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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

Abstract

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia's iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Introduction

Australian ecosystems sustain human life through the provision of services including food and fiber among many other dynamic interactions1. Ironically, it is human activity that operates as the dominant driver of ecosystem disruption through biodiversity change2. Habitat fragmentation, known as the process of dividing large continuous habitats into small patches of land, isolated from each other, is the major anthropogenic biodiversity change threatening Australian ecosystems2. Habitat fragmentation modifies the structure and diversity of species composition in any given area, thus reducing the ar....

Protocol

This project was performed under strict animal and human care guidelines. Animal ethics was granted by Western Sydney University (A12373). Additionally, a lab risk assessment and biosafety and radiation form were submitted and accepted by Western Sydney University to safely undertake this research (B12366).

NOTE: Koala fur samples for this project were obtained from the Adelaide Koala and Wildlife Hospital, located at 282 Anzac Highway, Plympton South Australia. Fur was taken from one koala wh.......

Representative Results

Assay detection of hormone metabolites of interest is determined using parallelism. Using a parallelism curve, the 50% binding point also determines the sample dilution factor on the standard curve (Figure 1). As shown in the parallelism graph (Figure 1), the 100% ethanol and 100% Isopropanol extracts did not provide parallel displacement against the cortisol standard. However, the 100% methanol extract provided parallel displacement against the cortisol standard. Dried extracts were run.......

Discussion

There are a number of studies that use a range of techniques to detect cortisol in mammalian fur. This study presents results for the detection of cortisol in fur collected from a wild koala exposed to current anthropogenic stress. This ground-breaking study used fur to test which of the three commonly used solvents are best at extracting cortisol, a measure of chronic stress, from koala fur. Results showed that 100% methanol was the recommended solvent for cortisol extraction in this type of mammalian fur.

Acknowledgements

This work was supported through start-up research funding for Edward Narayan through the Western Sydney University, School of Science and Health. The authors thank Jack Nakhoul for assistance with sample processing.

....

Materials

NameCompanyCatalog NumberComments
Centrifuge Tubesn/an/a1.5 mL
Chrome Steel Beadsn/an/a3.2 mm x 3
Cortisol KitArbor AssaysK003-H1WManufactured in Michigan USA
DetectX Cortisol Enzyme Immunoassay KitArbor AssaysK003-H5Used first-time for cortisol testing in koala fur
Ethanoln/an/aHPLC Grade
Isopropanoln/an/aHPLC Grade
Methanoln/an/aHPLC Grade
Micro Pipetten/an/an/a
Micro Precision Sieven/an/a0.5 mm
Microplate ReaderBio Radin/an/a
Microplate WasherBio Radin/an/a
Orbital ShakerBio Linen/an/a
Plastic Weighing Boatn/an/an/a
Plate Sealern/an/an/a
Precision Balancen/an/an/a
Vortex MixerEppendorfn/an/a

References

  1. Sandhu, H. S., Crossman, N. D., Smith, F. P. Ecosystem services and Australian agricultural enterprises. Ecological Economics. 74, 19-26 (2012).
  2. Martinez-Ramos, M., Ortiz-Rodriguez, I. A., Pinero, D., Dirzo, R., Sarukhan, J.

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