The presence of females induces elevated cortisol levels in an alpha male: Experimental evidence in chimpanzees.

OBJECTIVES: In group-living primates, it has been reported that the alpha male exhibits high concentrations of cortisol and testosterone in the context of mating competition. We investigated how the presence of females affected salivary cortisol and testosterone levels in males from a small captive group of chimpanzees (Pan troglodytes). Specifically, we assessed whether the presence of females resulted in a rapid increase in salivary cortisol and testosterone levels in the alpha male. MATERIALS AND METHODS: We compared the social behavior and salivary hormone concentrations of four males before and after the presentation of receptive females. Three times a day, we collected saliva samples, a useful matrix for investigating short-term hormonal changes, and measured cortisol and testosterone concentration by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The frequency of inter-male aggression increased in the presence of females, indicating intense competition among males. Salivary cortisol levels increased in all males in the presence of females; however, the increase was significantly more pronounced in the alpha male. We found a complex three-way interaction among the presence of females, sampling timings, and male dominance rank in the analysis of salivary testosterone. Contrary to our prediction, a post hoc analysis revealed that salivary testosterone levels decreased after female introduction and that the alpha male did not show a higher level of salivary testosterone. CONCLUSIONS: Our study provides experimental evidence suggesting that the presence of females plays a significant role in the rank-related variation in the cortisol levels in male chimpanzees. Furthermore, our findings demonstrate the usefulness of salivary hormones for detecting short-term physiological changes in studies of socioendocrinology.

Reactivation of lymphocryptovirus (Epstein-Barr virus chimpanzee) and dominance in chimpanzees.

Nine male chimpanzees originally reared in solitary cages were set up to form a group. Plasma viral load of the lymphocryptovirus (LCV) of chimpanzee [Epstein-Barr virus chimpanzee (EBVcmp)] was measured by real-time PCR. In the group formation (Form) period, the first-ranking male showed an imminent increase in plasma EBVcmp load compared with 1 week before (pre-Form) and 3 months after (post-Form) group formation. Other upper-ranking males such as the second-, third- and fourth-male also showed the highest level of viral load in the Form period. The kinetics of EBVcmp load in the Form period were statistically different from other periods (against pre-Form, t=-4.878, P<0.001; against post-Form, t=6.434, P<0.001). The effect of the male dominance rank did not differ between the pre-Form and post-Form periods (t=-1.557, P=0.12). Reactivation of LCV (EBV) as an immunological stress marker for humans might also be applied to chimpanzees.

Validation of salivary cortisol and testosterone assays in chimpanzees by liquid chromatography-tandem mass spectrometry.

Owing to its high temporal sensitivity, saliva has distinct advantages for measuring steroids, compared with other noninvasive samples such as urine and feces. Here, we report the validity of assaying salivary cortisol (C) and testosterone (T) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in captive male chimpanzees, Pan troglodytes. For both the C and T concentrations, we found positive relationships between saliva and plasma. The concentrations of C and T in saliva showed clear patterns of diurnal fluctuation, whereas those in urine and feces did not. These results suggest that the salivary steroid concentrations can be regarded as good indicators of circulating steroid levels. We also developed and validated an efficient method for collecting saliva samples from cotton rope. Although rope includes inherent steroid-like compounds and may affect the accuracy of steroid measurements, our rope-washing procedures effectively removed intrinsic steroidal materials. There was a significant association between the C and T concentrations measured from saliva collected from rope licked by the chimpanzees and those measured from saliva collected directly from the mouth. Salivary T values estimated by LC/MS-MS were similar to those measured by radioimmunoassay. The results indicate the usefulness of saliva as a noninvasive steroid measure and that steroids in the saliva of chimpanzees can be accurately measured by LC-MS/MS.

Social relationship and hair cortisol level in captive male chimpanzees (Pan troglodytes).

Understanding how social relationships affect long-term stress is important because stress has a profound impact on the welfare of animals and social relationships often exert a strong influence on their stress responses. The purpose of this study was to investigate the relationship between social behaviors and long-term stress levels as assessed by hair cortisol (HC) concentration. The subjects were 11 chimpanzees living in an all-male group (divided into two sub-groups) in Kumamoto Sanctuary, Kyoto University, Japan. Behavioral data were collected between December 2014 and March 2015. The total observation time was 129 h. Hair samples were collected in late March and early April 2015, and cortisol was extracted from the hair and measured with enzyme immunoassay. The hair growth rate was estimated to be 1.33 ± 0.06 cm/month. The results revealed that there was a positive correlation between the rate of receiving aggression and HC levels. We also found a significant negative correlation between the balance between giving and receiving grooming (grooming balance index: GBI), which was calculated by subtracting the rate with which grooming is given from that with which it is received, and the rate of receiving aggression and between the GBI and HC levels. Thus, individuals receiving higher levels of aggression also tended to give grooming for relatively long periods compared to the time they were being groomed. In contrast, the rate of initiating aggression did not have a relationship with either HC levels or any measure of social grooming. We did not find social buffering effects, as there was no correlation between mutual social grooming and HC levels. These results show that not only aggressive interactions but also overall social situations in which animals do not have balanced relationships with others might result in the long-term elevation of cortisol levels in captive male chimpanzees.

Effects of Relocation and Individual and Environmental Factors on the Long-Term Stress Levels in Captive Chimpanzees (Pan troglodytes): Monitoring Hair Cortisol and Behaviors.

Understanding the factors associated with the long-term stress levels of captive animals is important from the view of animal welfare. In this study, we investigated the effects of relocation in addition to individual and environmental factors related to social management on long-term stress level in group-living captive chimpanzees by examining behaviors and hair cortisol (HC). Specifically, we conducted two studies. The first compared changes in HC levels before and after the relocation of 8 chimpanzees (Study 1) and the second examined the relationship between individual and environmental factors and individual HC levels in 58 chimpanzees living in Kumamoto Sanctuary (KS), Kyoto University (Study 2). We hypothesized that relocation, social situation, sex, and early rearing conditions, would affect the HC levels of captive chimpanzees. We cut arm hair from chimpanzees and extracted and assayed cortisol with an enzyme immunoassay. Aggressive behaviors were recorded ad libitum by keepers using a daily behavior monitoring sheet developed for this study. The results of Study 1 indicate that HC levels increased during the first year after relocation to the new environment and then decreased during the second year. We observed individual differences in reactions to relocation and hypothesized that social factors may mediate these changes. In Study 2, we found that the standardized rate of receiving aggression, rearing history, sex, and group formation had a significant influence on mean HC levels. Relocation status was not a significant factor, but mean HC level was positively correlated with the rate of receiving aggression. Mean HC levels were higher in males than in females, and the association between aggressive interactions and HC levels differed by sex. These results suggest that, although relocation can affect long-term stress level, individuals' experiences of aggression and sex may be more important contributors to long-term stress than relocation alone.

Analysis of hair cortisol levels in captive chimpanzees: Effect of various methods on cortisol stability and variability.

Hair cortisol has been reported to be a useful measure of long-term hypothalamic-pituitary-adrenal (HPA) axis activation in several species. It serves as a practical tool for long-term stress assessment, but it is important to understand the methodological factors that can affects hair cortisol assays to avoid methodological artifacts. To that end, we tested several procedures for measuring cortisol levels in hair collected from captive chimpanzees. The results showed that reproducibility was high, and we found no differences in cortisol levels among the various storage, drying, and sampling methods. However, the fineness of homogenized hair, sample weight, and extraction time affected absolute hair cortisol concentration. Although hair cortisol levels were stable over time, factors that may influence measurement results should be kept constant throughout a study.•We modified and validated a methodology involving enzyme immunoassays to reliably measure the hair cortisol levels of captive chimpanzees.•The results revealed that the fineness of homogenized hair, sample weight, and extraction time caused variations in absolute hair cortisol concentrations in chimpanzees. In contrast, storage, drying, and sampling from similar body parts did not affect the results.

Identification of a protanomalous chimpanzee by molecular genetic and electroretinogram analyses.

We determined the structures of long (L)-wavelength-sensitive and middle (M)-wavelength-sensitive opsin gene array of 58 male chimpanzees and we investigated relative sensitivity to red and green lights by electroretinogram flicker photometry. One subject had protanomalous color vision, while others had normal color vision. Unlike in humans, a polymorphic difference in the copy number of the genes and a polymorphic base substitution at amino acid position 180 were not frequently observed in chimpanzees.

Body, head, and facial growth: comparison between macaques (Macaca fuscata) and chimpanzee (Pan troglodytes) based on somatometry.

Chimpanzees and macaques were compared in their growth of head, face and body, based on a large-scale somatometrical database. Their growth stages, i to v, were determined by inflection points in velocity curves. Sex differences in their growth are shown both by elongated stages in juvenile and adolescent and by the greater velocity in males in the stages. Chimpanzees need longer to get their full growth, especially in the later infantile and juvenile stages. The growth patterns are classified into three types of "sigmoid", "parabolic", and "fast & slow" in distance curves, and in velocity curves, they correspond to "convex", acceleration in mid-growth stage; "linear", linear deceleration with age; and "concave", rapid deceleration in earlier stages and slow velocity in later stages. Great differences between chimpanzees and macaques were found in their growth patterns of upper facial height and facial height, which are "linear" or intermediate of "linear" and "concave" in macaques and "concave" in chimpanzees. These differences in their growth patterns explain the characteristic development of craniofacial proportions.

Behavioral evidence of color vision deficiency in a protanomalia chimpanzee (Pan troglodytes).

Although color vision deficiency is very rare among Old World monkeys and apes, one male chimpanzee (Lucky) was identified as protanomalous by genetic and physiological analyses. This study assessed behavioral phenotypes of Lucky and four chimpanzees with normal color vision by discrimination task using the modified Ishihara pseudo-isochromatic plates. Lucky could not discriminate the stimuli that the other chimpanzees could. This is the first behavioral evidence of color vision deficiency in chimpanzees.

Advantage of dichromats over trichromats in discrimination of color-camouflaged stimuli in nonhuman primates.

Due to a middle- to long-wavelength-sensitive (M/LWS) cone opsin polymorphism, there is considerable phenotypic variation in the color vision of New World monkeys. Many females have trichromatic vision, whereas some females and all males have dichromatic vision. The selective pressures that maintain this polymorphism are unclear. In the present study we compared the performance of dichromats and trichromats in a discrimination task. We examined tri- and dichromatic individuals of two species: brown capuchin monkeys (Cebus apella) and long-tailed macaques (Macaca fascicularis). We also examined one protanomalous chimpanzee (Pan troglodytes). The subjects' task was to discriminate a circular pattern from other patterns in which textural elements differed in orientation and thickness from the background. After they were trained with stimuli of a single color, the subjects were presented with color-camouflaged stimuli with a green/red mosaic overlaid onto the pattern. The dichromatic monkeys and the protanomalous chimpanzee selected the correct stimulus under camouflaged conditions at rates significantly above chance levels, while the trichromats did not. These findings demonstrate that dichromatic nonhuman primates possess a superior visual ability to discriminate color-camouflaged stimuli, and that such an ability may confer selective advantages with respect to the detection of cryptic foods and/or predators.