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 con...

<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. 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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.

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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...

Watch this Scientific Journal Video about Cortisol Extraction from Sturgeon Fin and Jawbone Matrices at JoVE.com

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) ...

Our method introduces a novel alternative stress indicator that could be a platform for numerous future studies for measuring cortisol in new non plot and hair matrices. The advantages of this technique are its simplicity and its ability to be used to evaluate stress levels even in slaughtered animals. Establishing a relationship between high cortisol levels and healthy animals or humans using this technique can help with the diagnosis of some diseases.This method can also be used to study centuries old biological samples such as these old fossils to evaluate stress levels within the sample life span. For cortisol extraction, weigh out 300 plus or minus 10 milligrams of each sample on a digital analytical scale with a 0.0001 accuracy before placing the samples into individual 15 milliliter conical tubes. Add three milliliters of isopropanol to each sample tube and rotate the tubes at 80 rotations per minute for 2.5 minutes to wash out the cortisol and to remove any potential external contamination.After the last wash, air dry the samples at room temperature for seven days before washing the samples three more times as demonstrated using fresh ultra pure water for each wash. Weigh out 75 plus or minus five milligrams of dried fin or jawbone sample and use a bead beater to finally grind the sample at 50 hertz for 32 minutes. Then, deliver 1.5 milliliters of methanol to each tube of powdered fin or jawbone and place the samples on a tube rotator at 40 rotations per minute for 18 hours at room temperature.Following cortisol extraction, sediment the sample tissues by centrifugation. And transfer the top one milliliter of the yellowish, organic cortisol containing layer from each sample into individual 1.5 milliliter micro-centrifuged tubes. Dry the cortisol samples at 38 degrees Celsius in a fume hood overnight to evaporate the methanol.The next morning, add 400 microliters of PBS to each tube and vortex and centrifuge the samples to collect the cortisol. For ELISA Analysis, load 25 microliters of each extracted standard cortisol sample and control in duplicate into the appropriate wells of a 96 well ELISA plate. Load 25 microliters of Assay diluent into two wells to serve as the zero and into each non specific binding well.Mix 15 microliters of enzyme conjugate with 24 milliliters of Assay diluent and add 200 microliters of conjugate to each well. Mix the plate on a rotator for five minutes at 500 rotations per minute. Followed by a one hour incubation at room temperature.At the end of the incubation, wash the plate four times with 300 microliters of 1x Wash Buffer per well. Quickly inverting the plate over the sink after each wash to discard the buffer and blotting the plate on a stack of paper towels. After the last wash, add 200 microliters of TMB substrate solution to each well and place the plate onto the rotator for five minutes at 500 rotations per minute.After mixing, incubate the plate for 25 minutes at room temperature protected from light. At the end of the incubation, add 50 microliters of stop solution to each well. And mix the plate contents on the rotator for three minutes at 500 rotations per minute or until all of the wells have turned from green to yellow.Then, read the optical density of the samples and use the micro plate software to convert the cortisol levels in each sample to picograms per milligram. In this representative analysis, cortisol levels tended to be higher in fin samples washed with isopropanol than in those washed with water, but no significant differences in fin cortisol levels were observed among sturgeon species. There was no significant interaction between washing solvents and sturgeon species.Examination of cortisol from H.Huso jawbones to determine whether sturgeon jawbones might be used as an alternative matrix to fins revealed that the washing solvent had no significant effect on the cortisol level measured in H.Huso's sturgeon. Further, the data revealed a high similarity among the fins of three sturgeon species tested and in H.Huso jawbones. It is important to use the bead beater to grind the samples into fine powder and to use an appropriate commercial ELISA Assay Kit for a successful cortisol measurement.This method can also be used for detecting cortisol in teeth and other hard matrices as cortisol level measurements provide important information about how the environment affects animal biology. The method could be used as a new approach to assessing cortisol levels in human child milk teeth and in pediatric endocrinology, psycho biology, behavioral, archeological, and forensic studies.

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JoVE Journal

Environment

8.1K 조회수.  Kangwon National University. 우리의 방법은 새로운 비 플롯 및 머리 행렬에서 코티솔을 측정하기위한 수많은 미래 연구를위한 플랫폼이 될 수있는 새로운 대체 스트레스 지표를 소개합니다. 이 기술의 장점은 단순함과 도살 된 동물에서도 스트레스 수준을 평가하는 데 사용할 수있는 능력입니다. 이 기술을 사용하여 높은 코티솔 수준과 건강한 동물 또는 인간 사이의 관계를 확립하는 것은 일부 질병의 ...