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.

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...

<ol>
	<li>Sandhu, H. S., Crossman, N. D., Smith, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecosystem+services+and+Australian+agricultural+enterprises.">Ecosystem services and Australian agricultural enterprises.</a> Ecological Economics. 74, 19-26 (2012).</li><li>Martinez-Ramos, M., Ortiz-Rodriguez, I. A., Pinero, D., Dirzo, R., Sarukhan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthropogenic+disturbances+jeopardize+biodiversity+conservation+within+tropical+rainforest+reserves.">Anthropogenic disturbances jeopardize biodiversity conservation within tropical rainforest reserves.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (19), 5323-5328 (2016).</li><li>Aukema, J. E., Pricope, N. G., Husak, G. 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J., Williams, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+dynamics+of+physiological+impacts+of+environmental+stressors+on+Australian+marsupials,+focus+on+the+koala+(Phascolarctos+cinereus).">Understanding the dynamics of physiological impacts of environmental stressors on Australian marsupials, focus on the koala (Phascolarctos cinereus).</a> BMC Zoology. 1 (1), (2016).</li><li>Woinarski, J., Burbidge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phascolarctos+cinereus.">Phascolarctos cinereus.</a> The IUCN Red List of Threatened Species 2016. , (2016).</li><li>Gonzalez-Astudillo, V., Allavena, R., McKinnon, A., Larkin, R., Henning, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decline+causes+of+Koalas+in+South+East+Queensland,+Australia:+a+17-year+retrospective+study+of+mortality+and+morbidity.">Decline causes of Koalas in South East Queensland, Australia: a 17-year retrospective study of mortality and morbidity.</a> Scientific Reports. 7, 42587 (2017).</li><li>Hing, S., Narayan, E. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticosteroid+concentrations+in+feces+and+hair+of+captive+caribou+and+reindeer+following+adrenocorticotropic+hormone+challenge.">Glucocorticosteroid concentrations in feces and hair of captive caribou and reindeer following adrenocorticotropic hormone challenge.</a> General and Comparative Endocrinology. 172 (3), 382-391 (2011).</li><li>Macbeth, B. J., Cattet, M. R. L., Stenhouse, G. B., Gibeau, M. L., Janz, D. 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The authors have nothing to disclose.

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.
<p class=...

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 area of habitat necessary for these species to maintain viable populations2. The result of this is increased competition between species for resources including food, fuel, fiber, and water3. The destruction of Australian ecosystems through biodiversity change is having catastrophic consequences on many Australian native species1.
Australia&#39;s most iconic marsupial species, the koala (Phascolarctos cinereus), depends on Australian ecosystems remaining healthy for their survival4. The introduction of the European Settlement caused a rapid decline in Australian populations of koalas, as they were slaughtered for their pelts in pursuit of profit in a large export trade5. This practice was banned in the 1980&#39;s and populations of koalas were then able to stabilize5. However, exponential growth of the human population has resulted in this species competing for much of their habitat, and their survival is again under threat6. According to the International Union for the Conservation of Nature (IUCN), all populations of Australian koalas are listed as vulnerable to extinction with a decreasing population trend7. This listing is attributed to the uncertainty around relevant population parameters and marked variation in population trends for this species7. As the most iconic and endemic animals, koalas largely benefit the Australian economy through tourism (NSW Office of Environment and Heritage 2018). An estimation suggests that koala related tourism has generated approximately 9,000 jobs and contributes between $1.1 and $2.5 billion to the economy (NSW Office of Environment and Heritage 2018). The removal of any one species has the potential to be catastrophic, and can be seen in the steady decline of native Australian wildlife6. Additionally, the Australia economy will feel the ramifications if populations of Australian koalas continue to decline at the rate they are6.
It is suggested that prevalence of death and disease in response to habitat fragmentation is the result of chronic stress8. Already, twenty-four marsupial species have been declared extinct in Australia due to habitat fragmentation, with koalas following a similar trend8. The complexity of habitat fragmentation and biological systems is synergistic but can be unpacked through analysis of the stress response6. Generally, any disturbance in an animals natural surroundings activates a complex cascade of neurohormonal events, known as a &#39;fight or flight&#39;&#160;response9,10. This response to stress is a process that begins in the brain where the hypothalamic-pituitary-adrenal (HPA) axis is activated11. A component of the brain called the hypothalamus releases corticotrophin-releasing hormone (CRH), which then signals the anterior pituitary to release adrenocorticotrophic hormone (ACTH)11. This in turn stimulates the glucocorticoid secretion from the adrenal medulla. The body circulates glucocorticoids through the blood, which diverts the storage of glucose from glycogen and mobilizes glucose from stored glycogen11. This cascade of neurohormonal events is the response used by the animal to deal with unpredictable stimuli11. However, when glucocorticoids are being released and remain elevated for a prolonged period of time, the animal is considered to be experiencing chronic stress12,13. This process involves diverting energy away from other corporal bodily functions, as it is needed for ongoing glucocorticoid production13. As a result, chronic stress can prohibit growth, reproduction and immunity, all being key fitness traits required for survival14.
Measuring an animal&#39;s glucocorticoid production is a common indicator used to determine whether or not the animal is experiencing physiological stress15. To do so, glucocorticoids can be measured in blood plasma, serum, saliva, urine or faeces16. However, evidence suggests that hair is a much more effective indicator of chronic stress, as opposed to the aforementioned16. This is because hair is thought to incorporate blood-borne hormones during its growth phase; it is relatively stable; and any cortisol detected in hair reflects physiological stress experienced over the period of hair growth, which can be weeks through to months16. Furthermore, any collection of cortisol should be non-invasive in order to minimise the stress associated with capture and handling16. However, any stress experienced during this event would not impact glucocorticoid levels in hair16. There have been many studies that explore the proficiency of using hair to measure long-term stress in a number of animals, and include studies on reindeer, grizzly bears, rhesus monkeys, muskoxen, and brown bears17,18,19,20,21. Hair cortisol is usually extracted by first washing the sample to ensure sweat and sebum-derived cortisol deposited on the surface of the hair is not co-extracted with cortisol and then pulverizing the sample in a bead-beater22. After washing, the sample needs to be dried to ensure complete evaporation22. Finally, using a solvent, the sample can be extracted and reconstituted to facilitate the assay of cortisol22. The most common solvent used to extract cortisol from fur is methanol21,23; however, there are some studies that use ethanol and isopropanol in their cortisol extraction techniques. For example, a study that used ethanol was successful for extracting cortisol from human amniotic fluid24. Additionally, a study that used isopropanol was successful for extracting cortisol from human hair and nails25,26. For this reason, this study tested all three solvents (methanol, ethanol, and isopropanol) to determine which was the most successful for extraction of cortisol from samples of koala fur.
The primary objective of this study was to use current techniques to validate an optimal hormone extraction technique to be used as a non-invasive measure of cortisol from koala fur. This was achieved by testing three extraction solvents (methanol, ethanol, and isopropanol). We hypothesized that methanol will be the optimal solvent used for extracting cortisol from koala fur because it is the recommended solvent of extraction by Arbor assay cortisol kits27.&#160;&#160;&#160;&#160;&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:616px;" width="616">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Centrifuge Tubes</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Chrome Steel Beads</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">3.2 mm x 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cortisol Kit</td>
			<td style="width:72px;">Arbor Assays</td>
			<td style="width:119px;">K003-H1W</td>
			<td style="width:211px;">Manufactured in Michigan USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DetectX Cortisol Enzyme Immunoassay Kit</td>
			<td style="width:72px;">Arbor Assays</td>
			<td style="width:119px;">K003-H5</td>
			<td style="width:211px;">Used first-time for cortisol testing in koala fur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">HPLC Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isopropanol</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">HPLC Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Methanol</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">HPLC Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Micro Pipette</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Micro Precision Sieve</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">0.5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microplate Reader</td>
			<td style="width:72px;">Bio Radi</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microplate Washer</td>
			<td style="width:72px;">Bio Radi</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Orbital Shaker</td>
			<td style="width:72px;">Bio Line</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Plastic Weighing Boat</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Plate Sealer</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Precision Balance</td>
			<td style="width:72px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Vortex Mixer</td>
			<td style="width:72px;">Eppendorf</td>
			<td style="width:119px;">n/a</td>
			<td style="width:211px;">n/a</td>
		</tr>
	</tbody>
</table>

cortisol measurement in koala (phascolarctos cinereus) fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;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.

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...

Watch this Scientific Journal Video about Cortisol Measurement in Koala Phascolarctos cinereus Fur at JoVE.com

Cortisol Measurement in Koala Phascolarctos cinereus Fur

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.

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

This is a novel method for the assessment of the glucocorticoid hormone, cortisol, from koala fur. It provides a novel biomarker for the assessment of chronic stress in koalas. The main advantage of this technique is that it is a highly sensitive and reproducible method with a fairly quick turnaround time between sample preparation and hormone assay.This technique allows for the quantitative assessment of chronic stress in koalas, which is a highly useful clinical management tool. Helping me with this procedure is Dylan Fox, a grad student from my laboratory. To begin this procedure retrieve the fur from storage at minus 80 degrees Celsius and let it thaw at room temperature.Then, weigh the fur on a laboratory analytical precision balance. Place 60 milligrams of the fur into a pre-weighed and labeled 1.5-milliliter centrifuge tube and repeat this until 18 tubes are filled. Using a pipette, add one milliliter of 100%HPLC grade isopropanol to each tube.Vortex the samples for 30 seconds. Strain each sample with a 0.5-milliliter micro-precision sieve so as to achieve separation of liquid and fur, and discard the liquid into a waste container. Place each fur sample into a labeled plastic weighing boat.Then place the weighing boats into a vacuum desiccator and leave for three days to let the fur dry. Once the fur is completely dry, place each sample into a labeled 1.5-milliliter microcentrifuge tube. Place each sample in a bead mill with three chrome steel beads and pulverize for two minutes at 30 shakes per second.Repeat this transfer with 100%analytical grade methanol and 100%analytical grade isopropanol until 18 total tubes have been filled. After this, use a pipette to transfer one milliliter of the first extraction technique into six 1.5-milliliter centrifuge tubes containing the fur sample. Cap each tube, and use a shaker to incubate them at room temperature with constant pulsating for at least 12 hours.Then strain the samples with a 0.5-milliliter micro-precision sieve. Use a pipette to transfer the liquid into a new labeled 1.5-milliliter microcentrifuge tube, while ensuring that the fur is discarded appropriately. In a fume hood, completely try the solvent extract under a stream of nitrogen vapor.Then, reconstitute the dried sample with 400 microliters of assay buffer and 100 microliters of 100%analytical grade ethanol. To make the controls, first select samples from animals with known exposure to the stressor. To make an extract pool, take 20 microliters of extract from each sample until a total volume of 200 microliters is obtained.Store the extract pool at minus 80 degrees Celsius until ready to run assays. When ready, obtain the dilution factor for the 30%and 70%binding points from the parallelism graph for the extract against the cortisol standard, and use assay buffer to dilute the sample pool appropriately to create the high and low controls. Using a commercial cortisol kit set up a 96-well strip plate including the samples, controls, cortisol standards, nonspecific-binding wells, and maximum-binding wells according to the supplier's instructions.Use the plate layout sheet included in the kit booklet to list the positions of the samples, controls and standards. Next, follow the fur hormone extraction process described previously to obtain 100%methanol-extracted koala fur. Prepare the cortisol kit's reagents according to manufacturer's instructions.Pipette 50 microliters of samples or standards into the wells of the plate. Pipette 75 microliters of assay buffer into the nonspecific-binding wells, and 50 microliters of assay buffer into the maximum-binding wells. Using a repeater pipette add 25 microliters of the cortisol conjugate to each well.Then, pipette 25 microliters of the cortisol antibody into each well, except the nonspecific-binding wells. Gently tap the sides of the plate to ensure that the reagents are well mixed. Cover the plate with the plate sealer, and use an orbital shaker to shake at room temperature for one hour.After this, remove the plate seal, and aspirate the well plate by washing each well with 300 microliters of wash buffer four times. Then dry the plate by tapping it on clean absorbent towels. Pipette 100 microliters of tetramethylbenzidine substrate to each well.Place the plate sealer on the well plate, and incubate at room temperature for 30 minutes. After this, pipette 50 microliters of stop solution into each well. Transfer the plate into a plate reader capable to reading 450 nanometers, and calculate the final hormone concentration as outlined in the text protocol.In this study, the essay detection of hormone metabolites of interest is determined using parallelism. On the parallelism curve, the 50%binding point determines the sample dilution factor on the standard curve. As can be seen, the 100%ethanol and 100%isopropanol extracts did not provide parallel displacement against the cortisol standard.However, the 100%methanol extract does provide parallel displacement against the cortisol standard. Intra-and inter-assay coefficients of variation are determined from high-and low-binding sample extracts run in all the assays. The low-binding internal controls are seen to be neat koala extract pool, while the high-binding internal controls are seen to be one-to-two diluted koala extract pool.The association between each solvent extract and cortisol standard are then determined using a regression plot. The 100%methanol extract provides the best line of regression with the highest R-squared value compared to the 100%ethanol and 100%isopropanol extracts. It is important to remember that the sample wash step must be performed before grinding the samples to avoid the loss of steroid hormones.Following this procedure, we can measure other steroidal hormones, such as reproductive hormones to look at the interactions between stress and reproduction in koalas. This technique provides a new tool for the emerging field of conservation physiology, enabling the non-invasive assessment of cortisol in koala fur. The commercial cortisol kit provides reagents in minute quantity.However, it is important to wear personal protective equipment to minimize injury to oneself whilst conducting this laboratory procedure.

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Research

JoVE Journal

Biochemistry

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