This work was conducted with the support of the Cooperative Research Program for Agriculture Science &#38; Technology Development (Project title: Livestock productivity change analysis with climate change, Project No. PJ012771), Rural Development Administration, Republic of Korea. Also, this study was supported by a grant (No. PJ01344604) from the Animal Nutrition &#38; Physiology Team, National Institute of Animal Science, RDA, Seoul, Republic of Korea. The authors gratefully acknowledge Persian Gesture CEO Mohammad Hassan Salmanzadeh and his team, who provided fish from the three sturgeon species examined in this study.

The following experimental procedures and methods were approved by the Animal Welfare and Ethics Authority of Kangwon National University, Chuncheon, Republic of Korea.
1. Fin collection
<ol>
	<li>Capture the sturgeon gently using a net to minimize injury and stress.</li>
	<li>Rinse the fish carefully with fresh water and then wipe the body surface with an absorbent towel prior to euthanasia.</li>
	<li>Hit the head of the fish using a plastic hammer such that the fish is stunned or loses consciousness. Remove the head using a knife.</li>
	<li>Measure the body weight (g) and length (cm).</li>
	<li>After euthanasia, collect fin samples by cutting as close as the body as possible using sterilized surgical scissors. 
	NOTE: Individual, non-recycled absorbent towels must be used for each fish. Descriptive statistics for the species used in this study were as follows: beluga sturgeon (H. huso): age = 18 &#177; 2.1 months, body weight = 2,700 &#177; 300 g, and body length = 55 &#177; 5 cm; Siberian sturgeon (A. baerii): age = 9.6 &#177; 2.4 months, body weight = 1,750 &#177; 250 g, and body length = 45 &#177; 5 cm; sevruga sturgeon (A. stellatus): age = 14 &#177; 1.3 months, body weight = 1,000 &#177; 100 g, and body length = 65 &#177; 5 cm.</li>
</ol>
2. Fin preparation for cortisol extraction
<ol>
	<li>Place the fin samples (one sample per tissue: ~3 g) on laboratory weighing paper (107 mm &#215; 210 mm) and dry at room temperature for a few days until dry.</li>
	<li>Wrap samples in sheets of aluminum foil, place in labeled plastic bags, and transfer to the laboratory.</li>
	<li>Store samples in a refrigerator for further use, including washing, cortisol extraction, drying, and ELISA analysis (Figure 2).</li>
</ol>
3. Fin cortisol analysis
<ol>
	<li>Calibrate the digital analytical scale (accuracy: 0.0001) and weigh out 300 &#177; 10 mg samples with weighing paper on the scale pan.</li>
	<li>Wash the samples.
	<ol>
		<li>Transfer each sample into a 15 L conical polypropylene tube. Add 3 mL of isopropanol to each tube using a 5,000 &#181;L single-channel pipette.</li>
		<li>Rotate the tubes at 80&#160;rpm for 2.5 min to wash out cortisol and remove any potential external contamination. Repeat this procedure twice.</li>
		<li>Air-dry the washed samples at room temperature (22-28 &#176;C) for 7 days.</li>
		<li>Repeat the washing procedure using ultrapure water as the washing agent.</li>
	</ol>
	</li>
	<li>Extract the jawbone from the body tissue using bone-cutting forceps. Apply steps 1.5-3.2.4 to the jawbone samples.</li>
	<li>Weigh out (75 &#177; 5 mg) dried fin or jawbone samples and grind using a bead beater at 50 Hz for 32 min.
	<ol>
		<li>Deliver 1.5 mL of methanol into each tube containing powdered fin or jawbone using a 1000 &#181;L pipette. Place the samples on a tube rotator at slow rotation (40 rpm) for 18 h at room temperature to extract cortisol with continuous mixing.</li>
	</ol>
	</li>
	<li>Following cortisol extraction, centrifuge the samples at 9,500 x g for 10 min at room temperature. After centrifugation, collect the top organic layer containing cortisol (1 mL) from each sample and place it into a separate 1.5 mL microcentrifuge tube.
	<ol>
		<li>Dry the samples by incubation at 38 &#176;C to evaporate the methanol. Keep the extracted cortisol samples under a fume hood overnight to allow methanol to dissipate. 
		NOTE: The cortisol-containing layer is usually yellowish in color.</li>
	</ol>
	</li>
</ol>
4. Fin cortisol detection
<ol>
	<li>Thaw the dried fin or jawbone samples at room temperature for 1.5 h prior to using the ELISA kit.</li>
	<li>Add 400 &#181;L of phosphate buffer, vortex, and centrifuge at 1,500 x g for 15 min.</li>
	<li>Run each sample (25 &#181;L) in duplicate to improve assay accuracy and reliability. Remove any data outside the standard curve as outliers.</li>
	<li>Set a microplate reader to 450 nm, then set to &#181;g dL-1 and read the optical density of the plate.
	<ol>
		<li>Use the microplate software with a four-parameter non-linear regression curve fit. Convert the cortisol levels of the samples obtained from the software into pg mg-1 using the following equation: 
		F = 10,000E (A/B) (C/D), 
		where F = the final value of the fin cortisol level in (pg mg-1), E = the volume (mL) of the assay buffer used to reconstitute the dried extract, A = the concentration (&#181;g dL-1) provided by the assay output, B = the weight (mg) of the fin subjected to extraction, C = the volume (mL) of methanol added to the powdered fin, and D = the volume (mL) of methanol recovered from the extract and subsequently dried down3.</li>
	</ol>
	</li>
</ol>
5.Statistical analysis
<ol>
	<li>Divide each sample into two sub-samples prior to the washing procedure and then run in duplicate during the ELISA kit assay (2 &#215; 2 = 4 observations per sample) to improve the power of the test and reliability of the results.</li>
	<li>Compare the effects of the two washing solvents and their interactions by applying the general linear model (GLM) procedure in the SAS software environment to the measurement data18.</li>
	<li>Test differences between means using Tukey&#39;s test at a significance level of p &#60; 0.05. Accept 0.05 &#60; p &#60; 0.10 as evidence of a tendency rather than as a significant difference.</li>
</ol>

<ol>
	<li>Meyer, J. S., Novak, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cortisol:+a+novel+biomarker+of+hypothalamic-pituitary-+adrenocortical+activity.">Hair cortisol: a novel biomarker of hypothalamic-pituitary- adrenocortical activity.</a> Endocrinology. 153, 4120-4127 (2012).</li><li>Ghassemi Nejad, J., 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=A+cortisol+study:+facial+hair+and+nails.">A cortisol study: facial hair and nails.</a> Journal of Steroids Hormonal Sciences. 7, 1-5 (2016).</li><li>Meyer, J. S., Novak, M. A., Hamel, A., Rosenberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+analysis+of+cortisol+from+human+and+monkey+hair.">Extraction and analysis of cortisol from human and monkey hair.</a> Journal of Visualized Experiments. (83), e50882 (2014).</li><li>Davenport, M. D., Tiefenbacher, S., Lutz, C. K., Novak, M. A., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+endogenous+cortisol+concentrations+in+the+hair+of+rhesus+macaques.">Analysis of endogenous cortisol concentrations in the hair of rhesus macaques.</a> General and Comparative Endocrinology. 147, 255-261 (2006).</li><li>Ghassemi Nejad, J., Ataallahi, M., Park, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+validation+of+measuring+Hanwoo+hair+cortisol+concentration+using+bead+beater+and+surgical+scissors.">Methodological validation of measuring Hanwoo hair cortisol concentration using bead beater and surgical scissors.</a> Journal of Animal Science and Technology. 61, 41-46 (2019).</li><li>Ghassemi Nejad, J., 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=Wool+cortisol+is+a+better+indicator+of+stress+than+blood+cortisol+in+ewes+exposed+to+heat+stress+and+water+restriction.">Wool cortisol is a better indicator of stress than blood cortisol in ewes exposed to heat stress and water restriction.</a> Animal. 8, 128-132 (2014).</li><li>Brossa, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cortisol in skin mucus and scales as a measure of fish stress and habitat quality. Ph.D. dissertation. , (2018).</li><li>Carbajal, A., 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=Cortisol+detection+in+fish+scales+by+enzyme+immunoassay:+biochemical+and+methodological+validation.">Cortisol detection in fish scales by enzyme immunoassay: biochemical and methodological validation.</a> Journal of Applied Ichthyology. 34, 1-4 (2018).</li><li>Bertotto, D., 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=Alternative+matrices+for+cortisol+measurement+in+fish.">Alternative matrices for cortisol measurement in fish.</a> Aquaculture Research. 41, 1261-1267 (2010).</li><li>Ghassemi Nejad, J., Jeong, C., Shahsavarani, H., Sung, I. K., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embedded+dental+cortisol+content:+a+pilot+study.">Embedded dental cortisol content: a pilot study.</a> Endocrinology &amp; Metabolic Syndrome. 5, 240 (2016).</li><li>Pankhurst, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endocrinology+of+stress+in+fish:+an+environmental+perspective.">The endocrinology of stress in fish: an environmental perspective.</a> General and Comparative Endocrinology. 170, 265-275 (2011).</li><li>Ghassemi Nejad, J., Kim, W. B., Lee, B. H., Sung, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coat+and+hair+color:+hair+cortisol+and+serotonin+levels+in+lactating+Holstein+cows+under+heat+stress+conditions.">Coat and hair color: hair cortisol and serotonin levels in lactating Holstein cows under heat stress conditions.</a> Animal Science Journal. 88, 190-194 (2017).</li><li>Baker, M. R., Gobush, K. S., Vynne, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+factors+influencing+stress+hormones+in+fish+and+wildlife.">Review of factors influencing stress hormones in fish and wildlife.</a> Journal of Nature Conservation. 21, 309-318 (2013).</li><li>Aerts, J., 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=Scales+tell+a+story+on+the+stress+history+of+fish.">Scales tell a story on the stress history of fish.</a> PLoS One. 10, e0123411 (2015).</li><li>Heimb&#252;rge, S., Kanitz, E., Otten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+hair+cortisol+for+the+assessment+of+stress+in+animals.">The use of hair cortisol for the assessment of stress in animals.</a> General and Comparative Endocrinology. , 10-17 (2019).</li><li>Ghassemi Nejad, J., 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=Comparing+hair+cortisol+concentrations+from+various+body+sites+and+serum+cortisol+in+Holstein+lactating+cows+and+heifers+during+thermal+comfort+zone.">Comparing hair cortisol concentrations from various body sites and serum cortisol in Holstein lactating cows and heifers during thermal comfort zone.</a> Journal of Veterinary Behavior: Clinical and Application Research. 30, 92-95 (2019).</li><li>Bussy, U., Wassink, L., Scribner, K. T., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cortisol+in+lake+sturgeon+(Acipenser+fulvescens)+eggs+by+liquid+chromatography+tandem+mass+spectrometry.">Determination of cortisol in lake sturgeon (Acipenser fulvescens) eggs by liquid chromatography tandem mass spectrometry.</a> Journal of Chromatography B. 1040, 162-168 (2017).</li><li>SAS. 1999. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SAS. User's Guide (Version 8.01 Edition). , (1999).</li><li>Barton, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+in+fishes:+a+diversity+of+responses+with+particular+reference+to+changes+in+circulating+corticosteroids.">Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids.</a> Integrative and Comparative Biology. 42, 517-525 (2002).</li><li>Ghassemi Nejad, J., 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=Measuring+hair+and+blood+cortisol+in+sheep+and+dairy+cattle+using+RIA+and+ELISA+assay:+a+comparison.">Measuring hair and blood cortisol in sheep and dairy cattle using RIA and ELISA assay: a comparison.</a> Biological Rhythm Research. Accepted. , (2019).</li></ol>

The authors have no conflicts of interest to disclose.

Sturgeon is sometimes called a &#34;living fossil&#34; because it has exhibited few adaptations throughout past millennia. The sturgeon genus Acipenser contains 27 species that produce caviar; however, three species (beluga, baerii, and sevruga) produce most of the global caviar supply. Sturgeon are vulnerable to over-fishing and interference in their natural habitat and are therefore more critically endangered than any other group of species. Sturgeon belong to the oldest group of living vertebrates, which has ...

Cortisol is a reliable indicator of animal stress. Cortisol extraction provides a valid framework for researchers to monitor stress levels and general patterns in stressors. For example, previous studies have conducted methodological validation of hair cortisol measurements using various methods in humans1,2, monkeys3,4, cattle5, sheep6, and goldfish7,8. In fish species, cortisol measurements in matrices such as scales, skin mucus, feces, and blood9 have been shown to provide information on fish health. When blood sampling is problematic or scales are lacking, alternative matrices for cortisol extraction are needed. In fish, alternative matrices can include the jawbone, a hard tissue&#160;similar to the human tooth10.
The development of new matrices and validated techniques to determine fish stress levels is of particular interest to the caviar industry, where sturgeon can experience prolonged exposure to environmental stress factors11. The sex of sturgeon cannot be determined before 2 years of age, and sturgeon do not have scales. Because cortisol gradually accumulates in solid matrices during the growth stage2,7,12, long-term cortisol accumulation data from hard matrices such as fins and jawbones could provide insight into stress levels at different growth stages. In contrast, blood cortisol levels provide a snapshot of stress levels at the time of death and cannot accurately represent stress during long-term rearing conditions13,14. With increasing competition in the caviar market, new approaches to improve stress conditions for the production of healthier eggs among sturgeon species during long-term rearing (8-12 years or longer) are an increasingly important area of research. Due to the high cost of sturgeon, harvested samples are extremely costly ($8,000-15,000 per mature fish depending on species and growth stage), a limiting factor for research projects. However, the development of an appropriate technique for cortisol extraction from sturgeon fins and jawbones could be usefully applied both to fish farming systems and in wild fish to improve the quality and harvest of sturgeon eggs for both consumption and conservation.
As well as providing reliable results6, the selection of an appropriate cortisol extraction technique is of critical importance to ensure that other compounds present in the matrix during sample preparation do not confound the output, which might lead to inconsistent results. It is equally important to determine whether fin and jawbone cortisol levels are influenced by hormone levels in the surrounding water. Heimb&#252;rge et al.15 suggested that a number of factors may influence cortisol levels including age, sex, pregnancy, season, color12, and body region from which cortisol is extracted16. However, little information is available on the effects of washing solvents on cortisol extraction in fish body matrices8, and none on these effects in sturgeon, except for sturgeon eggs17.
Although analyzing baseline cortisol levels from the fins and jawbones of sturgeon requires that the fish be euthanized, this approach does not entail the invasive techniques required for blood sampling in live sturgeon. Fin and jawbone samples are easily collected, and extraction from these tissues can be performed rapidly. Similarly, hormone extraction and analysis are straightforward and require little specialized equipment.
In this study, we present a new and easily applied technique for the extraction, washing, and determination of cortisol from fish fins and jawbones, with the aim of determining whether cortisol levels measured from these matrices can be reliably used as stress indicators. The advantages of this technique include an easy and non-invasive8 approach, less data variation, and reliable output1,6,8,17; the technique is applicable to fish species without scales such as sturgeon. The technique requires slaughter of the fish, selection of appropriate washing solvents2,4, proper grinding of samples3,5, professional enzyme-linked immunosorbent assay (ELISA) application5,7, and extensive knowledge of the incorporation of cortisol sources into solid matrices6.
We applied two different washing solvents (ultrapure water and isopropanol) to obtain basal cortisol levels in fins from three sturgeon species: beluga (Huso huso), Siberian (Acipenser baerii), and sevruga (A. stellatus), under standard environmental conditions for each species. Jawbones of H. huso were also used to evaluate stress in sturgeon. This is the first study to measure cortisol levels in sturgeon jawbones. The results of this study will provide comparative cortisol data for sturgeon species in the early growth stage (~1 year) prior to sex determination.

<table><tbody><tr><td>Disposal latex surgical gloves</td><td>Ansell</td><td>63754090</td><td></td></tr><tr><td>Platform scale-electronic weighing 100kg</td><td>Baskoolnikoo</td><td>101 EM</td><td></td></tr><tr><td>Serological pipette to deliver up to 24 mL</td><td>Becton Dickinson Falcon</td><td>35-7550</td><td></td></tr><tr><td>Micro plate reader with 450 nm and 490 to 492 nm reference filters</td><td>BioTek</td><td>8041000</td><td></td></tr><tr><td>Reagent reservoirs</td><td>BrandTech</td><td>703459</td><td></td></tr><tr><td>Zipper storage plastic bag&amp;nbsp;</td><td>Cleanwrap</td><td>30cm x100m</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Daejung chemicals &amp;amp; Metals&amp;nbsp;</td><td>5035-4400</td><td></td></tr><tr><td>Methyl alcohol</td><td>Daejung chemicals &amp;amp; Metals&amp;nbsp;</td><td>5558-4100</td><td></td></tr><tr><td>Tube rotator- MX-RL-Pro</td><td>DLAB Scientific&amp;nbsp;</td><td>824-222217777</td><td></td></tr><tr><td>Precision pipette to deliver 1.5 and 10 mL</td><td>Eppendorf Research Plus</td><td>M21518D</td><td></td></tr><tr><td>&amp;nbsp; Precision pipette to deliver 15 and 25 &amp;mu;L</td><td>Eppendorf Research Plus</td><td>R25623C</td><td></td></tr><tr><td>Weighing paper (107 x 210 mm)</td><td>Fisherbrand</td><td>09-898-12B</td><td></td></tr><tr><td>Bead beater, 50/60 Hz 2A</td><td>GeneReach Biotechnology Corp</td><td>tp0088</td><td></td></tr><tr><td>Plate rotator with orbit capable of 500 rpm</td><td>Hangzhou Miu Instrument&amp;nbsp;</td><td>MU-E30-1044</td><td></td></tr><tr><td>Disposable polypropylene tubes to hold at least 24 mL</td><td>Hyundai Micro&amp;nbsp;</td><td>H20050</td><td></td></tr><tr><td>Fume hood</td><td>Kwang Dong Industrial</td><td>KD 901-22128175</td><td></td></tr><tr><td>Micro-centrifuge capable of 1500 x g</td><td>Labo Gene</td><td>&amp;nbsp;9.900.900.729</td><td></td></tr><tr><td>Mini vortex mixer</td><td>LMS</td><td>VTX-3000L&amp;nbsp;</td><td></td></tr><tr><td>Lotte aluminum foil roll&amp;nbsp;</td><td>Lotte Aluminum</td><td>B0722X5FK5</td><td></td></tr><tr><td>Digital scale</td><td>Mettler Toledo&amp;nbsp;</td><td>&amp;nbsp;ME204</td><td></td></tr><tr><td>Ultrapure water</td><td>MDM</td><td>MDM-0110</td><td></td></tr><tr><td>Pipette tips</td><td>Neptune Scientific</td><td>REF 2100.N</td><td></td></tr><tr><td>Large fish net</td><td>Pond H2O</td><td>Hoz135&amp;nbsp;</td><td></td></tr><tr><td>Salivary cortisol kit</td><td>Salimetrics</td><td>1-3002-4</td><td></td></tr><tr><td>Bone cutting forceps</td><td>Sankyo</td><td>26-188A</td><td></td></tr><tr><td>Precision multichannel pipette to deliver 50 &amp;mu;L and 200 &amp;mu;L</td><td>VITLAB</td><td>18A68756</td><td></td></tr><tr><td>Towel</td><td>Yuhan Kimberly</td><td>1707921546</td><td></td></tr><tr><td>Tissue paper (107 &amp;times; 210)</td><td>Yuhan Kimberly</td><td>41117</td><td></td></tr></tbody></table>

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.

The presented fin cortisol extraction technique was developed and confirmed in this study using three sturgeon species. Cortisol levels obtained using ultrapure water and isopropanol as washing solvents were compared (Figure 2). Cortisol from H. huso jawbones was examined to determine whether sturgeon jawbones might be used as an alternative matrix to fins. The effects of washing solvent, sturgeon species, and their interaction are shown in T...

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Cortisol Extraction from Sturgeon Fin and Jawbone 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) ...

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8.1K Views.  Kangwon National University. 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.

Video: Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicine

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

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 ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Developmental Biology

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

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

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

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

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 ...

Biology

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 ...

Neuroscience

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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

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

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