Developmental Psychobiology Research Group, Department of Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado, USA

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1 Individual differences in Macaques' respnses t stressrs based n scial and physilgical factrs: implicatins fr primate welfare and research utcmes Maria l. Bccia, Mark l. Laudenslager & Martin l. Reite Develpmental Psychbilgy Research Grup, Department f Psychiatry, University f Clrad Health Sciences Center, Denver, Clrad, USA Summary Primates are used extensively in a variety f research settings. Federal regulatins in the US mandate that caretakers prvide fr the 'psychlgical well-being f labratry primates'. One f the difficulties in implementing this law has been bth in the definitin f psychlgical well-being and in the need t deal with each primate species and, in sme cases, age r sex class, uniquely. Nn-human primates exhibit distinct individual differences in their behaviural and physilgical respnses t experimental challenges and caretaking prcedures. We have been investigating what factrs can predict sme f these individual differences, and have fund that factrs bth intrinsic and extrinsic are significant. Extrinsic factrs fund t predict individual differences in respnse t stressrs include the nature and prir experience with the challenge, the presence f familiar peers and availability f scial supprt. Intrinsic factrs include cgnitive interpretatins f the challenge and temperamental differences in reactivity. These studies highlight the imprtance f understanding the cntext and individual psychlgy f macaques in rder t prvide labratry envirnments cnducive t their welfare, and in rder t understand the impact experimental and caretaking prcedures are likely t have n the health and welfare f ur subjects. Keywrds Stress; mnkey; maternal separatin; scial supprtj autnmic reactivity; cardivascular systemj telemetryj temperament; animal welfare Understanding the factrs that cntribute t the differential effects f stressrs n labratry animals can have a significant impact n husbandry, handling and understanding research subjects, whether ne cnsiders the same stressr applied t different individuals r t the same individual n repeated ccasins. Factrs Based n presentatin during XIV Cngress f the Internatinal Primatlgical Sciety in Strasburg, France. August 1992 Crrespndence t: Maria L. Bccia. PhD. Frank Prter Graham Child Develpment Center. University f Nrth Carlina, CB 818, 15 Smith Level Rad, Chapel Hill, NC USA which cntribute t these differences may be classified as thse related t (ll the nnscial envirnment (such as feeding schedules, types f bedding, etc.), (2) the scial envirnment (such as levels f aggressiveness, availability f scial supprt, dminance status, etc.), and (3) factrs intrinsic t the subject [such as autnmic reactivity, temperament, individual cgnitive expectatins, develpmental histry, early experience, etc.). In ur labratry we have been investigating the ways in which sme f these factrs influence reactins t several kinds f stressrs in 2 species f macaques Labratry Animals (1995) 29,

2 Respnses t stressrs in macaques 251 (pigtail [Macaca nemistrina] and bnnet [M. radiata] macaquesl. Our labratry maintains several grups f pigtail and bnnet macaques, all f which are hused in large indr pens. These 2 species are very interesting frm a cmparative perspective because, althugh they are clsely related and can interbreed t sme extent, they are behaviurally quite distinct IRsenblum 1973; Kaufman &. Rsenblum 1966). The scial dynamics and intra-grup aggressiveness vary between these species, and therefre these species present a unique pprtunity fr cmparative studies f issues relevant t animal welfare and husbandry, as well as fr bimedical research questins. Where apprpriate, we have included herein species cmparisns which reveal interesting differences with implicatins fr animal welfare interventins. Nn-scial envirnment The first grup f factrs mentined abve which may influence individual differences in stress respnses are thse assciated with the nn-scial envirnment. Several studies have demnstrated that manipulating feeding schedules prduces significant behaviural and physilgical effects. We have examined the impact f fd distributin in the pen n behaviur and physilgy (Bccia 1989a,b, Bccia et , 1991a). The nrmal feeding prtcl invlves dispersing the fd thrughut the pen s that all r mst individuals can feed simultaneusly. We altered the prtcl s that fd was clumped in a small area. This was accmplished by attaching t the pen a small plexiglas bx the size f an individual transprt cage. When fd is placed in this bx, nly ne r 2 mnkeys culd feed at a time ('clumped feeding' paradigm). As a result f this manipulatin, mnkeys entered and fed in rder f their dminance hierarchy. At times ther than feeding, the behaviur f the grup was als altered: agnistic behaviurs increased and affiliative behaviurs, such as play, decreased during perids f clumped feeding (Bccia et ai. 1988). We have examined the effect f this manipulatin n several immune parameters, and fund that the degree f behaviural disturbance during clumped feeding was frequently assciated with changes in immunity (Bccia &. Laudenslager 1992). Fr example, fr lymphcyte respnses t T-cell mitgens (a marker f nn-specific immune cell activatin), the higher the level f behaviural disturbance the lwer the mitgen respnse. This was true acrss several grups f mnkeys In = 4), independent f their basal levels f aggressiveness. In a secnd set f studies, a different feeding manipulatin was used. In this case, we examined whether increased fraging pprtunities wuld reduce aggressiveness in grups f pigtail macques (Bccia 1989a,b). Fr this experiment, we prvided a mid-afternn fraging pprtunity by dispersing sunflwer seeds in the wd chip bedding f 2 grups f pigtail macaques. The sunflwer seeds are a valued fd item, and the animals readily searched thrugh the bedding fr them. Sme behaviural changes were cnsistent acrss grups. Fr example, bedding explratin significantly increased in bth grups during the fraging phase. In additin, stere typic hairpulling was dramatically reduced in bth grups (see Table 1). The effect f the fraging manipulatin differed} hwever, fr behaviurs fr which the grups had different baseline levels. Grup Tw was initially quite aggressive, and Grup One had very lw levels f agnistic behaviur. The fraging manipulatin resulted in greatly reduced levels f agnistic behaviurs fr Grup Tw, but nt Grup One [see Figure 1). Based n these results, we have altered the husbandry prcedures f the lab s that the sunflwer seed fraging pprtunity is given t all scial grups during weekday afternns. These studies indicate that a husbandry practice as simple as dispersal f fd can prfundly affect bth behaviural and physilgical respnses in macaques. These studies als highlight the fact that macaque

3 252 Bccia, Laudenslager & Reite Table 1 Incidence f hairpulling fllwing prvisin f sunflwer seeds in tw grups f pigtail macaques Grup 1 Grup 2 Phase" a.m. p.m. Ttal a.m. p.m. Ttal Baseline Tw weeks Tw mnths "Experimental phase refers t the baseline perid, the first 2 weeks f sunflwer seed prvisin, and a 2-week sample after 2 mnths f sunflwer seed prvisin 6 u w en > 3 u Z 1Il scial grups differ in terms f their behaviural prfile (perhaps as a functin f the persnalities f the individual mnkeys f which they are cmpsed). These grup differences are imprtant fr understanding the mnkeys' respnses t such manipulatins. ;:J 1Il a:... 6 U lu en 5 ~ 4 :> 3 u z ~ 2 lu a:... 2 GROUP 1 BASE 2 WKS 2 MOS ~ PHASE OF EXPERIMENT GROUP 2 BASE 2WKS 2MOS PHASE OF EXPERIMENT -- FIGHT Fig 1 Changes in aggressin fllwing prvisins f sunflwer seeds in 2 grups f pigtail macaques. Phase f experiment is as described in Table 1 _ SUBMIT ---- AGGAESS Scial envirnment Anther aspect f the envirnment which can influence individual respnses t stressrs is scial. Within any grup f macaques, f curse, individuals will have scial relatinships with ther grup members. These relatinships are a functin f a variety f factrs, including dminance status, kinship, grming partner preferences, etc. Hw any particular individual respnds t a stressr is influenced by their status in the grup, the availability f alternative scial relatinships, and ther dynamics which may be reflected in grming relatinships. Separatin f an infant frm his/her mther is ne f the mst prfund stressrs which a yung macaque can experience. The respnses f the pigtail and bnnet macaques t maternal separatin, hwever, are quite different (Kaufman & Rsenblum 1967, Reite et al. 1981, 1989, Laudenslager et al. 199). Bth species exhibit an initial agitatin phase, characterized by distress vcalizatins, high levels f lcmtin, and ther behaviurs which represent attempts by the infant t relcate and re-establish cntact with his/her mther. Only pigtail macaques, hwever, exhibit a secnd, depressive phase in this respnse. It has been suggested that bnnet macaques d nt becme depressed because f their particular scial envirnment. Because bnnet macaques exhibit lwer levels f aggressin and higher levels f scial cntact than pigtail macaques, mthers are less restrictive and permit their infants t freely interact with ther grup members. As a cnsequence, when separated frm their mthers, these infants are adpted by ne f these ther females. We directly tested this hypthesis by altering the early scial envirnment f sme bnnet macaque infants. We used the clumped feeding paradigm described abve, and allwed 2 infants t grw up in an envirnment f elevated agnism (Bccia et al. 1991al. One mther, wh was high ranking in her scial grup, did nt becme restrictive, while the ther, lw ranking, mther did. Scial netwrks

4 Respnses t stressrs in macaques 253 were examined by cnducting Rund Rbin Analyses f Variance (Warner et ) n the infants' interactins with ther grup members, including grming behaviur, passive scial cntact, prximity, and ther affiliative behaviurs. The scial netwrks f the 2 infants were quite different: the first had statistically significant attachments t 4 ther grup members representing ver 8% f the scial preferences, whereas the secnd had virtually nne, restricting his scial interactin preferences t the mther. When these infants were separated frm their mthers, the secnd (wh had n alternative attachment bjects in the grup), but nt the first (wh did), became prfundly depressed and spent 45% f his time during the separatin exhibiting a depressive sluched psture. Bth dminance status and scial supprt influenced the respnses f these infants t maternal lss, mediated by alteratins in maternal behaviurs. We als studied the influence f directly manipulating scial supprt n immune parameters during maternal separatin (Bccia et 1. submitted). In a scial grup cntaining 6 infants, we identified 3 wh had significant attachments with 3 juveniles in the grup. We remved all the mthers frm the grup, alng with all the juveniles except fr the three identified. Thus, we separated all 6 infants frm their mthers, while 3 retained scial supprt in the frm f a juvenile with whm they had a significant relatinship prir t the separatin. The availability f this supprt made a significant difference in the respnse t separatin. Behaviurally, infants with scial supprt shwed less evidence f depressin, as reflected in changes in play and eating behaviurs. They als shwed n change frm baseline in natural killer cell functin, cmpared t infants withut supprt, wh shwed a 4% decrease frm baseline t 2 h fllwing separatin. The number f affiliative behaviurs directed by the juveniles tward each infant was taken as an bjective measure f scial supprt. This measure crrelated significantly with natural killer cell functin at every pint in the separatin (with crrelatins ranging frm +.8 t +.98). Thus, the availability f ther scial resurces can prtect an infant against the behaviural and immunlgical cnsequences f maternal lss. The levels f cmpetitiveness and aggressiveness in these macaque species can als influence their respnses t a stressr (Bccia et al. 1992b). In a series f studies with 6 grups f macaques, the stressr was a 24 h water deprivatin perid, fllwed by access t a single water sput. In this situatin, mnkeys cmpete fr access t the water, and a clear dminance hierarchy can be determined by displacements and latency t the first drink btained by each mnkey. This technique has been used in ur labratry fr well ver a decade t assess dminance hierarchies. We have recently examined the impact f this manipulatin n the mnkeys' behaviural and physilgical respnses [Bccia et al. 1992b). In general, pigtail macaques exhibited changes in immune system parameters 48 h after this test, but bnnet mnkeys did nt. Changes included bth lymphcyte activatin by mitgens, and natural killer cell functin. The species difference appeared t be related t differences in the behaviural respnse f the 2 species t the water test. Pigtail macaques were mre aggressive than bnnet macaques, averaging twice as many agnistic behaviurs per subject during the cmpetitive water test. The mean number f displacements per individual during the actual water test was als higher in pigtail than bnnet macaques (7.5 versus 5.5), and the number f displacements ccurring during the water tests was significantly crrelated with the immune system changes bserved 48 h later. In fact, the impressin ne gets when bserving these grups being tested is that the 2 species perceive and respnd quite differently t the challenge presented by the water test. Pigtail macaques cmpete vigrusly fr access t the water when it is first turned n. Aggressin levels are elevated, there is

5 254 Bccia, Laudenslager & Reite extensive pushing and shving, and smetimes it is difficult t determine wh has pssessin f the sput because s many animals are clustered rund it. Bnnet macaques respnd quite differently. There is very little pushing and shving, and the animals seem t be cntent t simply wait, setting up a kind f queue in the rder f their dminance status. This suggests that individual differences in the perceptin f and the respnse t the stressr can be a significant predictr f the physilgical cnsequences f stressrs. All f the abve studies have related factrs extrinsic t individuals t their respnses t stressrs. We have als examined sme factrs intrinsic t the individual which may predict individual differences in respnse t stressrs. Intrinsic factrs We have cnsidered intrinsic factrs affecting stress respnses frm bth bilgical and psychlgical perspectives. Bilgically, it has been prpsed that autnmic nervus system activity (r reactivity) may predict sme f the individual differences in respnses t stressrs. Psychlgically, ne may fcus n the cgnitive expectatins which subjects develp in respnse t repeated participatin in experimental prtcls. We have examined bth f these phenmena in ur labratry. Heart rate can act as a marker f autnmic activity which may predict infants' reactins t maternal lss (Bccia et b). Heart rate was recrded via ur implantable bitelemetry system in apprximately 3 pigtail macaque infants. Baseline daytime heart rate was used t predict respnse t separatin by classifying infants int high r lw categries, based n a median split. Baseline heart rate predicted bth behaviural and physilgical respnse t maternal lss: bth distress vcalizatins n the first day f separatin and depressive sluched pstures during the remainder f the first week f separatin were mre frequent in infants with high baseline heart rates (see Figure 2). Sleep parameters, recrded frm 2 Q w >a: 15 w en III W 1 ::; ~ J ~ 5 ~ "#- COO 6 w en 5 LDWHR II HIGHHR 4 ~ a: w 3 D > 2 Z w j a 1 wa:... BASE SD1 SW1 EXPERIMENTAL PHASE ldwhr II HIGHHR SLOUCHED VOCALIZA lions POSTURE BASE SDt SWt EXPERIMENTAL PHASE Fig 2 Differences in distress vcalizatins (upper graph) and depressive pstures (lwer graph) by pigtail macaque infants in respnse t maternal separatin. as a functin f mean baseline heart rate. SDl =first day f separatin; SWl =first week f separatin all night EEG recrds, als differed significantly between infants with high and lw heart rate: infants with high heart rates shwed greater disruptin f REM sleep [as reflected in REM latency and number f REM perids) and less ttal sleep time. Thus, this marker f autnmic reactivity predicted bth behaviural and physilgical respnse t maternal separatin. The rle f cgnitive expectatins may be imprtant fr mnkeys' respnses t experimental manipulatins [Bccia et ai. 1992al. In the research described t this pint, we engage in a fair amunt f animal handling, fr bld sampling and ther manipulatins. These prcedures may themselves be perceived as stressrs by the mnkeys. Furthermre, as ur research designs are nt terminal, ur mnkeys tend t remain in ur labratry thrughut

6 Respnses t stressrs in macaques 255 their lives, and cnsequently participate in multiple experiments. It seemed likely, therefre, that the mnkeys with greater experience with experimental prtcls wuld develp expectatins abut new prtcls that yunger less experienced mnkeys wuld nt, and that these differences in the mnkeys' expectatins wuld influence the utcme f the research. The mnkeys in the cmpetitive water tests ranged in age frm 2.5 t 18 years f age, and frm having n prir experience with experimental prtcls t having participated in multiple studies. In ur animal handling prcedures, mnkeys are mved frm their living pen int a large 'gang cage' frm which they are individually remved fr testing. The mnkeys themselves determine the rder in which they exit bth the pen and the gang cage. We had nticed that the mnkeys tended t exit in a cnsistent rder, but had nt systematically examined this. We examined the impact f dminance status, age and experience n animals experiencing 5 repeated bld samples. Cntrary t expectatins, the mnkeys were nt exiting the cage in an rder based n dminance status. Rather, ver repeated samples, lder mnkeys came ut first and yunger mnkeys later. Interestingly, this was mre true fr the bnnet macaques than the pigtail macaques. This self-selectin n the part f the mnkeys was significant because hrmne measures (specifically crtisl) were crrelated with hw lng it tk t cllect the sample. The prir experience f the lder mnkeys may have taught them that the sner they cme ut the sner they return t their hme pen la strng reinfrcer fr their behaviur). Yunger animals, in cntrast, had nt yet made this cnnectin. This suggests that we must incrprate mre habituatin and baseline trials in experiments s that the mnkeys' behaviur stabilizes int a cnsistent pattern befre any experimental manipulatins begin. Experience with experimental prtcls early in develpment (e.g. maternal separatin) may als have behaviural and physilgical cnsequences which persist thrugh adulthd. We have fund that an early separatin experience has lng-term cnsequences fr bth scial and nn-scial behaviural respnses [Capitani &. Reite 1984, Capitani et ai. 1986). Cmpared t unseparated cntrls, adult pigtail macaques wh had experienced an early maternal separatin played less, had fewer scial partners, and less cmplex scial relatinships. In additin, these animals displayed higher levels f disturbance, reflected in steretypic and self-directed behaviurs, in the face f nvel stimuli, althugh crtisl levels between the grups did nt differ. These studies demnstrate that there are lng-term cnsequences f mnkeys' experiences with experimental prtcls, such that they may develp cgnitive expectatins abut prcedures that affect the data cllected cmpared with experimentally naive subjects. Cnclusins These studies indicate that factrs in bth the scial and nn-scial envirnment as well as thse intrinsic t the individual can prfundly affect individual respnses t bth experimental prtcls and husbandry manipulatins. They highlight the imprtance f being sensitive t the behaviur f the individual animals when making decisins abut changing labratry prcedures. Furthermre, prgrammes designed t enhance the well-being f labratry primates must take int accunt the species and individual differences as well as the cntext and husing f these animals. One set f guidelines will nt apply equally well t all species f primates. The scial and nn-scial envirnments influence basic physilgical prcesses and respnses, as d individual expectatins and temperament. These phenmena highlight the fact that primates are uniquely valuable fr certain types f bimedical research because the Same phenmena perate in human respnses. One imprtant questin raised by these studies is, what mediates the relatinship between these behaviural respnses and

7 256 Bccia, Laudenslager & Reite the immune system changes bserved here? The relatinship between the immune system and behaviur must be mediated thrugh the central nervus system, and in particular thrugh autnmic nervus system cnnectins with the immune system. These cnnectins have been demnstrated t ccur via circulating hrmnal factrs and direct sympathetic innervatin. The impact f behaviur n sympathetic nervus system functin may therefre prvide a clue as t hw the bserved behaviurs mediate immune changes. We have demnstrated that ne behaviur, grming, can impact a marker f autnmic nervus system arusal} heart rate (Bccia et ai. 1989), which may help explain the relatinship between behaviur and immune system changes. That is} receiving grming frm a scial partner can result in a significant decrease in heart rate. Fllwing aggressin fr example, heart rate declined an average f 25 beats per minute if grming was received frm thers (twice that seen fr initiate grming, r fr any ther behaviur). Thus, this behaviur appears directly t influence autnmic nervus system functin as reflected in heart rate. Recently, Gust and her clleagues (Gust et ai. 1993) have demnstrated that the impact f a grup frmatin n the immune system f female rhesus and pigtail macaques can be predicted frm the amunt f grming received by thse females in the weeks fllwing the grup frmatin. In the 'clumped feeding study described abve, we als fund that scial grming mderated effects n the immune system} as fr example seen fr lymphcyte respnse t a B-cell mitgen. These bservatins suggest that behaviur can impact health f labratry primates by mediating autnmic nervus system effects n immune system functin. Thus, we have seen that behaviur, envirnmental cntext, and physilgy are intimately cnnected. One cannt hpe t understand any f these levels thrughly withut cnsidering the thers. We cannt cnsider ur experime.ntal subjects t be unifrm test tubes t which we add ur experimental reagents. We must cnsider the scial cntext, species differences} and individual differences bth when we design welfare prgrammes fr ur labratry animals and when we design bimedical research prtcls. Failure t d s will result in welfare prcedures that d nt wrk and research results that are un interpretable. References Bccia ML [1989al Preliminary reprt n the use f a natural fraging task t reduce aggressin and steretypies in scially hused pigtail macaques. Labratry Primate Newsletter 28, 3-4 Bccia ML 11989b) Lng-term effects f a natural fraging task n aggressin and steretypies in scially hused pigtail macaques. Labratry Primate Newsletter 28, Bccia ML, Laudenslager ML (1992) Immunlgical and hrmnal cnsequences f alteratins in feeding regimens in bnnet mnkeys IMacaca ramata I. American Jurnal f Primatlgy 27, 18 Bccia ML, Laudenslager M, Reite M (19881 Fd distributin, dminance, and aggressive behavirs in bnnet macaques. American Jurnal f Primatlgy 16, Bccia ML, Reite M, Laudenslager M (19891 On the physilgy f grming in a pigtail macaque. Physilgy and Behavir 45, Bccia ML, Reite ML, Laudenslager ML [1991a) Early scial envirnment may alter the develpment f attachment and scial supprt: Tw case reprts. Infant Behavir and Develpment 14, Bccia ML, Laudenslager ML, Reite ML (1991bl Heart rate level predicts bibehaviural respnse t separatin in pigtail mnkey infants (Macaca nernestrina I. American Jurnal f Primatlgy 24, 9-1 Bccia ML, Brussard C, Scanlan 1, Laudenslager ML (1992a) Practice makes predictable: Repeated sampling differentially alters behaviral and physilgical respnses f yunger and lder mnkeys. In: The Inevitable Bnd: Examining Scientist-Animal Interactins (Balfur D, Davis H, eds), Cambridge University Press, pp Bccia ML, Laudenslager ML, Brussard C, Hijazi A [1992b) Immune respnses fllwing cmpetitive dminance tcsts in tw species f macaques. Brain Behavir and Immunity 6, Bccia ML, Scanlan 1, Laudenslager ML, Brussard CL, Hijazi AS, Reite ML [submitted) Presence f juvenile "friends" mitigates behaviral and immunlgical respnses t scial separatin in bnnet macaque infants. Physilgy etj Behaviur

8 Respnses t stressrs in macaques 257 Capitani JP, Reite ML (1984) The rles f early separatin experience and prir familiarity in the scial relatins f pigtail macaques: A descriptive multivariate study. Primates 25, Capitani JP, Rasmussen KLR, Snyder DS, Laudenslager M, Reite M Lng-term fllw-up f previusly separated pigtail macaques: Grup and individual differences in respnse t nvel situatins. Jumal f Child Psychlgy and Psychiatry 27, Gust DA, Grdn TP, Hambright MK, Wilsn ME (1993) Relatinship between scial factrs and pituitary-adrencrtical activity in female rhesus mnkeys (Macaca mulatta). Hrmnes and Behavir 27, Kaufman IC, Rsenblum LA (1966) A behaviral taxnmy fr Macaca nemestrina and Macaca Tadiata based n lngitudinal bservatin f family grups in the labratry. Primates 7, Kaufman IC, Rsenblum LA The reactin t separatin in infant mnkeys: Depressin and cnservatin-withdrawal. Psychsmatic Medicine 29, Laudenslager ML, Held PE, Bccia ML, Reite ML, Chen IT (199) Behaviral and immunlgical cnsequences f brief mther-infant separatin: A species cmparisn. Develpmental Psychbilgy 23, Reite M, Kaemingk K, Bccia ML (1989) Maternal separatin in bnnet mnkey infants: Altered attachment and scial supprt. Child Develpment 6, Reite M, Shrt R, Seiler C, Pauley JD (1981) Attachment, lss & depressin. Jumal f Child Psychlgy and Psychiatry 22, Rsenblum LA (1973) Maternal regulatin f infant behavir. In: Behaviral Regulatrs f Behavir in Primates. (Carpenter CR, ed.) Lewisburg, PA: Bucknell University Press, pp Warner RM, Kenny DA, Stt MA (1979) A new rund rbin analysis f variance fr scial interactin data. Jumal f Persnality and Scial Psychlgy 37,