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Seasonal and Social Correlates of Fecal Testosterone and Cortisol Levels in Wild Male Muriquis (Brachyteles arachnoides)
Hormones and Behavior 35, 125–134 (1999) Article ID hbeh.1998.1505, available online at http://www.idealibrary.com on
Seasonal and Social Correlates of Fecal Testosterone and Cortisol Levels in Wild Male Muriquis (Brachyteles arachnoides) Karen B. Strier,* ,1 Toni E. Ziegler,† and Daniel J. Wittwer† *Department of Anthropology, University of Wisconsin-Madison, 1180 Observatory Drive, Madison, Wisconsin 53706; and †Wisconsin Regional Primate Research Center, Madison, Wisconsin Received March 30, 1998; revised August 10, 1998; accepted December 7, 1998
Fecal testosterone and cortisol levels were analyzed from six wild male muriquis (Brachyteles arachnoides) over a 19-month period at the Estac¸a˜o Biolo´gica de Caratinga in Minas Gerais, Brazil, to investigate the hormonal correlates of seasonal sexual behavior and environmental conditions. Group mean testosterone levels based on weekly samples from the six males did not differ between copulatory and noncopulatory periods or between rainy and dry seasons. Cortisol levels did change with copulatory periods, and were significantly higher during the second dry season, when mating continued following an exceptionally heavy rainy season, than during the first dry season, when mating ceased. Males exhibited individual variation in the timing of their hormone shifts relative to their sexual activity, but neither hormone levels nor sexual activity were related to male age. Despite individual differences in the timing of testosterone fluctuations around the onset and offset of the copulatory season, all males exhibited elevated cortisol concentrations following a slight increase in testosterone at the beginning of the copulatory season. Both the lack of significant changes in testosterone levels with the onset of the rainy and copulatory season and the lack of prebreeding increases in cortisol may be related to the low levels of overt aggression displayed by male muriquis over access to mates. © 1999 Academic Press Key Words: muriquis; seasonality; testosterone; cortisol; fecal steroids.
Testosterone’s influence on male sexual behavior has been described in many vertebrate species. For instance, an increase in testosterone is associated with bird song production in juncos, Junco hyemalis (Ketter1 To whom correspondence and reprint requests should be addressed. E-mail: [email protected].
0018-506X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
son, Nolan, Wolf, Ziegenfus, Dufty, Ball, and Johnsen, 1991) and can promote territory aggression in birds (Wingfield and Ramenostsky, 1985). In male rats, mating behavior is dependent upon testosterone (Meisel and Sachs, 1994) and increased testosterone promotes a preference for female estrus odors (Stern, 1970). Testosterone, in conjunction with FSH, appears to be essential in spermatid maturation, and may influence fertility in males (Williams-Ashman, 1988; McLachlan, Wreford, O’Donnell, de Krester, and Robertson, 1996). Therefore, it is not surprising that environmental influences can promote an increase in testosterone in seasonally breeding animals. Testosterone changes can be regulated by seasonal cues such as changes in day length and patterns of rainfall (Wingfield and Kenagy, 1991). Males from a number of species of primates also show seasonal changes in testosterone, with peak levels occurring during the breeding season. Plasma testosterone levels in the lesser mouse lemur (Microcebus murinus) have been shown to increase and decrease in response to corresponding changes in photoperiod (Perret, 1992). Likewise, seasonality in testosterone levels has been reported in squirrel monkeys (Saimiri sciureus: Wiebe, Williams, Abee, Yeoman, and Diamond, 1988; Schiml, Mendoza, Saltzman, Lyons, and Mason, 1996), Hanuman langurs (Presbytis entellus: Lohiya, Sharma, Manivannan, and Anand Kumar, 1998), and several species of macaques (e.g., Macaca fuscata, Torii and Nigi, 1994; M. radiata, Kinger, Rajalakshmi, Kumar, Sharma, and Bajaj, 1995; and M. mulatta, Herndon, Mindi, Nordmeyer, and Turner, 1996). Both social and environmental stimuli may influence the onset of testosterone increases in primates. For captive rhesus males, Herndon et al. (1996) found
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that male testosterone levels fluctuated seasonally even without exposure to females, suggesting that environmental stimuli alone may be sufficient to activate male gonadal responses. By contrast, Gordon, Bernstein, and Rose (1978) found that visual contact with rhesus females was sufficient to maintain seasonality in testosterone. Without minimal distal contact with females, seasonal testosterone patterns were not maintained, suggesting that testosterone fluctuations may be socially facilitated. Exposure to sexually active females outside of the mating season can further stimulate testosterone elevations (Ruiz de Elvira, Herndon, and Wilson, 1982). Social stimuli are also thought to initiate increases in testosterone concentrations in other primates, including male squirrel monkeys (Schiml et al., 1996) and wild sifakas (Propithecus verreauxi: Brockman, Whitten, Richard, and Schneider, 1998). Like these primates, wild muriqui monkeys (Brachyteles arachnoides) display seasonal breeding and live in multimale, multifemale groups, but the hormonal correlates of male seasonal sexual activity have not previously been investigated. We compared male muriqui testosterone levels with the onset of the rainy/dry seasons and with the onset of the copulatory/noncopulatory periods. In species such as rhesus macaques and squirrel monkeys, testosterone elevations are preceded by cortisol elevations, which are thought to be associated with seasonal weight gains (Wiebe et al., 1988; Bercovitch, 1992; Schiml et al., 1996). We therefore expected to find a similar pattern in cortisol and testosterone concentrations in seasonally breeding male muriquis. Hormonal fluctuations in female muriquis are known to coincide with the copulatory and rainy seasons. Resumption of postpartum ovulation in wild females correlated with the resumption of postpartum sexual activity during the early rainy season months (Strier and Ziegler, 1997; Ziegler, Santos, Pissinatti, and Strier, 1997). The onset of postpartum cycling was not tightly synchronized among individual females, despite the temporal clumping of their prior and subsequent births during the annual dry season months of June–August, when two-thirds of all infants in our study group have been born (Strier and Ziegler, 1994; Strier, 1996, in press). Most mating takes place during the rainy season months, from October through March (Strier, 1997). However, we concluded that photoperiodicity, which varies by about 2 21 h throughout the year at our fieldsite, was unlikely to be the sole extrinsic factor responsible for seasonal ovulation in muriquis because infant mortality could result in the
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resumption of sexual activity (Strier, 1996) and ovarian cycling (Strier and Ziegler, 1997) within 2 months postpartum during the dry season. The apparent coincidence between annual fluctuations in rainfall and mating patterns, on the one hand, and the disparity between the timing of mating and conceptions, on the other hand, stimulated our interest in whether steroid levels in male muriquis exhibit seasonal patterns similar to those detected in females. By contrast to most other primates, muriquis lack overt evidence of dominance hierarchies, either among males or between males and females (Strier, 1992a,b). Females mate without harassment from males or inciting overt competition among males, and both sexes routinely mate with multiple partners (Strier, 1992b, 1997). Therefore, rank should not be a confounding variable affecting individual differences in male muriqui hormone levels. This paper presents the results of a 19-month investigation of the seasonal and social correlates of testosterone and cortisol levels in wild male muriquis.
MATERIALS AND METHODS Animals and Sample Collection Fecal samples were collected from January 1996 through July 1997 at 4- to 5-day intervals from six wild male muriquis inhabiting the 860 hectare forest at the Estac¸a˜o Biolo´gica de Caratinga, Minas Gerais, Brazil (Strier, 1992a). Our sampling schedule provided one or two fecal samples per week from each of the six males. We obtained from 73–77 weeks of samples for each male during our 82-week study period. The six male subjects were preselected from among the 13 sexually active males in the study group during this period based on their relative ages and known maternal kinship. Two of the males (CL and IV) were classified as mature based on their visible adult size in 1982 and their observed copulations during the 1983– 1984 rainy season. Male muriquis of known age may begin to copulate when they are 5– 6 years old (Strier, 1996), implying that our mature male subjects were at least 19 –20 years of age during the present study period. Two middle-aged males (DI and NI) were present as infants in the study group in 1982, and therefore 14 –15 years of age during this study. Two young males (DA and NE) were born in 1985 and 1987, respectively. All individuals in the study group were fully habituated to human observers and recognizable by their natural markings.
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Methods of collecting and preserving male fecal samples followed those we employed in a previous study of female muriqui fecal steroids, in which fresh fecal samples were stored on ice and extracted as wet feces at the field station (Strier and Ziegler, 1997). No statistical differences were found in levels of either testosterone (paired t test, t 5 0.49, n 5 5, P 5 0.65) or cortisol (t 5 0.12, n 5 5, P 5 0.91) between morning and afternoon fecal samples collected from the same individuals. Nonetheless, to reduce the potential confounding effects of diurnal testosterone and cortisol fluctuations on seasonal patterns, we restricted our collections of male fecal samples during the present study to the period between sunrise and 1100 h. Behavioral Data and Analyses All copulations involving members of the study group were recorded by two trained observers throughout the day during the course of near-daily opportunistic and systematic behavioral sampling during this study period (Strier and Ziegler, 1997). A total of 178 copulations were observed on 100 different days during the present study period. From 1 to 5 different females, and from 1 to 10 different males copulated per week during the 44 weeks in which copulations occurred. Because the initiation of the present study in January 1996 corresponded to the middle of the 1995–1996 rainy and mating season, we use the last observed copulation to define the end of the mating season this year. From 30 April–26 September 1996, no copulations were observed. The first observed copulations following this period of sexual quiescence occurred on 27 September 1996 and represent the onset of the 1996 –1997 mating season. The 1996 –1997 rainy season was atypical in that over 60% more rain fell (1855 mm) compared to the annual average of 1119 6 189 mm of rainfall from 1986 –1994 (Strier, 1996). Indeed, rainfall accumulation by January 1997 was already substantially greater (1285 mm) than the total annual rainfall of 1030 mm from 1995–1996. Based on monthly rainfall patterns, we distinguished four periods in our present analyses: first rainy season (January–April 1996); first dry season (May–August 1996); second rainy season (September 1996 –April 1997); and second dry season (May– July 1997). Mating patterns during the 1996 –1997 rainy season were correspondingly atypical. The muriquis continued to copulate throughout the second dry season, in contrast to their sexual inactivity during these same months in the previous year (Fig. 1). In contrast to
1996, there was no behavioral evidence to mark the end of the mating season in 1997. Muriqui mating patterns permitted us to divide our study into two copulatory periods, from mid January–late April 1996 and from late September 1996 –July 1997, and one noncopulatory period, from late April–late September 1996. Steroid Assays Sample preparation. All fecal samples were mixed, weighed (0.1 g), and extracted in the field according to the technique described by Strier and Ziegler (1997). A portion of the 5-ml water/ethanol steroid extraction, 500 ml, was subjected to solvolysis and further extracted with ethyl acetate to eliminate the single and multiple conjugation. Sequential hydrolysis and solvolysis of muriqui samples revealed that 64.5% of the fecal testosterone was bound by double conjugates, necessitating the solvolysis procedure described in Ziegler, Scheffler, Wittwer, Schultz-Darken, Snowdon, and Abbott (1996). After solvolysis, the samples were dried and resuspended in 500 ml ethanol. Aliquots of the ethanol sample were used for both testosterone and cortisol analyses. Testosterone analyses. No column chromatography separation was required for routine testosterone determination. However, two male muriqui samples were prepared as above and separated by HPLC with collection of the fractions for assay. The system consisted of a dual HPLC pump (Beckman Instruments, Schaumburg, IL) connected to a diode array analyzer for UV detection, which allowed visualization of the peaks as they were separated. Data were input through computer detection software (Beckman, System Gold, Version 8) and fractionated (Gilson, Middleton, WI) at 1 ml/min for the 40-min run. Samples reconstituted in 30 ml of mobile phase (40:60 acetonitrile:water) were injected into a 20-ml sample loop with a reversed phase precolumn (5 mm, 4.6 mm 3 4.5 cm, Beckman) and reversed phase HPLC column (ultrasphere, 5 mm, 4.6 3 25 cm, Beckman). The mobile phase remained constant at a flow rate of 1 ml/min for the first 25 min and then increased to 1.5 ml/min and 50:50 acetonitrile:water until the end of the run at 40 min. Half of each fraction was dried and assayed for cross-reactivity with the testosterone antibody. The fractions containing testosterone and, to a lesser degree, DHT (dihydrotestosterone) were the only fractions where activity occurred, indicating that there was no other steroid interference. The standard RIA for testosterone has been described (Robinson,
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Muriqui Male Fecal Steroids
Scheffler, Eisele, and Goy, 1975). After solvolysis, 50 ml of the ethanol suspension was used for the testosterone assay. The assay was validated for use with muriqui fecal steroid extracts by determining accuracy and parallelism. Mean accuracy in measuring testosterone was 103.44% 6 1.9 SE for nine standard curve points, and serial dilutions of a fecal pool from male muriquis were parallel to the testosterone standards with no differences in the slopes (t 5 21.56, P . 0.05). Intra- and interassay coefficients of variation for a muriqui fecal pool were 3.1 and 11.2, respectively (n 5 12). Cortisol analyses. Fractions from the remaining half of the HPLC separated samples were measured in the cortisol assay for determination of antibody crossreactivity with other steroids. Cross-reactivity only occurred at the retention times for cortisol and cortisone (collected in the same fraction and not separately identified), indicating that the cortisol assay would provide a true measurement of cortisol (and possibly cortisone) excretion in the muriqui. Cortisol was measured by an EIA (Ziegler, Scheffler, and Snowdon, 1995). Sample volumes of 100 ml of the ethanol solution were dried and resuspended in the assay buffer/ enzyme solution and aliquoted as 33% into each well. Standards (3.16 –1000 pg) were prepared the same way as samples with cortisol added in ethanol, dried, and resuspended in assay buffer/enzyme solution. Mean accuracy for the standard curve points was determined to be 124% 6 5.5 SE (n 5 6) and serially diluted muriqui feces were parallel to the standard curve with no difference in slopes (t 5 1.5, P . 0.05). Intra- and interassay coefficients of variation for a muriqui fecal pool were 3.8 and 9.4, respectively (n 5 18). Statistical Analyses Testosterone and cortisol concentrations were plotted for each of the six males on a weekly basis. We averaged the testosterone and cortisol concentrations from each individual with $ 1 sample in any week to obtain single weekly values per male. From visual inspection of their hormonal profiles, individual testosterone and cortisol levels appeared to increase after
the first day of observed copulation at the onset of the 1996 –1997 copulatory period. The day of the increase in testosterone concentration relative to the onset of the copulatory period was determined as the first day that the steroid level increased at least twofold and remained elevated. The onset of cortisol increase was also at least twofold and determined relative to the onset of the copulatory period. The timing of the sustained decrease in the concentrations of both steroids was also represented by at least a twofold change in these levels for each male. The twofold changes in hormonal levels were determined by comparing the mean of five preincrease samples representing a 3- to 4-week period to the mean of five postincrease samples for each male. To evaluate the possible relationships of copulatory periods or rainfall patterns on male steroid levels, the mean level of testosterone or cortisol was determined for each male per week, and averaged over each month. Mean testosterone and cortisol levels were calculated for the defined periods for each male. General Linear Model one-way ANOVAs were used to compare mean steroid levels per male across the copulatory–noncopulatory periods and the rainy-dry periods, as defined above. Post-hoc analyses were conducted with the Tukey test. Kruskal–Wallis tests were used to assess the possible effects of age on male hormone levels.
RESULTS Copulatory Behavior and Steroid Levels in Individual Males Copulation data indicated that the six males we used in our study accounted for 56% of the total 178 copulations observed during the 19 months. These males copulated on 85 of the 100 days in which $ 1 copulation was observed. Individual males averaged 16.7 6 9.8 copulations (median 5 16.5, range 5 5–33) across an average of 14.7 6 7.6 different days (median 5 15, range 5 5–26). Copulation frequencies among our six target males did not differ significantly by age class (X 2 5 4.3, df 5 2, P . 0.05). The 100 copulations
FIG. 1. Average monthly levels of rainfall are plotted in the top graph to compare with the copulation data that follows in the next three graphs. Weekly number of copulations, number of different females copulating, and number of different males copulating are plotted. Asterisks under the bottom graph indicate estimated conceptions based on documented births following a calculated 7-month gestation period (Strier and Ziegler, 1997). The only gap in behavioral observations . 10 days occurred from late December 1996 to early January 1997.
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FIG. 2. Profile of testosterone and cortisol levels by week for male CL. Shaded background indicates the noncopulatory period; white background indicates the copulatory periods. Asterisks indicate observed copulation dates for each male, respectively. The lack of correlation between muriqui male testosterone and cortisol levels is consistent with findings in rhesus macaques (Bercovitch and Clarke, 1995).
performed by these males were evenly distributed between the two older males (15 and 18%), but strongly skewed between the two middle aged males (5 and 20%) and younger males (9 and 33%). Age effects were not found for either steroid. No significant relationships were detected between male age and mean testosterone levels during the two copulatory periods (Kruskal–Wallis 5 3.43, df 5 2, P 5 0.18 for both) or during the noncopulatory period (Kruskal–Wallis 5 2.0, df 5 2, P 5 0.37). Older males showed a slight, but insignificant, trend for higher testosterone levels than younger males during the first copulatory period (r 2 5 0.63, n 5 6, P 5 0.06). Mean cortisol levels also showed no age differences during the combined copulatory periods
(Kruskal–Wallis 5 2.57, df 5 2, P 5 0.78) or during the noncopulatory period (Kruskal–Wallis 5 0.29, df 5 2, P 5 0.81). Individual males showed no significant correlations between weekly testosterone and cortisol levels (range of r values 5 0.07– 0.38). Figure 2 shows the hormonal profiles and observed copulation days of one of our study males. Each male showed a slight decline in testosterone levels within 2 months prior to the end of the first copulatory period in 1996, but the timing of these testosterone declines relative to each male’s last observed copulation was variable (Table 1). No clear pattern in declines in cortisol levels could be determined for individual males relative to either their last
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Muriqui Male Fecal Steroids
TABLE 1 Individual Hormonal Changes Relative to the Offset of the Copulatory Period a Group Offset
Individual Offset
Male
T
F
LOC
T
F
CL DA DI IV NE NI Mean SEM
241 260 257 260 255 26 246.50 8.60
16 None None None None 17
29 April 1996 29 April 1996 22 March 1996 29 April 1996 29 January 1996 3 March 1996
241 260 220 260 131 153 216.17 19.56
16 None None None None 156
The timing of $ 2-fold decreases in individual male testosterone (T) and cortisol (F) levels are shown relative to the last observed copulation (LOC) in the group, which occurred on 29 April 1996, and the LOC for each male, as indicated. The number of days before (2) or after (1) the last observed copulations are shown. The absence of detectable changes are indicated as none. a
observed copulation or the last observed copulation in the group during this period. A slight but consistent rise in testosterone levels occurred for four males from 0 to 4 days following the first observed copulation of the 1996 –1997 copulatory period (Table 2). No male showed an increase in cortisol levels prior to his testosterone surge during the 1996 –1997 breeding season.
levels (range of r values 5 0.18 – 0.35) or cortisol levels (range of r values 5 0.02– 0.20). Mean testosterone levels did not differ across copulatory and noncopulatory periods (F 5 2.22, df 5 2, 15, P 5 0.14). However, significant differences in cortisol levels were detected (F 5 7.39, df 5 2, 15, P 5 0.006). Post-hoc analyses indicated that mean cortisol levels were significantly higher during the second copulatory period (October 1996 –July 1997) than during the May–September 1996 noncopulatory period (P 5 0.007).
DISCUSSION Contrary to expectations based on other seasonally breeding primates, muriqui mean testosterone levels failed to exhibit significant seasonal changes correlating with either the onset of sexual behavior, as measured by behaviorally defined copulatory periods, or the onset of the rainy season. In fact, muriqui males failed to exhibit seasonal patterns in testosterone concentrations during the entire 19-month study period. By contrast, cortisol levels were significantly higher during the second rainy and dry seasons, when mating occurred, than during the first dry, noncopulatory, season. These data indicate that muriqui cortisol, but not testosterone, levels are responsive to seasonal stimuli. The absence of testosterone responsiveness associated with the onset of seasonal sexual activity in
Temporal Patterns in Testosterone and Cortisol Levels No significant differences were detected in mean testosterone levels among any of the rainy or dry periods (F 5 1.29, df 5 3, 20, P 5 0.30). Seasonal patterns in fecal cortisol levels differed from those of testosterone (Fig. 3). Significant differences in cortisol levels were found between the rainy and dry periods (F 5 5.09, df 5 3, 20, P 5 0.009). Post-hoc analyses indicated that cortisol levels were significantly lower during the first dry season than either the second dry season (P 5 0.01) or the second rainy season (P 5 0.03). All measures of sexual activity in the group exhibited seasonal patterns (Fig. 1), consistent with seasonal sexual activity reported for these muriquis in prior years (Strier, 1996, 1997). We did not find any significant relationships in weekly mean comparisons between the total number of copulations, number of copulation days, number of copulating females, or number of copulating males and mean testosterone
TABLE 2 Individual Hormonal Changes Relative to the Onset of the Copulatory Period a Group Onset Male
T
F
CL DA DI IV NE NI Mean SEM
14 0 14 0 157 157 120.33 11.62
160 17 169 169 157 174 156.00 10.13
Individual Onset FOC 30 27 12 13 10 20
October 1996 September 1996 October 1996 October 1996 October 1996 November 1996
T
F
230 0 211 217 144 14 21.67 10.40
134 17 157 151 144 121 135.67 7.74
The timing of $ 2-fold increases in individual male testosterone (T) and cortisol (F) levels are shown relative to the first observed copulation (FOC) in the group, which occurred on 27 September 1996, and the FOC for each male, as indicated. The number of days before (2) or after (1) the first observed copulations are shown. a
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FIG. 3. Weekly mean (and SEM) testosterone and cortisol levels over the 19-month study period. All six males are included. Shaded background indicates the noncopulatory period; white background indicates the copulatory periods. Gaps of .10 days in sample collections are indicated by discontinuous lines.
muriquis differs from that found in other seasonally breeding primates, such as Japanese macaques, where sexual activity was found to lag 1–2 months behind seasonal elevations in testosterone and DHT levels (Rostal, Glick, Eaton, and Resko, 1986). In male muriquis, the seasonal onset of sexual activity relative to individual testosterone levels was highly variable, which may have contributed to our inability to detect seasonal variation. Furthermore, the absence of overt aggression among male muriquis over sexual access to females (Strier, 1992b) may explain the absence of seasonal patterns in their testosterone profiles (Sapolsky, 1987). The fact that muriqui males copulated without consistent elevated levels of testosterone also suggests that they do not reduce their levels of testosterone
during the noncopulatory periods. In other multimale, multifemale groups of primates, males compete aggressively for access to females, and more aggressive males have higher levels of testosterone than less aggressive males (Rose, Holaday, and Bernstein, 1971). In muriquis, however, no overt signs of aggression or competition associated with access to mates have been observed (Strier, 1992b, 1997). The absence of significant seasonal increases in testosterone levels during the mating season is consistent with their unusually low levels of aggression. In contrast to their testosterone profiles, male muriquis exhibited significantly higher cortisol levels during the second copulatory period, which encompassed the second dry season following a year of atypically heavy rainfall than during the first dry sea-
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Muriqui Male Fecal Steroids
son. Additionally, cortisol levels in all males showed a sustained rise around the beginning of December 1996 at the time of the Brazilian summer solstice, which may indicate photoperiod influences, as has been suggested for amphibians (Pancak and Taylor, 1983; Zerani and Gobbetti, 1993). None of the six males showed an increase in cortisol levels prior to a twofold increase in his testosterone concentrations. This delay in the elevation of cortisol relative to testosterone levels differs from the relationship between these steroids in other primates living in multimale, multifemale groups, and resembles the temporal relationship between testosterone and glucocorticoids in seasonally breeding monogamous birds. In these birds (Ketterson et al., 1991), as well as muriquis, testosterone elevations precede those of adrenal hormones at the onset of the breeding season. In the squirrel monkey, peak increases in body weight and cortisol, dehydroepiandrosterone, and androstenedione levels (indicative of adrenal function) occur approximately 1 month prior to peak increases in testosterone levels and are thought to be related to the “fatting” response that males exhibit during the breeding season (Wiebe et al., 1988; Schiml et al., 1996). Male rhesus macaques also increase in body weight prior to the onset of the breeding season, when elevations in their estrogen levels also occur (Bercovitch, 1992). In these primates, fat storage may be part of the reproductive strategy males employ to increase their competitive ability in direct contests over access to potential mates and to liberate time and energy for reproductive activities that would otherwise be required for feeding (Bercovitch, 1992). Although captive male squirrel monkeys did not compete aggressively for access to mates during the mating season (Schiml et al., 1996), in the wild, larger, “fatter” male Costa Rican squirrel monkeys attained higher mating success (e.g., Boinski, 1987). In contrast to these primates, in which cortisol elevations associated with fat storage appear to prepare males for the breeding season, in muriquis, cortisol elevations appear to respond to seasonal breeding opportunities. The higher quality diet that muriquis obtain during the rainy season, when preferred fruits, flowers, and new leaves are more abundant at our field site (Strier, 1991) may be offset by their greater energetic expenditure, as evidenced by their significantly longer day ranges as they travel between these more patchily distributed food resources (Strier, 1987). Furthermore, unlike Peruvian male squirrel monkeys, whose social relationships may become increasingly hierarchical during the mating season (Mitchell, 1994), male muriquis maintain peaceful,
egalitarian relationships throughout the year, and female mate choices appear to be based on social preferences and avoidance of kin (Strier, 1997). The “fatting” responses that may enhance reproductive opportunities among primates such as squirrel monkeys or rhesus macaques might be less important among muriquis. Therefore, we suggest that annual fluctuations in cortisol concentrations may be associated with seasonal changes in diet, ranging, and metabolic profiles, while the lack of consistent seasonal changes in testosterone is due to the diminished value of aggressive competition among male muriquis. Proximate mechanisms accounting for, and associated with, the distinct birth clumping during the dry season remain to be determined.
ACKNOWLEDGMENTS Permission to conduct research in Brazil was provided by CNPq and IBAMA, with sponsorship during this period by Dr. G. A. B. Fonseca. The field research was supported by NSF Grants BNS 8619442, BNS 8959298, and SBR 9414129, the Seacon Fund of the Chicago Zoological Society, the Liz Claiborne and Art Ortenberg Foundation, the Scott Neotropic Funds of the Lincoln Park Zoo, and the Graduate School of the University of Wisconsin-Madison. N. Bejar, A. Carvalho, D. Carvalho, C. G. Costa, P. Coutinho, L. T. Dib, J. Gomes, M. A. Maciel, F. D. C. Mendes, F. Neri, S. Neto, C. P. Nogueira, A. Odalia Rı´moli, A. Oliva, L. Oliveira, R. Printes, J. Rı´moli, and W. Teixeira contributed to the long-term behavioral data and the fecal collections. Assay costs were supported by NSF Grant SBR 9414129 and the Wisconsin Regional Primate Research Center (NIH RROO, 167). S. L. Rust and G. Scheffler provided technical assistance; S. L. Rust provided comments on an earlier version of this manuscript, and we are grateful to two anonymous reviewers for their valuable suggestions. This is publication No. 39 – 028 of the WRPRC.
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Ketterson, E. D., Nolan, Jr. V., Wolf, L., Ziegenfus, C., Dufty, Jr., A. M., Ball, G. F., and Johnsen, T. S. (1991). Testosterone and avian life histories: The effect of experimentally elevated testosterone on corticosterone and body mass in dark-eyed juncos. Horm. Behav. 25, 489 –503. Kinger, S., Pal, P. C., Rajalakshmi, M., Kumar, P. K., Sharma, D. N., and Bajaj, J. S. (1995). Effects of testosterone buciclate on testicular and epididymal sperm functions in bonnet monkeys. Contraception 52, 121–127. Lohiya, N. K., Sharma, R. S., Manivannan, B., and Anand Kumar, T. C. (1998). Reproductive exocrine and endocrine profiles and their seasonality in male langur monkeys (Presbytis entellus entellus). J. Med. Primatol. 27, 15–20. McLachlan, R. I., Wreford, N. G., O’Donnell, O., de Krester, D. M., and Robertson, D. M. (1996). The endocrine regulation of spermatogenesis: Independent roles for testosterone and FSH. J. Endocrinol. 148, 1–9. Meisel, R. L., and Sachs, B. D. (1994). The physiology of male sexual behavior. In E. Knobil and J. D. Neill (Eds.), The Physiology of Reproduction, 2nd ed., pp. 3–105. Raven Press, New York. Mitchell, C. L. (1994). Migration alliances and coalitions among adult male South American squirrel monkeys (Saimiri sciureus). Behaviour 130, 169 –190. Pancak, M. K., and Taylor, D. H. (1983). Seasonal and daily plasma corticosterone rhythms in american toads, Bufo americanus. Gen. Comp. Endocrinol. 50, 490 – 497. Perret, M. (1992). Environmental and social determinants of sexual function in the male lesser mouse lemur (Microcebus murinus). Folia Primatol. 59, 1–25. Robinson, J. A., Scheffler, G., Eisele, G., and Goy, R. W. (1975). Effects of age and season on sexual behavior and plasma testosterone and dihydrotestosterone concentrations of laboratoryhoused male rhesus monkeys (Macaca mulatta). Biol. Reprod. 13, 203–210. Rose, R. M., Holaday, J. W., and Bernstein, I. S. (1971). Plasma testosterone, dominance rank and aggressive behavior in male rhesus monkeys. Nature 231, 366 –368. Rostal, D. C., Glick, B. B., Eaton, G. C., and Resko, J. A. (1986). Seasonality of adult male Japanese macaques (Macaca fuscata): Androgens and behavior in a confined troop. Horm. Behav. 20, 452– 462. Ruiz de Elvira, M. C., Herndon, J. G., and Wilson, M. E. (1982). Influence of estrogen-treated females on sexual behavior and male testosterone levels of a social group of rhesus monkeys during the nonbreeding season. Biol. Reprod. 26, 825– 834. Sapolsky, R. M. (1987). Stress, social status, and reproductive physiology in free-living baboons. In D. Crews (Ed.), Psychobiology of Reproductive Behavior, pp. 291–322. Prentice-Hall, Englewood Cliffs, NJ. Schiml, P. A., Mendoza, S. P., Saltzman, W., Lyons, D. M., and Mason, W. A. (1996). Seasonality in squirrel monkeys (Saimiri sciureus). Physiol. Behav. 60, 1105–1113. Stern, J. (1970). Responses of male rats to sex odors. Physiol. Behav. 5, 519 –524. Strier, K. B. (1987). Ranging behavior of woolly spider monkeys. Int. J. Primatol. 8, 575–591. Strier, K. B. (1991). Diet in one group of woolly spider monkeys, or muriquis (Brachyteles arachnoides). Am. J. Primatol. 23, 113–126.
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Strier, K. B. (1992a). Faces in the Forest: The Endangered Muriqui Monkeys of Brazil. Oxford Univ. Press, NY. Strier, K. B. (1992b). Causes and consequences on nonaggression in woolly spider monkeys. In J. Silverberg and J. P. Gray (Eds.), Aggression and Peacefulness in Humans and Other Primates, pp. 100 –116. Oxford Univ. Press, NY. Strier, K. B. (1996). Reproductive ecology of female muriquis (Brachyteles arachnoides). In M. A. Norconk, A. L. Rosenberger, and P. A. Garber (Eds.), Adaptive Radiations of Neotropical Primates, pp. 511–532. Plenum Press, NY. Strier, K. B. (1997). Mate preferences of wild muriqui monkeys (Brachyteles arachnoides): Reproductive and social correlates. Folia Primatol. 68, 120 –133. Strier, K. B. Predicting primate responses to “stochastic” demographic events. Primates, in press. Strier, K. B., and Ziegler, T. E. (1994). Insights into ovarian function in wild muriqui monkeys (Brachyteles arachnoides). Am. J. Primatol. 32, 31– 40. Strier, K. B., and Ziegler, T. E. (1997). Behavioral and endocrine characteristics for the reproductive cycle in wild muriqui monkeys, Brachyteles arachnoides. Am. J. Primatol. 42, 299 –310. Torii, R., and Nigi, H. (1994). Hypothalamo-pituitary-testicular function in male Japanese monkeys (Macaca fuscata) in non-mating season. Jikken Dobuts 43, 381–387. Wiebe, R. H., Williams, L. E., Abee, C. R., Yeoman, R. R., and Diamond, E. J. (1988). Seasonal changes in serum dehydroepiandrosterone, androstenedione, and testosterone levels in the squirrel monkey (Saimiri boliviensis boliviensis). Am. J. Primatol. 14, 285–291. Williams-Ashman, H. G. (1988). Perspectives on the male sexual physiology of eutherian mammals. In E. Knobil and J. D. Neill (Eds.), The Physiology of Reproduction, pp. 727–751. Raven Press, NY. Wingfield, J. C., and Kenagy, G. J. (1991). Natural regulation of reproductive cycles. In M. Screibman and R. E. Jones (Eds.), Vertebrate Endocrinology: Fundamental and Biomedical Implications, Vol. 4, Part B, pp. 181–241. Academic Press, New York. Wingfield, J. C., and Ramenofsky, M. (1985). Hormonal and environmental control of aggression in birds. In R. Gilles and J. Balthazart (Eds.), Neurobiology, pp. 92–104. Springer-Verlag, Berlin. Zerani, M., and Gobbetti, A. (1993). Corticosterone during the annual reproductive cycle and in sexual behavior in the crested newt, Triturus carnifex. Horm. Behav. 27, 29 –37. Ziegler, T. E., Scheffler, G., and Snowdon, C. T. (1995). The relationship of cortisol levels to social environment and reproductive functioning in female cotton-top tamarins, Saguinus oedipus. Horm. Behav. 29, 407– 424. Ziegler, T. E., Scheffler, G., Wittwer, D. J., Schultz-Darken, N., Snowdon, C. T., and Abbott, D. H. (1996). Metabolism of reproductive steroids during the ovarian cycle in two species of callitrichids, Saguinus oedipus and Callithrix jacchus and estimation of the ovulatory period from fecal steroids. Biol. Reprod. 54, 91–99. Ziegler, T. E., Santos, C. V., Pissinatti, A., and Strier, K. B. (1997). Steroid excretion during the ovarian cycle in captive and wild muriquis, Brachyteles arachnoides. Am. J. Primatol. 42, 311–321.
Seasonal and Social Correlates of Fecal Testosterone and Cortisol Levels in Wild Male Muriquis (Brachyteles arachnoides) Karen B. Strier,* ,1 Toni E. Ziegler,† and Daniel J. Wittwer† *Department of Anthropology, University of Wisconsin-Madison, 1180 Observatory Drive, Madison, Wisconsin 53706; and †Wisconsin Regional Primate Research Center, Madison, Wisconsin Received March 30, 1998; revised August 10, 1998; accepted December 7, 1998
Fecal testosterone and cortisol levels were analyzed from six wild male muriquis (Brachyteles arachnoides) over a 19-month period at the Estac¸a˜o Biolo´gica de Caratinga in Minas Gerais, Brazil, to investigate the hormonal correlates of seasonal sexual behavior and environmental conditions. Group mean testosterone levels based on weekly samples from the six males did not differ between copulatory and noncopulatory periods or between rainy and dry seasons. Cortisol levels did change with copulatory periods, and were significantly higher during the second dry season, when mating continued following an exceptionally heavy rainy season, than during the first dry season, when mating ceased. Males exhibited individual variation in the timing of their hormone shifts relative to their sexual activity, but neither hormone levels nor sexual activity were related to male age. Despite individual differences in the timing of testosterone fluctuations around the onset and offset of the copulatory season, all males exhibited elevated cortisol concentrations following a slight increase in testosterone at the beginning of the copulatory season. Both the lack of significant changes in testosterone levels with the onset of the rainy and copulatory season and the lack of prebreeding increases in cortisol may be related to the low levels of overt aggression displayed by male muriquis over access to mates. © 1999 Academic Press Key Words: muriquis; seasonality; testosterone; cortisol; fecal steroids.
Testosterone’s influence on male sexual behavior has been described in many vertebrate species. For instance, an increase in testosterone is associated with bird song production in juncos, Junco hyemalis (Ketter1 To whom correspondence and reprint requests should be addressed. E-mail: [email protected].
0018-506X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
son, Nolan, Wolf, Ziegenfus, Dufty, Ball, and Johnsen, 1991) and can promote territory aggression in birds (Wingfield and Ramenostsky, 1985). In male rats, mating behavior is dependent upon testosterone (Meisel and Sachs, 1994) and increased testosterone promotes a preference for female estrus odors (Stern, 1970). Testosterone, in conjunction with FSH, appears to be essential in spermatid maturation, and may influence fertility in males (Williams-Ashman, 1988; McLachlan, Wreford, O’Donnell, de Krester, and Robertson, 1996). Therefore, it is not surprising that environmental influences can promote an increase in testosterone in seasonally breeding animals. Testosterone changes can be regulated by seasonal cues such as changes in day length and patterns of rainfall (Wingfield and Kenagy, 1991). Males from a number of species of primates also show seasonal changes in testosterone, with peak levels occurring during the breeding season. Plasma testosterone levels in the lesser mouse lemur (Microcebus murinus) have been shown to increase and decrease in response to corresponding changes in photoperiod (Perret, 1992). Likewise, seasonality in testosterone levels has been reported in squirrel monkeys (Saimiri sciureus: Wiebe, Williams, Abee, Yeoman, and Diamond, 1988; Schiml, Mendoza, Saltzman, Lyons, and Mason, 1996), Hanuman langurs (Presbytis entellus: Lohiya, Sharma, Manivannan, and Anand Kumar, 1998), and several species of macaques (e.g., Macaca fuscata, Torii and Nigi, 1994; M. radiata, Kinger, Rajalakshmi, Kumar, Sharma, and Bajaj, 1995; and M. mulatta, Herndon, Mindi, Nordmeyer, and Turner, 1996). Both social and environmental stimuli may influence the onset of testosterone increases in primates. For captive rhesus males, Herndon et al. (1996) found
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that male testosterone levels fluctuated seasonally even without exposure to females, suggesting that environmental stimuli alone may be sufficient to activate male gonadal responses. By contrast, Gordon, Bernstein, and Rose (1978) found that visual contact with rhesus females was sufficient to maintain seasonality in testosterone. Without minimal distal contact with females, seasonal testosterone patterns were not maintained, suggesting that testosterone fluctuations may be socially facilitated. Exposure to sexually active females outside of the mating season can further stimulate testosterone elevations (Ruiz de Elvira, Herndon, and Wilson, 1982). Social stimuli are also thought to initiate increases in testosterone concentrations in other primates, including male squirrel monkeys (Schiml et al., 1996) and wild sifakas (Propithecus verreauxi: Brockman, Whitten, Richard, and Schneider, 1998). Like these primates, wild muriqui monkeys (Brachyteles arachnoides) display seasonal breeding and live in multimale, multifemale groups, but the hormonal correlates of male seasonal sexual activity have not previously been investigated. We compared male muriqui testosterone levels with the onset of the rainy/dry seasons and with the onset of the copulatory/noncopulatory periods. In species such as rhesus macaques and squirrel monkeys, testosterone elevations are preceded by cortisol elevations, which are thought to be associated with seasonal weight gains (Wiebe et al., 1988; Bercovitch, 1992; Schiml et al., 1996). We therefore expected to find a similar pattern in cortisol and testosterone concentrations in seasonally breeding male muriquis. Hormonal fluctuations in female muriquis are known to coincide with the copulatory and rainy seasons. Resumption of postpartum ovulation in wild females correlated with the resumption of postpartum sexual activity during the early rainy season months (Strier and Ziegler, 1997; Ziegler, Santos, Pissinatti, and Strier, 1997). The onset of postpartum cycling was not tightly synchronized among individual females, despite the temporal clumping of their prior and subsequent births during the annual dry season months of June–August, when two-thirds of all infants in our study group have been born (Strier and Ziegler, 1994; Strier, 1996, in press). Most mating takes place during the rainy season months, from October through March (Strier, 1997). However, we concluded that photoperiodicity, which varies by about 2 21 h throughout the year at our fieldsite, was unlikely to be the sole extrinsic factor responsible for seasonal ovulation in muriquis because infant mortality could result in the
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resumption of sexual activity (Strier, 1996) and ovarian cycling (Strier and Ziegler, 1997) within 2 months postpartum during the dry season. The apparent coincidence between annual fluctuations in rainfall and mating patterns, on the one hand, and the disparity between the timing of mating and conceptions, on the other hand, stimulated our interest in whether steroid levels in male muriquis exhibit seasonal patterns similar to those detected in females. By contrast to most other primates, muriquis lack overt evidence of dominance hierarchies, either among males or between males and females (Strier, 1992a,b). Females mate without harassment from males or inciting overt competition among males, and both sexes routinely mate with multiple partners (Strier, 1992b, 1997). Therefore, rank should not be a confounding variable affecting individual differences in male muriqui hormone levels. This paper presents the results of a 19-month investigation of the seasonal and social correlates of testosterone and cortisol levels in wild male muriquis.
MATERIALS AND METHODS Animals and Sample Collection Fecal samples were collected from January 1996 through July 1997 at 4- to 5-day intervals from six wild male muriquis inhabiting the 860 hectare forest at the Estac¸a˜o Biolo´gica de Caratinga, Minas Gerais, Brazil (Strier, 1992a). Our sampling schedule provided one or two fecal samples per week from each of the six males. We obtained from 73–77 weeks of samples for each male during our 82-week study period. The six male subjects were preselected from among the 13 sexually active males in the study group during this period based on their relative ages and known maternal kinship. Two of the males (CL and IV) were classified as mature based on their visible adult size in 1982 and their observed copulations during the 1983– 1984 rainy season. Male muriquis of known age may begin to copulate when they are 5– 6 years old (Strier, 1996), implying that our mature male subjects were at least 19 –20 years of age during the present study period. Two middle-aged males (DI and NI) were present as infants in the study group in 1982, and therefore 14 –15 years of age during this study. Two young males (DA and NE) were born in 1985 and 1987, respectively. All individuals in the study group were fully habituated to human observers and recognizable by their natural markings.
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Methods of collecting and preserving male fecal samples followed those we employed in a previous study of female muriqui fecal steroids, in which fresh fecal samples were stored on ice and extracted as wet feces at the field station (Strier and Ziegler, 1997). No statistical differences were found in levels of either testosterone (paired t test, t 5 0.49, n 5 5, P 5 0.65) or cortisol (t 5 0.12, n 5 5, P 5 0.91) between morning and afternoon fecal samples collected from the same individuals. Nonetheless, to reduce the potential confounding effects of diurnal testosterone and cortisol fluctuations on seasonal patterns, we restricted our collections of male fecal samples during the present study to the period between sunrise and 1100 h. Behavioral Data and Analyses All copulations involving members of the study group were recorded by two trained observers throughout the day during the course of near-daily opportunistic and systematic behavioral sampling during this study period (Strier and Ziegler, 1997). A total of 178 copulations were observed on 100 different days during the present study period. From 1 to 5 different females, and from 1 to 10 different males copulated per week during the 44 weeks in which copulations occurred. Because the initiation of the present study in January 1996 corresponded to the middle of the 1995–1996 rainy and mating season, we use the last observed copulation to define the end of the mating season this year. From 30 April–26 September 1996, no copulations were observed. The first observed copulations following this period of sexual quiescence occurred on 27 September 1996 and represent the onset of the 1996 –1997 mating season. The 1996 –1997 rainy season was atypical in that over 60% more rain fell (1855 mm) compared to the annual average of 1119 6 189 mm of rainfall from 1986 –1994 (Strier, 1996). Indeed, rainfall accumulation by January 1997 was already substantially greater (1285 mm) than the total annual rainfall of 1030 mm from 1995–1996. Based on monthly rainfall patterns, we distinguished four periods in our present analyses: first rainy season (January–April 1996); first dry season (May–August 1996); second rainy season (September 1996 –April 1997); and second dry season (May– July 1997). Mating patterns during the 1996 –1997 rainy season were correspondingly atypical. The muriquis continued to copulate throughout the second dry season, in contrast to their sexual inactivity during these same months in the previous year (Fig. 1). In contrast to
1996, there was no behavioral evidence to mark the end of the mating season in 1997. Muriqui mating patterns permitted us to divide our study into two copulatory periods, from mid January–late April 1996 and from late September 1996 –July 1997, and one noncopulatory period, from late April–late September 1996. Steroid Assays Sample preparation. All fecal samples were mixed, weighed (0.1 g), and extracted in the field according to the technique described by Strier and Ziegler (1997). A portion of the 5-ml water/ethanol steroid extraction, 500 ml, was subjected to solvolysis and further extracted with ethyl acetate to eliminate the single and multiple conjugation. Sequential hydrolysis and solvolysis of muriqui samples revealed that 64.5% of the fecal testosterone was bound by double conjugates, necessitating the solvolysis procedure described in Ziegler, Scheffler, Wittwer, Schultz-Darken, Snowdon, and Abbott (1996). After solvolysis, the samples were dried and resuspended in 500 ml ethanol. Aliquots of the ethanol sample were used for both testosterone and cortisol analyses. Testosterone analyses. No column chromatography separation was required for routine testosterone determination. However, two male muriqui samples were prepared as above and separated by HPLC with collection of the fractions for assay. The system consisted of a dual HPLC pump (Beckman Instruments, Schaumburg, IL) connected to a diode array analyzer for UV detection, which allowed visualization of the peaks as they were separated. Data were input through computer detection software (Beckman, System Gold, Version 8) and fractionated (Gilson, Middleton, WI) at 1 ml/min for the 40-min run. Samples reconstituted in 30 ml of mobile phase (40:60 acetonitrile:water) were injected into a 20-ml sample loop with a reversed phase precolumn (5 mm, 4.6 mm 3 4.5 cm, Beckman) and reversed phase HPLC column (ultrasphere, 5 mm, 4.6 3 25 cm, Beckman). The mobile phase remained constant at a flow rate of 1 ml/min for the first 25 min and then increased to 1.5 ml/min and 50:50 acetonitrile:water until the end of the run at 40 min. Half of each fraction was dried and assayed for cross-reactivity with the testosterone antibody. The fractions containing testosterone and, to a lesser degree, DHT (dihydrotestosterone) were the only fractions where activity occurred, indicating that there was no other steroid interference. The standard RIA for testosterone has been described (Robinson,
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Scheffler, Eisele, and Goy, 1975). After solvolysis, 50 ml of the ethanol suspension was used for the testosterone assay. The assay was validated for use with muriqui fecal steroid extracts by determining accuracy and parallelism. Mean accuracy in measuring testosterone was 103.44% 6 1.9 SE for nine standard curve points, and serial dilutions of a fecal pool from male muriquis were parallel to the testosterone standards with no differences in the slopes (t 5 21.56, P . 0.05). Intra- and interassay coefficients of variation for a muriqui fecal pool were 3.1 and 11.2, respectively (n 5 12). Cortisol analyses. Fractions from the remaining half of the HPLC separated samples were measured in the cortisol assay for determination of antibody crossreactivity with other steroids. Cross-reactivity only occurred at the retention times for cortisol and cortisone (collected in the same fraction and not separately identified), indicating that the cortisol assay would provide a true measurement of cortisol (and possibly cortisone) excretion in the muriqui. Cortisol was measured by an EIA (Ziegler, Scheffler, and Snowdon, 1995). Sample volumes of 100 ml of the ethanol solution were dried and resuspended in the assay buffer/ enzyme solution and aliquoted as 33% into each well. Standards (3.16 –1000 pg) were prepared the same way as samples with cortisol added in ethanol, dried, and resuspended in assay buffer/enzyme solution. Mean accuracy for the standard curve points was determined to be 124% 6 5.5 SE (n 5 6) and serially diluted muriqui feces were parallel to the standard curve with no difference in slopes (t 5 1.5, P . 0.05). Intra- and interassay coefficients of variation for a muriqui fecal pool were 3.8 and 9.4, respectively (n 5 18). Statistical Analyses Testosterone and cortisol concentrations were plotted for each of the six males on a weekly basis. We averaged the testosterone and cortisol concentrations from each individual with $ 1 sample in any week to obtain single weekly values per male. From visual inspection of their hormonal profiles, individual testosterone and cortisol levels appeared to increase after
the first day of observed copulation at the onset of the 1996 –1997 copulatory period. The day of the increase in testosterone concentration relative to the onset of the copulatory period was determined as the first day that the steroid level increased at least twofold and remained elevated. The onset of cortisol increase was also at least twofold and determined relative to the onset of the copulatory period. The timing of the sustained decrease in the concentrations of both steroids was also represented by at least a twofold change in these levels for each male. The twofold changes in hormonal levels were determined by comparing the mean of five preincrease samples representing a 3- to 4-week period to the mean of five postincrease samples for each male. To evaluate the possible relationships of copulatory periods or rainfall patterns on male steroid levels, the mean level of testosterone or cortisol was determined for each male per week, and averaged over each month. Mean testosterone and cortisol levels were calculated for the defined periods for each male. General Linear Model one-way ANOVAs were used to compare mean steroid levels per male across the copulatory–noncopulatory periods and the rainy-dry periods, as defined above. Post-hoc analyses were conducted with the Tukey test. Kruskal–Wallis tests were used to assess the possible effects of age on male hormone levels.
RESULTS Copulatory Behavior and Steroid Levels in Individual Males Copulation data indicated that the six males we used in our study accounted for 56% of the total 178 copulations observed during the 19 months. These males copulated on 85 of the 100 days in which $ 1 copulation was observed. Individual males averaged 16.7 6 9.8 copulations (median 5 16.5, range 5 5–33) across an average of 14.7 6 7.6 different days (median 5 15, range 5 5–26). Copulation frequencies among our six target males did not differ significantly by age class (X 2 5 4.3, df 5 2, P . 0.05). The 100 copulations
FIG. 1. Average monthly levels of rainfall are plotted in the top graph to compare with the copulation data that follows in the next three graphs. Weekly number of copulations, number of different females copulating, and number of different males copulating are plotted. Asterisks under the bottom graph indicate estimated conceptions based on documented births following a calculated 7-month gestation period (Strier and Ziegler, 1997). The only gap in behavioral observations . 10 days occurred from late December 1996 to early January 1997.
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FIG. 2. Profile of testosterone and cortisol levels by week for male CL. Shaded background indicates the noncopulatory period; white background indicates the copulatory periods. Asterisks indicate observed copulation dates for each male, respectively. The lack of correlation between muriqui male testosterone and cortisol levels is consistent with findings in rhesus macaques (Bercovitch and Clarke, 1995).
performed by these males were evenly distributed between the two older males (15 and 18%), but strongly skewed between the two middle aged males (5 and 20%) and younger males (9 and 33%). Age effects were not found for either steroid. No significant relationships were detected between male age and mean testosterone levels during the two copulatory periods (Kruskal–Wallis 5 3.43, df 5 2, P 5 0.18 for both) or during the noncopulatory period (Kruskal–Wallis 5 2.0, df 5 2, P 5 0.37). Older males showed a slight, but insignificant, trend for higher testosterone levels than younger males during the first copulatory period (r 2 5 0.63, n 5 6, P 5 0.06). Mean cortisol levels also showed no age differences during the combined copulatory periods
(Kruskal–Wallis 5 2.57, df 5 2, P 5 0.78) or during the noncopulatory period (Kruskal–Wallis 5 0.29, df 5 2, P 5 0.81). Individual males showed no significant correlations between weekly testosterone and cortisol levels (range of r values 5 0.07– 0.38). Figure 2 shows the hormonal profiles and observed copulation days of one of our study males. Each male showed a slight decline in testosterone levels within 2 months prior to the end of the first copulatory period in 1996, but the timing of these testosterone declines relative to each male’s last observed copulation was variable (Table 1). No clear pattern in declines in cortisol levels could be determined for individual males relative to either their last
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Muriqui Male Fecal Steroids
TABLE 1 Individual Hormonal Changes Relative to the Offset of the Copulatory Period a Group Offset
Individual Offset
Male
T
F
LOC
T
F
CL DA DI IV NE NI Mean SEM
241 260 257 260 255 26 246.50 8.60
16 None None None None 17
29 April 1996 29 April 1996 22 March 1996 29 April 1996 29 January 1996 3 March 1996
241 260 220 260 131 153 216.17 19.56
16 None None None None 156
The timing of $ 2-fold decreases in individual male testosterone (T) and cortisol (F) levels are shown relative to the last observed copulation (LOC) in the group, which occurred on 29 April 1996, and the LOC for each male, as indicated. The number of days before (2) or after (1) the last observed copulations are shown. The absence of detectable changes are indicated as none. a
observed copulation or the last observed copulation in the group during this period. A slight but consistent rise in testosterone levels occurred for four males from 0 to 4 days following the first observed copulation of the 1996 –1997 copulatory period (Table 2). No male showed an increase in cortisol levels prior to his testosterone surge during the 1996 –1997 breeding season.
levels (range of r values 5 0.18 – 0.35) or cortisol levels (range of r values 5 0.02– 0.20). Mean testosterone levels did not differ across copulatory and noncopulatory periods (F 5 2.22, df 5 2, 15, P 5 0.14). However, significant differences in cortisol levels were detected (F 5 7.39, df 5 2, 15, P 5 0.006). Post-hoc analyses indicated that mean cortisol levels were significantly higher during the second copulatory period (October 1996 –July 1997) than during the May–September 1996 noncopulatory period (P 5 0.007).
DISCUSSION Contrary to expectations based on other seasonally breeding primates, muriqui mean testosterone levels failed to exhibit significant seasonal changes correlating with either the onset of sexual behavior, as measured by behaviorally defined copulatory periods, or the onset of the rainy season. In fact, muriqui males failed to exhibit seasonal patterns in testosterone concentrations during the entire 19-month study period. By contrast, cortisol levels were significantly higher during the second rainy and dry seasons, when mating occurred, than during the first dry, noncopulatory, season. These data indicate that muriqui cortisol, but not testosterone, levels are responsive to seasonal stimuli. The absence of testosterone responsiveness associated with the onset of seasonal sexual activity in
Temporal Patterns in Testosterone and Cortisol Levels No significant differences were detected in mean testosterone levels among any of the rainy or dry periods (F 5 1.29, df 5 3, 20, P 5 0.30). Seasonal patterns in fecal cortisol levels differed from those of testosterone (Fig. 3). Significant differences in cortisol levels were found between the rainy and dry periods (F 5 5.09, df 5 3, 20, P 5 0.009). Post-hoc analyses indicated that cortisol levels were significantly lower during the first dry season than either the second dry season (P 5 0.01) or the second rainy season (P 5 0.03). All measures of sexual activity in the group exhibited seasonal patterns (Fig. 1), consistent with seasonal sexual activity reported for these muriquis in prior years (Strier, 1996, 1997). We did not find any significant relationships in weekly mean comparisons between the total number of copulations, number of copulation days, number of copulating females, or number of copulating males and mean testosterone
TABLE 2 Individual Hormonal Changes Relative to the Onset of the Copulatory Period a Group Onset Male
T
F
CL DA DI IV NE NI Mean SEM
14 0 14 0 157 157 120.33 11.62
160 17 169 169 157 174 156.00 10.13
Individual Onset FOC 30 27 12 13 10 20
October 1996 September 1996 October 1996 October 1996 October 1996 November 1996
T
F
230 0 211 217 144 14 21.67 10.40
134 17 157 151 144 121 135.67 7.74
The timing of $ 2-fold increases in individual male testosterone (T) and cortisol (F) levels are shown relative to the first observed copulation (FOC) in the group, which occurred on 27 September 1996, and the FOC for each male, as indicated. The number of days before (2) or after (1) the first observed copulations are shown. a
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FIG. 3. Weekly mean (and SEM) testosterone and cortisol levels over the 19-month study period. All six males are included. Shaded background indicates the noncopulatory period; white background indicates the copulatory periods. Gaps of .10 days in sample collections are indicated by discontinuous lines.
muriquis differs from that found in other seasonally breeding primates, such as Japanese macaques, where sexual activity was found to lag 1–2 months behind seasonal elevations in testosterone and DHT levels (Rostal, Glick, Eaton, and Resko, 1986). In male muriquis, the seasonal onset of sexual activity relative to individual testosterone levels was highly variable, which may have contributed to our inability to detect seasonal variation. Furthermore, the absence of overt aggression among male muriquis over sexual access to females (Strier, 1992b) may explain the absence of seasonal patterns in their testosterone profiles (Sapolsky, 1987). The fact that muriqui males copulated without consistent elevated levels of testosterone also suggests that they do not reduce their levels of testosterone
during the noncopulatory periods. In other multimale, multifemale groups of primates, males compete aggressively for access to females, and more aggressive males have higher levels of testosterone than less aggressive males (Rose, Holaday, and Bernstein, 1971). In muriquis, however, no overt signs of aggression or competition associated with access to mates have been observed (Strier, 1992b, 1997). The absence of significant seasonal increases in testosterone levels during the mating season is consistent with their unusually low levels of aggression. In contrast to their testosterone profiles, male muriquis exhibited significantly higher cortisol levels during the second copulatory period, which encompassed the second dry season following a year of atypically heavy rainfall than during the first dry sea-
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Muriqui Male Fecal Steroids
son. Additionally, cortisol levels in all males showed a sustained rise around the beginning of December 1996 at the time of the Brazilian summer solstice, which may indicate photoperiod influences, as has been suggested for amphibians (Pancak and Taylor, 1983; Zerani and Gobbetti, 1993). None of the six males showed an increase in cortisol levels prior to a twofold increase in his testosterone concentrations. This delay in the elevation of cortisol relative to testosterone levels differs from the relationship between these steroids in other primates living in multimale, multifemale groups, and resembles the temporal relationship between testosterone and glucocorticoids in seasonally breeding monogamous birds. In these birds (Ketterson et al., 1991), as well as muriquis, testosterone elevations precede those of adrenal hormones at the onset of the breeding season. In the squirrel monkey, peak increases in body weight and cortisol, dehydroepiandrosterone, and androstenedione levels (indicative of adrenal function) occur approximately 1 month prior to peak increases in testosterone levels and are thought to be related to the “fatting” response that males exhibit during the breeding season (Wiebe et al., 1988; Schiml et al., 1996). Male rhesus macaques also increase in body weight prior to the onset of the breeding season, when elevations in their estrogen levels also occur (Bercovitch, 1992). In these primates, fat storage may be part of the reproductive strategy males employ to increase their competitive ability in direct contests over access to potential mates and to liberate time and energy for reproductive activities that would otherwise be required for feeding (Bercovitch, 1992). Although captive male squirrel monkeys did not compete aggressively for access to mates during the mating season (Schiml et al., 1996), in the wild, larger, “fatter” male Costa Rican squirrel monkeys attained higher mating success (e.g., Boinski, 1987). In contrast to these primates, in which cortisol elevations associated with fat storage appear to prepare males for the breeding season, in muriquis, cortisol elevations appear to respond to seasonal breeding opportunities. The higher quality diet that muriquis obtain during the rainy season, when preferred fruits, flowers, and new leaves are more abundant at our field site (Strier, 1991) may be offset by their greater energetic expenditure, as evidenced by their significantly longer day ranges as they travel between these more patchily distributed food resources (Strier, 1987). Furthermore, unlike Peruvian male squirrel monkeys, whose social relationships may become increasingly hierarchical during the mating season (Mitchell, 1994), male muriquis maintain peaceful,
egalitarian relationships throughout the year, and female mate choices appear to be based on social preferences and avoidance of kin (Strier, 1997). The “fatting” responses that may enhance reproductive opportunities among primates such as squirrel monkeys or rhesus macaques might be less important among muriquis. Therefore, we suggest that annual fluctuations in cortisol concentrations may be associated with seasonal changes in diet, ranging, and metabolic profiles, while the lack of consistent seasonal changes in testosterone is due to the diminished value of aggressive competition among male muriquis. Proximate mechanisms accounting for, and associated with, the distinct birth clumping during the dry season remain to be determined.
ACKNOWLEDGMENTS Permission to conduct research in Brazil was provided by CNPq and IBAMA, with sponsorship during this period by Dr. G. A. B. Fonseca. The field research was supported by NSF Grants BNS 8619442, BNS 8959298, and SBR 9414129, the Seacon Fund of the Chicago Zoological Society, the Liz Claiborne and Art Ortenberg Foundation, the Scott Neotropic Funds of the Lincoln Park Zoo, and the Graduate School of the University of Wisconsin-Madison. N. Bejar, A. Carvalho, D. Carvalho, C. G. Costa, P. Coutinho, L. T. Dib, J. Gomes, M. A. Maciel, F. D. C. Mendes, F. Neri, S. Neto, C. P. Nogueira, A. Odalia Rı´moli, A. Oliva, L. Oliveira, R. Printes, J. Rı´moli, and W. Teixeira contributed to the long-term behavioral data and the fecal collections. Assay costs were supported by NSF Grant SBR 9414129 and the Wisconsin Regional Primate Research Center (NIH RROO, 167). S. L. Rust and G. Scheffler provided technical assistance; S. L. Rust provided comments on an earlier version of this manuscript, and we are grateful to two anonymous reviewers for their valuable suggestions. This is publication No. 39 – 028 of the WRPRC.
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