Is it for real? Structural differences between play and real fighting in adult chimpanzees (Pan troglodytes)

In primates, as well as in other mammals, play fighting (PF) is a complex form of playful activity that is structurally similar to real fighting (RF) and may also be used in a competitive way. Here, we verify the structural key differences that can distinguish PF from RF in adult chimpanzees (Pan troglodytes). We collected 962 h of video recording on 30 adult individuals belonging to four chimpanzee groups (Mona Chimpanzee Sanctuary, Spain; La Vallée des Singes and ZooParc de Beauval, France). We applied different indices—two of which were borrowed from the ecological measures of biodiversity—to test for structural differences between PF (345 sessions) and RF (461 sessions) in the levels of behavior repetition (Repeatability of Same Behavior Index, RSBI), distribution uniformity (Pielou Index, J), variability (Shannon Index, H′) and, symmetry (i.e., reciprocal exchange of offensive/defensive behaviors; Asymmetry Index, AI). Moreover, we compared the session duration between PF and RF. We found that duration and RSBI were higher in PF than RF while AI was higher in RF than PF. No difference was found between J and H′. Interestingly, both females and males maintained similar ranking positions (determined via Normalized David's scores) in RF and PF. Our study indicates that session duration, behavior repetition, and symmetry can be distinctive structural key features of PF whereas dominance role‐reversal, behavior variability, and distribution uniformity were not. PF in adult chimpanzees may have elements of serious contexts (e.g., absence of role‐reversal as in RF) which is in line with the view that play is a blended, multifunctional behavior deriving from the re‐combination of different behavioral systems. Our findings highlight the need to investigate play structure and manifestation in a nuanced way to better understand the actual motivation that underlies what appears to be play.


| INTRODUCTION
During play in primates and other animals, motor patterns from diverse behavioral domains can be recruited and combined in different ways (Fagen, 1981;Pellis & Burghardt, 2017). For this reason, it has been proposed that play may be a specific and independent behavioral system (Pellis & Burghardt, 2017;Pellis et al., 2019).
Based on the previous literature (Burghardt, 2005(Burghardt, , 2011, a behavior should satisfy five criteria to be recognized as play: (i) not having (for the observer) an evident resource (e.g. food, sexual partner) to compete for, (ii) being voluntary and rewarding, (iii) including patterns that are incomplete, exaggerated and variable in their form and timing, (iv) being composed of repeated but not stereotyped or abnormal patterns, and (v) occurring in absence of serious environmental/physical stressors.
Human and nonhuman primates can engage in different types of play (e.g., object play, solitary play; Burghardt, 2005;Cordoni & Palagi, 2011;Pellegrini & Smith, 1998), and one of the most complex is play fighting (or Rough-&-Tumble; Smith, 1989). During play fighting primates can engage in interactions that-although not entirely-largely reflect the performance of agonistic behaviors during real fighting (Cordoni et al., 2022a;Palagi et al., 2016;. Unless it escalates into aggression, play fighting is not usually associated with injuries and subjects do not protect an evident resource (Smith, 1997). Moreover, during play fighting primates can display specific facial expressions, body postures, or vocalizations to signal the "benign" intents of the subjects involved Iwaniuk et al., 2001;Kerney et al., 2017;Palagi et al., 2016;Pellis & Pellis, 1996;Pellis et al., 2015;Smith, 1989Smith, , 1997. Play fighting can be affected by individual (e.g., species, age, gender, and neural traits), social (e.g., affinitive and dominance relationships), and environmental (e.g., food abundance and predator presence) factors (Fagen, 1981;. Even though play fighting is more frequent in immature individuals, in some primate species it is also retained and variably expressed in adulthood (e.g., Propithecus verreauxi-sifakas, Antonacci et al., 2010; Pan paniscus-bonobos, Palagi, 2006; Pan troglodytes-chimpanzees, Yamanashi et al., 2018; Gorilla gorilla gorilla -lowland gorillas, Cordoni et al., 2022b). For example, in bonobos play fighting is maintained at relatively high levels between adults . As in juvenile individuals, in adult primates play fighting may serve both long-term benefits-such as social assessment and social status determination-and short-term benefits -such as conflict prevention (Antonacci et al., 2010;Norscia & Palagi, 2011;Paquette, 1994).
Play has been considered as a potential animal welfare indicator (Oliveira et al., 2010) because (i) it often disappears when individuals are under serious stressful conditions/states and fitness challenge (Burghardt, 2005;Fagen, 1981) and (ii) it is thought to be accompanied by a pleasurable emotional and rewarding experience (Panksepp, 2022;Pellis & Pellis, 2009;van Kerkhof et al., 2013;Vanderschuren et al., 2016;Vanderschuren, 2010). However, some studies revealed that-depending on the species-play fighting in adults can contribute in the short-term to buffer transient anxiety or be positively correlated with the levels of chronic stress and/or aggressive interactions (chimpanzees: Palagi et al., 2004;Yamanashi et al., 2018;bonobos: Palagi et al., 2006;monkeys: Norscia & Palagi, 2011;lemurs: Norscia & Palagi, 2016). Yamanashi et al. (2018) demonstrated that among adult chimpanzees play fighting levels correlated with aggression but not with grooming levels and that play fighting can be used for tension reduction. Hence, play is not always an indicator of individual positive emotional state. Under specific conditions, play fighting may be promoted by an adverse psychological or emotional state and may increase individual emotional resilience (Held & Špinka, 2011;Špinka et al., 2001) and the ability to cope with stressful and agonistic contests (Hausberger et al., 2012;Oliveira et al., 2010).
As in many mammals, primates perform many behaviors during "non-serious fighting" (i.e., play fighting; Bekoff & Allen, 1998;Bekoff, 2014) that are reflective of behaviors performed during "serious fighting" (i.e., aggression or real fighting). Play fighting may provide practice of tactics that can be similar to those used in real fighting even though play fighting does not completely mirror real fighting, especially in the way behavioral patterns are performed (Briffa & Lane, 2017;Burghardt, 2005;Symons, 1978). Hence, play fighting may represent an alternative way to acquire and test skills and knowledge when there are risks of doing so through direct experience (e.g., real fighting; Bock & Johnson, 2004). For example, in sifaka (Propithecus verreauxi) adult males can play with out-group members to overcome xenophobia and avoid conflict with nonfamiliar individuals (ice-breaker effect, short-term benefit; Antonacci et al., 2010). In howler monkeys (Alouatta palliata), play between adults may act as a tool to regulate competition and promote social tolerance (short-term benefit; Asensio et al., 2022). Through play fighting juvenile lowland gorillas (Gorilla gorilla gorilla) can manage the competition before the distribution of food thus avoiding direct confrontation (short-term benefit; Palagi et al., 2007). In chimpanzees (Pan troglodytes) play fighting acquires more competitive elements during juvenility thus permitting an efficient self-and socialassessment process (long-term benefit; Cordoni & Palagi, 2011;Paquette, 1994). In humans, several huntergatherer groups often performed coalitional play fighting that in the long term can improve skills involved in coalitional real fighting (Sugiyama et al., 2018).
The current study aims at evaluating in a quantitative and replicable way how fighting in the play context structurally differs from fighting in the aggressive context. To achieve this goal, we gathered behavioral data on chimpanzees because they (i) show adult-adult play fighting both in captive and wild conditions (Matsusaka, 2004;Palagi, 2006;Yamanashi et al., 2018) and (ii) are the species phylogenetically closest to humans (along with bonobos; Langergraber et al., 2012). Hence, studying chimpanzee behavior allows inferences about the evolutionary processes at the basis of the connection between play and real fighting in humans.
To quantify the possible structural key features that can allow the distinction between play fighting and real fighting in adult chimpanzees, we tested the following predictions.

| Prediction 1
Animals can interrupt and resume play in an atypical rhythm by generating different chains of actions with various mixed-up behavioral patterns (Pisula, 2008). Previous literature shows that play fighting-compared with real fighting-can show increased behavior repetition, variability, and distribution (Burghardt, 2005;2011;Cordoni et al., 2022a). Hence, we expected that in adult chimpanzees the general levels of behavioral pattern repetition (e.g. how many times the same behavior follows itself within a session), variability (e.g. how many types of behavioral patterns are performed in a session), and distribution uniformity (i.e., how many times the same type of behavioral pattern is performed within a session in relation to other pattern distribution) within sessions would be higher in play fighting rather than in real fighting (Prediction 1a).
Play is considered a pleasurable activity and may be rewarding for players (Burghardt, 2005(Burghardt, , 2011Pellis & Pellis, 2009;Trezza et al., 2010;van Kerkhof et al., 2013;Vanderschuren et al., 2016;Vanderschuren, 2010). As such players should be motivated to continue playing. Indeed, at the neural level the facilitation of the expression of play can increase the duration of playful session (e.g., van Kerkhof et al., 2013). On the other hand, aggression is not at all pleasurable and-as a social stressor-can cause an increase in cortisol levels with deleterious physical effects on subjects (Muller et al., 2021;Schrock et al., 2019). Individuals should therefore be motivated to reduce its duration as much as possible. Hence, even though either play fighting or real-fighting sessions can markedly vary in session length, we expected that the duration of the session would be higher in play fighting rather than in real fighting (Prediction 1b).

| Prediction 2
In primates, competition during play fighting can be balanced-at least up to a certain extent-by cooperation (e.g., via self-handicapping, rolereversal, and reciprocity; Bauer & Smuts, 2007;Pellis et al., 2010;Petrů et al., 2009). This balancing mechanism is necessary to sustain play and reduce the probability of escalation into serious aggression (Bekoff, 2014;Pellis & Pellis, 1988;Reinhart et al., 2010). During real fighting competition is not balanced by cooperation: individuals compete fiercely to gain an advantage over their competitors so as to acquire dominant positions and/or obtain priority access to resources (de Boer and Koolhaas (2017); de Koolhaas et al., 2013;Norscia & Palagi, 2016). Thus, we expected that in adult chimpanzees the levels of behavioral symmetry (i.e. in terms of reciprocity in offensive and defensive behavioral patterns) between interacting subjects would be higher in play fighting than in real fighting (Prediction 2a). If so, we also expected that individual dominance positions (deriving from the use of offensive vs defensive behaviors) in play fighting would not correlate with individual dominance positions in real fighting (Prediction 2b).

| The study groups
The present study was carried out on four groups of chimpanzees (Pan troglodytes; see Table 1) that are described in details below.
Mona Chimpanzee Sanctuary. Two of the study colonies were housed at the Mona Chimpanzee Sanctuary, a rehabilitation center managed by Fundaciòn MONA (Riudellots de la Selva, Spain). Each group-named Bilinga (BIL; N females = 3, N males = 4, age range = 18-37 years, mean age of adults = 29.9 ± 6.5 SD) and Mutamba (MUT; N females = 2, N males = 5, age range = 16-36 years, mean age of adults = 25.4 ± 8.1 SD), respectively-was composed of seven unrelated individuals. The females of both groups were treated with oral contraceptives and only one male (Victor) belonging to BIL group had been castrated before its arrival at the sanctuary. Both groups occupied similar indoor (50 m 2 BIL; 45 m 2 MUT) and outdoor (3220 m 2 BIL; 2420 m 2 MUT) facilities. Chimpanzees received food (i.e., fruits, vegetables, juice, dried fruits, seeds, and rice) scattered on the ground five times per day.
La Vallèe des Singes (Romagne, France). The group was composed of six unrelated chimpanzees (N females = 3, N males = 3, age range = 12-26 years, mean age of adults = 20.8 ± 6.5 SD). In October 2019 a new female-Lila-joined the group. Females were not treated with oral contraceptives and males were not castrated. The animals could freely move back and forth an indoor of about 200 m 2 and an island of about 3000 m 2 surrounded by a moat. Chimpanzees received food (i.e., vegetables, fruit, seeds, and pellets) scattered on the ground four times per day.
ZooParc de Beauval (Saint Aignan sur Cher, France). The group was composed of 16 individuals (N adult _ females = 8, N adult _ males = 2, age range = 15-46 years, mean age of adults = 35.6 ± 9.2 SD). Six females of the group (Baraka, Bonobo, Charlotte, Julie, Sangha, and Wamba) were treated with oral contraceptives and three males (Lukombé, Gamin, and Tumba) have got vasectomy. Chimpanzees occupied an indoor facility of about 300 m 2 and an island surrounded by a moat of 3000 m 2 . The animal received food (i.e., vegetables, seed/pellet cake, and fruits) scattered on the ground seven times per day.
The equipment of indoor and outdoor facilities of the four colonies was similar and included platforms, ropes, trunks, CORDONI ET AL. | 3 of 14 hammocks, straw, and vegetation. Environmental enrichments such as artificial termite nest and task maze were also provided. All the chimpanzees belonging to the four colonies were in continuous full contact (i.e., housed together and in complete tactile contact).
In our analyses we included only individuals that were sexually mature (Walker et al., 2018) and that showed both play and real fighting interactions (N = 24 adult individuals; Table 1). No difference was found in the age (in years) distribution of the individuals across groups (Kruskal-Wallis test N = 24, χ 2 = 5.034, df = 3, p = 0.169; mean age value ± SD: 28.93 ± 9.37; age range: 12-46).

| Data collection
Video-data on the four colonies were collected by five observers cameras (optical zoom 50x, frame rate: 60fps; precision 2csec).
Before the start of systematic video collection, all the observers were trained for 40 h by G.Co. in animal distinction, behavioral pattern identification (see Table 2) and video-recording procedures. Training finished when the interobserver reliability score-measured via Cohen's k-ranged from 0.80 to 0.90. The videos were then analyzed, frame-by-frame and or in slow-motion, by using the software freeware Avidemux 2.7.1. We collected a total of 962 h of videorecording.
Via the all occurrences sampling method (Altmann, 1974) we gathered all adult-adult play and real fighting sessions. In particular, for each event we recorded: (i) identity of the subjects involved and their features (i.e., sex, age, and group), (ii) behavioral patterns performed and their exact chronological order (see Table 2

| Operational definitions
A real fighting started when a chimpanzee directed a behavioral pattern exclusive to the aggressive domain (Table 2) towards a companion and it ended with one of the opponents moving or running away. A play fight started when a chimpanzee directed a behavioral pattern exclusive of the play domain (Table 2) towards a group mate and finished when both players stopped the interaction, one of them moved away or a third individual substituted one of the two players or interrupted the session by dividing the two players (Palagi, 2008). We never observed any polyadic (i.e., more than two players) sessions between adults; hence, our analyses focused on dyadic play fights.
For both play fighting and real fighting, two consecutive sessions were considered as different if the interaction stopped for more than 10 s. For both contexts, we defined decided for those interactions during which it was possible to clearly distinguish between winners T A B L E 1 The four chimpanzee colonies observed in this study. T A B L E 2 Behavioral patterns considered in this study for play fighting and real fighting in adult chimpanzees (see the text for definition of N, O and D categories).

Patterns exclusive of play fighting
Full play face E The chimpanzee opens her/his mouth with both upper and lower teeth exposed (Preuschoft & van Hooff, 1995) Laugh V Pant-like vocalization involves a series of low-frequency staccato grunts (Davila-Ross & Palagi, 2022;Gervais & Wilson, 2005) Peek a boo N The chimpanzee hides and suddenly pops out from a shelter Play eye cover O The chimpanzee covers with her/his hands the eyes of the playmate by making difficult playmate orientation in the environment Play face E The chimpanzee opens her/his mouth with only the lower teeth exposed (Preuschoft & van Hooff, 1995) Somersault N The chimpanzee flips over the ground or on vertical support in solitary or social manner

Patterns exclusive of real fighting
Avoid D The chimpanzee moves away from the path when another individual is approaching her/him or takes a less direct route around the other

Bared-teeth E
The chimpanzee's mouth corners are withdrawn and the lips retracted from teeth and gums. The mouth can be kept closed or slightly opened. It can be associated with screaming (Waller & Dunbar, 2005) Bob D The chimpanzees bends her/his back and weaves with head or whole body in a bowing position upwards or forward (Roberts et al., 2014). This is a typical submissive behaviors that can be performed by subordinates to avoid being attacked  Table 2).
In the analyses, we included sessions composed of at least two behavioral patterns to give both the interacting subjects the possibility to perform at least one behavior each.

RSBI = (times a same behavior follows itself) (total patterns composing the session − 1)
For example, in a play session where a behavior "A" is repeated seven times, resulting in the pattern "AAAAAAA," the RSBI is equal to six repetitions out of six total patterns (i.e. 7-1) resulting in 1.0 index value. Again, in a session composed of "AABBCCDDEE" there are five repetitions out of nine total patterns (i.e. 10-1) resulting in an index of 0.56. The index ranges from 0 to 1 for all session duration.

| Asymmetry Index (AI)
This index was used to quantify the level of play/real fighting symmetry in terms of reciprocity in offensive and defensive behaviors exchanged between subjects (Cordoni et al., , 2018.
It was calculated as follows: "the number of offensive behaviors by A towards B plus the number of defensive behaviors by B towards A" subtracted from "the number of offensive behaviors by B towards A plus the number of defensive behaviors by A towards B" divided by "the total number of behaviors performed by both individuals". The formula of AI is reported below: AI ranges from −1 to +1 with main values indicating (i) a complete symmetry of the session (zero), (ii) a complete asymmetry of the session in favor of A (+1), and (iii) a complete asymmetry of the session in favor of B (−1).

| Diversity indices
We employed two indices used for measuring biodiversity in ecological studies (Lakićević & Srđević, 2018;Morris et al., 2014;Türkmen & Kazanci, 2010) and we adapted them to play fighting and real fighting. Shannon index (H′; also known as Shannon's diversity index, Shannon-Wiener index, Shannon-Weaver index, and Shannon entropy) is the most common diversity index used in ecological studies and it focuses on species richness (Keylock, 2005;Shannon, 1948). The mathematical formula of Shannon index is:

Pielou index (J; also known as Species evenness) derives from
Shannon index and is the measure of the distribution of individuals among species within a specific ecosystem (Pielou, 1966). The mathematical formula of Pielou index is:

H′ is the observed value of Shannon index, H′ max is the lnS with S
representing the total number of species. The values of J vary between zero and one: when they are close to one it means that individuals are evenly distributed among species (Pielou, 1966

| Statistical analyses
We compared play fighting and real fighting indices (Prediction 1a and 2a) and durations (Prediction 1b) by carrying out paired analyses involving the same dyads in the two contexts. We tested the normality of data  , 2006;de Vries, 1993). We calculated two different sets of NDS for each chimpanzee, one by using decided real fighting sessions (NDS real_fighting ) and one by using decided play fighting sessions (NDS play_fighting ; see Operational definition). In the measure of NDS the observed proportion of wins was corrected for the chance occurrence of the observed outcome based on binomial distribution with each subject having an equal chance to win or lose in every play/real fighting. The correction is necessary when, as in the case of our study, the number of play/real fighting greatly differed between pairs. Then we checked for a possible correlation between individuals NDS real_fighting and NDS play_fighting values and carried out separated analyses for females and males. We employed Spearman correlation test for nonnormal distribution and the Pearson correlation test for normal distribution (Siegel & Castellan, 1988; Prediction 2b). The nonparametric analyses were performed by SPSS 28.0.
The significant threshold was set to α = 0.05.

| Prediction 2
By comparing the sessions performed by the same dyads, we found that the Asymmetry Index values were higher for real fighting than for play fighting (AI real_fighting > AI play_fighting ; ran-

| DISCUSSION
In the current study, we showed that in adult chimpanzees play fighting and real fighting can be structurally distinguished even though not always as expected following the indications of previous literature (Burghardt, 2005(Burghardt, , 2011Cordoni et al., 2022a;Smith, 1997). In our study, we found that during play fighting adult chimpanzees repeated the same behavioral patterns than in real fighting (RSBI, Figure 1). Because behavioral pattern repetitionbut not variability and distribution uniformity-was higher in play fighting than in real fighting sessions, we suggest that repetition can be confirmed as a key feature of play (sensu Burghardt, 2011)  | 9 of 14 respect on non-human primates, it has been suggested that in adult humans repetition is important for action training because movements become more stable as motor-skills improve (Barbado Murillo et al., 2017;Wynberg et al., 2021). In humans, the performance of a given activity is improved by repetition (Magallón et al., 2016;Willingham & Koroshetz, 1993). The process of acquisition and perfection of a new repertoire of movements is based on practice and induces neuronal plasticity, particularly at cerebellum level (Boyden et al., 2004;Kleim et al., 1997;Park et al., 2009). In rats, Whishaw et al. (2021) demonstrated that during play fighting adult individuals repeatedly used their hands and this was associated to motor training for the use of hands in many hand-related behaviors.
In our study colonies, play fighting sessions were more symmetrical than real fighting sessions, thus individuals reciprocated more frequently offensive and defensive patterns while playing ( Figure 3). As suggested for mutual grooming, in adult chimpanzees a reciprocal and symmetric exchange of behavioral patterns (i.e., attack and defensive maneuvers) during play fighting between individuals can serve as a signal of willingness to invest in the play bout and to prolong it (Immediate Investment Hypothesis; Allanic et al., 2020;Machanda et al., 2014). It has been posited that in chimpanzees the proximate cause of structural and temporal changes of play are partly explained by Heider's Balance Theory (Heider, 1946), according to which group-member would be motivated to change their unbalanced social interactions (in terms of reciprocity) into balanced ones to prolong these interactions (de Nooy et al., 2005;Krackhardt & Handcock, 2007;Moody, 2009;Shimada, 2013). Play fighting in adult chimpanzees can result as a blend of competition and cooperation (Pellis et al., , 2022) that have to always maintain a certain grade of reciprocity to allow the "non-serious" interaction to continue. Thus, in adult chimpanzees symmetry (i.e., reciprocity in offensive and defensive behavioral pattern exchange) may be a keystructural-feature that characterizes play fighting although different primate and non-primate species may incorporate reciprocity in different ways (Bauer & Smuts, 2007;Cordoni et al., 2016Cordoni et al., , 2018Cordoni et al., , 2021Pellis et al., 1993;Pellis et al., 2022).
Repetition and symmetry may work in prolonging the play session. Accordingly, we found that play fighting sessions lasted longer and were composed of more behavioral patterns (an indirect indicator of session length) than real fighting sessions. Indeed, in both primate and non-primate mammals play is supposed to be a pleasurable and rewarding activity, which would lead to its prolongation (Burghardt, 2011;Held & Špinka, 2011;Pellis et al., 2014;van Kerkhof et al., 2013). From a functional point of view, the duration can be a reliable measure of play success becauseby prolonging their interactions-individuals have more time for social assessment (Bertini et al., 2021;Palagi et al., 2019). In the longterm perspective, the period of time two subjects interact may shape the quality of their relationship (Hinde, 1979). Because chimpanzees invest in affiliative relationships throughout their lives and use play as social currency (Bray et al., 2021;Koski et al., 2012;Rosati et al., 2020;Schroepfer-Walker et al., 2015), we can assume that longer playfighting sessions can represent a valuable currency for social bond formation and maintenance. fighting. We can also suggest that play fighting has a highly competitive nature and can possibly replace aggression under certain circumstances Paquette, 1994).

| CONCLUSIONS
Overall, our results evaluated in a quantitative and replicable way that session duration and behavior repetition and symmetry can be key features of play fighting in adult chimpanzees whereas rolereversal and behavior variability and distribution uniformity cannot be. We can suggest that play fighting in adult chimpanzees may have elements of serious context (i.e., real fighting) such as the absence of role-reversal. To our knowledge few studies have identified play as a possible (and safe) substitute for aggression Paquette, 1994) or as a way to determine and/or maintain dominance relationships (Yamanashi et al., 2018) in chimpanzees. We may expect that also in immature chimpanzees play can include elements of serious context and can be competitive for obtaining benefits that partly overlap with those obtained by adult individuals (e.g., anxiety reduction, social assessment). In this view, we can suppose that play may be not always an indicator of positive animal welfare. Future research works are needed to investigate playful interaction structure and manifestation in a nuanced way with respect to facial expressions, individual/group features, and socio-environmental context so to better understand the motivation that underlies what appears to be play or rather motivational changes (e.g., from playful to aggressive) during sessions.

ACKNOWLEDGMENTS
The authors wish to thank the staff of Mona Chimpanzee Sanctuary, La Vallée des Singes, and the ZooParc de Beauval. Thank are also due to Baptiste Mulot, Jean Pascal Guéry, Miquel Llorente, and the chimpanzee keepers for allowing and facilitating this study. The authors also thank the two anonymous reviewers for their accurate revision of the manuscript and their precious suggestions.

CONFLICTS OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in the supplementary material of this article.

ETHICS STATEMENT
The current study was purely observational and non-manipulative, thus approval was not required by the authors' institutional animal care committees. The study adhered to both the American Society of Primatologists Principles for the Ethical Treatment of non-human primates and legal requirements of the country in which the research was conducted.