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Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques

Abstract

Since the non-invasive field endocrinology techniques were developed, several fecal preservation and extraction methods have been established for a variety of species. However, direct adaptation of methods from previous studies for use in crested macaques should be taken with caution. We conducted an experiment to assess the accuracy and stability of fecal estrogen metabolite (E1C) and glucocorticoid metabolite (GCM) concentrations in response to several preservation parameters: (1) time lag between sample collection and fecal preservation; (2) long-term storage of fecal samples in 80% methanol (MeOH) at ambient temperature; (3) different degrees of feces drying temperature using a conventional oven; and (4) different fecal preservation techniques (i.e., freeze-drying, oven-drying, and field-friendly extraction method) and extraction solvents (methanol, ethanol, and commercial alcohol). The study used fecal samples collected from crested macaques (Macaca nigra) living in the Tangkoko Reserve, North Sulawesi, Indonesia. Samples were assayed using validated E1C and GCM enzyme immunoassays. Concentrations of E1C and GCM in unprocessed feces stored at ambient temperature remained stable for up to 8 h of storage after which concentrations of both E1C and GCM changed significantly compared to controls extracted at time 0. Long-term storage in 80% MeOH at ambient temperature affected hormone concentrations significantly with concentrations of both E1C and GCM increasing after 6 and 4 months of storage, respectively. Drying fecal samples using a conventional oven at 50, 70, and 90 °C did not affect the E1C concentrations, but led to a significant decline for GCM concentrations in samples dried at 90 °C. Different fecal preservation techniques and extraction solvents provided similar results for both E1C and GCM concentrations. Our results confirm previous studies that prior to application of fecal hormone analysis in a new species, several preservation parameters should be evaluated for their effects on hormone metabolite stability. The results also provide several options for fecal preservation, extraction, and storage methods that can be selected depending on the condition of the field site and laboratory.

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Acknowledgements

We are grateful to all team members of the Macaca Nigra Project at Tangkoko for supporting data collection. We thank Andrea Heistermann for her valuable help during hormone training and analysis in the laboratory of endocrinology at the German Primate Centre, Göttingen, Germany, and to the head of the laboratory of hormone analysis at the Faculty of Veterinary Medicine, Bogor Agricultural University for hormone measurement. We gratefully acknowledge the permission granted by the Directorate General of Forest Protection and Nature Conservation (PHKA) and the Department of the Conservation of Natural Resources (BKSDA) in Manado and Indonesian Institute of Sciences (LIPI) to conduct research in the Tangkoko Batuangus Nature Reserve. This research was funded by the SEAMEO BIOTROP, Bogor Indonesia (grant number:060.20/PSRP/SPK-PNLT/III/2014), and Ministry of Research, Technology and Higher Education, Indonesia (Grant Number: 105/SP2H/LT/DPRM/IV/2017). The authors declare that they have no competing interests. All applicable international, national, and institutional guidelines for the care and use of animals were followed.

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Correspondence to Gholib Gholib.

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Gholib, G., Heistermann, M., Agil, M. et al. Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques. Primates 59, 281–292 (2018). https://doi.org/10.1007/s10329-018-0653-z

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  • DOI: https://doi.org/10.1007/s10329-018-0653-z

Keywords

  • Crested macaques
  • Fecal estrogen
  • Fecal glucocorticoid
  • Fecal preservation
  • Fecal extraction