Physiology & Behavior 98 (2009) 168–175

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Physiology & Behavior

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Seasonal and social influences on fecal androgen and glucocorticoid excretion in wild male long-tailed macaques (Macaca fascicularis)

C. Girard-Buttoz a, M. Heistermann a, S. Krummel a,b, A. Engelhardt a,c,⁎ a Department of Reproductive Biology, German Primate Centre, 37077 Göttingen, Germany b Faculty of Biology, Georg-August-University Göttingen, 37073 Göttingen, Germany c Institute for Human Biology and Anthropology, Free University of Berlin, 14195 Berlin, Germany article info abstract

Article history: Whereas it is well known that in strictly seasonal breeding primates (income breeders), alike other Received 28 November 2008 vertebrates, males show pronounced changes in testicular and adrenal levels concurrent with Received in revised form 7 May 2009 reproductive activity, hormonal patterns in males of non-strictly seasonal breeding primate species (capital Accepted 13 May 2009 breeders) and their relation to seasonal and social correlates remain largely unknown. In the present study, we examined the annual pattern of fecal androgen and glucocorticoid excretion and their relationship to Keywords: environmental (rainfall, temperature) and social factors (number of cycling females, male and Long-tailed macaques Macaca fascicularis rates, male dominance rank) in a group of wild long-tailed macaques (Macaca fascicularis), a Glucocorticoids species with a moderate degree of reproductive seasonality and classified as capital breeder. The study was Androgens carried out in the Gunung Leuser National Park, North Sumatra, Indonesia over a period of ten months Seasonality encompassing the conception and the season. Our results show that male long-tailed macaques exhibit a distinct annual variation in both androgen and glucocorticoid levels. Androgen (but not glucocorticoid) levels were significantly elevated during the conception period in association with elevated rates of male– male aggression and copulatory activity, both strongly related to the number of cycling females in the group. Neither glucocorticoid nor androgen levels were related to male dominance rank or to the environmental parameters investigated. Interestingly, levels of both started to increase in the late birth season and thus 1–2 months prior to the mating season, suggesting that male long-tailed macaques go through pre- breeding hormonal changes in preparation for prospective challenges. Our data thus provide the first evidence that males of a non-strictly seasonal breeding species/capital breeder show endocrine patterns generally similar to those found in strictly seasonal/income breeders. © 2009 Elsevier Inc. All rights reserved.

1. Introduction areviewsee[12]), which is typically linked to an increase in aggression rates [13,14] and higher energetic expenditures (“energy mobilization For males, the mating season is a particularly challenging period in hypothesis”: [12]) associated with increased competition for mate which they compete with each other for access to sexually receptive acquisition. In many of these species, male physiology during the mating females. In order to cope with the increased physical and energetic season, however, varies inter-individually and is often influenced by demands of male–male competition at this time, male vertebrates often dominance rank (e.g. [10,15,16]). undergo pronounced changes in body mass and physiology concurrent Seasonal reproductive activity is characteristic of many primate with changes in reproductive activity (e.g. mammals, [1,2]; reptiles, [3]; species, although there is large variation, ranging from species that and amphibians, [4]). Specifically, males of seasonally breeding species only breed during restricted times of the year with sharply delineated often show an increase in body and testes size and a marked rise in periods of mating (and thus birth) (strictly seasonal breeders, for levels during the mating season in association with an definition of primate reproductive seasonality see [17]) to a complete increase in aggression rate (e.g. mammals: [5];birds:[6]; reptiles: [7,8], non-seasonal reproduction with mating and being distributed see also the “challenge hypothesis”: [9]). In addition, males of these broadly throughout the year ([18,19]). Given that environmental species often show an elevation in glucocorticoid levels during the factors (such as photoperiod, climate and diet) as well as social factors mating season (e.g. mammals: [10]; amphibians and reptiles: [8,11];for (such as degree of male–male competition) are known to modulate male hormone secretion (e.g. [20–22]), it should be expected that primate species differ in their physiological and behavioral response to seasonal changes in environment depending on whether they ⁎ Corresponding author. Department of Reproductive Biology, German Primate Centre, Kellnerweg 4, 37077 Göttingen, Germany. Tel.: +49 5513851 202; fax: +49 5513851 288. exhibit a high or low degree of seasonality in breeding. In this respect E-mail address: [email protected] (A. Engelhardt). and in an attempt to further our understanding of seasonality and

0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.05.005 C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 169 reproductive function in primates, Brockman and van Schaik [23] have Thus, in order to further our understanding on the impact of season developed the concept of “income” vs. “capital” breeders [24] as a on male physiology and the relationship between male endocrine state framework for explaining patterns of seasonal breeding and the and reproductive behavior in a wild-living, non-strictly seasonally proximate mechanisms regulating them. Accordingly, strictly season- breeding primate species, we conducted a study on the long-tailed ally breeding species are classified as income breeders, which are macaque, a species classified by Brockman and van Schaik [23] as expected to rely principally on external cues such as photoperiod and capital breeder. Although female long-tailed macaques can conceive climate to activate reproductive activity, whereas less strictly year-round [48], birth peaks occur [48–50], the timing of which seems seasonally (and aseasonally) reproducing capital breeders are to depend primarily on availability of food [48]. Whether and to what expected to rely more on internal factors (e.g. fat stores, energy extent such moderate reproductive seasonality is associated with balance) for regulating onset and acceleration of reproduction. changes in male endocrine status and variation in sexual activity and Concerning endocrine responses in males, the model predicts that aggression rate and, assuming endocrine seasonal changes occur, males of income (strictly seasonal) breeders should show a marked whether these are related to external climatic conditions or are seasonal variation in testosterone levels in response to predominantly primarily socially mediated is, however, unknown. Studies conducted external cues. In contrast, males of capital (non-strictly seasonal or on males of this species in captivity so far generated inconsistent aseasonal) breeders are predicted to show less seasonal or no annual results, either showing an annual change in male testosterone levels variation in testosterone levels. In these species, male endocrine [46] or not [47]. In the present study we therefore set out to examine physiology is also expected to respond more to female reproductive the following questions under completely natural conditions: (i) what state and food abundance, rather than to photoperiod and climate are the endocrine (androgen and glucocorticoid) and behavioral changes. (aggression, sexual activity) changes across seasons in males of a non- Predictions made for the impact of season on endocrine patterns in strictly seasonal primate, the long-tailed macaque, (ii) what are the male primates have largely been confirmed for strictly seasonal/income temporal relationships involved, (iii) assuming that males show a breeders (e.g. rhesus macaques, Macaca mulatta: [25]; Japanese seasonal variation in endocrine status, what are the cues involved? In macaques, Macaca fuscata: [26];squirrelmonkeys,Saimiri boliviensis: this respect and according to Brockman and van Schaik [23] conceptual [27]; tufted capuchin monkeys, Cebus apella nigritus: [28], golden lion framework mentioned above, we posed the following predictions: tamarins, Leontopithecus rosalia: [29]; ring-tailed , catta: (i) male long-tailed macaques exhibit a seasonal variation in androgen [22]; Verreaux's sifakas, Propithecus verreauxi: [30,31]; redfronted and glucocorticoid levels which should, however, be less pronounced lemurs, Eulemur fulvus rufus: [32,33], lesser mouse lemur, Microcebus than in income breeders; (ii) endocrine profiles are not related to murinus [34]; and muriqui monkeys, Brachyteles arachnoides [35]). Here, external factors (e.g. rainfall, temperature) but can be alternatively the data generally show clear circannual fluctuations in male testoster- explained by endogenous factors related to the degree of reproductive one and/or cortisol levels with marked elevations of either or both competition (number of cycling females, level of male–male aggres- hormones during the mating season in association with heightened sion, level of sexual behavior). If the latter applies and as postulated by male–male aggression and sexual activity (but see [35]). The temporal the challenge hypothesis [9], we further predict that (iii) androgen relationship, magnitude and duration of these changes, however, vary levels are significantly elevated during the period when conceptions between species. In this respect, in some species, such as the sifaka occur, the period when male contest for reproduction should be most [30,31,36],squirrelmonkey[27], rhesus macaque [25] and Japanese pronounced and thus (iv) that male aggression and copulation rates macaque [21,26], male hormonal changes associated with breeding are elevated during the conception period and strongly related to the readiness precede the actual onset of the mating season (and thus of number of cycling females. reproduction and male–male competition) for up to 3 months. Given the Since male endocrine status can significantly be influenced by various promoting effects of testosterone and cortisol on bodily social status [36,51–54], particularly in a species like the long-tailed functions (e.g. sperm production, development of secondary sexual macaque where male reproductive success is clearly rank-related and characters, facilitation of sexual and aggressive behavior, promotion of high-ranking males engage heavily in energy-costly mate-guarding of muscle gain and fat storage [27,37–42]), such an early rise in both sex fertile females compared to low-ranking males [49,55], we also and adrenal steroids has been attributed to physiological preparations in investigate the impact of rank on male physiology and predict that anticipation of the behavioral and energetic demands of male high-ranking males exhibit significantly higher androgen and gluco- reproduction [40,43]. The observed time lag between the elevation of corticoid levels during the conception period. hormones and the onset of the breeding season in these species supports the prediction [23] that in income breeders, males rely on 2. Materials and methods external cues when getting prepared for the upcoming reproductive period. 2.1. Animals and study site By comparison, the impact of season on male physiology and the relationship between male behavioral patterns and endocrine status The study was carried out from February to November 2000 on are less clear in non-strictly seasonally/capital breeding primate long-tailed macaques living around the Ketambe Research Station species where the distinction between breeding and non-breeding (3°41′N, 97°39′E), Gunung Leuser National Park, North Sumatra, periods is less extreme and birth can occur year-round. To our Indonesia. The study area consists of primary lowland rainforest and knowledge the only studies carried out in this respect so far either has been described by Rijksen [56] and van Schaik and Mirmanto [57]. found no seasonal variation in male testosterone levels or testicular The long-tailed macaques in the area have been studied since 1979. In parameters (stumptailed macaque, Macaca arctoides: [44]; Hanuman this population, most births occur in the months July to November langur, Semnopithecus entellus: Lohiya et al. [45]) or results were with the mating season usually starting in December/January [48,58]. inconsistent (long-tailed macaques, Macaca fascicularis: [46,47]). We focused on group HA, since it had a size representative for this Furthermore, all of these studies were conducted in captivity, an species out of three groups (HA, HB and HD) originating from a split of environment which has often been demonstrated to substantially one large group (House group) prior to the onset of the study. All influence seasonal effects on reproductive physiology and behavior group members of group HA were individually known and well when compared to the situation in the wild (see [23]). Information on habituated to observers. The group consisted of eight adult females, seasonal endocrine patterns in primate males of capital breeders five adult males and several subadults/juveniles and, at the beginning living in the wild under natural ecological and social conditions is of the study, two infants. Seven infants were conceived between conspicuously absent. February and June 2000. The home range of group HA overlapped with 170 C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 those of groups HB and HD and with that of a third and a fourth during inter-group encounters. In total, fecal samples were thus group, KB and AT. Females of group HA engaged in sexual interactions collected from 10 males. 2–3 g fresh feces were collected directly after with males from all adjacent groups, which were all individually defecation and stored in 15 ml absolute ethanol at 4 °C until hormone known and also well habituated to observers. The alpha male of group analysis. Samples were usually collected between 7:00 a.m. and 2:00 HB joined group HA almost daily from beginning of March 2000 until p.m., with about two thirds of samples being collected in the morning mid-April 2000, but returned every night to sleep with his own group. hours (before 12 a.m.) in both periods (conception period: 59.0%; Another male joined group HA at the onset of the study, but often left non-conception period: 66.0%). For both, androgens and glucocorti- the group or stayed at its periphery. Depending on the specific coids, individual mean values for morning and afternoon samples did question, data were not only collected from HA males but also from not differ significantly from each other, suggesting no effect of males of the other groups (see below). collection time on fecal hormone concentration (androgens: morning: Information on the daily minimum, average, and maximum forest 1574.0±615.2; afternoon: 2072.5±1119.2; Wilcoxon signed rank temperature and amount of rainfall throughout the study was test: Z=−1.478, p=0.16; glucocorticoids: morning: 995.4±478.8; recorded daily using a standard outdoor thermometer and standard afternoon: 1200.3±613.0; Wilcoxon signed rank test: Z=−1.070, rain gauge. p=0.32). The study was conducted completely non-invasively and under the For hormone analysis, fecal samples were homogenized in their permission of the authorities of Indonesia. We adhered to the original ethanolic solvent and extracted twice by shaking overnight as Guidelines of the Use of Animals in Research, the legal requirements described by Ziegler et al. [61]. Following centrifugation of the final of Indonesia and the guidelines of the involved institutes. fecal suspension, the remaining fecal pellet was dried in a vacuum oven at 50 °C and the dry weight of each sample was subsequently 2.2. Behavioral observations determined. Efficiency of the extraction procedure, determined in a subset of samples by monitoring the recovery of tritiated hormone Continuous behavioral data were collected by A.E. and four added to the samples prior to homogenization, was 83.4±2.3% experienced Indonesian field-assistants. Two to three observers (n=48). For assessing male androgen concentrations, fecal extracts followed the study group every day from dawn until dusk (mean were measured for immunoreactive epiandrosterone (iEA) which has observation time per day: 11.1 h). Sexual behavior (copulations) and been previously identified as the quantitatively most abundant agonistic interactions between the 5 adult males were recorded using metabolite of testosterone in long-tailed macaque feces [62]. The the all occurrence sampling method [59] as for the vast majority of assay was biologically validated by demonstrating that the measure- observations, we were able to see all adult male and female group ment of iEA significantly discriminates in terms of androgen levels members. In long-tailed macaques, copulations are discrete events between age/sex classes, with levels in adult males being significantly which are easily distinguishable from each other since single higher (on average 3–4 fold) than those in subadult males and females copulations by a certain male are temporally clearly separated from (Fig. 1a). Furthermore, HPLC analysis performed on fecal extracts of each other and copulation attempts (which do not result in a full samples from two of the study males as described by Möhle [62] copulation) do rarely occur. Each full copulation was therefore counted as one event. Agonistic interactions (threatening, chasing, biting, grabbing, pulling etc.) were counted as separate events when their occurrence was separated by at least 5 min. Altogether 2582 copulations and 321 male–male agonistic interactions were recorded during 3144 total observation hours. Dominance ranks within sexes were determined by the display of the ‘bared-teeth-face’, a unidirectional submissive display [60], and with a sociometric matrix [55] in which the direction of aggression was entered. Both data sets concurred with each other in assigning rank of HA males. During inter-group encounters, agonistic interac- tions between males of adjacent groups were observed. Based on the ‘bared-teeth-face’ display (see above), alpha and beta males of these adjacent groups could be unambiguously determined from each other and from the lower ranking males of the groups.

2.3. Definition of periods

To investigate the overall influence of season on male behavior and hormone levels, the study was divided into a conception period (when all conceptions occurred) and a non-conception period. The occur- rence of conceptions was determined from fecal progestogen profiles of the group's adult females [49]. Thus, the conception period lasted from February 1 to June 15, 2000, while the non-conception period comprised the period from June 16 to November 30, 2000. The fecal progestogen profiles were also used to determine the number of cycling females (i.e. females being in their fertile phase, for definition and distribution of fertile phases, see [49]) in each study month.

2.4. Fecal sample collection and hormone analysis Fig. 1. Immunoreactive fecal epiandrosterone (iEA) levels (median,1st and 3rd quartile) in different age/sex classes of long-tailed macaques (a) and HPLC profiles of immunor- On average, one fecal sample was collected every 9 days (range 6– eactivity measured by the EA assay (b). Arrows in (b) indicate elution positions of reference 17 days) from each of the 5 resident males of group HA. In addition, standards: 1, cortisol; 2, corticosterone; 3, 11ß-dihydroxyetiocholanolone; 4, testosterone; samples from males of adjacent groups were collected ad libitum 5, androstenedione; 6, epiandrosterone; and 7, androsterone. C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 171 showed that the majority of immunoreactivity eluted as one major ordinate males (N=8). The second-ranking male of group HA, which peak at the position of authentic epiandrosterone (fractions 69–73; joined the group at the onset of the study, often left the group or Fig. 1b), thus confirming the presence of large amounts of EA in long- stayed at the periphery and was therefore excluded from any tailed macaque feces. individual analysis. Since the third-ranking male usually held the For monitoring changes in glucocorticoid levels, fecal extracts were functional beta position, he was considered the second-ranking male analyzed for immunoreactive 3α,11ß-hydroxyetiocholanolone and thus categorized as dominant. We calculated for each male the (3α,11ß-dihydroxy-CM), a group-specific measurement of 5-reduced median hormone level and compared values between dominant and 3α,11ß-dihydroxylated cortisol metabolites [63,64]. The assay has sub-ordinate males using the Mann–Whitney-U-test. been previously validated for assessing adrenocortical activity from To examine for a possible relationship between hormone levels feces in long-tailed macaques and other primate species using an and behavior, we correlated for each of the ten study months the ACTH challenge test and HPLC analysis (see [31,65]). median (see above) copulation rate and aggression rate for group HA Both hormone measurements were carried out by microtiter plate and the monthly median hormone level using the Spearman's rank enzyme immunoassay according to methods previously described correlation. We also correlated, using Spearman's rank correlation, [62,63]. In brief, fecal extracts were diluted in assay buffer (0.04 M monthly median androgen and glucocorticoid levels in group HA PBS, pH 7.2) and 50 μl aliquots in duplicate were assayed along with 50 μl males with the number of cycling females per month for those months aliquots of reference standard in doubling dilutions over the range of in which cycling females were available (February to September). 1.9–250 pg/well (EA) and 0.6–156 pg/well (3α,11ß-dihydroxy-CM), To evaluate the possible relationship between rainfall and respectively. Following incubation of the plates overnight at 4 °C, the temperature on male hormone levels, Spearman's rank correlation plates were washed four times and incubated with 150 μl streptavidin– tests were performed on the monthly median hormone levels of the peroxidase for 30 min in the dark at room temperature (RT) after which five resident males and (i) total monthly rainfall (in mm) and (ii) (following a second washing step) 150 μl of substrate solution was monthly measures of temperature (overall mean, mean of daily added to each well. Following substrate incubation (45–60 min, dark, minimum and maximum values, daily interval between minimum and

RT), the enzyme reaction was stopped with 50 μl2MH2SO4 to each well maximum values) across the ten study months. and absorbance was measured at 450 nm (reference 630 nm) on a plate All statistical tests were conducted with SPSS 15.0 for Windows reader. Serial dilutions of fecal extracts from different males gave and R 2.6.2 and used two-tailed probabilities (when not otherwise displacement curves parallel to those obtained for the standards. stated) with a significance level of 0.05. Sensitivities of the assays at 90% binding were 1.5 pg for EA and 1 pg for 3α,11ß-dihydroxy-CM. For the EA assay, intra- and inter-assay 3. Results coefficients of variation of high- and low value quality controls were 6.0% (n=16)and8.7%(n=10) (high) and 10.1% (n=16) and 17.3% (n=10) 3.1. Influence of period on sexual and agonistic behaviors (low), respectively. Respective figures for the 3α,11ß-dihydroxy-CM measurements were 7.3% (n=16) and 13.2% (n=10) (high) and 8.4% Monthly median male copulation and aggression rates both (n=16) and 16.2% (n=10) (low). All fecal hormone levels reported are showed highest levels during the conception period (February to expressed as mass hormone/g dry fecal weight. June; Fig. 2a) and decreased levels during the non-conception period,

2.5. Data analysis

Since the data were in many cases not normally distributed, non- parametric tests were performed throughout all statistical analyses. To investigate the effect of season on mating behavior, we tested, at the group level, for differences in copulation rates between the conception and non-conception periods with a chi-square test correcting the expected values for differences in observation time. The same test was performed for male–male aggression rate. In addition, to investigate the relationship between male aggres- sion and mating behavior, we calculated the Spearman's rank correlation coefficient for monthly median (calculated from the individual monthly means of the five males resident in group HA) copulation and aggression rates (N=10). In order to examine whether these two behaviors were related to the number of cycling females available and thus to the degree of male–male competition for mates, we correlated monthly median rates of aggression and copulation with the number of cycling females per month using Spearman's rank correlation. To examine the effect of period on EA and 3α,11β-dihydroxy-CM concentrations, we compared for each male for which we had at least two samples per period (N=10) the median hormone level for each of the two periods using the Wilcoxon signed rank test. Since, for both hormones, higher levels can be predicted for the conception period [22,28,33,36], one tailed probabilities were used. To examine the effect of male social status on EA and 3α,11β- dihydroxy-CM concentration during the conception period (the period when any rank effect on hormone level should be most pronounced), we classified males by their dominance rank. All males Fig. 2. Monthly variation in a) copulation (grey bars) and aggression (white bars) rates and fi that held alpha or beta rank position in their groups were classi ed as b) fecal androgen (iEA; grey dotted bars) and glucocorticoid (3α,11β-dihydroxy-CM; white dominant males (N=8) and all other males were classified as sub- bars) levels of the five resident males of group HA (median, 1st and 3rd quartile). 172 C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 particularly during the last three study months (September to November). In accordance with this, we found a significant effect of period on both behaviors (copulation rate: χ2 =1194, df=1,pb0.001; aggression rate: χ2 =112, df =1, pb0.001), with the copulation rate being five times and the agonistic interaction rate being three times higher during the conception period (median: 1.45 copulations/h, 0.17 agonistic interactions/h) compared to the non-conception period (median: 0.32 copulations/h and 0.05 agonistic interactions/h). There was furthermore a significant correlation between the monthly median male copulation and agonistic interaction rate (rs =0.661, pb0.038).

3.2. Influence of period on androgen and glucocorticoid output

Monthly levels of fecal iEA and 3α,11ß-dihydroxy-CM throughout the 10 months study period (Fig. 2b) followed a distinct seasonal pattern with highest concentrations occurring in February at the α beginning of the conception period (iEA: median 3600 ng/g, 3 ,11ß- Fig. 4. Fecal androgen (iEA; grey bars) and glucocorticoid (3α,11β-dihydroxy-CM; dihydroxy-CM: median 1651 ng/g) and in November, the last month white bars) levels for dominant (N=8) and sub-ordinate (N=8) males during the of the study period (non-conception period; iEA: median 3768 ng/g, conception period. Box plots show the median (horizontal line), the 1st and 3rd 3α,11ß-dihydroxy-CM: median 1364 ng/g). In-between, monthly quartiles (box) and the 10th and 90th percentiles (whiskers). levels of the two hormones were 2–4 times lower, with lowest concentrations in androgens occurring between May and September difference was not statistically significant for either hormone (last two months of the conception period and first three months of (3α,11β-dihydroxy-CM: median dominant males: 1493.07 ng/g; the non-conception period; median 1070 ng/g, range 981–1331 ng/g) median sub-ordinate males: 1138.54 ng/g; U=25, p =0.253; iEA: and minimum levels in glucocorticoids being found between April and median dominant males: 2254.87 ng/g; median sub-ordinate males: September; (median 810 ng/g, range 610–914 ng/g). 1494.75 ng/g; U=23, p=0.191). As expected from the monthly pattern, period had a significant effect on androgen levels, with concentrations being on average two times 3.4. Relationship between hormone levels, behavior and number of higher during the conception period compared to the non-conception cycling females period (conception period median: 2231.73 ng/g; non-conception period median: 1128.50 ng/g; Z=−1.27, p=0.032; Fig. 3). In contrast, Rates of copulation and aggressive behavior and monthly levels of glucocorticoid levels did not differ significantly between the two periods androgens and glucocorticoids followed a similar pattern throughout (conception period median: 1288.85 ng/g; non-conception period the conception period, but not the non-conception period (Fig. 2a, b). median: 808.39 ng/g; Z=−1.89, p=0.116; Fig. 3) although levels There is, therefore, no statistically significant correlation for these were also about 60% higher during the conception period. parameters when all ten study months are taken into analysis (iEA vs. copulation rate: r =−0.091, p=0.803; iEA vs. aggression rate: 3.3. Influence of rank on androgen and glucocorticoid output s rs =0.067; p =0.855; 3α,11ß-dihydroxy-CM vs. copulation rate: r = 0.127, p = 0.726; 3α,11ß-dihydroxy-CM vs. aggression rate: Although levels of both iEA and 3α,11ß-dihydroxy-CM were s r =0.152; p=0.676). Restricting correlation analyses to the months higher in dominant compared to sub-ordinate males (Fig. 4), the s from February to September (the months when cycling females were available [49]), yielded a strong and significant correlation between androgen levels and the median rate of aggressive male–male inter-

actions (rs =0.786; p=0.021). Monthly rates of male–male aggression and copulatory activity were both significantly correlated with the number of cycling females

per month (aggression: rs =0.850, pb0.002; copulation: rs =0.642, p=0.045). Furthermore, when the first 8 months of the study period where cycling females were available in the group were considered, the number of cycling females in each month also strongly correlated with

the monthly median levels of both androgen (rs =0.728, p=0.041)and glucocorticoid levels (rs =0.792, p=0.019) in the group males.

3.5. Seasonal variation in temperature and rainfall and their relationship to hormone levels

Average forest temperature fluctuated between 22.9 °C and 23.8 °C throughout the study months and thus did not show a marked seasonal variation (Fig. 5). The amount of rainfall, by comparison, was more variable, including a three month dry season between June and August. None of the climatic variables measured, however, showed a significant

correlation with either fecal androgen levels (rainfall: rs =0.127, Fig. 3. Fecal androgen (iEA; grey bars) and glucocorticoid (3α,11β-dihydroxy-CM; p=0.726; mean T°: r =0.115, p=0.751; min T°: r =0.225, p=0.532; white bars) levels during the conception and non-conception period (N=10 males). s s − − Box plots show the median (horizontal line), the 1st and 3rd quartiles (box) and the max T°: rs = 0.586, p=0.075; T° difference: rs = 0.532, p=0.113) 10th and 90th percentiles (whiskers). Dots indicate outliers. ⁎ p=0.032. or glucocorticoid concentrations (rainfall: rs =0.127,p=0.726;meanT°: C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 173

contest is lower should be adaptive in terms of offsetting the costs of heightened levels of both steroids [22,70]. The close association between androgen/glucocorticoid levels with the number of cycling females during periods when these were available is in line with an earlier study on captive male long-tailed macaques that showed that exposure to estrus females resulted in elevated levels of both testosterone and cortisol [71]. Together these findings clearly suggest that both hormones have an important function in the context of reproductive competition between males. In this respect, our finding is in line with the “challenge hypothesis” which predicts that in polygynous species, testosterone levels should be positively related to the degree of sexual competition for mates [9]. Thus long-tailed macaques seem to adapt to the challenges of reproduction in a similar way as many vertebrate and other primate species [25,30–32,72–74]. Our finding of a strong positive relationship

Fig. 5. Monthly variation in mean forest temperature (°C) and total amount of between male aggression rates and the number of cycling females as precipitation (mm) at the study site. predicted indicates that male–male contest for mating opportunities was highest when the majority of the group females displayed ovarian cycles [49] compared to later times. In accordance with this, elevated rs =0.442, p=0.2; min T°: rs =0.056, p=0.877; max T°: rs =−0.162, androgen and glucocorticoid levels were clearly higher during this p=0.655; T° difference: rs =−0.135, p=0.711). early conception period, a finding similar to those reported for ring- tailed lemurs [22] and Japanese macaques [26] and in line with [9] the 4. Discussion prediction that in animals with a certain seasonality in breeding, highest testosterone concentrations should occur during the early Our results show that wild male long-tailed macaques exhibit a stages of the mating season when reproductive contest between distinct seasonal variation in both, androgen and glucocorticoid levels, males is most pronounced. On a proximate level, the conception with concentrations of both hormones being highest at the beginning season elevations in androgens may sustain gametogenesis (and thus of the conception period and at the end of the non-conception period. facilitate males to mate repeatedly) and fighting ability (and thus the Overall, androgen (but not glucocorticoid) levels were significantly ability to monopolize cycling females). This would clearly make sense elevated during the conception period in association with elevated given that male reproductive success in long-tailed macaques is rates of male–male aggression and copulatory activity, both strongly mainly achieved through extended periods of mate-guarding or related to the number of cycling females in the group. The pattern in through post-copulatory sperm competition, both resulting in a high hormone levels was generally related to the degree of male–male degree of male–male reproductive competition [49,55]. reproductive competition (and thus socially modulated), rather than Interestingly, our data clearly show that following the conception being explained by changes in external environmental factors period, male hormones started to rise again during the late birth (temperature, rainfall). Interestingly, however, levels of both hor- season at a time when aggressive and sexual behavior was almost mones increased 1–2 months prior to the expected onset of the next absent. Thus, the rise in hormone levels clearly occurred prior to the mating season in the absence of male–male reproductive contest, onset of the next mating season suggesting, at least in part, a suggesting that male endocrine status does not solely respond to decoupling of male endocrine response to social parameters. As the social cues and that male long-tailed macaques go through pre- study ended in November (the time when the latest females gave breeding hormonal changes in preparation for the upcoming birth), we have no data on behavioral and endocrine changes in the challenges of male–male reproductive competition. Our data thus months thereafter that indicate when exactly the next mating season provide the first evidence that males of a primate species classified as started. Earlier studies [58,75] on the same population (as well as our capital (thus non-strictly seasonal) breeder show endocrine patterns own observations at the onset of the study), however, showed that generally similar to those found in income (strictly seasonal) breeders. mating activity and male–male aggression usually starts in December/ Given the classification of the long-tailed macaque as a capital January which is 1–2 months after the rise in male hormones observed breeder [23], we predicted male androgen and glucocorticoid levels to in the present study. It is therefore reasonable to assume that male show a seasonal variation in accordance with the moderate degree of long-tailed macaques go through pre-breeding season endocrine reproductive seasonality of the species [48–50]. Our finding of a changes similar to those so far only described for more strictly distinct seasonal pattern of both, androgens and glucocorticoids, with seasonally reproducing primate species (sifakas [30,31,36], squirrel an elevation of androgens during the conception period substantiates monkeys [27], rhesus macaques [25] and Japanese macaques [21,26]), our prediction. Our results for the long-tailed macaque therefore which all show a 1–3 months delay between seasonal increases in sex contrast with those reported for non-seasonally breeding captive and/or adrenal hormone secretion or changes in testes size and stumptailed macaques [44] and hanuman langurs [45], in which males activation of sexual and aggressive behavior. Given that sex and (studied in captivity though) do not show any seasonal variation in adrenal steroids play an important role for sperm production, muscle testosterone levels and other testicular parameters. Although endo- gain, fat storage and expression of sexual and aggressive behavior crine profiles in male long-tailed macaques thus principally resemble [27,37–42] and that pre-breeding season increases in these hormones the seasonal hormonal pattern reported for primate species with a likely serve to prepare males physiologically for the upcoming period much stronger degree of reproductive seasonality, the approximately of reproduction and male–male competition [40,43], it is most likely two-fold mating/conception season elevation in androgen and that the late birth season elevations in androgens and glucocorticoids glucocorticoid levels reported here is at the bottom of the range reported here also help male long-tailed macaques to enhance their (2.5–11 fold) generally found for more strictly seasonally breeding competitive ability, energy balance and overall fitness before the pre- species [21,28,32,34,36]. Given the potential fitness costs that high and post-copulatory contest actually starts. levels of testosterone and cortisol may carry [66–69],keeping Since the onset of elevations in androgens and glucocorticoids elevations of the two hormones to minimum levels required for during the late birth season was not associated with high levels of reproductive contest and reducing the levels during periods when aggression or sexual activity and also not with the availability of 174 C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 cycling females, the question arises, which factors may trigger Medan and Ketambe for providing an excellent research environment hormone production in male long-tailed macaques outside the and strong logistical support. We further thank Arwin, Azhar, Bahlias, conception period. Given that seasonal variations in male testosterone Dewi, Matplin, Rahimin, Samsu and Surya for assistance in the field, levels were not consistently found in captive long-tailed macaques Jutta Hagedorn and Andrea Heistermann for laboratory assistance and [46,47], it is unlikely that an inherent biological clock regulates Christof Neumann, Constance Dubuc, Keith Hodges and two anon- hormone production. Similarly, photoperiod, the most likely prox- ymous reviewers for very helpful comments on the manuscript. imate cue triggering increased activity of the hypothalamic-pituitary- Special thanks go to Carsten Niemitz for general support of the study. testicular axis in species living sufficiently far from equatorial This research was supported by the German Research Council (DFG) latitudes [21,34,76] is unlikely to be the trigger in our male long- (Ni186/14-1). A. Engelhardt was financially supported by the German tailed macaques since seasonal shifts in day length are minimal at our Academic Exchange Service (DAAD), the State of Berlin Graduate field site. In such environments, breeding cycles and associated Sponsorship (Nafög), the KKSG Fund, the Lucie-Burgers Foundation changes in physiology are often related to changes in precipitation and for Comparative Research, Arnhem, the Netherlands, and the food availability [77–79]. Rainfall and temperature, the two ecological Christian-Vogel-Fund. factors studied here, however, did not correspond with hormone levels. Instead, onset of elevations in androgens and glucocorticoids in References our study males followed the months when, at our field site, food is [1] Bercovitch FB. The physiology of male reproductive strategies. In: Dolhinov P, usually most abundant [48], suggesting that food availability (and Fuentes A, editors. The nonhuman primates. Mountain View, California: Mayfield; thus energetic condition) may trigger hormone output in male long- 1999. p. 237–44. tailed macaques. This is in line with the prediction that in capital [2] Santiago-Moreno J, Gomez-Brunet A, Toledano-Diaz A, Picazo R, Gonzalez-Bulnes A, Lopez-Sebastian A. Seasonal endocrine changes and breeding activity in breeders, to which long-tailed macaques belong, male endocrine Mediterranean wild ruminants. Reprod Domest Anim 2006;41:72–81. response is triggered by internal rather than external parameters [23]. [3] Wapstra E, Swain R. Reproductive correlates of abdominal fat body mass in Although male long-tailed macaques appear to change their Niveoscincus ocellatus, a skink with an asynchronous reproductive cycle. J Herpetol 2001;35:403–9. endocrine milieu in association with prospective challenges, there [4] Girgenrath M, Marsh RL. Season and testosterone affect contractile properties of was no rank effect neither on androgen nor on glucocorticoid levels fast calling muscles in the gray tree Hyla chrysoscelis. Am J Physiol-Regul during the conception period when contest between males for access Integr Comp Physiol 2003;284:R1513–20. to females was most pronounced. This was in contrast to our [5] Minter LJ, DeLiberto TJ. Seasonal variation in serum testosterone, testicular volume, and semen characteristics in the coyote (Canis latrans). Theriogenology prediction that high-ranking males should show higher hormonal 2008;69:946–52. levels than low-ranking ones given that they engage more heavily in [6] Johnsen TS. Behavioural correlates of testosterone and seasonal changes of steroids – energy-costly mate-guarding of and copulation with fertile females in red-winged blackbirds. Anim Behav 1998;55:957 65. [7] Smith LC, John-Alder HB. Seasonal specificity of hormonal, behavioral, and [49,55,58]. It is also in contrast to the observation that in captive- coloration responses to within- and between-sex encounters in male lizards housed individuals, dominant males displayed greater testosterone (Sceloporus undulatus). Horm Behav 1999;36:39–52. increases than sub-ordinate males when briefly exposed to estrous [8] Tokarz RR, McMann S, Seitz L, John-Alder H. Plasma corticosterone and testosterone levels during the annual reproductive cycle of male brown anoles females [71]. However, maintaining high levels of testosterone and/or (Anolis sagrei). Physiol Zool 1998;71:139–46. cortisol for prolonged periods can carry high costs in terms of [9] Wingfield JC, Hegner RE, Dufty AM, Ball GF. The “challenge hypothesis”: theoretical suppression of the immune system [80,81] and testicular function implications for patterns of testosterone secretion, mating systems, and breeding strategies. Am Nat 1990;136:829–46. [82,83], and detrimental effects on general health [81]. Males should [10] Mooring MS, Patton ML, Lance VA, Hall BM, Schaad EW, Fetter GA, et al. Glucocorticoids therefore usually keep hormone levels as low as possible and of bison bulls in relation to social status. Horm Behav 2006;49:369–75. therefore rank-related differences in male androgen and glucocorti- [11] Moore IT, Jessop TS. Stress, reproduction, and adrenocortical modulation in amphibians and reptiles. Horm Behav 2003;43:39–47. coid levels are mainly found during times of rank instabilities [12] Romero LM. Seasonal changes in plasma glucocorticoid concentrations in free- (reviewed in [84]; see also [26,74,75]). The lack of a rank-related living vertebrates. Gen Comp Endocr 2002;128:1–24. difference in the two endocrine measures in our study may therefore [13] Franceschini C, Siutz C, Palme R, Millesi E. Seasonal changes in cortisol and – be ascribed to the fact that male ranks remained relatively stable progesterone secretion in Common . Gen Comp Endocr 2007;152:14 21. [14] Strauss A, Mascher E, Palme R, Millesi E. Sexually mature and immature yearling throughout the conception period. Data on more wild long-tailed male European ground squirrels: a comparison of behavioral and physiological macaque groups during both stable and rank-challenging periods may parameters. Horm Behav 2007;52:646–52. further clarify this issue. [15] Li CW, Jiang ZG, Zeng Y, Yan C. Relationship between serum testosterone, dominance and mating success in Pere David's deer stags. Ethology 2004;110:681–91. Altogether, our results show that despite being able to breed year- [16] Creel S. Social dominance and stress hormones. Trends Ecol Evol 2001;16:491–7. round, wild male long-tailed macaques undergo distinct annual [17] van Schaik CP, van Noordwijk MA, Nunn CL. Sex and social evolution in primates. endocrine changes. Although androgen and glucocorticoid profiles In: Lee PC, editor. Comparative primate socioecology. Cambridge: Cambridge – fl University Press; 1999. p. 204 40. generally re ected the extent of sexual and aggressive activity in [18] Di Bitetti MS, Janson CH. When will the stork arrive? Patterns of birth seasonality males for most time of the year, our data also indicated a decoupling of in neotropical primates. Am J Primatol 2000;50:109–30. endocrine and behavioral variables prior to the upcoming mating [19] Janson C, Verdolin J. Seasonality of primate births in relation to climate. In: Brockman DK, van Schaik CP, editors. Seasonality in primates: studies of living and season, suggesting that males of this species prepare physiologically extinct human and non-human primates. Cambridge: Cambridge University Press; for the prospective challenges of male reproductive competition. This 2005. p. 307–50. observation of a physiological preparation in primate males of a non- [20] Seraphin SB, Whitten PL, Reynolds V. The interaction of hormones with ecological factors in male Budongo Forest chimpanzees. In: Newton-Fisher NE, et al, editor. strictly seasonal breeding species was, however, unexpected and may Primates of Western Uganda (developments in primatology: progress and mean that behaviorally independent preparation for prospective prospects). Berlin: Springer; 2006. p. 93–104. challenging situations is more common in primate males than [21] Rostal DC, Glick BB, Eaton GG, Resko JA. Seasonality of adult male Japanese fi previously thought. macaques (Macaca fuscata): androgens and behavior in a con ned troop. Horm Behav 1986;20:452–62. [22] Gould L, Ziegler TE. Variation in fecal testosterone levels, inter-male aggression, Acknowledgements dominance rank and age during mating and post-mating periods in wild adult male ring-tailed Lemurs (Lemur catta). Am J Primatol 2007;69:1325–39. [23] Brockman DK, van Schaik C. Seasonality and reproductive function. In: Brockman We gratefully acknowledge the cooperation and support of the DK, van Schaik C, editors. Seasonality in primates: studies of living and extinct Indonesian Institute of Sciences (LIPI), the General Directorate of human and non-human primates. Cambridge: Cambridge University Press; 2005. Forest Protection and Nature Conservation (PKA), Universitas p. 269–306. [24] Stearns S. Trade-offs in life-history evolution. Funct Ecol 1989;3:259–68. Nasional (UNAS), Jakarta, especially Tatang Mitra Setia, and the Leuser [25] Gordon TP, Rose RM, Bernstein IS. Seasonal rhythm in plasma testosterone levels in Management Unit (UML). We particularly thank the UML staff in rhesus monkeys (Macaca mulatta): a 3 year study. Horm Behav 1976;7:229–43. C. Girard-Buttoz et al. / Physiology & Behavior 98 (2009) 168–175 175

[26] Barrett GM, Shimizu K, Bardi M, Asaba S, Mori A. Endocrine correlates of rank, [54] Muller MN, Wrangham RW. Dominance, cortisol and stress in wild chimpanzees reproduction, and female-directed aggression in male Japanese macaques (Pan troglodytes schweinfurthii). Behav Ecol Sociobiol 2004;55:332–40. (Macaca fuscata). Horm Behav 2002;42:85–96. [55] De Ruiter JR, Van Hoof JARAM, Scheffrahn W. Social and genetic aspects of paternity [27] Wiebe RH, Williams LE, Abee CR, Yeoman RR, Diamond EJ. Seasonal changes in in wild long-tailed macaques (Macaca fascicularis). Behaviour 1994;129:203–24. serum dehydroepiandrosterone, androstenedione, and testosterone levels in the [56] Rijksen H. A field study on Sumatran orang-utans (Pongo pygmaeus abelli, Lesson squirrel monkey (Saimiri boliviensis boliviensis). Am J Primatol 1988;14:285–91. 1827): ecology, behavior and conservation. Wageningen: Veenman; 1978. [28] Lynch JW, Ziegler TE, Strier KB. Individual and seasonal variation in fecal [57] van Schaik CP, Mirmanto E. Spatial variation in the structure and litterfall of a testosterone and cortisol levels of wild male tufted capuchin monkeys, Cebus Sumatran rain forest. Biotropica 1985;17:196–205. apella nigritus. Horm Behav 2002;41:275–87. [58] van Noordwijk MA. Sexual behaviour of Sumatran long-tailed macaques (Macaca [29] Bales KL, French JA, McWilliams J, Lake RA, Dietz JM. Effects of social status, age, fascicularis). Z Tierpsychol 1985;70:277–96. and season on androgen and cortisol levels in wild male golden lion tamarins [59] Altmann J. Observational study of behavior: sampling methods. Behaviour (Leontopithecus rosalia). Horm Behav 2006;49:88–95. 1974;49:227–67. [30] Brockman DK, Whitten PL, Richard AF, Schneider A. Reproduction in free-ranging [60] van Hooff JARAM. The facial displays of the catarrhine monkeys and apes. In: male Propithecus verreauxi: the hormonal correlates of mating and aggression. Am Morris D, editor. Primate ethology. London: Weidenfeld & Nicolson; 1967. p. 7–68. J Phys Anthropol 1998;105:137–51. [61] Ziegler T, Hodges JK, Winkler P, Heistermann M. Hormonal correlates of [31] Fichtel C, Kraus C, Ganswindt A, Heistermann M. Influence of reproductive season reproductive seasonality in wild female Hanuman langurs (Presbytis entellus). and rank on fecal glucocorticoid levels in free-ranging male Verreaux's sifakas Am J Primatol 2000;51:119–34. (Propithecus verreauxi). Horm Behav 2007;51:640–8. [62] Möhle U, Heistermann M, Palme R, Hodges JK. Characterization of urinary and fecal [32] Ostner J, Kappeler PM, Heistermann M. Seasonal variation and social correlates of metabolites of testosterone and their measurement for assessing gonadal endocrine androgen excretion in male redfronted lemurs (Eulemur fulvus rufus). Behav Ecol function in male nonhuman primates. Gen Comp Endocr 2002;129:135–45. Sociobiol 2002;52:485–95. [63] Ganswindt A, Palme R, Heistermann M, Borragan S, Hodges JK. Non-invasive [33] Ostner J, Kappeler PM, Heistermann M. Androgen and glucocorticoid levels reflect assessment of adrenocortical function in the male African elephant (Loxodonta seasonally occurring social challenges in male redfronted lemurs (Eulemur fulvus africana) and its relation to musth. Gen Comp Endocr 2003;134:156–66. rufus). Behav Ecol Sociobiol 2008;62:627–38. [64] Möstl E, Palme R. Hormones as indicators of stress. Domest Anim Endocrin [34] Aujard F, Perret M. Age-related effects on reproductive function and sexual 2002;23:67–74. competition in the male prosimian primate, Microcebus Murinus. Physiol Behav [65] Heistermann M, Palme R, Ganswindt A. Comparison of different enzyme 1998;64:513–9. immunoassays for assessment of adrenocortical activity in primates based on [35] Strier KB, Ziegler TE, Wittwer DJ. Seasonal and social correlates of fecal testosterone fecal analysis. Am J Primatol 2006;68:257–73. and cortisol levels in wild male muriquis (Brachyteles arachnoides). Horm Behav [66] Wingfield JC, Jacobs JD, Tramontin AD, Perfito N, Meddle S, Maney DL, et al. Toward 1999;35:125–34. an ecological basis of hormone-behavior interactions in reproduction of birds. In: [36] Kraus C, Heistermann M, Kappeler PM. Physiological suppression of sexual Wallen K, Schneider J, editors. Reproduction in context. Cambridge, MA: MTI function of subordinate males: a subtle form of intrasexual competition among Press; 1999. p. 85–128. male sifakas (Propithecus verreauxi)? Physiol Behav 1999;66:855–61. [67] Wikelski M, Lynn S, Breuner C, Wingfield JC, Kenagy GC. Energy metabolism, [37] Sapolsky RM. Testicular function, social rank and personality among wild baboons. testosterone and corticosterone in white-crowned sparrows. J Comp Biochem A Psychoneuroendocrino 1991;16:1–13. 1999;185:463–71. [38] Matsubayashi K, Enomoto T. Longitudinal studies on annual changes in plasma [68] Klein SL. Hormones and affect sex and species differences in testosterone, body weight and spermatogenesis in adult Japanese monkeys immune function among vertebrates. Behav Processes 2000;51:149–66. (Macaca fuscata fuscata) under laboratory conditions. Primates 1983;24:521–9. [69] Pride RE. High faecal glucocorticoid levels predict mortality in ring-tailed lemurs [39] Gupta G, Maikhuri J, Setty B, Dhar JD. Seasonal variations in daily sperm (Lemur catta). Biol Lett 2005;1:60–3. production rate of rhesus and bonnet monkeys. J Med Primatol 2000;29:411–4. [70] Bercovitch FB, Harvey N. Reproductive life history. In: Thierry B, Singh M, [40] Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress Kaumanns W, editors. Macaque societies. Cambridge: Cambridge University Press; responses? Integrating permissive, suppressive, stimulatory, and preparative 2004. p. 61–83. actions. Endocr Rev 2000;21:55–89. [71] Glick BB. Male endocrine responses to females: effects of social cues in [41]SchimlPA,MendozaSP,SaltzmanW,LyonsDM,MasonWA.Seasonalityin cynomolgus macaques. Am J Primatol 1984;6:229–39. squirrel monkeys (Saimiri sciureus), social facilitation by females. Physiol [72] Beehner JC, Bergman TJ, Cheney DL, Seyfarth RM, Whitten PL. Testosterone Behav 1996;60:1105–13. predicts future dominance rank and mating activity among male chacma baboons. [42] Bercovitch FB. Estradiol concentrations, fat deposits, and reproductive strategies in Behav Ecol Sociobiol 2006;59:469–79. male rhesus macaques. Horm Behav 1992;26:272–82. [73] Rose RM, Holaday JW, Bernstein IS. Plasma testosterone, dominance rank and [43] Bercovitch FB, Ziegler TE. Current topics in primate socioendocrinology. Annu Rev aggressive behaviour in male rhesus monkeys. Nature 1971;231:366–8. Antrhropol 2002;31:45–67. [74] Teichroeb JA, Sicotte P. Social correlates of fecal testosterone in male ursinus [44] Nieuwenhuijsen K, Deneef KJ, Tenboschjjv, Slob AK. Testosterone, testis size, colobus monkeys (Colobus vellerosus): the effect of male reproductive competition seasonality, and behavior in group living stumptail macaques (Macaca arctoides). in aseasonal breeders. Horm Behav 2008;54:417–23. Horm Behav 1987;21:153–69. [75] Ostner J, Kappeler P, Heistermann M. Androgen and glucocorticoid levels reflect [45] Lohiya NK, Sharma RS, Manivannan B, Kumar MA. Reproductive exocrine and seasonally occurring social challenges in male redfronted lemurs (Eulemur fulvus endocrine profiles and their seasonality in male langur monkeys (Presbytis entellus rufus). Behav Ecol Sociobiol 2008;62:627–38. entellus). J Med Primatol 1998;27:15–20. [76] Lindburg DG. Seasonality of reproduction in primates. In: Mitchell G, Erwin J, [46] Dang DC, Meusy-Dessolle N. Annual plasma testosterone cycle and ejaculatory editors. Comparative primate biology. New York: Alan R. Liss; 1987. p. 167–218. ability in the laboratory-housed crab-eating macaque (Macaca fascicularis). [77] Butynski TM. Guenon birth seasons and correlates with rainfall and food. In: Reprod Nutr Dev 1981;21:59–68. Gautier-Hion A, et al, editor. A primate radiation: evolutionary biology of the [47] Shimizu K, Kojima C, Otsuka-Itoh M, Hayashi M, Watanabe G, Taya K. Seasonal African guenons. Cambridge: Cambridge University Press; 1988. p. 284–322. variations in circulating leptin and reproductive hormones in seasonal breeding [78] Dixson AF. Primate sexuality. New York: Oxford University Press; 1998. macaques. Folia Primatol 2004;75:412–3. [79] Bronson FH, Heideman PD. Seasonal regulation of reproduction in mammals. In: [48] van Schaik CP, van Noordwijk MA. Interannual variability in fruit abundance and Knobils E, Neill JD, editors. The physiology of reproduction, 2nd ed., vol. 2. the reproductive seasonality in Sumatran long-tailed macaques (Macaca fascicu- New York: Raven Press; 1994. p. 541–83. laris). J Zool 1985;206:533–49. [80] Grossman CJ. Interactions between the gonadal steroids and the immune system. [49] Engelhardt A, Heistermann M, Hodges JK, Nurnberg P, Niemitz C. Determinants of Science 1985;227:257–61. male reproductive success in wild long-tailed macaques (Macaca fascicularis): [81] Sapolsky RM. Endocrinology of the stress response. In: Becker JB, et al, editor. male monopolisation, female mate choice or post-copulatory mechanisms? Behav Behavioural endocrinology. 2nd edition. Cambridge, Massachusets: MIT Press; 2002. Ecol Sociobiol 2006;59:740–52. p. 409–50. [50] Kavanagh M, Laursen E. Breeding seasonality among long-tailed macaques, Macaca [82] Sapolsky RM. Stress-induced suppression of testicular function in the wild fascicularis, in peninsular Malaysia. Int J Primatol 1984;5:17–29. baboon: role of glucocorticoids. Endocrinology 1985;116:2273–8. [51] Creel S. Social dominance and stress hormones. Trends Ecol Evol 2001;16:491–7. [83] Hardy MP, Gao HB, Dong Q, Ge RS, Wang Q, Chai WR, et al. Stress hormone and [52] Abbott DH, Keverne EB, Bercovitch FB, Shively CA, Mendoza SP, Saltzman W, et al. male reproductive function. Cell Tissue Res 2005;322:147–53. Are subordinates always stressed? A comparative analysis of rank differences in [84] Sapolsky RM. The physiology of dominance in stable versus unstable social cortisol levels among primates. Horm Behav 2003;43:67–82. hierarchies. In: Manson WA, Mendoza SP, editors. Primate social conflicts. Albany, [53] Muller MN, Wrangham RW. Dominance, aggression and testosterone in wild NY: University of New York Press; 1993. p. 171–204. chimpanzees: a test of the ‘challenge hypothesis’. Anim Behav 2004;67:113–23.