Name: fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids
Uploaded: 2016-04-25T13:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

Ethics statement: procedures involving field sampling and animal subjects have been approved by the School of Animal, Rural and Environmental Science (ARES) at Nottingham Trent University.
1. Collection of Fecal Samples
NOTE: Gloves should be worn when handling fecal samples and methanol. If there is a strong suspicion that an animal could be suffering from a zoonotic disease, protective clothing such as a lab coat should also be worn.
<ol>
	<li>Collect the fecal samples as soon as possible (within min to a few hr) following defecation and place them into a sample bag. Take three to five subsamples of any defecated sample with a total weight of approximately 10 g. Label the bag with the time and date of collection, individual ID, and freeze the sample at -20 &#176;C until analysis.</li>
	<li>To minimize error, freeze all samples at -20 &#176;C until the study is complete then gather the samples together for analysis as a single batch.</li>
</ol>
2. Fecal Hormone Extraction
<ol>
	<li>Leave the fecal samples to thaw at RT.</li>
	<li>Ensure that the samples are homogenized by manually mixing each sample in its own sample bag.</li>
	<li>For each sample weigh 0.50 (&#177; 0.003 g) of fecal material on a microbalance in a weigh boat then transfer the feces to a 8 ml glass vial. Use a new weigh boat for each sample and clean the tweezers with 30% methanol between each sample.</li>
	<li>Combine the 0.5 g of fecal material with 5 ml of 90% methanol: water, and shake O/N&#160;on an orbital shaker at RT.</li>
	<li>The following morning, vortex the sample and then centrifuge for 20 min at 600 g.</li>
	<li>Decant the methanol fraction into a 16 x 125 mm2 glass tube and place in a rack in a water bath set to a temperature of 37 &#176;C. Evaporate to dryness using air in a fume cupboard. Rinse the extraction vial and place the remaining fecal pellet into a clinical waste bag for disposal.</li>
	<li>Re-suspend the fecal extracts in 100%, 1 ml methanol and transfer to a 12 x 75 mm2 polypropylene tube. Immediately cap and store at -20 &#176;C until analysis.</li>
</ol>
3. Hormone Analysis
<ol>
	<li>Carry out the analysis with an enzyme linked-immunoassay (EIA) using polyclonal corticosterone CJM006 antiserum. 
	NOTE: A second EIA was tested (Cortisol R4866 antiserum, but this failed to achieve the biochemical validation step (See Section 4.1). The Polyclonal corticosterone CJM006 antiserum and Cortisol R4866 antiserum and the respective HRP&#39;s are supplied by CJ Munro, University of California, Davis, CA.</li>
	<li>Dilute the antiserum at 1:15,000 in coating buffer (0.05 M NaHCO3, pH 9.6) using a repetitive pipette and a 50 &#181;l pipette tip, load a 96-well microtitre plate with 50 &#181;l/well. Cover the plate with a plate sealer and incubate O/N at 4 &#176;C. Leave two wells uncoated to represent the non-specific binding wells (NSB).</li>
	<li>Immediately before use wash the microtitre plates five times using an automated microtitre plate washer (wash solution; 0.15 M NaCl, 0.05% Tween 20).</li>
	<li>Load the plates with 50 &#181;l per well for the NSB&#39;s and &#39;zero&#39; wells [EIA buffer (0.1 M NaPO4, 0.149 M NaCl, 0.1% bovine serum albumin, pH 7.0) only], standards (corticosterone, 3.9 - 1,000 pg/well) or fecal extract samples (appropriately diluted at 1:20 EIA buffer, see section 4.1). Standards and zeros should be run in triplicate and quadruplicate, respectively. Diluted fecal extract samples and NSB&#39;s should be run in duplicate.</li>
	<li>Follow this immediately with addition of 50 &#181;l/well of the label horseradish peroxidase conjugate&#7547; diluted in EIA buffer to 1:70,000.</li>
	<li>Incubate the microtitre plates in the dark for 2 hr at RT.</li>
	<li>Wash the microtitre plates with wash solution 5 times and incubate the plates at RT in the dark with 100 &#181;l/well of RT substrate [0.4 mM 2,2&#8242;-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt, 1.6 mM H2O2, 0.05 M citrate, pH 4.0]. Prepare the substrate immediately prior to use.</li>
	<li>Use a spectrophometer at a wavelength of 405 nm to read the microtitre plates. Incubation of plates is considered complete when the optical density of the zero wells reaches 0.8 to 1.0.</li>
	<li>To insure repeatability, use controls [EIA standards or a biological sample diluted in EIA buffer to bind at approximately 30% (EIA standard), 50% (a biological sample e.g., pool of domestic horse fecal extract) and 70% (EIA standard)] to demonstrate an intra- and inter-assay coefficients of variation of &#60; 10% and &#60; 15% respectively.</li>
</ol>
4. Enzyme Linked-immunoassay: Validation for the Study Species and Sex
<ol>
	<li>Biochemical Validation (Parallelism)
	<ol>
		<li>Using EIA buffer, perform a serial dilution on pooled fecal extract, ideally taken from suspected high and low hormone concentration samples from several individuals (range: neat, 1:2 to 1:8192) and run on the EIA as described in section 3. 
		NOTE: A parallelism is considered achieved when the serial dilutions of fecal extracts yield a displacement curve parallel to the standard curve and linear regression results demonstrate R2 &#62; 0.90, a slope &#62; 0.50 and p &#60; 0.05. The serial diluted fecal extract that binds around 50% on the standard curve should be considered the ideal dilution to run all fecal extracts (e.g., 1:20). There is no interference considered on the assay when the linear regression results demonstrate R2 &#62; 0.90 and p &#60; 0.05.</li>
	</ol>
	</li>
	<li>Biochemical Validation (Interference)
	<ol>
		<li>Spike 200 &#181;l of serially diluted standards (zero, 3.9 to 1000 ng/well corticosterone) with 200 &#181;l of the appropriately diluted pooled fecal extract [diluted at the 50% binding determined by parallelism (1:20)] and run on the EIA as in section 3. Following EIA analysis plot the observed concentration minus the background (concentration of the zero sample) versus the expected concentration. 
		NOTE: If the addition of diluted pooled fecal extract to standards does not significantly alter the amount expected and linear regression results yields R2 &#62; 0.90, a slope close to 1.0 and p &#60; 0.05. No interference with the EIA has been achieved.</li>
	</ol>
	</li>
	<li>Biological Validation: 
	NOTE: A demonstration that the chosen EIA has the ability to measure biologically relevant changes of the hormone of interest [e.g., the rise in glucocorticoid concentrations following a pharmacological (ACTH is often a licenced procedure) or biological challenge (e.g., psychological and/or environmental; more likely to be an opportunistic unregulated event)].
	<ol>
		<li>Select, extract and analyse fecal samples for glucocorticoid metabolite concentrations using the process detailed in sections 1 - 3 before and after a challenging event. 
		NOTE: A challenging event will be species specific. An example method is provided below for the domestic horse. Full examples of challenging events for horses have been published previously including the use of management9 and husbandry procedures16. A significant rise in glucocorticoid metabolite concentrations following the challenging event must be observed and confirmed using statistical analysis. This must incorporate where appropriate, the effect of repeated measures, sample date and individual. Biological validation is then considered achieved.</li>
	</ol>
	</li>
</ol>

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R., Wiggans, G. R., Van Soest, P. 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The authors have nothing to disclose.

Fecal corticosterone analysis provides a means of assessing long term patterns of adrenal activity in horses. The non-invasive nature of the method overcomes the confounding effects of other sampling methods used to assess adrenal activity including saliva and plasma analysis9. In addition the technique has a clear non-invasive advantage if studying free ranging horses.
There are several key points to discuss regarding this method and its appropriate use. A critical step in the protocol is the validation of the assay for the species in question and appropriate antibody choice. If the antibody used cross-reacts with metabolites of structurally similar but functionally different glucocorticoids this could confound results17. Therefore, assays to measure hormone metabolites require a careful physiological or biological validation, details of which exist in the literature18,19 and within the protocol described.
The condition of the fecal sample needs to be considered including freshness of the sample and environmental exposure which have both been shown to affect hormonal metabolite levels20. Excessive rain or sunshine exposure are known to cause increases or decreases in metabolite concentrations due to bacterial metabolization21. Researchers who wish to collect samples of varying degrees of freshness must investigate the effects of time on sample integrity, in addition to environmental impact. This can be done prior to the study by setting up a small experiment and periodically sampling faeces from male and female individuals, with varying degrees of freshness and exposure to climatic variables.
In most research scenarios, samples from known individuals will be helpful or necessary10. In domestic horses this can be achieved by monitoring a group for defaecation or using ingested food markers to identify feces from individuals in larger groups12. In free ranging species this may prove time consuming as individual tracking will be required.
When selecting the method of glucocorticoid analysis it must be considered that the results of fecal corticosterone assay represent a pooled sample over time rather than a point in time measure; therefore, there will be less fluctuation in the data. If the researcher is interested in long term effects of a situation, husbandry technique or management practice then fecal analysis offers an appropriate technique. If assessment of short term patterns in hormone concentration is required then plasma or salivary analysis would be the preferred method.

The method described aims to analyse corticosterone concentrations in equine feces in order to provide a non-invasive evaluation of adrenal activity. Measuring hypothalamic-pituitary-adrenal (HPA) axis activity is an accepted approach to study the response to potentially aversive situations in both captive and domestic species. The reference technique and the most widely used method is the use of blood plasma1 however, alternate methods such as fecal analysis have been developed in order to overcome the stress induced by blood sampling itself and allow the ability to monitor free ranging species.
During an aversive situation, physiological homeostasis is disrupted. The hypothalamus in the brain releases corticotrophin releasing hormone (CRH) which acts on the anterior pituitary gland and stimulates release of adrenocorticotrophic hormone (ACTH). ACTH enters the bloodstream and stimulates the adrenal cortex to secrete species specific glucocorticoids (GC). Glucocorticoids are closely linked to stressful events rather than being consistently produced in all energy heightened states therefore they are often measured in preference over other stress linked hormones2. Glucocorticoids are responsible for several adaptive effects in horses. Energy is rapidly mobilised from storage sites in the body in the form of fatty acids and glucose, oxygen intake is increased, sensory function is enhanced3 and blood flow is decreased to areas not necessary for movement4. As well as acting as a coping mechanism, the stress induced rise in Glucocorticoids may also help to prepare the animal for the next stressor5.
Assessing hormone levels in the plasma and saliva involves measuring the actual circulating hormone however, measuring the metabolites in the faeces measures the metabolic end product of the hormone. Circulating steroids are catabolized in the liver before excretion in to the bile where they undergo further changes facilitated by the enzymatic activities of bacterial flora within the intestinal track6. Therefore, immunoassays directed towards blood glucocorticoids may not be suitable for analysis of fecal glucocorticoid metabolites7.
As fecal collection can be carried out with no disturbance to the horse, analysis of feces for corticosterone, has been used extensively to monitor HPA activity in a number of circumstances. Elevated corticosterone in the feces of horses has been reported in response to potentially aversive situations including during post-operative veterinary treatment8 and in restrictive housing9. Fecal sampling reflects a pooled glucocorticoid level over time rather than the point in time sampling offered by plasma and saliva making it appropriate for monitoring long term, chronic or seasonal patterns10. Due to the non-invasive nature of the method, samples can be collected repeatedly for an individual without the need for capture or restraint11. However, species specific gut transit time must be taken into account when planning a sampling protocol. In horses, gut transit time is around 18 hr12 therefore, adrenal response and subsequent corticosterone metabolites can be detected in the feces one day after initial activation of the HPA axis.
When utilising non-invasive immunoassay techniques a careful validation for the species being investigated is essential13. In addition, sex differences in hormone metabolite excretion have been reported probably due to differences in metabolic rate and type of corticosterone metabolite excreted in various species including mice14, and chickens15. It was therefore important as part of this method that the assay was validated for use in both male and female domestic horses as is detailed in the protocol. This difference in hormone metabolism between genders has consequences for data quality yet it is rarely addressed and included as part of assay validation.
This non-invasive method allows long term assessment of adrenal activity in domestic horses. The protocol details both the validation of the assay and the assay technique itself.

<table><tbody><tr><td>Corticosterone antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Cortisol antibody &amp;amp; HRP kit</td><td>Coralie Munro - UC Davis</td><td></td><td>No longer available through UC Davis - please see Arbor Assays</td></tr><tr><td>Corticosterone synthetic standard hormone</td><td>Sigma Aldrich</td><td>50-23-7</td><td>Harnful if ingested or with skin contact. Use in fume cupboard</td></tr><tr><td>Cortisol synthetic standard hormone</td><td>Sigma Aldrich</td><td>15087-01-1</td><td>Harmful if ingested or with skin contact. Use in fume cupboard</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>67-56-1</td><td>Irritant. Use in fume cupboard</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>144-55-8</td><td>Irritant</td></tr><tr><td>Sodium Carbonate Anhydrous</td><td>Sigma Aldrich</td><td>497-19-8</td><td>Irritant</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>7558-79-4</td><td>Irritant</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma Aldrich</td><td>10049-21-5</td><td>Irritant</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>9048-46-8</td><td>Irritant</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>9005-64-5</td><td>Irritant</td></tr><tr><td>Citric Acid</td><td>Sigma Aldrich</td><td>77-92-9</td><td>Irritant</td></tr><tr><td>ABTS</td><td>Sigma Aldrich</td><td>30931-67-0</td><td>Irritant</td></tr><tr><td>Hydrogen Peroxide 30%</td><td>Sigma Aldrich</td><td>7722-84-1</td><td>Irritant</td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>7647-14-5</td><td>Irritant</td></tr><tr><td>Buffer capsules - pH 4</td><td>VWR</td><td>332732B</td><td></td></tr><tr><td>Buffer capsules - pH 7</td><td>VWR</td><td>332742D</td><td></td></tr><tr><td>Buffer capsules - pH 10</td><td>VWR</td><td>332762H</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Sigma Aldrich</td><td>435570</td><td>Irritant. Use in fume cupboard</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Irritant</td></tr><tr><td>Analytical balance</td><td>Fisher Scientific</td><td>BFS-525-010A</td><td></td></tr><tr><td>Air compressor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Computer +printer</td><td></td><td></td><td></td></tr><tr><td>fridge-freezer</td><td></td><td></td><td></td></tr><tr><td>Drying apparatus</td><td></td><td></td><td></td></tr><tr><td>+tubing</td><td></td><td></td><td></td></tr><tr><td>Flammable liquid storagecabinet</td><td>VWR</td><td>649-002</td><td></td></tr><tr><td>Fume cupboard</td><td></td><td></td><td></td></tr><tr><td>Hot-plate stirrer</td><td>VWR</td><td>640-282</td><td></td></tr><tr><td>Microplate reader</td><td>VWR</td><td></td><td></td></tr><tr><td>Microplate washer</td><td>VWR</td><td></td><td></td></tr><tr><td>pH meter</td><td>VWR</td><td></td><td></td></tr><tr><td>Eppendorf Research&amp;reg; pipettes - multipack option 2</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 1,000 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 200 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette - 20 &amp;micro;l</td><td>VWR</td><td></td><td></td></tr><tr><td>Repeater pipette</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette filler</td><td>VWR</td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Progen Scientific</td><td></td><td></td></tr><tr><td>Sonicator</td><td>Hilsonic</td><td></td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td></td><td></td></tr><tr><td>Warm water bath</td><td></td><td></td><td></td></tr><tr><td>Water purification system</td><td>Millipore</td><td></td><td></td></tr></tbody></table>

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.

Domestic horses (n = 16, 8 mares, 8 geldings) with a mean age of 15 years (&#177; 3) were grouped according to gender and subjected to four housing designs with increasing levels of social isolation (n = 4 horse/treatment). Housing 1 involved horses living in a herd environment, closely simulating their natural habitat. Housing 2 involved horses living in pairs in an indoor barn. Housing 3 involved horses housed alone in stables but with visual contact to other horses and housing 4 involved total isolation of the horses.
Exposure to each treatment was in a randomized block design for a period of five days. Following this the horses were turned out into grass paddocks in their experimental groups for two days before exposure to the next housing treatment. Fecal samples were collected once per day on days 1, 2, and 3 spent within each housing treatment from n = 8 horses (n = 2 horses within each treatment). Fecal samples were collected as soon as possible and within 1 hr after defecation. All samples were collected after 1,200 hr on the first full day of stabling meaning that the first sample on day one was collected at least 20 hr after the horse was introduced to the housing treatment. Samples from day one, two and three (total of 24 samples per housing design) were assessed for fecal corticosterone levels reflective of the past 18 hr due to rate of passage of digesta12. The same individual horses were used for fecal analysis throughout the study. For full details of this biological challenge, including additional measured parameters please refer to the published work9 Alternate examples of challenging events for domestic horses can also be found in the literature16.
This section provides example results of a biochemical validation (parallelism) for both corticosterone and cortisol in male and female domestic horse feces. This section also provides representative data obtained from fecal extraction and EIA analysis. Results were obtained as part of a larger study that investigated the impact of social isolation caused by housing design, upon equine behaviour and physiology9.
The results of the biochemical validation revealed that the corticosterone EIA was appropriate to measure adrenal activity [Figure 1A male sample % binding = 25.349 + 0.729 (standard % binding), R2 = 0.9545, F (1, 7) = 146.710, p less than 0.001); Figure 1B female sample % binding = 29.989 + 0.7198 (standard % binding), R2 = 0.95594, F (1, 7) = 151.865, p less than 0.001] but not the cortisol EIA [Figure 1C male sample %binding = 79.089 + 0.0629 (standard % binding), R2 = 0.175, F (1, 7) = 1.485, p =0.262); Figure 1D female sample %binding = 81.652 + 0.0772 (standard % binding), R2 = 0.257, F (1, 7) = 2.422, p = 0.164]. As the corticosterone EIA demonstrated parallelism unlike the cortisol EIA and interference test on the corticosterone EIA revealed no evidence of matrix interference, as addition of diluted faecal extract to corticosterone standards did not alter the amount expected (male: Observed = 1.302 + 1.017 (Expected), R2 = 0.9972, F1, 7 = 1287.128, p &#60;0.001; female: Observed = 0.3169 + 1.0931(Expected), R2 = 0.9972, F1, 7 = 2432.65, p &#60;0.001;).
The results of the EIA revealed that there was no significant difference in corticosterone levels between male horses (M = 37.7, SD = 14.1) and female horses (M = 33.8, SD = 12.7; t (70) = 1.23, p = 0.22). The level of fecal corticosterone increased as the level of isolation increased. Isolated horses (housing design 4) had significantly higher levels of fecal corticosterone compared to all other housing designs (Wilks Lambda = 0.58, F (3, 18) = 4.29, p = 0.01, multivariate partial eta squared = 0.42; p &#8804;0.02). The level of fecal corticosterone was higher for all horses on all three sample days during the most isolated housing when compared to the other housing treatments. The concentrations of mean fecal corticosterone for each housing design for each day are presented in Table 1.
<img alt="Figure 1" src="/files/ftp_upload/53479/53479fig1.jpg" /> 
Figure 1. Biochemical Validation (Parallelism) of a Corticosterone and Cortisol Enzyme Linked-Immunoassay for Male and Female Horses. This figure details the percent binding of serial diluted pools of male and female domestic horse fecal extracts against increasing corticosterone and cortisol standard curve concentrations on an EIA. The results demonstrate that the corticosterone (A, male; B, female) and not the cortisol EIA (C, male; D, female) yielded a successful displacement curve parallel with the corticosterone EIA standard curve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53479/53479fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td rowspan="3"></td>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">MEAN (&#177;SD) FECAL CORTICOSTERONE (ng/g) IN EACH HOUSING TREATMENT</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HOUSING TREATMENT</td>
			<td>Day 1</td>
			<td>Day 2</td>
			<td>Day 3</td>
			<td>OVERALL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1</td>
			<td>31.73 &#177; 10.2</td>
			<td>32.18 &#177; 8.0</td>
			<td>29.22 &#177; 5.9</td>
			<td>31.05 &#177; 7.8</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>32.75 &#177; 10.0</td>
			<td>33.66 &#177; 12.9</td>
			<td>34.67 &#177; 9.3</td>
			<td>33.69 &#177; 10.3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>35.06 &#177; 14.6</td>
			<td>35.14 &#177; 15.9</td>
			<td>33.13 &#177; 12.5</td>
			<td>34.44 &#177; 13.6</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>*</td>
		</tr>
		<tr>
			<td>4</td>
			<td>38.16 &#177; 17.8</td>
			<td>42.00 &#177; 16.7</td>
			<td>41.52 &#177; 17.6</td>
			<td>40.56 &#177; 16.5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Mean (&#177; SD) Fecal Corticosterone (ng/g) for each Housing Treatment for Days One, Two and Three and Mean Fecal Corticosterone (ng/g) for all Three Days (Overall) in each Housing Treatment. This table shows the mean values for fecal corticosterone (ng/g) &#177; standard deviation for each of the housing treatments during day one, two and three. Samples were collected at least 20 hr after horses entered the housing. It also shows mean fecal corticosterone (ng/g) &#177; standard deviation for all three days (overall) in each housing treatment. The fecal corticosterone concentration was significantly higher (*) during the isolated housing design. The lowest concentration of fecal corticosterone for all days was found in the group housed treatment (treatment 1). Adapted from Yarnell et al. (2015), with permission from the Journal of Physiology and Behaviour.

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

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. 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, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Nanyang Technological University

Research

JoVE Journal

Biology

9.6K Views.  Nottingham Trent University. 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.

Video: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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.

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.

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

Biochemistry

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

A Novel Method: Super-selective Adrenal Venous Sampling

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts of aldosterone and cortisol, respectively. The conventional adrenal venous sampling (cAVS) method collects blood samples from both adrenal central veins and is useful for identifying the laterality of excess hormone production in a unilateral lesion(s), as documented in PA cases. In cAVS, plasma cortisol concentrations (PCCs) are used to normalize plasma aldosterone concentrations (PACs). A novel &#34;super-selective&#34; adrenal venous sampling (ssAVS) method was developed using a micro-catheter, which collects blood samples from adrenal tributary veins (TVs). PACs in ssAVS samples do not require PCC normalization because samples contain a limited amount of systemic venous blood, if any. The ssAVS method enabled segmental lesion(s) to be detected in both adrenal glands, which may be treated by bilateral adrenalectomy, thereby sparing lesion-free segment(s). Right and left adrenals typically have three TVs each, i.e., the superior, lateral, and inferior TVs in the right adrenal as well as the superior-median, superior-lateral, and lateral TVs in the left adrenal. In the ssAVS method, specific parent catheters and a technique to handle them are required, and have been described herein. Furthermore, ssAVS results from three cases of PA are presented: bilateral aldosterone-producing adenoma (APA) (Case #1), left APA and right possible cortisol-producing adenoma causing SCS (Case #2), and idiopathic hyperaldosteronism in which bilateral adrenal segments produced excessive amounts of aldosterone (Case #3). The ssAVS method is not difficult for expert angiographers, and, thus, is recommended worldwide to treat PA cases for which cAVS does not represent a viable surgical treatment option.

&#39;Super-selective&#39; adrenal venous sampling (ssAVS, also called segmental adrenal venous sampling: sAVS)&#160;was performed using a micro-catheter to identify adrenal segment(s) that produce excessive amounts of hormones. The ssAVS technique was described, cases in which adrenal segmental lesion(s) were identified by ssAVS were presented, and the usefulness of ssAVS in future adrenal research was discussed.

Primary aldosteronism (PA) and subclinical Cushing&#39;s syndrome (SCS) are conditions in which the adrenal glands autonomously produce excessive amounts ...

Medicine

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

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 ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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