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...

<ol>
	<li>Morm&#232;de, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+the+hypothalamic-pituitary-adrenal+function+as+a+tool+to+evaluate+animal+welfare.">Exploration of the hypothalamic-pituitary-adrenal function as a tool to evaluate animal welfare.</a> Physiology and Behaviour. 92 (3), 317-339 (2007).</li><li>Lane, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+non-invasive+glucocorticoid+measures+be+used+as+reliable+indicators+of+stress+in+animals?.">Can non-invasive glucocorticoid measures be used as reliable indicators of stress in animals?.</a> Animal Welfare. 15 (4), 331-342 (2006).</li><li>Morgan, K. N., Tromborg, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sources+of+stress+in+captivity.">Sources of stress in captivity.</a> Applied Animal Behaviour Science. 102, 262-302 (2007).</li><li>Nelson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An introduction to behavioural endocrinology (3rd Ed). , 670-671 (2005).</li><li>Sapolsky, R. M., Romero, L. M., Munck, A. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Do+Glucocorticoids+Influence+Stress+Responses?+Integrating+Permissive,+Suppressive,+Stimulatory,+and+Preparative+Actions.">How Do Glucocorticoids Influence Stress Responses? Integrating Permissive, Suppressive, Stimulatory, and Preparative Actions.</a> Endocrine Reviews. 21 (1), 55-89 (2000).</li><li>Macdonald, K. M., Macdonald, I. A., Bokkenheuser, V. D., Winter, J., McLernon, A. M., Mosbach, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+steroids+in+the+human+gut.">Degradation of steroids in the human gut.</a> Journal of Lipid Research. 24, 675-700 (1983).</li><li>Young, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+monitoring+of+adrenocortical+activity+in+carnivores+by+fecal+glucocorticoid+analysis.">Non-invasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analysis.</a> General and Comparative Endocrinology. 137, 148-165 (2004).</li><li>Merl, S., Scherzer, S., Palme, R., Mostl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+causes+increased+concentrations+of+glucocorticoid+metabolites+in+horse+faeces.">Pain causes increased concentrations of glucocorticoid metabolites in horse faeces.</a> Journal of Equine Veterinary Science. 20, 586-590 (2000).</li><li>Yarnell, K., Hall, C., Royle, C., Walker, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domesticated+horses+differ+in+their+behavioural+and+physiological+responses+to+isolated+and+group+housing.">Domesticated horses differ in their behavioural and physiological responses to isolated and group housing.</a> Physiology and Behaviour. 143, 51-57 (2015).</li><li>Wielebnowski, N., Watters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+fecal+endocrine+monitoring+to+conservation+and+behaviour+studies+of+wild+mammals:+important+considerations+and+preliminary+tests.">Applying fecal endocrine monitoring to conservation and behaviour studies of wild mammals: important considerations and preliminary tests.</a> Israel journal of ecology and evolution. 53, 439-460 (2007).</li><li>Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fecal+steroids:+guidelines+for+a+practical+application.">Measuring fecal steroids: guidelines for a practical application.</a> Annals of the New York Academy of Sciences. 1046, 75-80 (2005).</li><li>Uden, P., Rounsaville, G. R., Wiggans, G. R., Van Soest, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+liquid+and+solid+digesta+retention+in+ruminants,+equines+and+rabbits+given+timothy+hay.">The measurement of liquid and solid digesta retention in ruminants, equines and rabbits given timothy hay.</a> British Journal of Nutrition. 48, 329-339 (1982).</li><li>Goymann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+monitoring+of+hormones+in+bird+droppings:+biological+validations,+sampling,+extraction,+sex+differences+and+the+influence+of+diet+on+hormone+metabolite+levels.">Non-invasive monitoring of hormones in bird droppings: biological validations, sampling, extraction, sex differences and the influence of diet on hormone metabolite levels.</a> Annals of the New York Academy of Sciences. 1046, 35-53 (2005).</li><li>Touma, C., sachser, N., Mostl, E., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sex+and+time+of+day+on+metabolism+and+excretion+of+corticosterone+in+urine+and+feces+of+mice.">Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice.</a> General and comparative Endocrinology. 130, 267-278 (2003).</li><li>Rattenbacher, S., Mostl, E., Hackl, R., Ghareeb, K., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+corticosterone+metabolites+in+chicken+droppings.">Measurement of corticosterone metabolites in chicken droppings.</a> British Poultry Science. 45, 704-711 (2004).</li><li>Yarnell, K., Hall, C., Billett, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assessment+of+the+aversive+nature+of+an+animal+management+procedure+using+behavioural+and+physiological+measures.">An assessment of the aversive nature of an animal management procedure using behavioural and physiological measures.</a> Physiology &amp; Behaviour. 118, 32-39 (2013).</li><li>Goymann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+non-invasive+hormone+research+in+uncontrolled,+natural+environments:+the+problem+with+sex,+diet,+metabolic+rate+and+the+individual.">On the use of non-invasive hormone research in uncontrolled, natural environments: the problem with sex, diet, metabolic rate and the individual.</a> Methods in Ecology and Evolution. 3, 757-765 (2012).</li><li>Sheriff, M. J., Dantzer, B., Delehanty, B., Palme, R., Boonstra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+stress+in+wildlife:+techniques+for+quantifying+glucocorticoids.">Measuring stress in wildlife: techniques for quantifying glucocorticoids.</a> Oecologia. 166, 614-619 (2011).</li><li>Watson, R., Munro, C. J., Edwards, K. L., Norton, V., Brown, J. L., Walker, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+versatile+enzyme+immunoassay+for+non-invasive+assessment+of+glucocorticoid+metabolites+in+a+diversity+of+taxonomic+species.">Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.</a> General Comparative Endocrinology. 186, 16-24 (2013).</li><li>Touma, C., Palme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fecal+glucocorticoid+metabolites+in+mammals+and+birds:+the+importance+of+validation.">Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation.</a> Annals of the New York Academy of Sciences. 1046, 54-74 (2005).</li><li>Millspaugh, J. J., Washburn, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fecal+glucocorticoid+metabolite+measures+in+conservation+biology+research:+considerations+for+application+and+interpretation.">Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation.</a> General and Comparative Endocrinology. 138, 189-199 (2004).</li></ol>

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 prot...

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 &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Cortisol antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</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>Harnful 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&#174; pipettes - multipack option 2</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 1000ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 200ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 20ul</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 involv...

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, ...

The overall goal of this procedure is to demonstrate how adrenal activity can be assessed non-invasively in equines through the extraction and analysis of fecal glucocortocoid metabolites. This method can help us answer key questions in the area of welfare assessment by looking at the physiological impacts of husbandry protocols on domestic horses, all the way through to wild populations of African zebras. The main advantage of this technique is it offers a non-invasive option for measuring the long-term adrenal activity in both domestic and free-ranging horses.On the day of the experiment, after thawing at room temperature, manually homogenize the fecal samples by mixing within each individual sample bag. Then, for each sample, measure out the appropriate amount of feces on a micro balance in a clean weigh boat, and transfer the samples into individual eight milliliter glass vials. Next, mix five milliliters of 90%methanol in water with each sample, and shake the samples overnight on an orbital shaker at room temperature.The following morning, vortex the samples and spin them down. At the end of the centrifugation, decant the methanol fractions into individual 16 by 125 millimeter glass tubes. Then briefly place the tubes in a 37-degree Celsius water bath, followed by evaporation of the methanol under air in a fume cupboard.When all of the methanol has dispersed, resuspend the fecal extracts in one milliliter of 100%methanol. Then transfer the extracts into individual 12 by 75 millimeter polypropylene tubes, cap the tubes, and store the samples at minus 20 degrees Celsius until their analysis. Before beginning the analysis, use a repetitive pipette with a 50 microliter pipette tip to load 50 microliters of polyclonal corticosterone CJM006 antiserum, diluted in coating buffer, per well, in a 96 well microtiter plate.Then cover the plate with a plate sealer and incubate the antiserum overnight at four degrees Celsius. The next day, immediately before beginning the analysis, use an automated microtiter plate washer to wash the plate five times with wash solution. Then load the nonspecific binding and zero wells with 50 microliters of enzyme-linked immunoassay, or EIA buffer, per well, the standard wells with the appropriate concentrations of corticosterone, and the experimental wells with the fecal extract samples diluted in EIA buffer.After loading the last sample, immediately add 50 microliters of horseradish perodixase conjugate diluted in EIA buffer to each well, and incubate the microtiter plate in the dark for two hours at room temperature. At the end of the incubation, wash the plate with wash solution five times. Then incubate the samples with 100 microliters per well of freshly prepared, room temperature substrate in the dark at room temperature, with periodic reading of the plate at 405 nanometers on a spectrophotometer.The incubation is considered complete when the optical density of the zero wells reaches 0.8 to 1.0. To perform a biochemical validation for parallelism, perform a serial dilution on pooled fecal extracts, ideally taken from suspected high-and low-hormone concentration samples from several individuals. Then run the samples on the EIA as just demonstrated.A parallelism is considered achieved when the serial dilutions of the fecal extracts yield a displacement curve parallel to the standard curve, and the linear regression results demonstrate an R squared greater than 0.9, a slope greater than 0.5, and a P less than 05. To perform a biochemical validation for interference, spike 200 microliters of serial diluted standards with 200 microliters of the appropriate pooled fecal extract, diluted at the 50%binding as determined by the parallelism assay. Then run the samples on the EIA as demonstrated.Following the EIA analysis, plot the observed concentration minus the background concentration of the zero sample versus the expected concentration. No interference is considered achieved if the addition of diluted pooled fecal extract to the standards does not significantly alter the amount expected, and linear regression results yields R squared greater than 0.9, a slope close to one, and P less than 05. To perform a biological validation, run samples extracted before, and after a challenging event on the EIA as just demonstrated.The biological validation is a critical step for the validation of EIA, and is considered achieved when a significant rise in glucocorticoid metabolites is observed after a challenging event. The data in graph A and B demonstrate the biochemical validation of adult male and female domestic horses, by demonstrating parallelism with a corticosterone standard curve. No biochemical validation with the cortisol EIA was achieved, as there was no parallelism between the pooled fecal extract and the cortisol standard curve.These data from challenged male and female domestic horses demonstrate biological validation, as the concentration of fecal corticosterone increased as the level of isolation increased in these animals. Indeed, the horses in the isolated housing situation exhibited significantly higher levels of fecal corticosterone, compared to all of the other housing designs. So assessing hormone concentrations in saliva or plasma usually involves us looking at the native hormone, but when we're assessing it in fecal material, we're looking at a breakdown of that product and looking at the metabolic end products of that hormone.Indeed, fecal sampling offers a pooled glucocortocoid concentration over time, rather than the single point reflected by saliva or plasma. This makes it more appropriate for measuring long term, chronic or seasonal adrenal activity. So as fecal sample collection is non-invasive to the animal, it's been used extensively to monitor equine adrenal activity in numerous natural and experimental circumstances.When running EIA, it is important to minimize the time of loading the microtiter plate to under 10 minutes, as longer times will result in drift. It is also important to ensure that duplicate samples have a coefficient of variation of less than 10%and controls have an intra-and inter-assay coefficient variation of 10%and 15%respectively. So after watching this video, you should have a really good understanding of how to extract fecal glucocortocoid metabolites, analyze those extracts in an enzyme immunoassay, and also perform the required validation steps.

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

Research

JoVE Journal

Biology

9.5K ビュー.  Nottingham Trent University. The overall goal of this procedure is to demonstrate how adrenal activity can be assessed non-invasively in equines through the extraction and analysis of fecal glucocortocoid metabolites. This method can help us answer key questions in the area of welfare assessment by looking at the physiological impacts of husbandry protocols on domestic horses, all the way through to wild populations of African zebras. The main advantage of this technique is it offers a non-invasive option for measuring t...

ビデオ: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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 ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.