RESEARCH ARTICLE Effects of Freely Accessible Computerized Test Systems on the Spontaneous Behaviors and Stress Level of Guinea Baboons (Papio papio)

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1 American Journal of Primatology 9999:1 9 (2013) RESEARCH ARTICLE Effects of Freely Accessible Computerized Test Systems on the Spontaneous Behaviors and Stress Level of Guinea Baboons (Papio papio) JOËL FAGOT 1,2 *, JULIE GULLSTRAND 1,2, CARALYN KEMP 2, CÉLINE DEFILLES 3, AND MOURAD MEKAOUCHE 3 1 Laboratory of Cognitive Psychology, CNRS and Aix Marseille University, Marseille, France 2 Brain and Language Research Institute, Marseille, France 3 Center for Research in Neurobiology and Neurophysiology of Marseille, CNRS and Aix Marseille University, Marseille, France Fagot and Paleressompoulle [Fagot and Paleressompoulle (2009) Behav Res Methods 41: ] described a new automated learning device for monkeys (ALDM) to test the cognitive functions of nonhuman primates within their social groups. However, the impact of the ALDM procedure on animal well being needs to be investigated. The present study assessed the consequences of ALDM testing on the behavioral repertoire of Guinea baboons (Papio papio) and their stress levels as inferred from measurements of saliva cortisol. Accessibility to ALDM test computers reduced the number of resting periods as well as the number of stereotypies. Lower cortisol levels were also found during ALDM testing. These findings and others demonstrate that ALDM testing has a positive impact on animal wellbeing and can be considered as a means for behavioral enrichment in captive primates. 9999:1 9, Wiley Periodicals, Inc. Key words: primate; cognition; welfare; automatic testing INTRODUCTION During recent decades, efforts have been made towards improving the housing and care of nonhuman primates in laboratories. These efforts reflect the growing concerns of the scientific community and general public for animal well being, as well as enhanced governmental requirements regarding laboratory practices [e.g., Jennings et al., 2009; Rennie & Buchanan Smith, 2006]. Several approaches have been undertaken with the general goal of reducing stress and number of abnormal behaviors observed in laboratory animals, and to enhance flexibility in animal husbandry and reliability in the collection of scientific data. To improve well being in nonhuman primates in laboratories, a major approach has been to enrich their living conditions with physical or social enrichments. Certain types of inanimate environmental enrichments have been shown to positively affect the behavior of laboratory primates. For example, by providing opportunities to forage for artificial fruits, Bayne et al. [1992] showed well being benefits in rhesus monkeys (Macaca mulatta) and the reduction of abnormal behaviors, while Renquist & Judge [1985] demonstrated that the provision of climbing structures or manipulable objects encourage a rich repertoire of behaviors in a variety of nonhuman primate species. Social housing is also known to be one of the best enrichment strategies for captive monkeys, allowing them the opportunity to display a richer repertoire of social behaviors which they cannot express when housed in isolation [Schapiro et al., 1996]. Furthermore, individuals socially housed (in pairs or in groups) display improved immune responses over their solo conspecifics [Schapiro et al., 2000]. Positive reinforcement techniques, which are based on operant conditioning procedures, have also achieved wide recognition as a valuable tool for improving animal well being in laboratories [e.g., Prescott & Buchanan Smith, 2003]. These techniques have been mostly used to train animals to cooperate with experimental procedures, that is, to accept injections [Priest, 1991] or to be transferred from the home cage to the test cage by way of poles or transport cages [see Laule et al., 2003]. Using positive Contract grant sponsor: EU Euprim Net II project; contract grant number: #RII ; contract grant sponsor: Fédération de Recherche 3C; contract grant sponsor: Provence Regional (PACA) Council (Volet Exploratoire 2008) Conflicts of interest: None. Correspondence to: Joël Fagot, Laboratory of Cognitive Psychology, CNRS and Aix Marseille University, 3 Place Victor Hugo, Bât 9, Case C, Marseille cedex 3, France. E mail: joel.fagot@univ-amu.fr Received 13 May 2013; revised 11 July 2013; revision accepted 22 July 2013 DOI: /ajp Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com) Wiley Periodicals, Inc.

2 2/Fagot et al. reinforcement techniques, Evans et al. [2008] were able to train capuchin monkeys (Cebus apella) to be voluntarily isolated from their groups to perform computerized tasks. In furthering laboratory animal welfare, we recently developed an experimental device for studying cognition in Guinea baboons (Papio papio) in our laboratory [Fagot & Bonté, 2010; Fagot & Paleressompoulle, 2009]. Briefly, the automated learning device for monkeys (ALDM) allows free and voluntary access to touch screen computers, displaying tasks that subjects can choose to solve. This approach seems beneficial for the animals as capture, relocation, and isolation of the monkeys is not required to run the experimental protocols. Moreover, the monkeys can maintain visual, auditory, and even tactile contact with conspecifics at all times, and are therefore not deprived of social interactions. Recent studies have revealed the benefits and efficiency of the ALDM procedure. As the monkeys have voluntary access to the test system, they perform trials at a high frequency (hundreds to thousands of trials per day), pay close attention to the task, and consequently, demonstrate excellent performance on cognitive tasks with a high level of complexity [see Fagot & Bonté, 2010]. The recent demonstration that P. papio can solve analogical reasoning tasks [Fagot & Maugard, in press; Fagot & Thompson, 2011] and can discriminate words from non word categories of four letter strings [Grainger et al., 2012] are clear illustrations of the procedure from a scientific standpoint. Studies from our and other research groups have suggested that computerbased tasks might serve as efficient environmental enrichments for monkeys and apes. For instance, Washburn & Rumbaugh [1992, 1998] showed that the number of stereotypic and aggressive behaviors decreased in rhesus monkeys (M. mulatta) as a consequence of computerized testing. In the same species, Platt & Novak [1997] reported a higher rate of activity in socially and individually housed animals which could use video games. More recently, Yamanashi & Hayashi [2011] compared the activity budget of experimental chimpanzees (Pan troglodytes) participating in computer controlled cognitive tasks to that of wild and captive groups of chimpanzees with no access to such tasks. During computerized testing, the feeding and resting times of the subjects were roughly identical as those of the wild chimpanzees during the experimental days. However, these two components of the budget time were more similar to those of captive chimpanzees when access to computers was prevented. In addition, baboons with access to the computers display a significant increase in activity levels [Fagot & Bonté, 2010], but the effect of the ALDMs on well being needs to be established more firmly. In this context, the aim of the present study was to measure the effect of ALDM systems on the baboons spontaneous behaviors and stress levels. To achieve this goal, we combined and compared behavioral and physiological measures of stress in two conditions: when baboons had access to the ALDM test systems and when these systems were closed and thus inaccessible. Our first goal was to describe to which extent the time budget of the baboons can be affected by their use of the ALDM test systems. Using scan sampling observational methods, we quantified their activity in the two test conditions regarding six main categories of social and non social behaviors. During that process, special attention was paid to the abnormal behaviors known to be indicators of poor well being and stress, such as self directed behaviors [Castles & Whiten, 1998; Maestripieri et al., 1992] and stereotypies [Mason, 1991]. Our second goal was to obtain physiological indicators of animal well being during testing. For that purpose, we collected saliva samples from the baboons and analyzed the level of cortisol they contained. In primates, including baboons [e.g., Papio hamadryas: Taranov & Goncharov, 1981; Papio anubis: Sapolsky, 1982], the hormonal system generates a cortisol spike after a stressful event. Cortisol level is therefore a physiological measure that can be used to infer stress in experimental conditions. That spike inthecortisollevel canbemeasured inblood [e.g., M. mulatta: Maestripieri et al., 2008; P. anubis: Sapolsky et al., 1997], feces [Bergman et al., 2005], urine [e.g., Callithrix kuhlii: Smith & French, 1997], saliva [e.g., M. mulatta: Lutz et al., 2000; P. hamadryas: Pearson et al., 2008], and even hair samplings [e.g., M. mulatta: Davenport et al., 2006]. In the current study, we trained baboons with positive reinforcement techniques to chew a cotton rope in order to collect and measure the saliva cortisol. This procedure was favored as it avoided stress induced by the sampling procedure that may affect baseline measurements. METHODS Subjects and Housing The participants were nine male Guinea baboons (P. papio) ranging from 2 to 27 years old (Table I), living within the Rousset sur Arc CNRS primate Center (France). Only males were considered as subjects because of known cortisol variations in females, such as when they are gestating or lactating [Weingrill et al., 2008], or in estrus periods [Pearson et al., 2008]. The male baboons were housed in three different social groups in separate enclosures. Four baboons (aged 2 12 years) were housed in a group of 30 individuals (hereafter referred to as Group_30 ). This group included other males, females, and their offspring of varying ages. Group_30 was housed in a 25 m 30 m outdoor enclosure connected by tunnels to indoor housing (6 m 4 m) used at night. Four other subjects (5 7 years old) were housed together (Group_4) in a 4.7 m 6.4 m outdoor enclosure

3 Computerized Testing and Welfare in Baboons /3 TABLE I. Name and Age in Years of the Participants in Each Social Group Name Age Group_30 PIP 12.9 VIV 7.7 DAN 4.3 FEL 2.1 Group_4 ART 6.7 BAR 6.0 CLO 5.4 CAU 5.3 Group_1 B connected to indoor housing (2 m 4 m). Finally, our last subject (27 years old; Group_1) was housed alone (in a facility similar to Group_4) as a consequence of social incompatibility with other group members. All groups had visual and auditory contact with each other. Water was provided ad libitum within each enclosure. Feeding (fruits, vegetables, and monkey chows) occurred once daily at 5 PM in order to limit interference with computerized testing. All baboons had free access to one or several ALDM computerized test systems, which were installed in trailers adjacent to the outdoor enclosures. The members of Group_30 had simultaneous access to a battery of 10 ALDM systems. Group_4 had access to three such systems and the single based subject had access to one test system. The ALDM test systems have been described in Fagot & Bonté [2010] and Fagot & Paleressompoulle [2009]. Briefly, each ALDM test system consists of an operant conditioning test chamber equipped with a 19 in. touch screen, a food dispenser and a radio frequency identification (RFID) reader that identifies each baboon via a microchip implanted in each arm. All research conducted adhered to the American Society of Primatologists Principles for the Ethical Treatment of Nonhuman Primates and received approval from Provence Alpes Côte d Azur ethics committee for animal experimental research. Computerized Task A test program, written with E Prime Version 2 professional, uses the subjects identity to determine its last stopping point in the sequence of trials, and assigns the independent variables to be experienced by each subject during the trial. During the present study, the task presented to the subjects was a visual search requiring selection of a T shape target presented among seven L shape distracters. The baboons received a food reward when they successfully completed a trial or received a three second time out otherwise. Experimental Design The study was conducted over a period of 16 days in July On each day, behavioral observations (see procedure below) were conducted using the scan sampling method over three 30 min periods, during which behavioral data were recorded each minute on each of the nine subjects. Observation periods started at 9 AM, 11 AM, and 2.30 PM. Cortisol levels are known to follow a robust circadian rhythm and to decrease during the day [e.g., Kirschbaum & Hellhammer, 2000]. In addition, plasma cortisol due to punctual stress starts to transfer into saliva within 2 3 min, and the saliva cortisol peaks are observed approximately min after the event began [Kirschbaum & Hellhammer, 2000]. Due to these timelines, saliva was collected immediately after each scan sampling period (i.e., 9.30 AM, AM, and 3 PM; see the procedure below). This timing for saliva collection ascertained that the cortisol levels measured for each sampling reflected the stress level of the subject during the preceding period of behavioral observations. Saliva samples and behaviors were collected in two test conditions: when the ALDM systems were accessible (Open condition) and when access to these test systems was denied (Closed condition) by closing the door to each ALDM test system. For all baboons, the experiment was organized in two periods of recording following an ABBA BAAB design. In this design, the A condition indicated daily access to the test systems and the B condition indicated inaccessibility. Therefore, for each baboon, behavioral and cortisol measurements were collected during a total of 16 days (8 days in the Open condition and 8 days in the Closed condition). Due to laboratory management constraints, breaks of 2 4 days were introduced between the two ABBA and BAAB test periods. During these breaks, the monkeys had permanent access to the test computers but data were not recorded. The opening and closing of the ALDMs occurred at 5 PM, immediately after the monkeys were fed. Closing and opening the test system at that period had the advantage to limit the stress of the subjects, which were occupied eating in the enclosure. Procedure for the Recording of Behavioral Data Within the Enclosures Six behavioral categories were recorded: Resting (e.g., inactive, sleeping), Self directed behaviors (e.g., scratching, self grooming), Social behaviors (e.g., grooming, social playing, conflicts), behaviors directed towards an Object (e.g., exploration, foraging), Locomotion (e.g., walking, running), and any Other behavior that are not been listed in the previous categories. Occurrences of stereotypic behavior were scored as a subclass of the Other category. These behaviors were rare but included pacing, rhythmic jumping, and rocking. Whenever

4 4/Fagot et al. locomotion was observed, the behavior was considered as a being stereotypic when it was rhythmic and repetitive; for instance, during pacing. Non rhythmic, purposeful locomotion was otherwise classified within the general Locomotion category. No behavior was recorded when the monkey was in the ALDM test system; instead, the monkey was coded as being not visible. Use of the ALDMs was only time that the baboons could be out of view. In each session, a minimum of three observers simultaneously encoded (by hand) the behaviors, one observer per social group. On a regular basis, a fourth observer also double encoded the behaviors in one of these the groups in order to assess the reliability of the coding procedure. The group assigned to each observer rotated on a daily basis. Altogether, twentyfour 30 min sessions were double encoded during the experiment resulting in a total of 2,160 behaviors. Cohen s kappa coefficients were computed to measure inter rater agreements. These tests revealed excellent inter observer agreements (mean K ¼ 92.0, range ). Procedure for the Collection and Analysis of Saliva Samplings A device inspired from Lutz et al. [2000] and Pearson et al. [2008] was used to collect the saliva. It consisted of a 1 m hollow length of metal piping, with a 5 cm piece of cotton rope (Salimetrics Europe Ltd., Suffolk, UK) tied at the end of the tube. The cotton rope was flavored with a solution of Invisible Kool Aid (Kraft Food, Northfield, IL; two parts Kool Aid for one part water), in order to attract the baboons and elicit salivation. The Kool Aid did not alter the assay and therefore did not interfere with the measurement of cortisol levels [e.g., Lutz et al., 2000]. To collect the saliva, the experimenter introduced the end of the pipe into the enclosure. The nine baboons were trained to approach the tube and to lick the cotton rope without biting it, while avoiding touching it with their hands. The sweetness of the Kool Aid made the pole attractive to the monkeys. If a non focal baboon approached the tube, or when one of the subjects moved to touch the tube with their hand, the device was gently removed. This extinction procedure proved efficient as, by the end of the training period, only the focal animals licked the rope and used only their mouth to touch it. The four subjects from each of Group_30 and Group_4 were sampled simultaneously by the four experimenters. Only one experimenter collected the saliva of the isolated male in Group_1. When the cotton rope was sufficiently damp with saliva, it was removed from the sampling device and the portion licked by the baboon was cut and placed in a labeled swab storage tube (Salimetrics Europe Ltd.). Immediately after collection of the saliva, the storage tubes were centrifuged at 3,500 rpm for 15 min. The saliva samples were then divided into aliquots of 100 ml and stored at 80 C until assayed. Assays were conducted at Hopital Nord, Faculté de Médecine (Marseille, France) using commercially available Enzyme Immuno Assay kit (Salivary cortisol EIA Kit; Salimetrics Europe Ltd.). Each sample was analyzed as duplicates. On average, 480 ml (SD ¼ 267 ml) of saliva was collected from each sample. This volume was sufficient for analyses as a minimum of 75 ml was required for measuring the cortisol level. The absorbance was read at 450 nm using a plate reader (Mithras, Berthold Technologies, Germany), strictly following the instructions provided in the EIA Kit to determine cortisol levels. RESULTS Use of the ALDM Test Systems Results obtained in the computerized task are not presented here, as this research was concerned with the effect of the ALDM system on well being [for a detailed description of the computerized task and results, see Goujon & Fagot, in press]. Note, however, that the baboons performed an average of 1,520 trials per day (range 614 2,354 trials) during the study period. As inferred from time recorded data, the conduction of these trials were organized as numerous series of short runs of 3.13 min on average (SD 3 sec), which corresponded to an average cumulative use of the test systems of 177 min per day (range min). These long cumulative periods of interactions with the test computers suggest that the ALDM systems are a very effective form of environmental enrichment. As a burst of activity occurred immediately after the ALDMs were opened, the description of the use of the ADLM systems needs to distinguish the activity of the baboons during the first day after reopening from the behavior observed during the following day, depending on the ABBA procedure, when the baboons had access to the ALDMs. This activity is presented in Figure 1. During the day following the opening of access to the ALDM systems (i.e., from 6 AM to 5 PM), the activity of the baboons showed a unimodal curve that peaked around 12 PM (see Fig. 1). However, the numbers of trials performed with the computers between 6 AM and 5 PM did not differ between the first and second days after the opening [mean first day ¼ 1,112 trials (SD 89); mean second day ¼ 1,044 trials (SD 90)]; one way analysis of variance (ANOVA), F (1,8) ¼ 0.65, ns. Therefore, the analyses of the spontaneous behaviors and cortisol levels rejected Day as a factor. Spontaneous Behaviors Within the Enclosure The number of observations was, of course, smaller in the Open than in the Closed condition,

5 Computerized Testing and Welfare in Baboons /5 Fig. 1. Number of trials performed on average by the nine baboons in the Open condition, during the first day immediately after ALDM reopening (solid circles) and the second day of reopening (lined circles). because the monkeys were out of view at times during their interactions with the ALDMs in the Open condition. One tailed t tests confirmed that the mean number of observations in the Open periods were statistically lower than 30 (i.e., maximum number of observations for each individual in each scan period) in the three sampling periods (9 AM: mean ¼ SD 1.45, t (8) ¼ 6.43, P ¼ ; 11 AM: SD SD 1.23, t (8) ¼ 7.69, P < ; mean 2.30 PM ¼ SD 1.08, t (8) ¼ 7.62, P < ). As the number of observations differed reliably in the Open and Closed conditions, we computed the total number of occurrences of each behavioral category (Resting, Selfdirected, Social, Object, Locomotion, and Other) obtained in each test condition and converted these values to proportions. Data obtained in the Open and Closed conditions were then compared using a twotailed test for significance of difference between the two proportions [Bruning & Kintz, 1987]. The P value was Bonferroni corrected in these tests and adjusted to P ¼ to avoid spurious positive results due to the large number of tests (n ¼ 54). Wilcoxon signedrank tests (P < 0.05) were computed to compare the mean proportions obtained for each behavioral category in the two test conditions. Table II reveals remarkably consistent results across the individuals. For the Resting category, six of the baboons showed reliably fewer resting behaviors in the Open than in the Closed condition. This effect was reliable at the group level (Wilcoxon signed rank test: Z ¼ 0, P ¼ , N ¼ 9). The opposite result was obtained for the category Locomotion. Only two baboons showed reliably more instances of Locomotion in the Closed condition. However, as most of the baboons showed more instances of Locomotion in the Open condition, this effect emerged as significant at the group level (Table II, Wilcoxon signed rank test: Z ¼ 32, P ¼ , N ¼ 9). No significant differences emerged at the group level for the four remaining behavioral TABLE II. Proportion of the Six Main Behavioral Categories for Each Individual and Test Condition (Open vs. Closed) Resting Self Social Object Locomotion Other Open Closed Open Closed Open Closed Open Closed Open Closed Open Closed PIP VIV DAN FEL ART BAR CAU CLO B Mean Wilcoxon P ¼ P ¼ P ¼ P ¼ P ¼ P ¼ For each baboon and behavioral category, reliable differences between the test conditions are indicated in bold. They were inferred from Bonferroni corrected tests of differences between two proportions. The bottom two lines report the grand means in each test condition, and the results of Wilcoxon signed rank tests comparing these group means for each behavioral category.

6 6/Fagot et al. categories (Self: Wilcoxon signed rank test: Z ¼ 15, P ¼ 0.426, N ¼ 9; Social: Wilcoxon signed rank test: Z ¼ 32, P ¼ 0.301, N ¼ 9; Object: Wilcoxon signedrank test: Z ¼ 37, P ¼ 0.098, N ¼ 9; Other: Wilcoxon signed rank test: Z ¼ 7, P ¼ 0.074, N ¼ 9). However, when reliable differences emerged at the individual level, they corresponded to a reduced proportion of Self directed behaviors (significant in one individual) and to an increased proportion of Social behaviors (significant in three individuals) and Object orientated behaviors (significant in five individuals). In short, these findings suggest that the use of the ALDM test system tended to reduce the number of Resting and Self directed behaviors but increased the number of Social, Object, and Locomotion orientated behaviors. During the observation sessions, we observed eight social conflicts at the group level in the Open condition and seven in the Closed condition, suggesting that the closing or opening of the test systems did not induced social conflicts. Only four instances of agonistic behaviors were captured with our scanning procedure. That number precluded conclusions on how these behaviors varied as a consequence of ALDM testing. Concerning the frequencies of abnormal behaviors, the four males of Group_30 were never observed to exhibit stereotypies. However, a total of 51 occurrences of stereotypies were observed in Group_4; these were expressed by only three of the baboons. A comparison of the individual frequencies revealed significantly fewer stereotypic behaviors when the ALDM systems were available than when the baboons were denied access (ART: Open ¼ 1, Closed ¼ 30, Binomial test, P < 0.05; BAR: Open ¼ 0; Closed ¼ 17, P < 0.05; CLO: Open ¼ 0, Closed ¼ 3, not testable due to small sample size). The single baboon of Group_1 was observed performing a greater number of stereotypic behaviors during the study than the other baboons (n ¼ 245); however, no difference was found between the Open and Closed conditions (Open ¼ 127, Closed ¼ 118, two tailed binomial test, ns). stress levels (after a conflict: mean 9.30 AM ¼ 1.24 ml SD 0.14, n ¼ 14; mean AM ¼ 1.36 ml SD 0.37, n ¼ 8; mean 3 PM ¼ 1.34 ml SD 0.17, n ¼ 15; without conflict: mean 9.30 AM ¼ 0.79 ml SD 0.06, n ¼ 130; mean AM ¼ 0.68 ml SD 0.06, n ¼ 136; mean 3 PM ¼ 0.62 ml SD 0.04, n ¼ 129). As this variation in cortisol level could affect our baseline measurements in the Open and Closed conditions, the saliva samples obtained after a social conflict were disregarded for statistical analyses. Mean cortisol levels are reported in Figure 2. For each subject, Mann Whitney U tests (P < 0.05) compared the cortisol levels in the Open and Closed conditions, independently for each sampling period (9.30 AM; AM, and 3 PM). As shown in Figure 2, these tests revealed very few reliable differences at the individual level (but see FEL in all three sampling periods and DAN at 3 PM), although most Salivary Cortisol Level We collected and analyzed 48 saliva samples for each baboon (24 in each of the Open and Closed conditions). As cortisol levels may increase drastically after a social conflict [e.g., Pearson et al., 2008], we separately considered the saliva samples (N ¼ 37) obtained immediately after a 30 min observation period during which social conflicts were observed within the social group (only seven social conflicts were observed during the study in the Closed condition and eight in the Open condition). As expected, cortisol levels were much higher in these samples than in absence of conflicts, thereby confirming the efficiency of our procedure to measure Fig. 2. Mean cortisol levels in the Open and Closed condition for each baboon and sampling period. Reliable differences between the two conditions as inferred from Mann Whitney U tests (P < 0.05).

7 Computerized Testing and Welfare in Baboons /7 of the subjects expressed higher cortisol levels in the Closed than in the Open Condition (9.30 AM: 7 out of 9; AM: 8 out of 9; 3 PM: 5 out of 9; see Fig. 2). Cortisol levels were also analyzed at the group level with a two way ANOVA, considering the test condition (Open vs. Closed) and the sampling period (9.30 AM, AM, and 3 PM) as the two main factors. This analysis revealed that the main effect of test condition was reliable (F (1.8) ¼ 7.40, P ¼ 0.026). On average, salivary cortisol levels were lower when the ALDM systems were accessible (mean Open ¼ 0.59 mg/dl; mean Closed ¼ 0.81 mg/dl; Fig. 3). The cortisol levels declined slightly from the morning to the afternoon (mean 9.30 AM ¼ 0.79 mg/dl; mean AM ¼ 0.69 mg/dl; mean 3 PM ¼ 0.62 mg/dl), but the main effect of sampling period did not emerge as significant (F (2.16) ¼ 0.429, P ¼ 0.12). Interestingly, the effect of test condition by sampling period approached significance (F (2.16) ¼ 2.224, P ¼ 0.06). This interaction is illustrated in Figure 3. Cortisol levels declined strongly during the day when the test systems were inaccessible. This effect was reduced in amplitude when the baboons could interact with the ALDM test systems. An independent one way AN- OVA confirmed that cortisol levels were significantly lower at 9.30 AM (F (1,8) ¼ 6.79, P ¼ 0.031) and AM (F (1,8) ¼ 7.26, P ¼ 0.027) when the ALDMs were accessible (in comparison to inaccessibility), but failed to reveal reliable differences between the conditions at 3 PM (F (1,8) ¼ 2.48, ns.). DISCUSSION In the current study, the baboons demonstrated a high motivation to interact with the test computers. They performed an average of 1,500 trials per day in the Open condition and used the test systems an average of approximately 3 hr. With such a high motivation to interact with the ALDM computers, the question arises as to what extent ALDM testing affects the general behavior of the baboons, and if the ALDM test system has an overall positive or negative Fig. 3. Mean cortisol levels (and standard errors) of the nine baboons in the Open and Closed condition and for each sampling period. impact on well being. The current study combined behavioral observations and measures of cortisol levels to address this issue. Table II revealed that the access to test systems had some effect on the overall time budget, but this was relatively small in amplitude. Indeed, the most salient effect corresponded to a reduced frequency of Resting behaviors in the Open condition of 14% from those observed in the Closed condition (see values in Table II). Similarly, the number of Locomotion behaviors increased statistically in the Open condition from the number observed in the Closed condition. However, the difference between these two conditions was only 4.38% (see Table II). Together, these findings demonstrate that the use of the ALDMs does not drastically alter the time budget of the participants, and that they continue to express a rich behavioral repertoire when the ALDM systems are accessible. To further infer the potential positive or negative effects of ALDM testing, it is important to investigate how the proportion of each category varied in the two test conditions. On average, use of the ALDMs significantly reduced the proportion of Resting behaviors (see Table II), demonstrating that the baboons were more active in their enclosure when they had access to the test systems. Alternatively, findings indicate that ALDM testing increased the frequency of the three main behavioral categories Locomotion (reliable at the group level), Social behaviors (reliable in DAN, FEL, and CLO), and Object (reliable in VIV, DAN, BAR, and CAU but see FEL, see Table II). In nonhuman primates, stress is often indicated by a reduction in overall activity level in the social as well non social domains [e.g., Marriner & Drickamer, 1994]. The pattern of behavioral modifications obtained when the ALDM systems were accessible, corresponding to an increased activity level, strongly suggests a positive effect of ALDM testing on stress level. Only the individuals in Group_4 and Group_1 exhibited stereotypies. In the former group, stereotypies were most often observed when access to the ALDM was prevented, but virtually disappeared when the ALDM systems were accessible. This finding further confirms the positive impact of ALDM testing from the standpoint of animal wellbeing. The individual of Group_1 showed the largest numbers of stereotypies which could be a consequence of social isolation [Mason, 1991]. For that baboon, ALDM testing had no reliable effect on the number of stereotypies; we presume that it is due to more deeply ingrained stereotypies in this older subject. Note, however, that access to the ALDM reduced the number of occurrences of resting periods for this subject, suggesting that the ALDM was beneficial, even if it had no detectable consequence on stereotypies.

8 8/Fagot et al. The final advantage of this study is in the provision of quantitative measures of saliva cortisol levels in our two test conditions. In primates [e.g., P. anubis: Sapolsky, 1982], the hormonal system generates cortisol spikes as a consequence of stressful events. Cortisol level is thus a biological indicator of stress levels in experimental conditions. The current study replicated the well known finding that cortisol levels are at their maximum in the morning and decline progressively during the day [Kirschbaum & Hellhammer, 2000]. This sole finding validates the procedure used to analyze the assays. Comparison of the saliva cortisol levels in the Open and Closed conditions revealed a main effect of test condition corresponding to lower cortisol levels when the ALDM systems were accessible. In humans, stressors have greater effects on cortisol levels when they occurred in the morning, at a time of high circulating levels of corticosteroids, rather than in the afternoon [Maheu et al., 2005]. This finding was replicated in our study, as revealed by greater differences of mean cortisol levels in the two conditions for the 9.30 AM samplings. This difference between the two test conditions decreased during the afternoon, possibly due to floor effects limiting possible variations. In our laboratory, the action of closing the ALDM test system is a daily routine used, for instance, to clean the test chambers. This action was therefore not a stressful event in itself that may account for an increased cortisol level in the Closed condition. More likely, access to the test system had a positive impact on the general stress of the subjects because they had access to a larger palette of potential behaviors. A lower cortisol level in the Open condition is a decisive demonstration that ALDM testing has the main advantage to reduce baseline stress levels in the participants. Early studies have already underlined several advantages of ALDM testing for scientific purposes [see Fagot & Bonté, 2010; Fagot & Paleressompoulle, 2009]. These advantages include, among others, the possibility to efficiently collect large volumes of data in a substantially larger number of subjects than with more traditional tests, as well as the possible consideration of the social and non social aspects of cognition permitted by the testing of individuals in social groups. In this context, the current study reveals that ALDM testing induced a coherent pattern of behavioral (e.g., increased activity level) and physiological (i.e., salivary cortisol) modifications suggestive of improved animal well being. We conclude from these findings that ALDM testing might also be conceived as a means for behavioral enrichment in captive primates. ACKNOWLEDGMENTS This study was conducted at the CNRS Primate Center, Rousset sur Arc, France. The authors thank the staff of the CNRS Primate Center (Roussetsur Arc, France) as well as Auryane Lharidon, Benoît Rosay, Laetitia Ruppé, and Quentin Guillory for technical support. More general support was also provided by the Fédération de Recherche 3C as well as a research grant to J.F. (Volet Exploratoire 2008) from the Provence Regional (PACA) council. REFERENCES Bayne K, Dexter S, Mainzer H, McCully C, Campbell G, Yamada F The use of artificial turf as a foraging substrate for individually housed Rhesus monkeys (Macaca mulatta). Anim Welf 1: Bergman TJ, Beehner JC, Cheney DL, Seyfarth RM, Whitten PL Correlates of stress in free ranging male chacma baboons (Papio hamadryas ursinus). Anim Behav 70: Bruning JL, Kintz BL Computational handbook of statistics. 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