, Volume 58, Issue 2, pp 285–294 | Cite as

Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans

  • Taufiq Purna Nugraha
  • Michael Heistermann
  • Muhammad Agil
  • Bambang Purwantara
  • Iman Supriatna
  • Gholib Gholib
  • Carel P. van Schaik
  • Tony Weingrill
Original Article


Measuring hormone metabolites from feces is the most often used method to assess hormonal status in wildlife. Although immediate freezing of fecal samples collected in the field is the best method to minimize the risk of degradation of hormones over time, this is often not possible in remote field sites. Therefore, alternative storage and preservation methods for fecal samples are required in these conditions. We conducted an experiment to investigate if fecal glucocorticoid (FGCM) and progesterone metabolite (pregnanediol-3-glucuronide; PdG) levels measured from samples that were extracted with a simple, field-friendly methodology correlate with those generated from frozen samples. We also evaluated whether storing fecal samples in alcohol is a suitable alternative to preserve FGCM and PdG concentrations long-term (i.e. over a 9-month period) at locations where fecal extraction is not feasible. Finally, we tested if the hormone concentrations in unpreserved fecal samples of orangutans change over 14 h when stored at ambient conditions, representing the maximum duration between sample collection and return to the camp. FGCM and PdG levels measured from samples that were extracted with the field-friendly method showed strong correlations with those generated from frozen samples, and mean levels did not differ significantly between these methods. FGCM concentrations showed no significant change compared to control samples when fecal samples were stored for up to 6 months in alcohol at ambient temperature and PdG concentrations even remained stable for up to 9 months of storage. FGCM concentrations of fecal samples kept at ambient temperature for up to 14 h post-defecation did not significantly differ compared to control samples frozen immediately after collection. These results provide the basis for the successful monitoring of the physiological status of orangutans living in remote natural settings, like those included in the Indonesian reintroduction programs.


Pongo spp. Fecal glucocorticoids PdG Sample storage Hormone degradation 



This study was part of the project Building Indonesia’s Research Capacity for Orangutan Conservation Biology (joint research project IZ70Z0_131309) and funded by the program Research Partnerships with Developing Countries, a joint initiative of the Swiss National Science Foundation and the Swiss Agency for Development and Cooperation. Further generous funding was provided by the A.H. Schultz Foundation and the Claraz Foundation. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. The authors have no conflicts of interest to declare. We thank the orangutan keepers, vet and staff at Batu Mbelin Orangutan Care Centre and Ragunan Zoo for their help in collecting samples for this study. We also thank Dr. Ian Singleton from SOCP and YEL for giving us permission to collect samples, drh. Ricko Lany Jaya and drh. Yenny Saraswati for their help during sample collection in Medan, and Andrea Heistermann for her expert help during hormone training in Göttingen. We are also grateful to Keith Hodges and the German Primate Centre as well as the University of Zurich for their long-standing support of the endocrinology lab at the Faculty of Veterinary Medicine, Bogor Agricultural University.


  1. Ancrenaz M, Marshall A, Goossens B, van Schaik C, Sugardjito J, Gumal M, Wich S (2008) Pongo pygmaeus. In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.4Google Scholar
  2. Ashley NT, Barboza PS, Macbeth BJ, Janz DM, Cattet MRL, Booth RK, Wasser SK (2011) Glucocorticosteroid concentrations in feces and hair of captive caribou and reindeer following adrenocorticotropic hormone challenge. Gen Comp Endocrinol 172:382–391CrossRefPubMedGoogle Scholar
  3. Barelli C, Heistermann M (2009) Monitoring Female reproductive status in white-handed gibbons (Hylobates lar) using fecal hormone analysis and patterns of genital skin swellings. In: Whittaker D, Lappan S (eds) The gibbons new perspectives on small ape socioecology and population biology. Springer, New York, pp 313–325Google Scholar
  4. Brown JL, Wasser SK, Wildt DE, Graham LH (1994) Comparative aspects of steroid hormone metabolism and ovarian activity in felids, measured noninvasively in feces. Biol Reprod 51:776–786CrossRefPubMedGoogle Scholar
  5. Fichtel C, Kraus C, Ganswindt A, Heistermann M (2007) Influence of reproductive season and rank on fecal glucocorticoid levels in free-ranging male Verreaux’s sifakas (Propithecus verreauxi). Horm Behav 51:640–648CrossRefPubMedGoogle Scholar
  6. Galama WT, Graham LH, Savage A (2004) Comparison of fecal storage methods for steroid analysis in black rhinoceroses (Diceros bicornis). Zoo Biol 23:291–300CrossRefGoogle Scholar
  7. Ganswindt A, Palme R, Heistermann M, Borragan S, Hodges JK (2003) Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth. Gen Comp Endocrinol 134:156–166CrossRefPubMedGoogle Scholar
  8. Goymann W (2005) Noninvasive monitoring of hormones in bird droppings: physiological validation, sampling, extraction, sex differences, and the influence of diet on hormone metabolite levels. Ann N Y Acad Sci 1046:35–53CrossRefPubMedGoogle Scholar
  9. Goymann W (2012) On the use of non-invasive hormone research in uncontrolled, natural environments: the problem with sex, diet, metabolic rate and the individual. Methods Ecol Evol 3:757–765CrossRefGoogle Scholar
  10. Heistermann M, Hodges JK (1995) Endocrine monitoring of the ovarian cycle and pregnancy in the saddle-back tamarin (Saginus fuscicollis) by measurement of steroid conjugates in urine. Am J Primatol 35:117–127CrossRefGoogle Scholar
  11. Heistermann M, Finke M, Hodges JK (1995) Assessment of female reproductive status in captive-housed hanuman langurs (Presbytis entellus) by measurement of urinary and fecal steroid excretion patterns. Am J Primatol 37:275–284CrossRefGoogle Scholar
  12. Heistermann M, Ademmer C, Kaumanns W (2004) Ovarian cycle and effect of social changes on adrenal and ovarian function in Pygathrix nemaeus. Int J Primatol 25:689–708CrossRefGoogle Scholar
  13. Hodges JK, Heistermann M (2011) Field endocrinology: monitoring hormonal changes in free-ranging primates. In: Setchell JM, Curtis DJ (eds) Field and laboratory methods in primatology: a practical guide, 2nd edn. Cambridge University, Cambridge, pp 353–370CrossRefGoogle Scholar
  14. Hodges K, Brown JL, Heistermann M (2010) Endocrine monitoring of reproduction and stress. In: Kleiman DG, Thompson KV, Kirk Baer C (eds) Wild mammals in captivity: principles and techniques for zoo management. The University of Chicago Press, Chicago, pp 447–468Google Scholar
  15. Hulsman A, Dalerum F, Ganswindt A, Muenscher S, Bertschinger HJ, Paris M (2011) Non-invasive monitoring of glucocorticoid metabolites in brown hyaena (Hyaena brunnea) feces. Zoo Biol 30:451–458CrossRefPubMedGoogle Scholar
  16. Hunt KE, Wasser SK (2003) Effect of long-term preservation methods on fecal glucocorticoid concentrations of grizzly bear and African elephant. Physiol Biochem Zool 76:918–928CrossRefPubMedGoogle Scholar
  17. Jenni L, Keller N, Almasi B, Duplain J, Homberger B, Lanz M, Korner-Nievergelt F, Schaub M, Jenni-Eiermann S (2015) Transport and release procedures in reintroduction programs: stress and survival in grey partridges. Anim Conserv 18:62–72CrossRefGoogle Scholar
  18. Kalbitzer U, Heistermann M (2013) Long-term storage effects in steroid metabolite extracts from baboon (Papio sp.) faeces—a comparison of three commonly applied storage methods. Methods Ecol Evol 4:493–500CrossRefGoogle Scholar
  19. Kalbitzer U, Heistermann M, Cheney D, Seyfarth R, Fischer J (2015) Social behavior and patterns of testosterone and glucocorticoid levels differ between male chacma and Guinea baboons. Horm Behav 75:100–110CrossRefPubMedGoogle Scholar
  20. Khan MZ, Altmann J, Isani SS, Yu J (2002) A matter of time: evaluating the storage of fecal samples for steroid analysis. Gen Comp Endocrinol 128:57–64CrossRefPubMedGoogle Scholar
  21. Lynch JW, Khan MZ, Altmann J, Njahira MN, Rubenstein N (2003) Concentrations of four fecal steroids in wild baboons: short-term storage conditions and consequences for data interpretation. Gen Comp Endocrinol 132:264–271CrossRefPubMedGoogle Scholar
  22. Marty PR, van Noordwijk MA, Heistermann M, Willems EP, Dunkel LP, Cadilek M, Agil M, Weingrill T (2015) Endocrinological correlates of male bimaturism in wild Bornean orangutans. Am J Primatol 77:1170–1178CrossRefPubMedGoogle Scholar
  23. Millspaugh JJ, Washburn BE (2003) Within-sample variation of fecal glucocorticoid measurements. Gen Comp Endocrinol 132:21–26CrossRefPubMedGoogle Scholar
  24. Millspaugh JJ, Washburn BE (2004) Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation. Gen Comp Endocrinol 138:189–199CrossRefPubMedGoogle Scholar
  25. Möstl E, Messmann S, Bagu E, Robia C, Palme R (1999) Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. J Vet Med A 46:621–631CrossRefGoogle Scholar
  26. Muehlenbein MP, Ancrenaz M, Sakong R, Ambu L, Prall S, Fuller G, Raghanti MA (2012) Ape conservation physiology: fecal glucocorticoid responses in wild Pongo pygmaeus morio following human visitation. PLoS One 7(3):e33357CrossRefPubMedPubMedCentralGoogle Scholar
  27. Murray CM, Heintz MR, Lonsdorf EV, Parr LA, Santymire RM (2013) Validation of a field technique and characterization of fecal glucocorticoid metabolite analysis in wild chimpanzees (Pan troglodytes). Am J Primatol 75:57–64CrossRefPubMedGoogle Scholar
  28. Ozella L, Anfossi L, Di Nardo F, Pessani D (2015) Non-invasive monitoring of adrenocortical activity in captive African Penguin (Spheniscus demersus) by measuring faecal glucocorticoid metabolites. Gen Comp Endocrinol 224:104–112CrossRefPubMedGoogle Scholar
  29. Palme R (2005) Measuring fecal steroids: guidelines for practical application. Ann N Y Acad Sci 1046:75–80CrossRefPubMedGoogle Scholar
  30. Palme R, Touma C, Arias N, Dominchin M, Lepschy M (2013) Steroid extraction: get the best out of faecal samples. Wien Tierarztl Monatsschr 100:238–246Google Scholar
  31. Rimbach R, Heymann EW, Link A, Heistermann M (2013) Validation of an enzyme immunoassay for assessing adrenocortical activity and evaluation of factors that affect levels of fecal glucocorticoid metabolites in two New World primates. Gen Comp Endocrinol 191:13–23CrossRefPubMedGoogle Scholar
  32. Sheriff MJ, Dantzer B, Delehanty B, Palme R, Boonstra R (2011) Measuring stress in wildlife: techniques for quantifying glucocorticoids. Oecologia 166:869–887CrossRefPubMedGoogle Scholar
  33. Shutt K, Setchell JM, Heistermann M (2012) Non-invasive monitoring of physiological stress in the Western lowland gorilla (Gorilla gorilla gorilla): validation of a fecal glucocorticoid assay and methods for practical application in the field. Gen Comp Endocrinol 179:167–177CrossRefPubMedGoogle Scholar
  34. Singleton I, Wich S, Griffiths M (2008) Pongo abelii. IUCN Red List of Threatened Species. In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.4Google Scholar
  35. Soehartono T, Susilo H, Andayani N, Atmoko S, Sihite J, Saleh C, Sutrisno A (2008) Orangutan Indonesia conservation strategies and action plan. Ministry of Forestry, JakartaGoogle Scholar
  36. Teixeira CP, de Azevedo CS, Mendl M, Cipreste CF, Young RJ (2007) Revisiting translocation and reintroduction programmes: the importance of considering stress. Anim Behav 73:1–13CrossRefGoogle Scholar
  37. Terio KA, Brown JL, Moreland R, Munson L (2002) Comparison of different drying and storage methods on quantifiable concentrations of fecal steroids in the cheetah. Zoo Biol 21:215–222CrossRefGoogle Scholar
  38. Touma C, Palme R (2005) Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation. Ann N Y Acad Sci 1046:54–74CrossRefPubMedGoogle Scholar
  39. Washburn BE, Millspaugh JJ (2002) Effects of simulated environmental conditions on glucocorticoids metabolite measurements in white-tailed deer feces. Gen Comp Endocrinol 127:217–222CrossRefPubMedGoogle Scholar
  40. Wasser SK, Risler L, Steiner RA (1988) Excreted steroids in primate feces over the menstrual cycle and pregnancy. Biol Reprod 39:862–872CrossRefPubMedGoogle Scholar
  41. Weingrill T, Willems EP, Zimmermann N, Steinmetz H, Heistermann M (2011) Species-specific patterns in fecal glucocorticoid and androgen levels in zoo-living orangutans (Pongo spp.). Gen Comp Endocrinol 172:446–457CrossRefPubMedGoogle Scholar
  42. Whitten PL, Brockman DK, Stavisky RC (1998) Recent advances in noninvasive techniques to monitor hormone-behavior interactions. Yearb Phys Anthropol 41:1–23CrossRefGoogle Scholar
  43. Winter J, Bokkenheuser VD (1978) 21-dehydroxylation of corticoids by anaerobic bacteria isolated from human fecal flora. J Steroid Biochem 9:379–384CrossRefPubMedGoogle Scholar
  44. Yeo R, Sawdon M (2013) Hormonal control of metabolism: regulation of plasma glucose. Anaesth Intensive Care Med 14:296–300CrossRefGoogle Scholar
  45. Ziegler TE, Wittwer DJ (2005) Fecal steroid research in the field and laboratory: improved methods for storage, transport, processing, and analysis. Am J Primatol 67:159–174CrossRefPubMedGoogle Scholar

Copyright information

© Japan Monkey Centre and Springer Japan 2016

Authors and Affiliations

  • Taufiq Purna Nugraha
    • 1
    • 2
    • 5
  • Michael Heistermann
    • 3
  • Muhammad Agil
    • 1
  • Bambang Purwantara
    • 1
  • Iman Supriatna
    • 1
  • Gholib Gholib
    • 4
  • Carel P. van Schaik
    • 5
  • Tony Weingrill
    • 5
  1. 1.Faculty of Veterinary ScienceBogor Agricultural UniversityBogorIndonesia
  2. 2.Laboratory of Reproduction Division of Zoology, Research Center for BiologyIndonesian Institute of Sciences (LIPI)CibinongIndonesia
  3. 3.German Primate CentreEndocrinology LaboratoryGöttingenGermany
  4. 4.Laboratory of Physiology, Faculty of Veterinary MedicineUniversity of Syiah KualaBanda AcehIndonesia
  5. 5.Department of AnthropologyUniversity of ZurichZurichSwitzerland

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