J Med Primatol doi:10.1111/jmp.12061
ORIGINAL ARTICLE
Evaluation of the fecal steroid concentrations in Alouatta belzebul (Primates, Atelidae) in the National Forest of , Brazil Tapirape-Aquiri in Para Frederico Ozanan Barros Monteiro1, Tatiana Kugelmeier2, Rodrigo del Rio do Valle3,4, Alexandre Bastos Fernandes Lima5, Felipe Ennes Silva6, Simone de Souza Martins5, Luciana Guedes Pereira5, Karen Lopes Dinucci5 & Priscila Viau7 1 2 3 4 5 6 7
^nia (UFRA), Bele m, PA, Brazil de e Producß~ Instituto da Sau ao Animal (ISPA), Universidade Federal Rural da Amazo de (PIBS), Rio de Janeiro, RJ, Brazil Fundacß~ ao Oswaldo Cruz (FIOCRUZ), Programa Institucional Biodiversidade & Sau ^ncias da Sau de, Universidade Paulista (UNIP), S~ao Paulo, SP, Brazil Instituto de Cie Wildlife Management Consultoria Veterin aria Ltda, S~ ao Paulo, SP, Brazil Empresa Habtec Engenharia Ambiental, Rio de Janeiro, RJ, Brazil , AM, Brazil Instituto de Desenvolvimento Sustent avel Mamirau a (IDSM), Tefe rio de Hormo ^nios (PROVET), S~ Laborato ao Paulo, SP, Brazil
Keywords howler monkey – radioimmunoassay – scan sampling method Correspondence Prof. Frederico Ozanan Barros Monteiro, ^nia Universidade Federal Rural da Amazo (UFRA)/Instituto da Sa ude e Producß~ ao Animal (ISPA), Avenida Presidente Tancredo Neves, nº 2501, Bairro: Terra Firme m, Par Cep: 66.077-901, Bele a, Brazil. Tel.: +55 91 32105139; fax: +55 91 32105139; e-mail: [email protected]; [email protected] Accepted July 10, 2013.
Abstract Background The studies on fecal steroid metabolites published with freeliving primates are limited mainly by the difficulty in obtaining samples. Methods A radioimmunoassay was used to measure the fecal steroid concentrations in Alouatta belzebul in the National Forest of Tapirape-Aquiri in Brazil. Results and Conclusions Androgens were significantly higher for the adult males from the Area of Influence (AI-I group) when compared to those from the Control Area (CA group) (P < 0.05). Progestin and estrogen concentrations were higher in the females from the CA group than in those from the AI-I for both the adult females and females with offspring; however, P < 0.05 was only observed in the concentrations of fecal progestins from the adult females. The physiological differences between the AI-I and CA groups suggest that the cause was a sum of factors, such as an exposure to sound waves, feeding habits, daily activity patterns, and the habituation of the animals.
Introduction Within neotropical primates, the members of the Alouatta genus have some of the largest body sizes and have a wider geographical distribution [24], with a range that encompasses the north of Argentina and south of Brazil to the south of Mexico [11]. They form small family groups, generally with a dominant adult male, adult reproducing females, juveniles, and babies. They are essentially folivores who dedicate a high percentage of their time to resting, which is generally more than 50% [10, 21]. The Alouatta belzebul species, which is known as the red-handed howler monkey, shows a specific geographical J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
distribution, as follows: to the south of the Amazon river as far as the state of Maranh~ ao, including the islands of Maraj o and Mexiana in the state of Par a, and to the northeast of the Atlantic Forest. This species is in the main list of threatened species, being classified in the categories ‘Critically Endangered’ [22] and ‘Vulnerable’ [16]. Their greatest threats are the destruction of their habitat, deforestation, and hunting, which is a strong threat in some areas of distribution. Therefore, the implementation of monitoring programs and management of the wild population to prevent the extinction of the species become paramount. The analysis of steroid hormones in feces and urine is an important tool in the understanding of the
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relationship between physiology and the environment in which the animal is inserted [33], thereby contributing to in situ and ex situ conservation programs. These methods allow researchers to evaluate and measure the variations in duration and phases of the ovarian cycle [13, 18], detect lactational anestrus [14], monitor the presence of seasonal influences [20, 31, 35], and measure the stress or welfare of wild species in conditions of captivity and/or free living [26]. There are several significant studies published on Alouatta and reproductive conditions [4, 5, 9]. However, endocrine studies are under-represented in this genus [15, 18, 32], this would be the first endocrine study on A. belzebul to our knowledge. According to Carlstead and Shepherdson [3], machine sounds may be an inadvertent source of aversion to many species. Various aspects of persistent acoustic stimuli such as intensity, frequency distribution, infrasound and content may be common stressors for captive animals. However, very little is known about these aspects in free-living animals, especially in nonhuman
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primates. Based on our literature review, this was the first study to evaluate acoustic stress in free-living monkeys. We aimed to evaluate the fecal steroid (corticosteroids, androgens, progestin, and estrogen) concentrations of A. belzebul that were monitored under an anthropic pressure, mainly sound noises, due to the implementation of a mining company in the National Forest of Tapirape-Aquiri in Par a, Brazil. Therefore, the hypothesis is that noise disturbance can be stressful and may influence both glucocorticoids and reproductive hormones. Material and methods Two groups of A. belzebul were monitored in the National Forest of Tapirape-Aquiri (05°46′17.84″ to 05°48′42.68″ South and 50º33′59.67″ to 50°31′42.21″ West) in the state of Par a, Brazil, where the Salobo mining project that belongs to the Vale mining company takes place (Fig. 1).
Fig. 1 The geographic location of the Control Area (CA) and the Area of Influence (AI-I) and the route followed by the animals in the CA (red -Aquiri. dots) and individuals in the AI-I (yellow dots) in the National Forest of Tapirape
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The groups were monitored from December 2010 to September 2011, during which the first 2 months were used for the habituation of the animals and the following months were used for the effective data collection. The researchers remained on site for approximately 15 days each month, with the same time interval between campaigns. Two areas of study were sampled (Fig. 1), one of which was under a direct influence from the activities generating sound waves from the Salobo Project, named the Area of Influence (AI-I). In this area, the presence of the AI-I group was observed, which consisted of animals categorized in the following way: one adult male and three females (one adult, one subadult, and one juvenile). In the June campaign, a birth was observed, thereby making it a group of five individuals. The second area of study was termed the Control Area (CA). This area was considered free from the influences of activities and was located between the streams ‘Igarape’ Mano and Salobo. The group from this area was composed of an adult male, a subadult male, three adult females (one of which had a baby), a juvenile male, and three juveniles of similar ages and nonidentified sex. Subsequently, this group was seen without the subadult male and one of the juvenile males, thereby totaling eight individuals. Collection and processing of the fecal sample For the reproductive endocrine study, only the adult animals were considered. The procedures for the collection and processing of fecal samples were performed as described by Ziegler and Wittwer [36] for field studies based on the measurement of fecal steroids. All monkeys were identified with the aid of binoculars. When defecation occurred, the fecal pellet was removed from the forest floor. Thus, samples of fresh feces were collected (immediately after defecation) in both areas of study (AI-I and CA) between 6 AM and 5 PM. After collection, the samples were appropriately placed in plastic bags, sealed, and identified with the date, time, place of collection, and the sex–age category of each animal, as follows: adult male (AM), adult female (AF), pregnant female or female with offspring (FWO), and non-identified (NI). Soon after collection, the samples were placed in a polystyrene receptacle with ice. At the end of the day, they were transferred to and stored in a freezer at 20°C until processing. The samples were brought from the field site to the Physiology Laboratory at the Universidade Federal Rural da Amaz^ onia, and the first stage of processing was the drying of the samples in a benchtop lyophilizer with a vacuum pump, model Enterprise I (Terroni J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Scientific Equipment, Sao Carlos, SP, Brazil). After lyophilization, the samples were again kept at 20°C until the extractions. For the hormonal extraction, 0.1 g of lyophilized feces was weighed in a semi-analytical balance (model BL 320H; Shimadzu Corporation, Japan), and then, 5 ml of 50% ethanol was added. The samples were then mixed on a vortex mixer (Phoenix, model AP-56; SP Labor, Sao Paulo, SP, Brazil) for 30 s and, subsequently, kept in a mechanical homogenizer (Phoenix AP-22; SP Labor, Sao Paulo, SP, Brazil) for 16 hours. After complete homogenization, the samples were centrifuged at 3000 rpm for 10 minutes, and 2 ml of the supernatant was divided into two polypropylene microtubes. The fecal extracts were kept in a freezer at 20°C until the hormonal measurements were performed at the Endocrinology Laboratory of Provet Diagnostic Veterinary Medicine in S~ ao Paulo, SP, Brazil. Hormone analysis All of the samples were assayed for the fecal metabolite concentrations of the following four steroid hormones: corticosteroids, androgens, progestin, and estrogens. The technique employed was a radioimmunoassay in a computerized gamma counter (Anser; Analytic Services Inc., Arlington, VA, United States), with the use of solid-phase commercial diagnostic kits for the progestins and androgens. For the estrogens and corticosteroids, double-antibody liquid-phase kits were used. The kits were manufactured for measuring these hormones in human serum or plasma, and the hormonal assays were performed according to the protocols provided by the manufacturers (Siemensâ, Los Angeles, CA, USA) for the estrogens, androgens, and progestins (MP Biomedicals, LLC, Solon, OH, USA) for the corticosteroids. The parameters for the quality of the assays, such as sensitivity, minimum dose, binding percentage, and coefficients of intra- and interassay variation for the low and high values of the calibration curve for each analyte, were analyzed. The laboratory validation of the commercial diagnostic kits for the estrogens double antibody and androgens was performed using a ‘pool’ of the fecal samples with a low concentration, that is, close to zero (matrix). Known quantities of labeled hormones were added to this matrix in dilutions, so that the final concentrations would be similar to the points in the standard curve of the diagnostic kit. This method indicated whether the fecal extract (matrix) interfered with the antigen reaction (labeled hormone and sample 3
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hormone) with the antibody from the commercial diagnostic kit used. The values of the curve thus obtained were correlated with the values of the standard curve from the diagnostic kit. The comparison was made using the linear regression coefficient, which was expressed as chi-squared only for the expression of positive values and for the Pearson’s correlation coefficient. The assay was validated when the correlation coefficient was close to 1 or 100%. The commercial diagnostic kits for measuring the fecal metabolites of progestins and corticosteroids in the genus Alouatta sp. were previously validated for the genus by our study group [18].
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or proportions, by dividing the number of records of the category of interest by the total records for the group. To test whether the level of each behavior was equally distributed along the hours of the day, the Kolmogorov–Smirnov test was used. To test the null hypothesis about the difference between the noise intensity between the two areas, a nonparametric Mann–Whitney U-test was used. A level of significance of 0.05 was adopted for all tests, which was calculated with the aid of the BioEstat software (version 5.0; Sociedade Civil de Mamirau a, Belem, Par a, Brazil). Results
Behavior monitoring For the collection of behavioral data, the scan sampling method was used [1, 6]. The sampling happened for five minutes, with 15-minute intervals to reduce samplingdependent error [29]. For each scan, the activity for each individual in sight was recorded, and the general pattern of the activities was divided into four main categories of exclusionary behaviors: 1—resting; 2—movement; 3— feeding; and 4—social interaction. The noise intensity in the areas of study was measured in decibels (dB), with a precision of 1 dB, using the DL-4000 decibel meter (Icel Gubintec©, Manaus, AM, Brazil) and using the curves of auditory perception for the human ear and a response of 200 msec as a standard. The sampling of noise intensity for each ecological–behavioral sample unit was taken. Statistical analysis For the hormone analyses, the basic parameters of the descriptive statistics (mean, standard error of the mean (mean SE), and minimum and maximum values) were calculated for all the variables analyzed. The comparison of means was performed for the metabolites of corticosteroids, androgens, progestins, and estrogens in the CA and AI-I between the AM, AF, FWO, and NI groups. A nonparametric analysis of variance (Kruskal–Wallis) was used. Considering the difference in the number of behavioral records in each group, a test for the comparison of two binomial proportions was used to test the null hypothesis on the difference between the quantities of each behavior between the two groups (two independent samples). This same test was used to assess the null hypothesis on the difference between the amounts of food items consumed between the two groups. With the intention of standardizing the samples for the activity patterns, the records were then transformed into rates, 4
Eighty-nine samples of feces distributed by categories (AM, AF, FWO, and NI) in each area of study were collected, with 56 samples from the CA group and 29 from the AI-I group. The higher number of samples for the CA group occurred due to problems of access to the AI-I, which caused difficulties in the search, the habituation of the AI-I group, and, consequently, feces collection. The hormone assays were divided according to each hormone. For corticosteroids, the sensitivity was 1.96 ng/ml, the low intra-assay coefficient of variation (CV) was 3.74%, and the high intra-assay CV was 0.85%. In the assay for androgen measurement, we obtained a sensitivity of 1.82 ng/dL, with a low intraassay CV of 4.17% and a high intra-assay CV of 1.17%. For the progestin assay, we obtained a sensitivity of 0.004 ng/ml, with an intra-assay CV lower than 4.21% and interassay CVs of 2.45% and 0.55% for the low and high values, respectively. In the double-antibody estrogen assay, the sensitivity was 0.42 pg/ml, with intraassay CVs of 3.12% and 0.90% for the low and high values, respectively. The validation of the commercial diagnostic kit (Siemensâ, Los Angeles, CA, USA) for the ‘solid phase’ of androgens and for the double-antibody estrogens for use in fecal samples from primates of the genus Alouatta was carried out. There was an agreement between the curve from the commercial diagnostic kit and the matrix dilution curves studied. We observed that in all tests performed, we obtained a high linear regression. The correlation (Pearson’s test) between the modified curve and the standard curve provided by the kit for the androgen and estrogen assays was r = 0.982, P = 0.0010, and r = 0.970, P < 0.001, respectively. The results showed a good linearity for the androgen and estrogen assays r = 0.99 and r = 0.98, respectively, in the studied concentration range. Based on these results, it was possible to suggest a laboratory validation of the assays. J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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The concentrations of fecal steroid metabolites showed a large individual variation, which was also within the same group and the same category. Table 1 shows the comparison of the means, standard error of the mean, and minimum and maximum values of the concentrations of fecal corticosteroids. The animals from the AI-I group showed higher concentrations of fecal steroids than those from the CA group for all of the categories; however, there was no significant difference (Kruskal–Wallis: H = 0.25; 1.05; 1.72; 0.55 df = 1; P = 0.62 for AF; 0.31 for FWO; 0.19 for AM; and 0.46 for NI). The data from Table 2 show the mean concentrations, standard error of the mean, and minimum and maximum values of fecal metabolites for androgens, progestins, and estrogens for the AM, AF, and FWO groups from the CA and AI-I. The concentrations of androgens were significantly higher for the AM from the AI-I in relation to those from the CA (Kruskal– Wallis: H = 4.2; df = 1; P < 0.05). The analyses showed that the fecal progestins and estrogens were higher in the females from the CA group than those from the AI-I group for both the AF and FWO; however, there was only a significant difference in the concentrations of
fecal progestins in the AF (Kruskal–Wallis: H = 6.75; df = 1; P < 0.05). In total, 3272 behavioral records were obtained; of these, 2358 were for the CA group, and 914 were for the AI-I group. These records were obtained from 609 instantaneous scans, among which 332 were taken for the CA group, and 277 were for the AI-I group. As described for the collection of fecal samples, the higher number of records for the CA group was due to problems of access to the AI-I region, which caused difficulty for the search and habituation of the AI-I group and the subsequent data collection despite a higher number of days (40) and hours (338) spent on-site for this group. Comparatively, there were no significant differences between the proportion of resting (Z = 0.9376; P = 0.1742), movement (Z = 0.1812; P = 0.4281), feeding (Z = 0.2927; P = 0.3849), and social interactions (Z = 0.2283; P = 0.4097) between the two study groups. In relation to the feeding behavior between the observed items, leaves were consumed most by the two groups (AI-I: 76%; CA: 59%), and there was no significant difference between the groups regarding the consumption of leaves (Z = 0.5074; P = 0.3059). However, a considerable difference between the groups
Table 1 Mean concentrations, standard error of mean (mean SE), and minimum and maximum values of fecal corticosteroid (ng/g dry feces) for categories (adult males, adult females, female with offspring, and non-identified) in Control Area (CA) and Area of Influence (AI-I) Control Area (CA) Category
Mean SE
N
Adult males Adult females Female with offspring Non-identified
Area of Influence (AI-I)
12 28 7 4
43.27 39.10 32.46 33.65
Minimum/maximum a
21.77/114.12 25.92/90.96 19.68/45.29 26.28/36.84
7.15 2.34a 3.28a 2.48a
Mean SE
N 6 6 3 14
59.47 39.68 40.68 44.84
Minimum/maximum a
18.49 3.87a 7.81a 5.65a
35.76/151.21 24.96/54.04 27.2/54.24 19.37/84.31
N, number of samples per category. Lowercase superscript letters indicate mean comparisons between groups conducted in the same category (Kruskal–Wallis: H = 0.25; 1.05; 1.72; 0.55; df = 1, P = 0.62; 0.31; 0.19; 0.46, respectively).
Table 2 Mean concentrations, standard error of mean (mean SE), and minimum and maximum values of fecal metabolites of androgens, estrogens, and progestins for categories (adult males—AM, adult females—AF, and female with offspring—FWO) in Control Area (CA) and Area of Influence (AI-I) Control Area (CA) Hormones
Category I
Androgens (lg/g dry feces) ProgestinsII (lg/g dry feces) EstrogensIII (ng/g dry feces)
AM AF FWO AF FWO
N 12 28 7 28 7
Area of Influence (AI-I)
Mean SE 34.96 1.42 0.80 189.63 126.70
Minimum/maximum b
4.64 0.52a 0.68a 65.83a 105.99a
8.46/69.94 0.00/14.04 0.01/4.89 1.11/1524.9 1.00/761.05
N 6 6 3 6 3
Mean SE 49.38 0.05 0.21 23.93 30.21
Minimum/maximum a
4.43 0.02b 0.16a 9.48a 10.14a
34.08/65.38 0.00/0.11 0.03/0.52 0.43/57.73 9.95/40.92
N, number of samples per category. Lowercase superscript letters indicate mean comparisons between groups conducted in the same category. Kruskal–Wallis: (H = 4.2, df = 1, P < 0.05)I; (H = 6.75, 0.00, df = 1, P < 0.05, P = 1, respectively)II; (H = 0.90, 0.01, df = 1, P = 0.34; 0.91, respectively)III. J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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was observed in the proportion of the remaining consumed items, such as fruit (CA: 30%; AI-I: 1%; Z = 5.7380; P < 0.0001), flowers (CA: 10%; AI-I: 5%; Z = 1.7015; P = 0.0444), and earth (AI-I: 18%; CA: 1% Z = 5.5366; P < 0.0001). The consumption of fruit and flowers was higher for the CA group, and the consumption of earth from termitaria was significantly higher for the AI-I group. A greater noise intensity was encountered by the group in AI-I, with the highest mean (56.95 dB) and maximum value (65 dB) both in the month of April. The results confirmed that the AI-I group was significantly exposed to more noise from the mining activities (U = 3422.00; Z = 12.8826; P < 0.0001). The detonations in the mines did not occur regularly and were recorded on eight occasions during the campaign, especially in the visits to the AI-I area, and they always occurred around midday. The most intense noises were from the machines and the movement of the workers in the study area and the vicinities. Discussion The studies on fecal steroid metabolites published with free-living animals, in particular with neotropical primates, are limited mainly by the difficulty in obtaining samples. Therefore, the results from this study offer important data to aid in the understanding of the relationship between the physiology and the environment where these animals live. The large variation in fecal steroid concentrations observed in this study was also reported by Palme [25]. This author stated that such variations are expected when one works with fecal matrix, given that many factors may contribute to this variation, such as intraand interspecific differences, circadian rhythm of hormone secretion, seasonal variations, the age, sex, and reproductive status of the studied individuals, diet, and the habituation of the animals. Therefore, there are many factors to be considered in processing fecal steroids. Steroids are excreted into the feces at different lag times due to processing of the steroids in the liver and reabsorption in the gut. Some steroids, such as estradiol, are highly conjugated and may be delayed in their excretion into the feces [36]. One way to reduce the variation from the circadian rhythm of hormone secretion would be to always collect the samples at the same time or within a small time interval [23]. However, we had difficulty in standardizing the collection time as the present study dealt with free-living animals. Therefore, to guarantee as high a number of samples per group as possible, we opted for collecting the samples throughout the day while always 6
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respecting the main requirement of collecting fresh samples of feces to avoid hormone degradation, as suggested by Ziegler and Wittwer [36]. The high folivory of the genus Alouatta [10, 21] may result in the increased amount of fiber and moisture in their diet. These factors may cause an underestimation of the concentration of fecal steroid metabolites because they increase the transit and volume of the fecal bolus, thereby interfering with hormone excretion [27, 28, 34]. Due to this feeding habit, the first stage of processing was a drying of the samples (lyophilization) in an attempt to obtain more homogenous and consistent results. Despite the levels of fecal corticosteroids not showing a significant difference between the animals from the AI-I and CA groups, the analysis of the behavior observations allowed us to infer that the group from the CA was more stable. Therefore, the significantly higher mean fecal androgen metabolite concentrations for the males from the AI-I than those from the CA may be related to stress, with the consequential release of steroids by the adrenal glands [12]. Based on the behavioral data for the groups, it was observed that by the end of the study, the CA group, in addition to being in an area with a lower incidence of anthropic influences generated by mining activities, also found themselves more accustomed to the presence of the researchers, which may have been directly reflected in the hormone analyses, thereby demonstrating stability. Alternatively, the animals from the AI-I group, as they were not properly accustomed and were under the influence of anthropic activities, showed a high degree of geophagia, which in general indicates a need for supplemental minerals or some degree of parasite infestation. Nevertheless, this can also be indirectly related to some level of stress or a change of habit [2, 30]. Although the proposed experimental design had used a group that was outside the area of impact as the control group, it was not possible to affirm that the variations observed were due to the differences in sound waves between the study areas. Despite the significant difference found for the noise intensity in the two areas, other factors may have interfered with the results, such as the type of noise (machinery, detonation, etc.), the habituation, and the natural adaptation of the howlers themselves to the anthropic interferences. To understand this finding, it is important to consider that these animals are resistant to some degree of change in the landscape [17], acclimatizing themselves more easily than other primate species. However, the Alouatta populations have been suffering a strong impact as a consequence of the increasing fragmentation of their habitats; therefore, it is extremely relevant that the J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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changes caused by anthropic actions are monitored. Factors such as the composition, density, and ecology of the species, their reproductive cycle, the floristic and faunistic composition of the studied areas, and the climatic conditions must be known, so that they become important tools for preservation [7, 8, 19]. Therefore, studies to monitor the population of primates, before, during, and after the human activities and enterprises become necessary to obtain information that can aid in the protection or management of the species. Construction noise has also been identified as a potential source of stress although the few studies conducted to date have failed to demonstrate anything other than short-term responses to specific auditory events such as blasting noise [3]. Therefore, although our results point to physiological differences between
the CA and AI-I groups, these data only suggest that these differences were caused by the sum of several factors, such as the exposure to noises from the activity of the enterprises, feeding habits, the patterns of daily activities, and habituation. Acknowledgments We would like to thank the Universidade Federal Rural da Amaz^ onia and National Primate Center (CENP) for their support. We also would like to thank Dr. Christina Wippich Whiteman, DVM, Ph.D. (Brazilian Institute of the Environment and Natural Renewable Resources— IBAMA) for suggestions and a technical English review.
References 1 Altmann J: Observational study of behavior: sampling methods. Behaviour 1974; 49:227–47. 2 Bicca-Marques JC, CalegaroMarques C: A case of geophagy in the black howling monkey Alouatta caraya. Neotrop Primates 1994; 2: 7–9. 3 Carlstead K, Shepherdson D: Alleviating stress in zoo animals with environmental enrichment. In: The Biology of Animal Stress: Basic Principles and Implications for Animal Welfare. Moberg GP & Mench JA (eds). New York: CABI, 2000; 337–50. 4 Crockett CM, Rudran R: Red howler monkey birth data II: interannual, habitat, and sex comparisons. Am J Primatol 1987; 13:369–84. 5 Crockett CM, Sekulic R: Gestation length red howler monkeys. Am J Primatol 1982; 3:291–4. 6 Fortes VB, Bicca-Marques JC: Ecologia e comportamento de primatas: metodos de estudo de campo. Caderno La Salle XI 2005; 2:207–18. 7 Fujita S, Mitsunaga F, Sugiura H, Shimizu K: Measurement of urinary and fecal steroid metabolites during the ovarian cycle in captive and wild japanese macaques, Macaca fuscata. Am J Primatol 2001; 53:167–76.
8 Fujita S, Sugiura H, Mitsunaga F, Shimizu K: Hormone profiles and reproductive characteristics in wild female Japanese macaques (Macaca fuscata). Am J Primatol 2004; 64:367–75. 9 Glander KE: Reproduction and population growth in free-ranging mantled howling monkeys. Am J Phys Anthropol 1980; 53:25–36. 10 Glander KE: Habitat description and resource utilization: A preliminary report on mantled howling monkeys ecology. In: Socioecology and Psychology of Primates. Tuttle R (ed.) Mouton: The Hague, 1975; 37–57. 11 Groves CP: Primate taxonomy. In: Smithsonian Series in Comparative Evolutionary Biology. Washington, DC: Smithsonian Books, 2001. 12 Guyton AC, Hall JE: Textbook of Medical Physiology. Philadelphia: WB Saunders, 2006. 13 Heistermann M, M€ ohle U, Verv€acke H, Van Elsacker L, Hodges JK: Application of urinary and fecal steroid measurements for monitoring ovarian function and pregnancy in bonobo (Pan paniscus) and evaluation of perineal swelling patterns in relation to endocrine events. Biol Reprod 1996; 55:844–53.
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14 Heistermann M, Uhrigshardt J, Husung A, Kaumanns W, Hodges JK: Measurement of faecal steroid metabolites in the lion-tailed macaque (Macaca silenus): a non-invasive tool for assessing female ovarian function. Primate Report 2001; 59:27–42. 15 Herrick JR, Agoramoorthy RRG, Harder JD: Urinary progesterone in free-ranging red howler monkeys (Alouatta seniculus). Preliminary observations of the estrous cycle and gestation. American. J Med Primatol 2000; 51:257–63. 16 IUCN [International Union for Conservation of Nature]: The Red List of Threatened Species, Available at http://www.iucnredlist.org/ (accessed on July 27 2013). 17 Kowalewski MM, Zunino GE: Impact of deforestation on population of Alouatta caraya in Northern Argentina. Folia Primatol 1999; 70:163–6. 18 Kugelmeier T, Valle RDR, Guimar~aes MABV, Muniz JAPC, Monteiro FOB, Oliveira CA: Tracking the ovarian cycle in black-and-gold howlers (Alouatta caraya) by measuring fecal steroids and observing vaginal bleeding. Int J Primatol 2011; 32:605– 15.
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19 L€ ottker P, Huck M, Heymann EW, Heistermann M: Endocrine correlates of reproductive status in breeding and non-breeding wild female moustached tamarins. Int J Primatol 2004; 4:919–37. 20 Lynch JW, Ziegler TE, Strier KB: Individual and seasonal variation in fecal androgens and cortisol levels of wild male tufted capuchin monkeys, Cebus apella nigritus. Horm Behav 2002; 41:275–87. 21 Milton K: The Foranging Strategy of Howler Monkeys. New York: Columbia University Press, 1980. 22 MMA [Ministerio do Meio Ambiente: Lista nacional das especies da fauna brasileira ameacßadas de extincß~ao]. Instrucß~ao Normativa Nº 003 de 26 de maio de 2003. 23 M€ ostl E, Palme R: Hormones as indicators of stress. Domest Anim Endocrin 2002; 23:67–74. 24 Neville MK, Glander KE, Braza F, Rylands AB: The howling monkeys, genus Alouatta. In: Ecology and Behavior of Neotropical Primates 2. Mittermeier RA, Rylands AB, Coimbra-Filho AF, Fonseca GAB (eds). Washington DC: World Wildlife Fund, 1988; 349–453. 25 Palme R: Measuring fecal steroids guidelines for practical application. Ann N Y Acad Sci 2005; 1046:75– 80.
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26 Pereira RJG: Metodos n~aoinvasivos para analises hormonais aplicadas aos estudos de ecologia e etologia. R Bras Zootec 2007; 36:71–6. 27 Rabiee AR, Macmillan KL, Schwarzenberger F: The effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows. Anim Reprod Sci 2001; 67:205–14. 28 Schwarzenberger F, M€ ostl E, Palme R, Bamberg E: Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Anim Reprod Sci 1996; 42:515–26. 29 Setz EZF, Hoyos A: Particß~ao do tempo: o problema da depend^encia ~es comportamentais entre observacßo sucessivas. In: A Primatologia no Brasil. Vol. 2. Mello MT (Ed.). Campinas: Imprensa Universitaria/ UFMG, 1986:191–201. 30 Souza LL, Ferrari SF, Costa ML, Kern DC: Geophagy as a correlate of folivory in red-handed howler monkeys (Alouatta belzebul) from eastern Brazilian Amazonia. J Chem Ecol 2002; 28:1613–21. 31 Strier KB, Ziegle TE, Wittwer DJ: Seasonal and social correlates of fecal androgens and cortisol levels in wild male muriquis (Brachyteles arachnoides). Horm Behav 1999; 35:125–34.
32 Van Belle S, Estrada A, Ziegler TE, Strier KB: Sexual behavior across ovarian cycle in wild black howler monkeys (Alouatta pigra): male mate guarding and female mate choice. Am J Primatol 2009; 71:153–64. 33 Wasser SK, Monfort SL, Wildt DE: Rapid extraction steroids for measuring reproductive cyclicity and early pregnancy in free-ranging yellow baboons (Papio cynocephalus cynocephalus). J Reprod Fertil 1991; 92:415–23. 34 Wasser SK, Thomas R, Nair PP, Guidry C, Southers J, Lucas J, Wildt DE, Monfort L: Effects of dietary fibre on faecal steroid measurements in baboons (Papio cynocephalus cynocephalus). J Reprod Fertil 1993; 97:569–74. 35 Ziegler T, Hodges K, Winkler P, Heistermann M: Hormonal correlates of reproductive seasonality in wild female hanuman langurs (Presbytis entellus). Am J Primatol 2000; 51:119–34. 36 Ziegler TE, Wittwer DJ: Fecal steroid research in the field and laboratory: improved methods for storage, transport, processing, and analysis. Am J Primatol 2005; 67:159–74.
J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
ORIGINAL ARTICLE
Evaluation of the fecal steroid concentrations in Alouatta belzebul (Primates, Atelidae) in the National Forest of , Brazil Tapirape-Aquiri in Para Frederico Ozanan Barros Monteiro1, Tatiana Kugelmeier2, Rodrigo del Rio do Valle3,4, Alexandre Bastos Fernandes Lima5, Felipe Ennes Silva6, Simone de Souza Martins5, Luciana Guedes Pereira5, Karen Lopes Dinucci5 & Priscila Viau7 1 2 3 4 5 6 7
^nia (UFRA), Bele m, PA, Brazil de e Producß~ Instituto da Sau ao Animal (ISPA), Universidade Federal Rural da Amazo de (PIBS), Rio de Janeiro, RJ, Brazil Fundacß~ ao Oswaldo Cruz (FIOCRUZ), Programa Institucional Biodiversidade & Sau ^ncias da Sau de, Universidade Paulista (UNIP), S~ao Paulo, SP, Brazil Instituto de Cie Wildlife Management Consultoria Veterin aria Ltda, S~ ao Paulo, SP, Brazil Empresa Habtec Engenharia Ambiental, Rio de Janeiro, RJ, Brazil , AM, Brazil Instituto de Desenvolvimento Sustent avel Mamirau a (IDSM), Tefe rio de Hormo ^nios (PROVET), S~ Laborato ao Paulo, SP, Brazil
Keywords howler monkey – radioimmunoassay – scan sampling method Correspondence Prof. Frederico Ozanan Barros Monteiro, ^nia Universidade Federal Rural da Amazo (UFRA)/Instituto da Sa ude e Producß~ ao Animal (ISPA), Avenida Presidente Tancredo Neves, nº 2501, Bairro: Terra Firme m, Par Cep: 66.077-901, Bele a, Brazil. Tel.: +55 91 32105139; fax: +55 91 32105139; e-mail: [email protected]; [email protected] Accepted July 10, 2013.
Abstract Background The studies on fecal steroid metabolites published with freeliving primates are limited mainly by the difficulty in obtaining samples. Methods A radioimmunoassay was used to measure the fecal steroid concentrations in Alouatta belzebul in the National Forest of Tapirape-Aquiri in Brazil. Results and Conclusions Androgens were significantly higher for the adult males from the Area of Influence (AI-I group) when compared to those from the Control Area (CA group) (P < 0.05). Progestin and estrogen concentrations were higher in the females from the CA group than in those from the AI-I for both the adult females and females with offspring; however, P < 0.05 was only observed in the concentrations of fecal progestins from the adult females. The physiological differences between the AI-I and CA groups suggest that the cause was a sum of factors, such as an exposure to sound waves, feeding habits, daily activity patterns, and the habituation of the animals.
Introduction Within neotropical primates, the members of the Alouatta genus have some of the largest body sizes and have a wider geographical distribution [24], with a range that encompasses the north of Argentina and south of Brazil to the south of Mexico [11]. They form small family groups, generally with a dominant adult male, adult reproducing females, juveniles, and babies. They are essentially folivores who dedicate a high percentage of their time to resting, which is generally more than 50% [10, 21]. The Alouatta belzebul species, which is known as the red-handed howler monkey, shows a specific geographical J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
distribution, as follows: to the south of the Amazon river as far as the state of Maranh~ ao, including the islands of Maraj o and Mexiana in the state of Par a, and to the northeast of the Atlantic Forest. This species is in the main list of threatened species, being classified in the categories ‘Critically Endangered’ [22] and ‘Vulnerable’ [16]. Their greatest threats are the destruction of their habitat, deforestation, and hunting, which is a strong threat in some areas of distribution. Therefore, the implementation of monitoring programs and management of the wild population to prevent the extinction of the species become paramount. The analysis of steroid hormones in feces and urine is an important tool in the understanding of the
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relationship between physiology and the environment in which the animal is inserted [33], thereby contributing to in situ and ex situ conservation programs. These methods allow researchers to evaluate and measure the variations in duration and phases of the ovarian cycle [13, 18], detect lactational anestrus [14], monitor the presence of seasonal influences [20, 31, 35], and measure the stress or welfare of wild species in conditions of captivity and/or free living [26]. There are several significant studies published on Alouatta and reproductive conditions [4, 5, 9]. However, endocrine studies are under-represented in this genus [15, 18, 32], this would be the first endocrine study on A. belzebul to our knowledge. According to Carlstead and Shepherdson [3], machine sounds may be an inadvertent source of aversion to many species. Various aspects of persistent acoustic stimuli such as intensity, frequency distribution, infrasound and content may be common stressors for captive animals. However, very little is known about these aspects in free-living animals, especially in nonhuman
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primates. Based on our literature review, this was the first study to evaluate acoustic stress in free-living monkeys. We aimed to evaluate the fecal steroid (corticosteroids, androgens, progestin, and estrogen) concentrations of A. belzebul that were monitored under an anthropic pressure, mainly sound noises, due to the implementation of a mining company in the National Forest of Tapirape-Aquiri in Par a, Brazil. Therefore, the hypothesis is that noise disturbance can be stressful and may influence both glucocorticoids and reproductive hormones. Material and methods Two groups of A. belzebul were monitored in the National Forest of Tapirape-Aquiri (05°46′17.84″ to 05°48′42.68″ South and 50º33′59.67″ to 50°31′42.21″ West) in the state of Par a, Brazil, where the Salobo mining project that belongs to the Vale mining company takes place (Fig. 1).
Fig. 1 The geographic location of the Control Area (CA) and the Area of Influence (AI-I) and the route followed by the animals in the CA (red -Aquiri. dots) and individuals in the AI-I (yellow dots) in the National Forest of Tapirape
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The groups were monitored from December 2010 to September 2011, during which the first 2 months were used for the habituation of the animals and the following months were used for the effective data collection. The researchers remained on site for approximately 15 days each month, with the same time interval between campaigns. Two areas of study were sampled (Fig. 1), one of which was under a direct influence from the activities generating sound waves from the Salobo Project, named the Area of Influence (AI-I). In this area, the presence of the AI-I group was observed, which consisted of animals categorized in the following way: one adult male and three females (one adult, one subadult, and one juvenile). In the June campaign, a birth was observed, thereby making it a group of five individuals. The second area of study was termed the Control Area (CA). This area was considered free from the influences of activities and was located between the streams ‘Igarape’ Mano and Salobo. The group from this area was composed of an adult male, a subadult male, three adult females (one of which had a baby), a juvenile male, and three juveniles of similar ages and nonidentified sex. Subsequently, this group was seen without the subadult male and one of the juvenile males, thereby totaling eight individuals. Collection and processing of the fecal sample For the reproductive endocrine study, only the adult animals were considered. The procedures for the collection and processing of fecal samples were performed as described by Ziegler and Wittwer [36] for field studies based on the measurement of fecal steroids. All monkeys were identified with the aid of binoculars. When defecation occurred, the fecal pellet was removed from the forest floor. Thus, samples of fresh feces were collected (immediately after defecation) in both areas of study (AI-I and CA) between 6 AM and 5 PM. After collection, the samples were appropriately placed in plastic bags, sealed, and identified with the date, time, place of collection, and the sex–age category of each animal, as follows: adult male (AM), adult female (AF), pregnant female or female with offspring (FWO), and non-identified (NI). Soon after collection, the samples were placed in a polystyrene receptacle with ice. At the end of the day, they were transferred to and stored in a freezer at 20°C until processing. The samples were brought from the field site to the Physiology Laboratory at the Universidade Federal Rural da Amaz^ onia, and the first stage of processing was the drying of the samples in a benchtop lyophilizer with a vacuum pump, model Enterprise I (Terroni J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Scientific Equipment, Sao Carlos, SP, Brazil). After lyophilization, the samples were again kept at 20°C until the extractions. For the hormonal extraction, 0.1 g of lyophilized feces was weighed in a semi-analytical balance (model BL 320H; Shimadzu Corporation, Japan), and then, 5 ml of 50% ethanol was added. The samples were then mixed on a vortex mixer (Phoenix, model AP-56; SP Labor, Sao Paulo, SP, Brazil) for 30 s and, subsequently, kept in a mechanical homogenizer (Phoenix AP-22; SP Labor, Sao Paulo, SP, Brazil) for 16 hours. After complete homogenization, the samples were centrifuged at 3000 rpm for 10 minutes, and 2 ml of the supernatant was divided into two polypropylene microtubes. The fecal extracts were kept in a freezer at 20°C until the hormonal measurements were performed at the Endocrinology Laboratory of Provet Diagnostic Veterinary Medicine in S~ ao Paulo, SP, Brazil. Hormone analysis All of the samples were assayed for the fecal metabolite concentrations of the following four steroid hormones: corticosteroids, androgens, progestin, and estrogens. The technique employed was a radioimmunoassay in a computerized gamma counter (Anser; Analytic Services Inc., Arlington, VA, United States), with the use of solid-phase commercial diagnostic kits for the progestins and androgens. For the estrogens and corticosteroids, double-antibody liquid-phase kits were used. The kits were manufactured for measuring these hormones in human serum or plasma, and the hormonal assays were performed according to the protocols provided by the manufacturers (Siemensâ, Los Angeles, CA, USA) for the estrogens, androgens, and progestins (MP Biomedicals, LLC, Solon, OH, USA) for the corticosteroids. The parameters for the quality of the assays, such as sensitivity, minimum dose, binding percentage, and coefficients of intra- and interassay variation for the low and high values of the calibration curve for each analyte, were analyzed. The laboratory validation of the commercial diagnostic kits for the estrogens double antibody and androgens was performed using a ‘pool’ of the fecal samples with a low concentration, that is, close to zero (matrix). Known quantities of labeled hormones were added to this matrix in dilutions, so that the final concentrations would be similar to the points in the standard curve of the diagnostic kit. This method indicated whether the fecal extract (matrix) interfered with the antigen reaction (labeled hormone and sample 3
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hormone) with the antibody from the commercial diagnostic kit used. The values of the curve thus obtained were correlated with the values of the standard curve from the diagnostic kit. The comparison was made using the linear regression coefficient, which was expressed as chi-squared only for the expression of positive values and for the Pearson’s correlation coefficient. The assay was validated when the correlation coefficient was close to 1 or 100%. The commercial diagnostic kits for measuring the fecal metabolites of progestins and corticosteroids in the genus Alouatta sp. were previously validated for the genus by our study group [18].
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or proportions, by dividing the number of records of the category of interest by the total records for the group. To test whether the level of each behavior was equally distributed along the hours of the day, the Kolmogorov–Smirnov test was used. To test the null hypothesis about the difference between the noise intensity between the two areas, a nonparametric Mann–Whitney U-test was used. A level of significance of 0.05 was adopted for all tests, which was calculated with the aid of the BioEstat software (version 5.0; Sociedade Civil de Mamirau a, Belem, Par a, Brazil). Results
Behavior monitoring For the collection of behavioral data, the scan sampling method was used [1, 6]. The sampling happened for five minutes, with 15-minute intervals to reduce samplingdependent error [29]. For each scan, the activity for each individual in sight was recorded, and the general pattern of the activities was divided into four main categories of exclusionary behaviors: 1—resting; 2—movement; 3— feeding; and 4—social interaction. The noise intensity in the areas of study was measured in decibels (dB), with a precision of 1 dB, using the DL-4000 decibel meter (Icel Gubintec©, Manaus, AM, Brazil) and using the curves of auditory perception for the human ear and a response of 200 msec as a standard. The sampling of noise intensity for each ecological–behavioral sample unit was taken. Statistical analysis For the hormone analyses, the basic parameters of the descriptive statistics (mean, standard error of the mean (mean SE), and minimum and maximum values) were calculated for all the variables analyzed. The comparison of means was performed for the metabolites of corticosteroids, androgens, progestins, and estrogens in the CA and AI-I between the AM, AF, FWO, and NI groups. A nonparametric analysis of variance (Kruskal–Wallis) was used. Considering the difference in the number of behavioral records in each group, a test for the comparison of two binomial proportions was used to test the null hypothesis on the difference between the quantities of each behavior between the two groups (two independent samples). This same test was used to assess the null hypothesis on the difference between the amounts of food items consumed between the two groups. With the intention of standardizing the samples for the activity patterns, the records were then transformed into rates, 4
Eighty-nine samples of feces distributed by categories (AM, AF, FWO, and NI) in each area of study were collected, with 56 samples from the CA group and 29 from the AI-I group. The higher number of samples for the CA group occurred due to problems of access to the AI-I, which caused difficulties in the search, the habituation of the AI-I group, and, consequently, feces collection. The hormone assays were divided according to each hormone. For corticosteroids, the sensitivity was 1.96 ng/ml, the low intra-assay coefficient of variation (CV) was 3.74%, and the high intra-assay CV was 0.85%. In the assay for androgen measurement, we obtained a sensitivity of 1.82 ng/dL, with a low intraassay CV of 4.17% and a high intra-assay CV of 1.17%. For the progestin assay, we obtained a sensitivity of 0.004 ng/ml, with an intra-assay CV lower than 4.21% and interassay CVs of 2.45% and 0.55% for the low and high values, respectively. In the double-antibody estrogen assay, the sensitivity was 0.42 pg/ml, with intraassay CVs of 3.12% and 0.90% for the low and high values, respectively. The validation of the commercial diagnostic kit (Siemensâ, Los Angeles, CA, USA) for the ‘solid phase’ of androgens and for the double-antibody estrogens for use in fecal samples from primates of the genus Alouatta was carried out. There was an agreement between the curve from the commercial diagnostic kit and the matrix dilution curves studied. We observed that in all tests performed, we obtained a high linear regression. The correlation (Pearson’s test) between the modified curve and the standard curve provided by the kit for the androgen and estrogen assays was r = 0.982, P = 0.0010, and r = 0.970, P < 0.001, respectively. The results showed a good linearity for the androgen and estrogen assays r = 0.99 and r = 0.98, respectively, in the studied concentration range. Based on these results, it was possible to suggest a laboratory validation of the assays. J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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The concentrations of fecal steroid metabolites showed a large individual variation, which was also within the same group and the same category. Table 1 shows the comparison of the means, standard error of the mean, and minimum and maximum values of the concentrations of fecal corticosteroids. The animals from the AI-I group showed higher concentrations of fecal steroids than those from the CA group for all of the categories; however, there was no significant difference (Kruskal–Wallis: H = 0.25; 1.05; 1.72; 0.55 df = 1; P = 0.62 for AF; 0.31 for FWO; 0.19 for AM; and 0.46 for NI). The data from Table 2 show the mean concentrations, standard error of the mean, and minimum and maximum values of fecal metabolites for androgens, progestins, and estrogens for the AM, AF, and FWO groups from the CA and AI-I. The concentrations of androgens were significantly higher for the AM from the AI-I in relation to those from the CA (Kruskal– Wallis: H = 4.2; df = 1; P < 0.05). The analyses showed that the fecal progestins and estrogens were higher in the females from the CA group than those from the AI-I group for both the AF and FWO; however, there was only a significant difference in the concentrations of
fecal progestins in the AF (Kruskal–Wallis: H = 6.75; df = 1; P < 0.05). In total, 3272 behavioral records were obtained; of these, 2358 were for the CA group, and 914 were for the AI-I group. These records were obtained from 609 instantaneous scans, among which 332 were taken for the CA group, and 277 were for the AI-I group. As described for the collection of fecal samples, the higher number of records for the CA group was due to problems of access to the AI-I region, which caused difficulty for the search and habituation of the AI-I group and the subsequent data collection despite a higher number of days (40) and hours (338) spent on-site for this group. Comparatively, there were no significant differences between the proportion of resting (Z = 0.9376; P = 0.1742), movement (Z = 0.1812; P = 0.4281), feeding (Z = 0.2927; P = 0.3849), and social interactions (Z = 0.2283; P = 0.4097) between the two study groups. In relation to the feeding behavior between the observed items, leaves were consumed most by the two groups (AI-I: 76%; CA: 59%), and there was no significant difference between the groups regarding the consumption of leaves (Z = 0.5074; P = 0.3059). However, a considerable difference between the groups
Table 1 Mean concentrations, standard error of mean (mean SE), and minimum and maximum values of fecal corticosteroid (ng/g dry feces) for categories (adult males, adult females, female with offspring, and non-identified) in Control Area (CA) and Area of Influence (AI-I) Control Area (CA) Category
Mean SE
N
Adult males Adult females Female with offspring Non-identified
Area of Influence (AI-I)
12 28 7 4
43.27 39.10 32.46 33.65
Minimum/maximum a
21.77/114.12 25.92/90.96 19.68/45.29 26.28/36.84
7.15 2.34a 3.28a 2.48a
Mean SE
N 6 6 3 14
59.47 39.68 40.68 44.84
Minimum/maximum a
18.49 3.87a 7.81a 5.65a
35.76/151.21 24.96/54.04 27.2/54.24 19.37/84.31
N, number of samples per category. Lowercase superscript letters indicate mean comparisons between groups conducted in the same category (Kruskal–Wallis: H = 0.25; 1.05; 1.72; 0.55; df = 1, P = 0.62; 0.31; 0.19; 0.46, respectively).
Table 2 Mean concentrations, standard error of mean (mean SE), and minimum and maximum values of fecal metabolites of androgens, estrogens, and progestins for categories (adult males—AM, adult females—AF, and female with offspring—FWO) in Control Area (CA) and Area of Influence (AI-I) Control Area (CA) Hormones
Category I
Androgens (lg/g dry feces) ProgestinsII (lg/g dry feces) EstrogensIII (ng/g dry feces)
AM AF FWO AF FWO
N 12 28 7 28 7
Area of Influence (AI-I)
Mean SE 34.96 1.42 0.80 189.63 126.70
Minimum/maximum b
4.64 0.52a 0.68a 65.83a 105.99a
8.46/69.94 0.00/14.04 0.01/4.89 1.11/1524.9 1.00/761.05
N 6 6 3 6 3
Mean SE 49.38 0.05 0.21 23.93 30.21
Minimum/maximum a
4.43 0.02b 0.16a 9.48a 10.14a
34.08/65.38 0.00/0.11 0.03/0.52 0.43/57.73 9.95/40.92
N, number of samples per category. Lowercase superscript letters indicate mean comparisons between groups conducted in the same category. Kruskal–Wallis: (H = 4.2, df = 1, P < 0.05)I; (H = 6.75, 0.00, df = 1, P < 0.05, P = 1, respectively)II; (H = 0.90, 0.01, df = 1, P = 0.34; 0.91, respectively)III. J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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was observed in the proportion of the remaining consumed items, such as fruit (CA: 30%; AI-I: 1%; Z = 5.7380; P < 0.0001), flowers (CA: 10%; AI-I: 5%; Z = 1.7015; P = 0.0444), and earth (AI-I: 18%; CA: 1% Z = 5.5366; P < 0.0001). The consumption of fruit and flowers was higher for the CA group, and the consumption of earth from termitaria was significantly higher for the AI-I group. A greater noise intensity was encountered by the group in AI-I, with the highest mean (56.95 dB) and maximum value (65 dB) both in the month of April. The results confirmed that the AI-I group was significantly exposed to more noise from the mining activities (U = 3422.00; Z = 12.8826; P < 0.0001). The detonations in the mines did not occur regularly and were recorded on eight occasions during the campaign, especially in the visits to the AI-I area, and they always occurred around midday. The most intense noises were from the machines and the movement of the workers in the study area and the vicinities. Discussion The studies on fecal steroid metabolites published with free-living animals, in particular with neotropical primates, are limited mainly by the difficulty in obtaining samples. Therefore, the results from this study offer important data to aid in the understanding of the relationship between the physiology and the environment where these animals live. The large variation in fecal steroid concentrations observed in this study was also reported by Palme [25]. This author stated that such variations are expected when one works with fecal matrix, given that many factors may contribute to this variation, such as intraand interspecific differences, circadian rhythm of hormone secretion, seasonal variations, the age, sex, and reproductive status of the studied individuals, diet, and the habituation of the animals. Therefore, there are many factors to be considered in processing fecal steroids. Steroids are excreted into the feces at different lag times due to processing of the steroids in the liver and reabsorption in the gut. Some steroids, such as estradiol, are highly conjugated and may be delayed in their excretion into the feces [36]. One way to reduce the variation from the circadian rhythm of hormone secretion would be to always collect the samples at the same time or within a small time interval [23]. However, we had difficulty in standardizing the collection time as the present study dealt with free-living animals. Therefore, to guarantee as high a number of samples per group as possible, we opted for collecting the samples throughout the day while always 6
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respecting the main requirement of collecting fresh samples of feces to avoid hormone degradation, as suggested by Ziegler and Wittwer [36]. The high folivory of the genus Alouatta [10, 21] may result in the increased amount of fiber and moisture in their diet. These factors may cause an underestimation of the concentration of fecal steroid metabolites because they increase the transit and volume of the fecal bolus, thereby interfering with hormone excretion [27, 28, 34]. Due to this feeding habit, the first stage of processing was a drying of the samples (lyophilization) in an attempt to obtain more homogenous and consistent results. Despite the levels of fecal corticosteroids not showing a significant difference between the animals from the AI-I and CA groups, the analysis of the behavior observations allowed us to infer that the group from the CA was more stable. Therefore, the significantly higher mean fecal androgen metabolite concentrations for the males from the AI-I than those from the CA may be related to stress, with the consequential release of steroids by the adrenal glands [12]. Based on the behavioral data for the groups, it was observed that by the end of the study, the CA group, in addition to being in an area with a lower incidence of anthropic influences generated by mining activities, also found themselves more accustomed to the presence of the researchers, which may have been directly reflected in the hormone analyses, thereby demonstrating stability. Alternatively, the animals from the AI-I group, as they were not properly accustomed and were under the influence of anthropic activities, showed a high degree of geophagia, which in general indicates a need for supplemental minerals or some degree of parasite infestation. Nevertheless, this can also be indirectly related to some level of stress or a change of habit [2, 30]. Although the proposed experimental design had used a group that was outside the area of impact as the control group, it was not possible to affirm that the variations observed were due to the differences in sound waves between the study areas. Despite the significant difference found for the noise intensity in the two areas, other factors may have interfered with the results, such as the type of noise (machinery, detonation, etc.), the habituation, and the natural adaptation of the howlers themselves to the anthropic interferences. To understand this finding, it is important to consider that these animals are resistant to some degree of change in the landscape [17], acclimatizing themselves more easily than other primate species. However, the Alouatta populations have been suffering a strong impact as a consequence of the increasing fragmentation of their habitats; therefore, it is extremely relevant that the J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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changes caused by anthropic actions are monitored. Factors such as the composition, density, and ecology of the species, their reproductive cycle, the floristic and faunistic composition of the studied areas, and the climatic conditions must be known, so that they become important tools for preservation [7, 8, 19]. Therefore, studies to monitor the population of primates, before, during, and after the human activities and enterprises become necessary to obtain information that can aid in the protection or management of the species. Construction noise has also been identified as a potential source of stress although the few studies conducted to date have failed to demonstrate anything other than short-term responses to specific auditory events such as blasting noise [3]. Therefore, although our results point to physiological differences between
the CA and AI-I groups, these data only suggest that these differences were caused by the sum of several factors, such as the exposure to noises from the activity of the enterprises, feeding habits, the patterns of daily activities, and habituation. Acknowledgments We would like to thank the Universidade Federal Rural da Amaz^ onia and National Primate Center (CENP) for their support. We also would like to thank Dr. Christina Wippich Whiteman, DVM, Ph.D. (Brazilian Institute of the Environment and Natural Renewable Resources— IBAMA) for suggestions and a technical English review.
References 1 Altmann J: Observational study of behavior: sampling methods. Behaviour 1974; 49:227–47. 2 Bicca-Marques JC, CalegaroMarques C: A case of geophagy in the black howling monkey Alouatta caraya. Neotrop Primates 1994; 2: 7–9. 3 Carlstead K, Shepherdson D: Alleviating stress in zoo animals with environmental enrichment. In: The Biology of Animal Stress: Basic Principles and Implications for Animal Welfare. Moberg GP & Mench JA (eds). New York: CABI, 2000; 337–50. 4 Crockett CM, Rudran R: Red howler monkey birth data II: interannual, habitat, and sex comparisons. Am J Primatol 1987; 13:369–84. 5 Crockett CM, Sekulic R: Gestation length red howler monkeys. Am J Primatol 1982; 3:291–4. 6 Fortes VB, Bicca-Marques JC: Ecologia e comportamento de primatas: metodos de estudo de campo. Caderno La Salle XI 2005; 2:207–18. 7 Fujita S, Mitsunaga F, Sugiura H, Shimizu K: Measurement of urinary and fecal steroid metabolites during the ovarian cycle in captive and wild japanese macaques, Macaca fuscata. Am J Primatol 2001; 53:167–76.
8 Fujita S, Sugiura H, Mitsunaga F, Shimizu K: Hormone profiles and reproductive characteristics in wild female Japanese macaques (Macaca fuscata). Am J Primatol 2004; 64:367–75. 9 Glander KE: Reproduction and population growth in free-ranging mantled howling monkeys. Am J Phys Anthropol 1980; 53:25–36. 10 Glander KE: Habitat description and resource utilization: A preliminary report on mantled howling monkeys ecology. In: Socioecology and Psychology of Primates. Tuttle R (ed.) Mouton: The Hague, 1975; 37–57. 11 Groves CP: Primate taxonomy. In: Smithsonian Series in Comparative Evolutionary Biology. Washington, DC: Smithsonian Books, 2001. 12 Guyton AC, Hall JE: Textbook of Medical Physiology. Philadelphia: WB Saunders, 2006. 13 Heistermann M, M€ ohle U, Verv€acke H, Van Elsacker L, Hodges JK: Application of urinary and fecal steroid measurements for monitoring ovarian function and pregnancy in bonobo (Pan paniscus) and evaluation of perineal swelling patterns in relation to endocrine events. Biol Reprod 1996; 55:844–53.
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14 Heistermann M, Uhrigshardt J, Husung A, Kaumanns W, Hodges JK: Measurement of faecal steroid metabolites in the lion-tailed macaque (Macaca silenus): a non-invasive tool for assessing female ovarian function. Primate Report 2001; 59:27–42. 15 Herrick JR, Agoramoorthy RRG, Harder JD: Urinary progesterone in free-ranging red howler monkeys (Alouatta seniculus). Preliminary observations of the estrous cycle and gestation. American. J Med Primatol 2000; 51:257–63. 16 IUCN [International Union for Conservation of Nature]: The Red List of Threatened Species, Available at http://www.iucnredlist.org/ (accessed on July 27 2013). 17 Kowalewski MM, Zunino GE: Impact of deforestation on population of Alouatta caraya in Northern Argentina. Folia Primatol 1999; 70:163–6. 18 Kugelmeier T, Valle RDR, Guimar~aes MABV, Muniz JAPC, Monteiro FOB, Oliveira CA: Tracking the ovarian cycle in black-and-gold howlers (Alouatta caraya) by measuring fecal steroids and observing vaginal bleeding. Int J Primatol 2011; 32:605– 15.
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Fecal steroid concentrations in A. belzebul
19 L€ ottker P, Huck M, Heymann EW, Heistermann M: Endocrine correlates of reproductive status in breeding and non-breeding wild female moustached tamarins. Int J Primatol 2004; 4:919–37. 20 Lynch JW, Ziegler TE, Strier KB: Individual and seasonal variation in fecal androgens and cortisol levels of wild male tufted capuchin monkeys, Cebus apella nigritus. Horm Behav 2002; 41:275–87. 21 Milton K: The Foranging Strategy of Howler Monkeys. New York: Columbia University Press, 1980. 22 MMA [Ministerio do Meio Ambiente: Lista nacional das especies da fauna brasileira ameacßadas de extincß~ao]. Instrucß~ao Normativa Nº 003 de 26 de maio de 2003. 23 M€ ostl E, Palme R: Hormones as indicators of stress. Domest Anim Endocrin 2002; 23:67–74. 24 Neville MK, Glander KE, Braza F, Rylands AB: The howling monkeys, genus Alouatta. In: Ecology and Behavior of Neotropical Primates 2. Mittermeier RA, Rylands AB, Coimbra-Filho AF, Fonseca GAB (eds). Washington DC: World Wildlife Fund, 1988; 349–453. 25 Palme R: Measuring fecal steroids guidelines for practical application. Ann N Y Acad Sci 2005; 1046:75– 80.
8
Monteiro et al.
26 Pereira RJG: Metodos n~aoinvasivos para analises hormonais aplicadas aos estudos de ecologia e etologia. R Bras Zootec 2007; 36:71–6. 27 Rabiee AR, Macmillan KL, Schwarzenberger F: The effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows. Anim Reprod Sci 2001; 67:205–14. 28 Schwarzenberger F, M€ ostl E, Palme R, Bamberg E: Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Anim Reprod Sci 1996; 42:515–26. 29 Setz EZF, Hoyos A: Particß~ao do tempo: o problema da depend^encia ~es comportamentais entre observacßo sucessivas. In: A Primatologia no Brasil. Vol. 2. Mello MT (Ed.). Campinas: Imprensa Universitaria/ UFMG, 1986:191–201. 30 Souza LL, Ferrari SF, Costa ML, Kern DC: Geophagy as a correlate of folivory in red-handed howler monkeys (Alouatta belzebul) from eastern Brazilian Amazonia. J Chem Ecol 2002; 28:1613–21. 31 Strier KB, Ziegle TE, Wittwer DJ: Seasonal and social correlates of fecal androgens and cortisol levels in wild male muriquis (Brachyteles arachnoides). Horm Behav 1999; 35:125–34.
32 Van Belle S, Estrada A, Ziegler TE, Strier KB: Sexual behavior across ovarian cycle in wild black howler monkeys (Alouatta pigra): male mate guarding and female mate choice. Am J Primatol 2009; 71:153–64. 33 Wasser SK, Monfort SL, Wildt DE: Rapid extraction steroids for measuring reproductive cyclicity and early pregnancy in free-ranging yellow baboons (Papio cynocephalus cynocephalus). J Reprod Fertil 1991; 92:415–23. 34 Wasser SK, Thomas R, Nair PP, Guidry C, Southers J, Lucas J, Wildt DE, Monfort L: Effects of dietary fibre on faecal steroid measurements in baboons (Papio cynocephalus cynocephalus). J Reprod Fertil 1993; 97:569–74. 35 Ziegler T, Hodges K, Winkler P, Heistermann M: Hormonal correlates of reproductive seasonality in wild female hanuman langurs (Presbytis entellus). Am J Primatol 2000; 51:119–34. 36 Ziegler TE, Wittwer DJ: Fecal steroid research in the field and laboratory: improved methods for storage, transport, processing, and analysis. Am J Primatol 2005; 67:159–74.
J Med Primatol (2013) 1–8 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
