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 which had been admitted to the hospital and euthanized due to their severe injuries. The deceased koala had been stored in a freezer within a body bag soon after death. After removing the deceased koala from the body bag, 1.2 g of fur was shaved from the nape of the neck using standard animal clippers. The fur was shaved as close as possible to the skin, so as to ensure the skin was not cut. Once shaved, the deceased koala was put back into the body bag and placed in the freezer. The fur was then placed in a pouch made of aluminum foil and stored below -20 &#176;C. In transit, the fur was kept at ambient temperature, and on arrival to the laboratory, the fur was stored at -80 &#176;C.
1. Koala fur cortisol extraction
<ol>
	<li>Remove the fur from storage at -80 &#176;C and allow time to thaw.</li>
	<li>Weigh the fur on a laboratory analytical precision balance.</li>
	<li>Place 60 mg of the fur into a pre-weighed and labelled 1.5 mL centrifuge tube and repeat until 18 tubes are filled. 
	NOTE: 18 fur sub-samples were used for this validation study.</li>
	<li>Add 1 mL of 100% high-performance liquid chromatography (HPLC) grade isopropanol to each tube using a pipette.</li>
	<li>Vortex samples for 30 s.</li>
	<li>Strain each sample using a 0.5 mm micro precision sieve so as to achieve separation of liquid and fur.</li>
	<li>Discard the liquid into a waste container.</li>
	<li>Place each fur sample into a labelled plastic weighing boat, then place into a vacuum desiccator, leave the fur to dry for 3 days.</li>
	<li>Once completely dry, place each sample into a labelled 1.5 mL microcentrifuge tube.</li>
	<li>Place each sample in a bead mill with 3 chrome steel beads (3.2 mm) and pulverize for 2 min at 30 shakes per second.</li>
	<li>Pipette 1.5 mL of the first extraction technique (100% analytical grade ethanol) into 6 1.5 mL microcentrifuge tubes containing the fur sample.</li>
	<li>Perform the same for 100% analytical grade methanol and 100% analytical grade isopropanol until eighteen 1.5 mL microcentrifuge tubes are filled.</li>
	<li>Cap each 1.5 mL microcentrifuge tube and incubate at room temperature (RT) with constant pulsating using a shaker for 3 h.</li>
	<li>Remove and strain samples using a 0.5 mm micro precision sieve.</li>
	<li>Transfer the liquid into a new, labelled 1.5 mL microcentrifuge tube with a pipette while ensuring that the fur is discarded appropriately.</li>
	<li>Completely dry solvent extract under a stream of N2 vapor under a fume-cupboard.</li>
	<li>Reconstitute the dried sample extract using 400 &#181;L of assay buffer (composition provided in the commercial cortisol kit; see Table of Materials) and 100 &#181;L of 100% analytical grade ethanol. 
	NOTE: Sample extracts can be stored at -80 .</li>
</ol>
2. Internal controls
<ol>
	<li>To make controls, make a pool of extracted samples with high hormone levels. To make this pool, select samples from animals with known exposure to stressor. For example, select samples from koalas that have been rescued from environmental trauma as they will generally display high cortisol hormone levels6.
	<ol>
		<li>To make the extract pool, take 20 &#181;L of extract from each sample (n = 10) until a total volume of 200 &#181;L is obtained. The extract pool can be stored at -80 C until assays. Run the pool in each assay as low or high internal controls (see step 2.2).</li>
	</ol>
	</li>
	<li>For the assay, use the pool to make stocks for low and high controls that bind at 70% (C1) and 30% (C2), respectively. Obtain the dilution factor for the 30% and 70% binding points from the parallelism graph for the extract against the cortisol standard (Figure 1). Use the assay buffer for dilution of sample pool. For example, use 60 &#181;L of the pool extract and 60 &#181;L of assay buffer for 1:2 dilution. 
	NOTE: For the methanol extract, the 30% binding point was at neat while 70% binding point was approximately 1:2 as per Figure 1. Thus, these provided the dilution factor for the internal controls (C1 and C2 respectively) for running within the assay.</li>
</ol>
3. Cortisol analysis in koala fur extracts
<ol>
	<li>Use a commercial cortisol kit (Table of Materials) and set up the 96 well strip plate including the samples, controls, cortisol standards, non-specific binding wells, and maximum binding wells following the supplier&#8217;s instructions. Use the plate layout sheet provided in the kit booklet to list the positions of samples, controls, and standards on the plate map. 
	NOTE: It is recommended that all samples, controls, and standards are run in duplicate to allow accuracy of results.</li>
	<li>Prepare samples. Follow the fur hormone extraction (section 1) to obtain 100% methanol extracted koala fur.</li>
	<li>Prepare reagents. Follow the procedure described in the commercial cortisol kit to prepare the reagents including (1) assay buffer, (2) wash Buffer, and (3) standards (compositions provided in the cortisol kit, Table of Materials).</li>
	<li>As per the instructions provided in the cortisol kit, pipet 50 &#181;L of samples or standards into wells in the plate. Pipet 75 &#181;L and 50 &#181;L of assay buffer into the non-specific binding (NSB) wells and the maximum binding (B0 or zero standard) wells, respectively.</li>
	<li>Add 25 &#181;L of the cortisol conjugate to each well using a repeater pipet. Then pipet 25 &#181;L of the cortisol antibody into each well, except the NSB wells. Gently tap the sides of the plate to ensure that the reagents are well mixed.</li>
	<li>Cover the plate with the plate sealer and shake at room temperature for 1 h (at slow speed) using an orbital shaker.</li>
	<li>Remove the plate sealer and aspirate the well plate by washing each well with 300 &#181;L of wash buffer 4 times.</li>
	<li>Dry the plate by tapping the plate on clean absorbent towels.</li>
	<li>Pipette 100 &#181;L of tetramethylbenzadine (TMB) substrate (composition provided in the cortisol kit, Table of Materials) to each well.</li>
	<li>Place the plate sealer on the well plate and incubate at RT for 30 min.</li>
	<li>Pipette 50 &#181;L of stop solution to each well.</li>
	<li>Place the well plate in a plate reader capable of reading 450 nm.</li>
	<li>To calculate the final hormone concentration, derive the final fur cortisol concentration in ng/mg of sample by multiplying the pg/mL hormone concentration with the final extract volume (0.5 mL) and dividing by the fur sample mass (60 mg).</li>
</ol>

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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.
Ethanol, methanol and isopropanol are all primary alcohols that are bonded by hydrogen molecules and are commonly used as solvents in hormone extraction experiments28. Generally, polar substances dissolve best in other polar substances, whereas non-polar substances dissolve best in other non-polar substances. The alcohol group containing methanol is very polar, whereas the alcohol group containing isopropanol is very non-polar. Due to its molecular build, alcohol group containing ethanol has the advantage of being both a polar and non-polar solvent. Steroid hormones such as cortisol are considered non-polar, meaning that cortisol should have a strong binding association with polar solvents.
For a more comprehensive analysis of extraction solvents used to assess physiological stress in koala fur, future research projects should attempt identical methods in that order as described in Figure 3. Similar studies have historically performed the wash before grinding22, so as to ensure there is no unintended sweat and/or sebum derived cortisol deposited into the fur sample. Furthermore, it is important that measuring cortisol alone cannot guarantee a complete indication of chronic stress. Hair cortisol readings are a valuable tool when attempting to understand physiological stress experienced by an animal, but elevated HPA activity can occur under a variety of conditions including physical exercise, metabolic abnormalities and the presence of infectious disease22. Other important factors that should be taken into consideration to main integrity of hormone data include the following. (1) Acceptable level of random error - the coefficients of variation obtained from internal controls (CV1 and CV2) should be averaged to &#60;15% for all assays. (2) Random error within sample assay&#8213;duplicate samples run on each plate should have a CV% of &#60;15%; otherwise the sample will need to be re-run. (3) Assay detection limit - concentration of hormone quantified within each assay should be within the assay detection limit (between readings for highest dilution and neat standard); otherwise samples may require further dilution (if levels detected for samples are greater than the concentration of neat standard) or may not be analyzed within the assay (if levels detected for samples are less than the concentration of the highest diluted standard). (4) Assay sensitivity - this can be affected by background reading (non-specific binding), therefore it is important to maintain the highest level of quality assurance for the assay (e.g., equipment such as plate washer and plate reader must be serviced regularly). (5) Sample extract drying - this step could result in potential cross-contamination or loss of samples. It is recommended that samples be dried under steam of N2 gas individually and to replace the Pasteur pipette used for extraction between each sample.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59216/59216fig3.jpg" /> 
Figure 3: Conceptual flow diagram showing the key steps involved in the koala fur cortisol enzyme-immunoassay (EIA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59216/59216fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The procedure outlined in this study (Figure 3) is one that can be easily replicated as it is relatively easy to perform, step-by-step methodology which incorporates readily available chemicals, reagents, and supplies with equipment that is likely to be found in a standard analytical laboratory. The application of this study enables a non-invasive technique to be used to assess physiological stress in both wild and captive koalas.&#160;

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><tbody><tr><td>Centrifuge Tubes</td><td>n/a</td><td>n/a</td><td>1.5 mL</td></tr><tr><td>Chrome Steel Beads</td><td>n/a</td><td>n/a</td><td>3.2 mm x 3</td></tr><tr><td>Cortisol Kit</td><td>Arbor Assays</td><td>K003-H1W</td><td>Manufactured in Michigan USA</td></tr><tr><td>DetectX Cortisol Enzyme Immunoassay Kit</td><td>Arbor Assays</td><td>K003-H5</td><td>Used first-time for cortisol testing in koala fur</td></tr><tr><td>Ethanol</td><td>n/a</td><td>n/a</td><td>HPLC Grade</td></tr><tr><td>Isopropanol</td><td>n/a</td><td>n/a</td><td>HPLC Grade</td></tr><tr><td>Methanol</td><td>n/a</td><td>n/a</td><td>HPLC Grade</td></tr><tr><td>Micro Pipette</td><td>n/a</td><td>n/a</td><td>n/a</td></tr><tr><td>Micro Precision Sieve</td><td>n/a</td><td>n/a</td><td>0.5 mm</td></tr><tr><td>Microplate Reader</td><td>Bio Radi</td><td>n/a</td><td>n/a</td></tr><tr><td>Microplate Washer</td><td>Bio Radi</td><td>n/a</td><td>n/a</td></tr><tr><td>Orbital Shaker</td><td>Bio Line</td><td>n/a</td><td>n/a</td></tr><tr><td>Plastic Weighing Boat</td><td>n/a</td><td>n/a</td><td>n/a</td></tr><tr><td>Plate Sealer</td><td>n/a</td><td>n/a</td><td>n/a</td></tr><tr><td>Precision Balance</td><td>n/a</td><td>n/a</td><td>n/a</td></tr><tr><td>Vortex Mixer</td><td>Eppendorf</td><td>n/a</td><td>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 neat through dilution in assay buffer (100 &#181;L of 100% ethanol and 400 &#181;L of assay buffer).
Intra- (within) and inter- (between) assay coefficients of variation (CV) were determined from high- (approximately 70%) and low- (approximately 30%) binding sample extracts run in all the assays. Based on the parallelism graph (Figure 1), the 30% (low) binding internal controls were neat koala extract pool while the 70% (high) binding internal controls were 1:2 diluted koala extract pool. CV% for internal high and low internal controls were &#60;15%.
Error margin within the assay can be determined using quality control including the intra- and inter- assay coefficients of variation, which should be &#60;15%. Assay sensitivity was calculated as the value 2 standard deviations from the mean response of the blank (zero binding) samples, and expressed as 81.26 pg/well.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59216/59216fig1.jpg" /> 
Figure 1: Parallelism of pooled koala fur extracted using 3 different solvents (100% ethanol, 100% isopropanol or 100% methanol) against cortisol standard curve under a cortisol enzyme-immunoassay. B/TB is the percentage of binding over total binding. The serial dilution factor (e.g., 1:2X mean dilution factor of 2) has been provided together with the concentration of each standard. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59216/59216fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Secondly, the association between each solvent extract and cortisol standard was determined using a regression plot (Figure 2). As shown in Figure 2, the 100% methanol extract provided the best line of regression with the highest R2 value compared to the 100% ethanol and 100% isopropanol extracts.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59216/59216fig2.jpg" /> 
Figure 2: Regression plots for percentage binding of the cortisol standard against each of the 3 solvents (ethanol, methanol, and isopropanol) used to extract koala fur. The R2 value was obtained from the line of best fit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59216/59216fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Furthermore, sub-set of koala fur extracted using each of the three solvents were assayed and the results are provided in Table 1 below. As shown in Table 1, the observed concentration of cortisol standard was within the range of 2879.61-125.70 pg/well. Neither the ethanol or isopropanol extraction method could achieve consistency in the result as the fur extract concentrations obtained using either of the methods resulted in very high min-max range of hormone concentrations (see Table 1 numbers marked in red), which were beyond the detection limit of the cortisol assay. However, the methanol extracts resulted in cortisol concentrations within the range of the cortisol standard (as shown in bold black numbers in Table 1). Furthermore, the concentrations of fur cortisol detected using methanol extraction method was highly consistent compared to the results obtained using the other two methods (see Table 1). Thus, we accept the null hypothesis that methanol is the most suitable solvent for koala fur hormone extraction compared to ethanol and isopropanol.
<img alt="Table 1" src="/files/ftp_upload/59216/59216table1.jpg" /> 
Table 1: The cortisol concentration (ng/mg) for koala fur (n = 18) extracted using 3 different solvents (ethanol, isopropanol or methanol) and run against cortisol standard curve under a cortisol enzyme-immunoassay. Bold red numbers show inconsistent concentrations for ethanol and isopropanol extracts which were beyond the assay range (pg/well). Bold black numbers show the concentrations for fur cortisol extracted using methanol which fell within the range of the cortisol standards (pg/well). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59216/59216table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

cortisol-measurement-in-koala-phascolarctos-cinereus-fur

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.6K Views.  Western Sydney University. 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.

Video: Cortisol Measurement in Koala Phascolarctos cinereus Fur

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 the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.

This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) &plusmn; capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from 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 skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

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

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Environment

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report formats, a growing number of studies now incorporate biological stress-related hormones and catalysts, such as cortisol and &#945;-amylase, to measure levels of stress. Presented here is a protocol to examine the effects on levels of biophysical and psychological stress following visitation to three different locations with differing levels of nature. Biophysical and self-reported psychological stress levels are measured immediately upon entering the selected locations and just prior to the visitors leaving the site. Using a &#34;drool&#34;&#160;method, the biophysical measure consists of 1-2 mL samples of saliva provided by study subjects upon entry to one of three study locations. As prescribed by extant literature, the saliva is collected within a 45 minute time frame following the end of the visitor&#8217;s engagement at the location. Following saliva collection, the samples are labeled and transported to a biological lab. Cortisol is the biophysical variable of interest in this study and measured using an ELISA process with a TECAN plate reader. To measure self-reported stress, the Perceived Stress Questionnaire (PSQ), which reports levels of worry, tension, joy, and perceived demands. Data are collected at all three sites in the late afternoon through early evening. When compared across all three settings, stress levels, as measured by both the biological markers and self-reports, are significantly lower after visitation to the most natural setting.

The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, &#945;-amylase, and a psychological self-report instrument.&#160;

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report ...

Behavior

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Neuroscience

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

Medicine

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Biology

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...