Name: cortisol measurement in koala (phascolarctos cinereus) fur
Uploaded: 2019-08-23T12:30:00
Description: Watch this Scientific Journal Video about Cortisol Measurement in Koala Phascolarctos cinereus Fur at JoVE.com

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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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.
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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 ...

Research

JoVE Journal

Biochemistry

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Measurement of Fatty Acid ÃƒÅ½Ã‚Â²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Time-resolved FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...