-cycle differences in fertility but do not accurately discriminate inter-individual differences. Males may be more stimulated to mate with females at the appropriate time due to behavioral and visual cues (e.g. female behavior and sexual swelling size), while sexual skin color and auditory cues (i.e. copulation calls) seem to be uninformative concerning the intra-cycle probability of fertility. Males already in consort with a fully swollen female may have access to additional olfactory cues, which could allow them to time their mating to ovulation more accurately. These results are consistent with a multimodal framework for ovulatory signaling in olive baboons.

The role of female sexual signals in indicating the fertile phase

Females displayed several informative clues of ovulatory state. Sexual swelling size acted as a visual signal that correlated with the fertile phase [see also [START_REF] Brauch | Female sexual behavior and sexual swelling size as potential cues for males to discern the female fertile phase in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar[END_REF][START_REF] Daspre | Signals of female reproductive quality and fertility in colony-living baboons (Papio h. anubis) in relation to ensuring paternal investment[END_REF][START_REF] Deschner | Female sexual swelling size, timing of ovulation, and male behavior in wild West African chimpanzees[END_REF][START_REF] Gesquiere | Mechanisms of sexual selection: sexual swellings and estrogen concentrations as fertility indicators and cues for male consort decisions in wild baboons[END_REF]Higham et al. 2008a;[START_REF] Möhle | Patterns of anogenital swelling size and their endocrine correlates during ovulatory cycles and early pregnancy in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar[END_REF]. Females also exhibited more approaches and spent more time close to males during their fertile phase, suggesting that proceptive behaviors were potentially informative to males about female fertility as seen in a number of other species [e.g. long-tailed macaques: [START_REF] Engelhardt | Female sexual behaviour, but not sex skin swelling, reliably indicates the timing of the fertile phase in wild long-tailed macaques (Macaca fascicularis)[END_REF]humans: Gangestad et al. 2002[START_REF] Miller | Ovulatory cycle effects on tip earnings by lap dancers: economic evidence for human estrus?[END_REF]howler monkeys: Van Belle et al. 2009]. These results contrast with those from other studies of some species of non-human primates, in which such behaviors have not been found to increase around the fertile phase [Barbary macaques [START_REF] Brauch | Female sexual behavior and sexual swelling size as potential cues for males to discern the female fertile phase in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar[END_REF]; chimpanzees: [START_REF] Thompson | Male mating interest varies with female fecundity in Pan troglodytes schweinfurthii of Kanyawara, Kibale National Park[END_REF]; Japanese macaques: [START_REF] Garcia | Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)[END_REF]olive baboons: Higham et al. 2009]. In [START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF] baboon study, some proceptive behaviors, such as presentations, were related to sex hormones but there was no relationship between these behaviors and the timing of ovulation specifically. However, [START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF] did not have data on female initiations and approaches as in the present study. Besides visual and behavioral signals, we also found that olfactory cues, as indexed by male olfactory inspections, tended to be higher during the fertile and postfertile phases [Cerda-Molina et al. 2006b;[START_REF] Clarke | What role do olfactory cues play in chacma baboon mating?[END_REF][START_REF] Van Belle | Sexual behavior across ovarian cycles in wild black howler monkeys (Alouatta pigra): male mate guarding and female mate choice[END_REF], i.e. when consortship is already established. Olfactory signals may allow consorting males to detect subtle changes in vaginal secretions which may encode information concerning the timing of ovulation. These olfactory signals could have proximate effects on mating: they could stimulate male sexual interest and behaviors, especially ejaculatory mounts, by promoting sperm production [START_REF] Keverne | Sex attractants in primates[END_REF][START_REF] Matsumoto-Oda | Vaginal fatty acids produced by chimpanzees during menstrual cycles[END_REF][START_REF] Wallen | Sex and context: hormones and primate sexual motivation[END_REF]] and mate guarding [START_REF] Smith | Individual olfactory signatures in common marmosets (Callithrix jacchus)[END_REF]].

These cues could also facilitate penis intromission and ejaculation by lubricating the vagina during the most fertile phase. The role of olfactory cues in ovulatory communication still remains unclear but they could be crucial to influencing mating choices.

Females also displayed other clues which may be uninformative concerning the timing of the fertile phase. As with previous studies [long-tailed macaques: [START_REF] Engelhardt | Assessment of female reproductive status by male longtailed macaques, Macaca fascicularis, under natural conditions[END_REF][START_REF] Engelhardt | Information content of female copulation calls in wild long-tailed macaques (Macaca fascicularis)[END_REF]Barbary macaques: Pfefferle et al. 2011], auditory signals, i.e. copulation calls, did not indicate the likely fertile phase. The most commonly cited and best-supported hypotheses for the function of copulation calls focus on their role in inducing male-male competition, and potentially sperm competition, but also in increasing mate guarding by the copulating male, by informing other males that copulation has occurred [START_REF] Maestripieri | Primate copulation calls and postcopulatory female choice[END_REF]Maestripieri et al. 2005;[START_REF] Pfefferle | Female Barbary macaque (Macaca sylvanus) copulation calls do not reveal the fertile phase but influence mating outcome[END_REF]]. However, in our captive context, male competition was low (i.e. only two adult males with preferred mating females) and the distances between males and females were relatively small, such that females may not need to advertise their ovulatory status by long-distance clues. Therefore, these auditory signals may be of lesser important to indicating ovulation. We also found no sexual swelling color variation specifically associated with the differences in cycle phases, consistent with previous findings of olive baboons [Higham et al. 2008b]. Baboons differ from species with reproductive state changes in facial skin color, such as macaques, where facial signal variation is suggested to contain reliable information about the timing of ovulation [e.g .rhesus macaques: [START_REF] Dubuc | Sexual skin color contains information about the timing of the fertile phase in free-ranging Macaca mulatta[END_REF][START_REF] Higham | Color signal information content and the eye of the beholder: a case study in the rhesus macaque[END_REF]]. In species where sexual swellings are small or lacking [e.g. rhesus macaques: [START_REF] Dubuc | Sexual skin color contains information about the timing of the fertile phase in free-ranging Macaca mulatta[END_REF]Japanese macaques: Garcia et al. 2009], face coloration could be a highly visible cue available to males at a considerable distance to gauge the timing of the fertile phase. In baboons, on the other hand, with large advertisements of fertility, e.g. exaggerated sexual swelling, sexual skin color variation may be of lesser important to indicating the likely fertile phase.

Female sexual signals of inter-individual differences

Swelling size was not correlated with female rank or age and thus sexual swellings did not seem to advertise a female's reproductive value as predicted by the reliableindicator hypothesis [START_REF] Domb | Sexual swellings advertise female quality in wild baboons[END_REF][START_REF] Pagel | Evolution of conspicuous estrus advertisement in Old World monkeys[END_REF]], but rather were in line with the graded-signal hypothesis [START_REF] Nunn | The evolution of exaggerated sexual swellings in primates and the graded-signal hypothesis[END_REF][START_REF] Nunn | Do exaggerated sexual swellings function in female mating competition in primates? A comparative test of the reliable indicator hypothesis[END_REF], representing probabilistic intra-cycle signals of the likelihood of ovulation. Our results contrast with another study in baboons showing that sexual swelling size was a reliable indicator of maturation stage fertility differences [START_REF] Gesquiere | Mechanisms of sexual selection: sexual swellings and estrogen concentrations as fertility indicators and cues for male consort decisions in wild baboons[END_REF]; and the question of whether sexual swellings represent a sexually selected signal of enduring individual-level differences remains unanswered.

That sexual skin color did not indicate female age or rank again contrasts with studies of mandrills [START_REF] Setchell | Sexual swelling in mandrills (Mandrillus sphinx): a test of the reliable indicator hypothesis[END_REF][START_REF] Setchell | Signal content of red facial coloration in female mandrills (Mandrillus sphinx)[END_REF], and the signaling functions of baboon sexual swellings are still far from being fully understood.

Intra-specific variability is extraordinarily important in papionins, raising the possibility that swellings may have different functions in different species and even populations [START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF]]. Further comparative studies in this taxon are required.

Neither copulation calls nor olfactory signals were found to be informative of female characteristics, again in contrast to previous studies [copulation calls: Guinea baboons: Maestripieri et al. 2005;Chacma baboons: O'Connell and Cowlishaw 1994;olfactory cues: mandrills: Setchell et al. 2010]. The only phenotypic trait conveying information about female characteristics, e.g. dominance status, was female behavior.

Male response to female signals: are males discriminating in mating choices?

Adult males were able to discriminate intra-cycle differences in fertility. They displayed more sexual behaviors and tended to direct more ejaculatory mounts toward females during the expected time of ovulation [as in [START_REF] Daspre | Signals of female reproductive quality and fertility in colony-living baboons (Papio h. anubis) in relation to ensuring paternal investment[END_REF][START_REF] Higham | Female reproductive signaling, and male mating behavior, in the olive baboon[END_REF], which suggests that male olive baboons can use female sexual signals to discriminate intra-cycle differences in fertility. Two mechanisms could explain the relationship between male sexual behaviors and cycle phase: a passive response to changes in female sexual signals and an active concentration of male mating effort. Since proceptive behaviors varied in relation to the timing of ovulation, we suggest that these female behaviors may mediate the temporal links between male mating activities and period of the cycle. Moreover, the fact that we only found an increase in the number of ejaculatory mounts during the fertile period and not in the number of non-ejaculatory mounts suggests that males may actively concentrate their reproductive effort during the period of highest probability of conception. It could also be argued that different female signals (e.g. female behaviors and olfactory cues) may stimulate male responses.

Males tended to bias their sexual behaviors toward high-ranking females [START_REF] Garcia | Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)[END_REF][START_REF] Muller | Male chimpanzees prefer mating with old females[END_REF][START_REF] Proctor | Male chimpanzees' grooming rates vary by female age, parity, and fertility status[END_REF][START_REF] Zumpe | Social factors modulate the effects of hormones on the sexual and aggressive behavior of macaques[END_REF],

suggesting that female mating success was dominance-rank related. As visual, auditory and olfactory cues were not found to convey information about female characteristics, we suggest that males could use behavioral cues to discriminate among females based on rank differences. Contrary to previous studies [yellow baboons [START_REF] Gesquiere | Mechanisms of sexual selection: sexual swellings and estrogen concentrations as fertility indicators and cues for male consort decisions in wild baboons[END_REF]chimpanzees: Muller et al. 2006[START_REF] Proctor | Male chimpanzees' grooming rates vary by female age, parity, and fertility status[END_REF], we found that males did not preferentially mate with older females.

Multimodal signaling of fertility

A variety of elements acted in concert to provide information to males about the timing of the fertile phase. A multimodal framework of signals provides clues as to female reproductive status in olive baboons, with signals being displayed in several sensory modalities [START_REF] Candolin | The use of multiple cues in mate choice[END_REF]]. The use of multiple cues (swelling size, behavioral traits and olfactory cues) is consistent with the back-up hypothesis [START_REF] Johnstone | The evolution of animal signals[END_REF][START_REF] Møller | Why have birds got multiple sexual ornaments?[END_REF] which proposes that multiple signals allow a more accurate assessment of mate quality and a decrease in mate choice errors.

Our results are also consistent with previous suggestions that all males present could have information about the fertile window, through female behavioral signaling and sexual swelling size, but that only consorting males could accurately determine the timing of highest ovulation probability through precise olfactory signals that are only accessible from close range [baboons: [START_REF] Clarke | What role do olfactory cues play in chacma baboon mating?[END_REF], Higham et al. 2009;longtailed macaques: Engelhardt et al. 2004]. They are also in agreement with previous suggestions that differential distribution and access to ovulatory clues, due to the different "active spaces" of different signals [START_REF] Candolin | Sexual signaling in the European bitterling: females learn the truth by direct inspection of the resource[END_REF][START_REF] Lopez | Pheromonal recognition of females takes precedence over the chromatic cue in male Iberian wall lizards[END_REF], may allow females and males to establish different mating strategies.

The use of multiple sexual signals by females may consist of a tradeoff between costs of sexual signaling and benefits from mating. As previously suggested [Higham et al. 2009], by combining long-distance signals such as swelling size, and short-distance cues such as olfactory components, for which different males (i.e. consorting and nonconsorting males) have differential access, females may assure (by promoting mate guarding) and confuse (by increasing male and sperm competition) paternity simultaneously [START_REF] Nunn | The evolution of exaggerated sexual swellings in primates and the graded-signal hypothesis[END_REF][START_REF] Van Schaik | Social counterstrategies against infanticide by males in primates and other mammals[END_REF]. Moreover, females may decouple these two signals (e.g. swelling size and olfactory cues) and provide slightly misleading information about their reproductive status to non-consorting males through slight changes in swelling size while they are more fertile, whereas the consorting male could still use olfactory cues to detect ovulation. If only a subset of males is able to get close enough to key in on cues available only at close-range (such as olfactory or tactile signals), indirect female choice may be operating.

On the other hand, males may combine information from multiple cues and signals of the probability of ovulation in order to discriminate in mating choices and to reduce the cost of mating (sperm production and active mate guarding). By increasing the number of sensory modalities, males may obtain more information. Moreover, a sequential use of multiple female signals may allow males to reduce mate choice costs.

When multiple cues are used sequentially, a first cue (e.g. morphological traits of the sexual swelling) may indicate which females are worth inspecting for a second cue that is more costly to evaluate than the first but that gives more precise information about mate quality (olfactory and tactile cues). This strategy could reduce the number of females that are inspected more closely and, thus, mate-choice costs. That olfactory inspections were reduced, rather than increased, when swelling height was maximal, does not invalidate this suggestion, but rather may imply that the strength of the olfactory cues is higher during this phase of the cycle, such that males need not directly inspect females to detect them.

Table 1. Definitions of female and male sexual behaviors recorded.

Figure 1 :

 1 Figure 1: Picture of a sexual swelling with Gretag color standard, showing measures of

Figure 2 :

 2 Figure 2: Comparison between the prefertile, fertile, and postfertile phases concerning

Figure 3 :

 3 Figure 3: Comparison between the prefertile, fertile, and postfertile phases concerning

Figure 4 :

 4 Figure 4: Comparison between the prefertile, fertile, and postfertile phases concerning

Table 1 .

 1 Definitions of female and male sexual behaviors recorded.

		Female behaviors	Number of events recorded during focal sampling
	Approaches	Female comes close (less than 1m) or in contact with a male	120
	Sexual presentations	Female directs her anogenital area toward a sexual swelling male, the tail is generally raised to expose	45
	Copulation calls	Female utters special vocalizations during the last stage of the mount	21
		Male behaviors	Number of events recorded during focal sampling
	Approaches	Male comes close (less than 1m) or in contact with a female	212
	Holding behaviors	Male grabs female hips, generally observed just before mating	52
		-Ejaculatory mounts: male mounts a
		female, with intromission and ejaculation	34
		(i.e. successful)
	Mounts	-Non-ejaculatory mounts: male mounts a
		female, with intromission but without	4
		ejaculation (i.e. unsuccessful)
		Deliberate placing of the nostrils to within 5
	Olfactory inspections	cm of a female's anogenital area [cf. Clarke et al. 2009]. Male sniffs female's anogenital area or licks his finger after a tactile
		inspection.

Acknowledgments

The authors thank Guy Dubreuil for allowing our research to be carried out at the CNRS Primatology Station. Thanks also to the animal keepers for their field assistance. We thank Amélie Chimènes for her help with the preparation of images, Martin Stevens for providing MatLab scripts, and Julien Renoult for kindly sending us his script for the Govardosvskii et al. rhodopsin template. We greatly thank Roger Mundry for kindly sending us his script for permutation analyses and Fisher's omnibus tests. The authors especially thank Prof. Fred Bercovitch for helpful comments on the manuscript. The comments of three anonymous reviewers and editors also greatly improved an earlier draft of this paper. This study was funded by CNRS grant from the UPR 2147 to C.

Garcia. The authors declare no conflict of interest.


Claudia Terlouw

Pourquoi et comment réduire le stress des animaux à l'abattage

Introduction

Tout au long de l'élevage et pendant l'abattage, les animaux peuvent être confrontés à des situations de contrainte. Ils peuvent être maintenus par des cornadis ou des boxes pour faciliter leur gestion, subir un manque de confort (températures ambiantes ou conditions de repos non adaptées par exemple), ou être confrontés à des problèmes de santé ou des douleurs (boiteries par exemple). Le regard de la société sur ces aspects de l'élevage et d'abattage est de plus en plus critique ; beaucoup pensent que les animaux subissent des stress et sont capables de souffrir. En réponse à ces réactions, le cadre légal évolue progressivement. Le rôle des recherches en science animale est d'analyser ces questions et de produire des connaissances les plus objectives possibles. La présente contribution a pour but d'apporter des réponses scientifiques à certaines questions relatives au stress des animaux, notamment pendant la période de l'abattage.

Réduire le stress à l'abattage : pourquoi ?

Il y a trois arguments majeurs pour réduire le stress à l'abattage qui sont présentés ci-dessous : l'éthique, la sécurité des hommes et des animaux et la qualité des viandes.

La question d'éthique

Le stress a souvent été décrit comme l'état de l'animal lorsqu'il est incapable de s'adapter, par son comportement et sa physiologie, aux contraintes environnementaux ou physiques [START_REF] Fraser | The term "stress" in a veterinary context[END_REF][START_REF] Broom | Applications of neurobiological studies to farm animal welfare[END_REF]. Cette question a son importance, mais si on veut savoir si le stress animal pose un problème d'éthique, il est indispensable de se poser la question de la capacité des animaux d'éprouver des émotions [START_REF] Dawkins | Animal suffering: the science of animal welfare[END_REF][START_REF] Duncan | Animal welfare defined in terms of feelings[END_REF][START_REF] Dantzer | Can farm animal welfare be understood without taking into account the issues of emotion and cognition?[END_REF][START_REF] Désiré | Emotions in farm animals: a new approach to animal welfare in applied ethology[END_REF]. La question d'éthique ne se pose que si les animaux sont capables de souffrir, c'est-à-dire s'ils sont capables d'éprouver des émotions négatives. Le fer qui constitue la Tour Eiffel subit des contraintes, comme de la compression, de l'étirement et des contraintes thermiques, mais il n'y pas de question d'éthique, parce que l'on n'attribue pas des émotions à la Tour. En revanche, les études sur le comportement, sur la physiologie et sur l'anatomie et le fonctionnement du cerveau ont montré que les animaux sont capables d'éprouver des émotions négatives et positives [START_REF] Paul | Measuring emotional processes in animals: the utility of a cognitive approach[END_REF][START_REF] Boissy | Emotions and cognition: a new approach to animal welfare[END_REF]. Par exemple, le système limbique, connu pour son implication dans les émotions chez l'homme, existe également dans le cerveau des mammifères non humains [START_REF] Ledoux | Emotion circuits in the brain[END_REF]. Le système limbique est impliqué dans de nombreuses réactions comportementales lors de situations stressantes chez les mammifères [START_REF] Panksepp | Affective consciousness: Core emotional feelings in animals and humans: Neurobiology of Animal Consciousness[END_REF][START_REF] Damasio | Emotion in the perspective of an integrated nervous system[END_REF]. Ainsi, alors que certains événements environnementaux et physiques contraignent l'animal à s'adapter à la situation, ils sont également susceptibles de provoquer des émotions négatives chez lui. Par conséquent, on considère que le stress, y compris chez les animaux, fait référence à la présence d'émotions négatives. Ces émotions se produisent lorsque l'animal se sent menacé, que la menace soit réelle ou non. Afin de s'adapter à cette menace, l'animal répond par son comportement, par des réactions de fuite ou de défense s'il a peur par exemple, et par sa physiologie, avec une augmentation de la fréquence cardiaque et la sécrétion de certaines hormones pour permettre, entre autres, les efforts physiques (par exemple pour fuir).

En conclusion, les animaux sont capables d'éprouver des émotions positives, mais aussi négatives. Les humains qui ont des animaux sous leur responsabilité, sont également responsables de leur état émotionnel. Beaucoup considèrent que les humains ont donc l'obligation morale de s'occuper de ces animaux le mieux possible, pour éviter qu'ils éprouvent des émotions négatives (Thompson, 2012). Même si les émotions positives jouent un rôle essentiel dans le bien-être animal, dans le contexte de l'abattage, à l'heure actuelle, on s'intéresse surtout à la question de comment limiter les émotions négatives chez les animaux.

La sécurité des hommes et des animaux

Pendant la période d'abattage, la peur est une cause majeure de stress chez les animaux. Les réactions de peur peuvent varier selon le facteur anxiogène (présence de l'Homme, isolement, événements inattendus…), l'animal (vécu, âge, genre, patrimoine génétique…) et le contexte (absence de congénères, accumulation de facteurs anxiogènes…). Ils peuvent impliquer des réactions de défense (attaque, menace), d'évitement (fuite, évasion, dissimulation) ou une inhibition des mouvements, pour rester inaperçu. L'animal peut aussi excréter de l'urine et des fèces, facilitant ainsi les réactions physiques (attaque ou fuite) tout en véhiculant des phéromones liées à la peur et pouvant être perçues par les autres individus [START_REF] Boissy | Presence of cues from stressed conspecifics increases reactivity to aversive events in cattle: evidence for the existence of alarm substances in urine[END_REF]. Les animaux peuvent également vocaliser ; comme pour les phéromones, ceci informe les membres du groupe de la menace subie [START_REF] Boissy | Fear and fearfulness in animals[END_REF][START_REF] Boissy | Presence of cues from stressed conspecifics increases reactivity to aversive events in cattle: evidence for the existence of alarm substances in urine[END_REF]. Les réactions comportementales de peur sont accompagnées des réactions physiologiques classiques de stress, telles que l'augmentation de la fréquence cardiaque et la sécrétion de certaines hormones, nécessaires pour les efforts physiques.

Plus l'animal a peur, plus il est difficile à conduire. Les réactions de fuite peuvent l'amener dans la mauvaise direction. Souvent, l'animal refuse d'avancer vers le camion ou dans les couloirs de l'abattoir. Ces difficultés peuvent entraîner l'animal mais parfois aussi l'opérateur dans un cercle vicieux, avec une excitabilité physique et du stress supplémentaires, rendant les opérations encore plus difficiles. Les réactions de peur pouvant être inattendues et violentes, comme des coups de pattes et des mouvements abrupts, elles augmentent les risques en termes de sécurité pour les opérateurs comme pour les animaux (Terlouw et al. 2014 ;Grandin 2013). Par exemple, une étude dans un abattoir français a montré que des réactions de fuite dans les couloirs étaient à l'origine de glissades et parfois même de chutes des bovins. Dans ce dernier cas, l'animal pouvait rester coincé sous les barrières et se faire piétiner par d'autres animaux [START_REF] Bourguet | Effects of food deprivation on behavioral reactivity and physiological status in Holstein cattle[END_REF]. Des animaux ayant reçu des impacts douloureux (choc avec les barrières, coup de bâton…) étaient également plus difficiles à manipuler ensuite [START_REF] Bourguet | Effects of food deprivation on behavioral reactivity and physiological status in Holstein cattle[END_REF].

En conclusion, la peur et d'autres événements négatifs provoquent des réactions potentiellement dangereuses, pour l'animal lui-même et pour l'homme qui le conduit. Il est donc souhaitable de concevoir un environnement sans facteurs anxiogènes ni autres sources de stress, où l'animal avance sans difficulté là où l'homme souhaite l'amener.

Les qualités des viandes

On sait depuis longtemps que les conditions d'abattage peuvent avoir un impact significatif sur la qualité de la viande. Les mécanismes sous-jacents sont bien décrits [START_REF] Bendall | Post-mortem changes in muscle[END_REF][START_REF] Hambrecht | Preslaughter handling effects on pork quality and glycolytic potential in two muscles differing in fiber type composition[END_REF]. Après l'abattage, les réactions biochimiques se poursuivent, mais comme le sang ne circule plus, il n'y a plus d'apport en oxygène et en nutriments. En conséquence, le glycogène (source d'énergie) stocké localement dans le muscle est catabolisé par voie anaérobie. En raison de l'absence de circulation sanguine, les produits de ces réactions, notamment des ions d'hydrogène (H + ), s'accumulent dans le muscle et entraînent une acidification. Cette acidification est initialement rapide, puis ralentit et se stabilise à une valeur appelée pH ultime, atteinte environ 24 heures après la mort. Cette acidification est nécessaire pour la transformation du muscle en viande. L'ampleur de l'acidification dépend fortement des réserves musculaires de glycogène avant l'abattage. L'activité physique et le stress accrus le jour ou les heures précédant l'abattage nécessitent de l'énergie et réduisent les réserves de glycogène dans les muscles. Ceci peut entraîner un manque d'acidification, et la viande risque d'être moins bonne, avec un mauvais goût et un faible pouvoir de conservation. La vitesse d'acidification dépend de l'activité métabolique musculaire immédiatement avant l'abattage [START_REF] Bendall | Post-mortem changes in muscle[END_REF][START_REF] Hambrecht | Preslaughter handling effects on pork quality and glycolytic potential in two muscles differing in fiber type composition[END_REF]. Une activité physique associée à du stress entraîne une activité métabolique élevée. Lorsqu'elle a lieu juste avant l'abattage, elle continuera après la mort de l'animal, entraînant une augmentation de la vitesse d'acidification. Ceci augmente également le risque de la production d'une moins bonne viande : elle est plus claire et perd son eau à la cuisson. Le stress peut également influer sur la tendreté de la viande. Dans une étude menée sur des vaches, la viande était plus tendre chez les vaches ayant une fréquence cardiaque relativement faible, c'està-dire un niveau de stress bas, juste avant l'abattage. On ne connait pas encore les mécanismes sousjacents à ce phénomène.

Les études montrent donc que la réduction du stress des animaux permet d'obtenir des viandes d'une meilleure qualité.

Réduire le stress à l'abattage : Comment ?

Ces dernières années, des efforts considérables ont été déployés sur le terrain pour réduire le stress des animaux pendant la période de l'abattage. Le nouveau règlement européen (Règlement (CE) No1099/2009 du Conseil de l'Union Européenne) a imposé de nouvelles contraintes en matière de protection animale lors de l'abattage (la formation des opérateurs, l'établissement de mode opératoire pour les procédures et la vérification de l'efficacité de l'étourdissement sont des exemples). De plus, les directeurs et personnels de beaucoup d'abattoirs, ainsi que des groupes de grandes distribution sont sensibilisés à la question de la protection animale et mettent en place des audits internes et externes pour surveiller au quotidien les procédures et les process. Ces avancées sont appuyées par les recherches scientifiques. Ainsi, le règlement européen est basé sur l'avis scientifique du groupe sur la santé animale et le bien-être des animaux à la suite d'une demande de la Commission Européenne (EFSA, 2004). Les acteurs du terrain font eux aussi très régulièrement appel aux chercheurs spécialisés dans les questions de stress à l'abattage.

Ci-dessous sont décrits quelques exemples d'approches scientifiques utilisées pour comprendre les causes du stress et pour apporter des solutions concrètes.

Comprendre le point de vue de l'animal

Souvent, les opérations liées à l'abattage, telles que le transport ou l'attente en abattoir, sont décrites comme stressantes pour l'animal, mais ce qui importe est de comprendre quels aspects de ces opérations sont réellement stressantes du point de vue de l'animal. L'état émotionnel de l'animal dépend de ce qu'il perçoit de son environnement puis par l'interprétation qu'il en fait. Il est donc essentiel de comprendre comment l'animal perçoit et interprète son environnement pour pouvoir identifier les facteurs de stress et interpréter les réponses des animaux.

Au cours du processus d'abattage, les animaux sont soumis à de nombreux événements, simultanément et successivement, dans une série de contextes environnementaux différents [START_REF] Terlouw | Pre-slaughter conditions, animal stress and welfare: current status and possible future research[END_REF]Grandin 2013). Avant de partir pour l'abattoir, les animaux peuvent être rassemblés sur une plate-forme de chargement ou dans un enclos pour faciliter le chargement ultérieur dans le camion qui les transportera à l'abattoir. Avant le départ, la nourriture peut être supprimée pour éviter le mal des transports, pour des raisons pratiques ou pour des raisons financières. Les conditions de chargement, de transport et de déchargement dépendent des installations au départ (à la ferme) et à l'arrivée (à l'abattoir), ainsi que de la disposition du camion, du style de conduite et de la distance parcourue [START_REF] Cockram | Effect of driver behaviour, driving events and road type on the stability and resting behaviour of sheep in transit[END_REF]). Après le déchargement, les animaux peuvent attendre plusieurs heures, souvent toute la nuit, dans la zone de stabulation de l'abattoir. Lors des étapes finales du processus, l'animal sera conduit dans le box d'étourdissement, où il sera étourdi avant d'être saigné.

Ces événements peuvent être associés à différents facteurs de stress psychologiques ou physiques. Certains facteurs de stress, tels que la non familiarité de la situation, la présence humaine ou la perturbation du groupe social, ont une origine psychologique [START_REF] Terlouw | Pre-slaughter conditions, animal stress and welfare: current status and possible future research[END_REF]. La présence d'un groupe social stable est un aspect important de la vie normale des animaux de ferme et la séparation du groupe provoque du stress comme le montrent les vocalisations et l'augmentation du rythme cardiaque [START_REF] Boissy | Behavioral, cardiac and cortisol responses to brief peer separation and reunion in cattle[END_REF]Boissy et al., 2005). D'autres facteurs de stress, tels que la privation de nourriture, la fatigue ou la douleur, ont, eux, une origine physique. Ces formes de stress ont probablement aussi une composante psychologique. Par exemple, des vaches privées de nourriture depuis 30 h présentaient plus de réactions de peur à des situations anxiogènes que des vaches nourries normalement. Ceci montre que la faim, qui a une origine physique, influence l'état psychologique [START_REF] Bourguet | Effects of food deprivation on behavioral reactivity and physiological status in Holstein cattle[END_REF]. Certaines pratiques de transport et d'abattage donnent lieu à différents stress simultanément. Par exemple, le mélange des animaux avant le transport ou à l'abattoir peut s'accompagner d'interactions agressives entre les animaux pour établir une nouvelle hiérarchie de dominance. Elles provoquent ainsi des émotions négatives chez l'animal, dues à la fois à la peur (psychologique) et à la douleur causée par les lésions tissulaires engendrées par les combats (physique et psychologique, cf. section suivante).

Une fois les facteurs de stress conceptualisés, il est possible de les étudier séparément ou en les combinant. Ainsi, il a été démontré que les porcs sont plus stressés à l'abattage lorsqu'un humain avec lequel ils ont eu des expériences négatives auparavant est présent [START_REF] Terlouw | Repeated handling of pigs during rearing. II. Effect of reactivity to humans on aggression during mixing and on meat quality[END_REF]. Chez les bovins, grâce à des tests de réactivité réalisés au cours de l'élevage, nous avons pu identifier des individus qui sont plus stressés que la moyenne par la séparation d'avec le groupe de congénères ou le changement d'environnement. Nous avons ensuite montré que ces mêmes individus sont également plus stressés à l'abattage. Ceci indique que pendant l'abattage, le stress social et la non familiarité du contexte sont des sources de stress pour ces bovins. Les résultats montrent également qu'il est possible d'identifier pendant la période d'élevage les bovins qui seront les plus sujets au stress au moment de l'abattage [START_REF] Bourguet | Characterising the emotional reactivity of cows to understand and predict their stress reactions to the slaughter procedure[END_REF].

Connaitre le fonctionnement du cerveau

Comme précédemment indiqué, la douleur (Encadré 1) et la peur sont des sources fréquentes de stress à l'abattage. Pour limiter le stress, l'abattage se fait le plus souvent en deux étapes : une première consiste à étourdir l'animal pour provoquer une perte de conscience et la deuxième consiste à le saigner pour induire la mort. L'animal inconscient est insensible aux stimulations venant de son environnement : son cerveau n'est plus en état de traiter les informations sensorielles. L'induction de la perte de conscience a donc pour but d'empêcher que l'animal ne ressente des douleurs ou de la peur pendant la mise à mort. L'inconscience doit durer jusqu'à la mort de l'animal.

Le cerveau est un organe dont le fonctionnement est extrêmement complexe et la science est très loin de connaitre tous ses secrets. Il est cependant important de connaitre certaines bases neurobiologiques pour comprendre le fonctionnement des différentes méthodes d'étourdissement et pour pouvoir évaluer si l'animal a été correctement étourdi.

Encadré 1 : La douleur est une source de stress

La douleur peut être engendrée par des stimulations mécaniques, chimiques ou thermiques. Une rupture tissulaire provoque la libération de molécules qui sont normalement contenues dans les cellules et le sang et qui sont libérées suite à la lésion. Le système immunitaire libère également des substances dans la plaie, pour favoriser la cicatrisation. La douleur provoquée par des lésions est en partie liée à la présence de toutes molécules, c'est-à-dire par une stimulation chimique. De ces stimulations mécaniques, chimiques ou thermiques naît un message nerveux dit « nociceptif », c'està-dire porteur d'une information de la présence d'une nocivité. Il est transmis au cerveau où il atteint différentes structures, dont les cortex somesthésiques et les cortex limbiques. Les cortex somesthésiques permettent une interprétation du signal en termes de type de stimulation, de localisation et d'intensité. Les cortex limbiques sont impliqués dans les dimensions émotionnelle et cognitive du message nociceptif. On parle uniquement de douleur si les deux dimensions, sensodiscriminative (cortex somesthésiques) et émotionnelle (cortex limbiques), sont présentes. La sensation de douleur est donc une expérience sensorielle associée à une émotion négative. En raison de la dimension émotionnelle négative, la douleur est considérée comme source potentielle de stress. Comme l'interprétation du signal nociceptif est faite par le cortex, chez un animal inconscient il n'y a pas de perception de douleur car le cortex ne fonctionne pas.

Antonio Damasio a défini la conscience comme « un état d'esprit dans lequel il existe une connaissance de sa propre existence et de l'existence de son environnement » (Damasio, 2010). Ces connaissances nécessitent une intégration complexe des informations sensorielles (celles qui renseignent sur l'état du corps et sur l'environnement), des émotions (qui sont le moteur des motivations), et de la mémoire (où sont stockées des connaissances apprises). Cette association des informations fait appel aux processus de la conscience. Le cortex cérébral est la couche supérieure du cerveau, a une apparence plissée et joue un rôle central dans les processus impliqués dans l'expérience consciente. Les cortex visuel, auditif, somesthésiques, gustatif et olfactif sont les cortex primaires, recevant des informations provenant des sens. Leur rôle est de réaliser le premier décryptage des signaux sensoriels. Le cortex moteur primaire envoie des signaux aux muscles, permettant à l'animal d'agir face à ce qu'il a perçu. Mais avant de réagir, le cerveau doit intégrer toutes les informations, comme indiqué ci-dessus. C'est le rôle des cortex associatifs : ils interprètent les informations principales, les intègrent dans un contexte plus large et planifient les réponses appropriées. Le bon fonctionnement des cortex primaires et associatifs est nécessaire pour connaître, comprendre et donner un sens à ce qui est perçu, avoir une perception consciente de l'environnement et de soi [START_REF] Crick | A framework for consciousness[END_REF][START_REF] Laureys | Death, unconsciousness and the brain[END_REF]. L'absence de fonctionnement de l'ensemble du cortex est liée à un état d'inconscience.

La formation réticulée est située à la base du cerveau, dans le tronc cérébral (Parvizi et Damasio, 2001). Les projections issues de la formation réticulée et des structures proches activent le cortex lui permettant de fonctionner correctement et d'avoir une perception consciente : on les appelle le système d'activation réticulaire ascendant. Il joue un rôle essentiel dans le maintien de l'éveil et la défaillance du système d'activation réticulaire ascendant abolit la conscience (Terlouw et al., 2015a).

Le thalamus est situé au centre du cerveau. C'est un relais essentiel pour les informations visuelles, auditives, gustatives, somesthésiques et motrices entre le tronc cérébral et le cortex et vice versa. Aussi, des lésions importantes du thalamus abolissent également la conscience (Terlouw et al., 2015a).

Les techniques de l'étourdissement

Les bovins sont généralement étourdis par une tige perforante. Le principe est la percussion, associée à une destruction mécanique partielle du cerveau, notamment du thalamus et/ou de la formation réticulée. Chez les autres espèces (moutons, volailles et porcs), l'étourdissement électrique est le plus souvent utilisé. L'étourdissement électrique peut également être utilisé pour les veaux ou même les bovins adultes dans certains pays. Le principe est de faire passer un courant électrique d'intensité suffisante dans le cerveau, provoquant une dépolarisation massive des neurones dans les deux hémisphères cérébraux. Enfin, l'étourdissement au gaz n'est utilisé que par quelques abattoirs de porcs en France, mais l'est dans de nombreux abattoirs de porcs et de volailles dans d'autres pays européens. Le principe consiste à plonger les animaux dans une atmosphère contenant de fortes concentrations de CO 2 qui, après inhalation, se dissout dans le sang. La présence de forte concentration de CO 2 acidifie le sang, mais également le cerveau. Toutes ces techniques empêchent le bon fonctionnement des neurones dans le cerveau, y compris le cortex, et induisent ainsi un état d'inconscience. Celui-ci est réversible ou irréversible en fonction des techniques et paramètres utilisés. Après l'application de l'étourdissement, les animaux doivent être saignés dès que possible au niveau du cou (moutons, volailles, bovins) ou du thorax (porcs, bovins).

L'analyse de l'efficacité de l'étourdissement

Dans des conditions de terrain, l'efficacité de l'étourdissement peut être évaluée à l'aide de certains indicateurs : l'effondrement immédiat, l'absence du réflexe cornéen1 et l'absence de respiration rythmique sont les indicateurs de l'inconscience décrits comme parmi les plus fiables (Terlouw et al., 2015a). Lorsque le réflexe cornéen est présent, l'animal n'est pas nécessairement conscient. En revanche, son absence est considérée comme le signe d'un état d'inconscience. L'utilisation exacte de ces indicateurs dépend de la technique de l'étourdissement utilisée. D'autres signes ont un pouvoir discriminant moins forts mais peuvent également être utilisés dans certain cas, comme la rotation oculaire ou les nystagmus (oscillation rapide du globe oculaire). D'autres signes ou mouvements sont peu fiables. Par exemple, après l'étourdissement, les bovins présentent souvent des pédalages et des mouvements du cou. Nous avons conduit deux études pour savoir si ces mouvements et réactions peuvent être utilisés comme indicateurs de présence de conscience (Terlouw et al., 2015b). Les résultats montrent que les pédalages et mouvements du cou post-étourdissement étaient présents chez la majorité des animaux sans lien avec l'efficacité du tir. Chez un groupe de bovins profondément inconscients suite à l'étourdissement, nous avons sectionné la moelle épinière. Les mouvements étaient toujours présents, c'est-à-dire même lorsque le cerveau était déconnecté du reste du corps. Ces résultats montrent que ces mouvements sont possibles en l'absence de connexion entre le cerveau et la moelle épinière et qu'ils dépendent donc de circuits nerveux impliquant la moelle épinière (mouvements réflexes), mais pas le cerveau. Il faut en conclure que la simple présence de pédalages n'est pas indicatrice d'un mauvais étourdissement. Ce sont des mouvements involontaires liés aux centres générateurs de rythme localisés dans le tronc cérébral et dans la moelle épinière (Terlouw et al., 2015b). Ces centres permettent la production de mouvements moteurs rythmiques, tels que la marche [START_REF] Guertin | The mammalian central pattern generator for locomotion[END_REF][START_REF] Frigon | Central Pattern Generators of the Mammalian Spinal Cord[END_REF] et leur fonctionnement est indépendant de la conscience. Cependant, il ne faut pas inverser le raisonnement, car un animal mal étourdi présentera aussi des pédalages, en raison de son état de conscience.

Dans cette étude, certains animaux ont présenté un mouvement du cou dans la direction ventrale en réponse à la coupure de la peau et des vaisseaux sanguins lors de la saignée. Cette réaction était plus faible chez les animaux après des intervalles d'étourdissement / saignée plus longs. Cette réaction est une réponse réflexe nociceptive (c.-à-d. engendré par une stimulation nocive) basée sur un circuit neural qui traverse la moelle épinière, mais pas le cerveau. La réponse plus forte chez les animaux saignés plus tôt après l'étourdissement indique que les nerfs impliqués dans la réponse arc-réflexe avaient toujours un certain degré de fonctionnalité. Quand l'animal est inconscient, la nociception génère le mouvement réflexe du cou sans pour autant que l'animal ressente de la douleur puisque le cerveau ne fonctionne pas (encadré 1). La réaction à la coupure de la peau et des tissus ne peut donc pas être utilisée comme indicateur de conscience.

Toutes ces connaissances ne présentent pas de solutions concrètes en soi, mais permettent de donner des réponses claires aux questions qui sont posées. Elles ont aidé à avoir des approches pratiques et pragmatiques pour trouver des solutions à des problématiques du terrain, qui concernent l'ensemble des acteurs : les industriels, les vétérinaires et les chercheurs qui accompagnent les abattoirs sur le terrain et les autorités politiques.

Conclusion

Il est inévitable que le sujet de l'abattage éveille des sentiments forts, sur la protection des animaux, la religion, la liberté, le bien-être, la consommation, l'argent. Les échanges entre les chercheurs, les ONG, les représentants politiques, les éducateurs, les philosophes, les industriels, les autorités religieuses, les exploitants, les juristes, les éleveurs, les étudiants et les journalistes aident chacun à mieux comprendre les nombreuses questions essentielles qui entourent le sujet. La science joue un rôle particulier dans le débat. La plupart du temps, la simple description factuelle de ce qui cause le stress chez les animaux, le fonctionnement des différentes techniques d'étourdissement et, plus généralement, de ce que nous savons aujourd'hui et des questions sur lesquelles plus de connaissances scientifiques sont nécessaires, permet de dépassionner les débats et facilite des discussions fructueuses. Les nombreuses questions, éthiques, religieuses et philosophiques, restent cependant difficiles à résoudre. Souvent, des critères communs sont difficiles à établir, mais la science peut aider à identifier des points de départ communs, pour lesquels un consensus existe. Un exemple est qu'une grande majorité de personnes s'accorde pour dire que la douleur chez les animaux devrait être évitée; ceci pourrait être un objectif commun à tous les acteurs.

Le réflexe cornéen est la réponse immédiate et involontaire de l'oeil (fermeture de la paupière), provoquée par l'effleurement de la cornée (partie antérieure et centrale de l'oeil, translucide, laissant voir l'iris et la pupille).

Remerciements

Je remercie vivement Dr Raphaëlle Botreau pour sa relecture et ses commentaires pertinents sur une version antérieure de ce texte.