Jalil Ghassemi Nejad1,2, Mohammad Ataallahi1, Mohammad H. Salmanzadeh3, Kyung T. Park3, Hong G. Lee2,4, Alireza Shoae5, Kyung I. Sung1, Kyu H. Park1 Atieh Rahimi6,
1- College of Animal Life Sciences, Kangwon National University
2- Team of An Educational Program for Specialists in Global Animal Science, Brain Korea 21 Plus Project, Konkuk University
3- Persian Gesture
4- Department of Animal Science and Technology, College of Animal Bioscience and Technology, Konkuk University
5- Department of Animal Science, Animal Physiology, AgResearch
6- Faculty of Agriculture, Animal Science Department, Ferdowsi University of Mashhad
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 onducted 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 nvironment.
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 ± 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80 rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 °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 ± 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 °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.
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 humans, monkeys, cattle, sheep, and goldfish. In fish species, cortisol measurements in matrices such as scales, skin mucus, feces, and blood 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 similar to the human tooth.
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 factors. The sex of sturgeon cannot be determined before years of age, and sturgeon do not have scales. Because cortisol gradually accumulates in solid matrices during the growth stage, 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 conditions. 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 results, 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. Heimbürge et al. suggested that a number of factors may influence cortisol levels including age, sex, pregnancy, season, color, and body region from which cortisol is extracted. However, little information is available on the effects of washing solvents on cortisol extraction in fish body matrices, and none on these effects in sturgeon, except for sturgeon eggs.
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-invasive approach, less data variation, and reliable output the technique is applicable to fish species without scales such as sturgeon. The technique requires slaughter of the fish, selection of appropriate washing solvents proper grinding of samples professional enzyme-linked immunosorbent assay (ELISA) application and extensive knowledge of the incorporation of cortisol sources into solid matrices.
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.
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 Table 1. Cortisol levels tended to be higher in fin samples washed with isopropanol than in those washed with water
(p = 0.089). There were no significant differences in fin cortisol levels (p = 0.525) among sturgeon species. There was no significant interaction between washing solvents and sturgeon species (p = 0.947). Washing solvent had no significant effect on cortisol level in H. huso sturgeon (p = 0.45) (Table 2). Intra-assay and inter-assay coefficients of variation were 14.15 and 7.70, respectively. The data showed high similarity among fins of the three sturgeon species (Table 1) and in H. huso jawbones (Table 2). We did not investigate correlations between cortisol levels in
jawbones and those in fins of different sturgeon species because we obtained jawbone samples only from H. huso. These relationships should be explored in a future study.
Figure 1. (A) Photograph of Huso huso sturgeon (10 years old). (B) Morphological characteristics of sturgeon.
Figure 2. Infographic of fin cortisol analysis performed in the laboratory. All photographs presented in the infographic abstract were taken in the laboratory.
Sturgeon is sometimes called a “living fossil” 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 existed for 150 million years. Acipenser species mature and
grow slowly; some (e.g., H. huso) can live for 100 years and exceed 2,000 kg in weight. Sturgeon are cartilaginous fish without scales, and are characterized by five rows of large, bony plates called scutes and tactile barbels located at the front of the mouth (Figure 1). Physiological differences between these species and other fish include decreased plasma (corticosteroid) responses to environmental stressors. Our fin cortisol measurements provide evidence that the sturgeon jawbone accumulates cortisol in proportion to circulating concentrations.
Fish display numerous responses to physical, chemical, and perceived stressors. These reactions are well known as adaptive mechanisms that allow the fish to cope with environmental disturbances and maintain a homeostatic state. If a stressor is sufficiently prolonged or serious that the fish is unable to regain homeostasis using its natural responses, then the fish may experience adverse effects, endangering its overall health and/or life.
The sex of sturgeon can be determined from around 2 years of age. Therefore, to determine whether cortisol levels and sturgeon sex are correlated, it is necessary to document long-term cortisol accumulation in fins and jawbones (as a new approach and alternative matrix) of sturgeon. This study is the first to report fin and jawbone cortisol levels in sturgeon.
he role of a cortisol wash solvent is to remove external cortisol sources from the skin mucus. Aerts et al. used distilled water to remove external cortisol contamination from fish skin; in previous studies we compared the effects of using isopropanol and water as a solvent to examine hair cortisol content.
The effects of wash solvent can vary among samples due to differences in the properties of sturgeon eggs, skin, fins, and jawbones. Brossa reported that cortisol level in scales of goldfish (Carassius auratus) remained constant when isopropanol was used as the solvent regardless of the number of washes, whereas cortisol levels varied when water was used. Our results showed that washing solvent had no effect on jaw cortisol levels. Differences between these studies include the number of washes, shaking vs. vortexing, isopropanol purity, and importantly, sensitivity or resistance of scales/skin to external liquid penetration. Ghassemi Nejad et al. demonstrated that the application of different assays such as RIA and ELISA can lead to differences in output. Steroids are more soluble in lower molecular mass alcohols (e.g., methanol) than in alcohols of higher molecular weight such as isopropanol4. Methanol extraction denatures protein by breaking non-covalent bonds, thus allowing hair cortisol release. Methanol also modifies hormone structure by breaking non-covalent bonds, resulting in the release of cortisol from tissues. To effectively homogenize sturgeon fins and jawbones prior to methanol extraction, a bead beater can be used to efficiently break down the tissue structure.
This procedure requires time to completely grind fin and jawbone samples; therefore, the process must be repeated to ensure complete pulverization and homogenization prior to cortisol extraction. Slow rotation for 18 h permits the gradual removal of cortisol by washing.
As suggested in previous studies of mammal hair external or internal sources of cortisol content in fins and jaws, other than blood, should not be neglected. Although this study was not specifically designed to investigate how cortisol diffuses from blood to fins or jaws, it highlights the need to expand our knowledge of this process to better interpret fluctuations in cortisol from these matrices. The properties of fins and jawbones differ from those of scales and skin. Bussy et al. quantified cortisol levels in lake sturgeon (A. fulvescens) eggs to investigate environmental effects on maternal physiological condition and egg quality.
They used methyl tert-butylether (MTBE), ethyl acetate (AcOEt) MTBE, and diethyl ether (Et2O) as washing solvents and concluded that ethyl acetate was the best extraction solvent in terms of recovery and matrix effect. In the present study, isopropanol removed greater amounts of external cortisol from skin mucus during washing, leading to a slight overestimation of cortisol from sturgeon fins, which must be carefully considered when interpreting extraction results. It is possible that isopropanol was able to wash out the skin of the fin, as has been reported in a previous study. Isopropanol is known to penetrate hair follicles and fish scales. The results of the present study indicate that the choice of solvent had no significant effect on cortisol levels, suggesting that cortisol extraction may be more difficult in some parts of the fin than in others using ultrapure water; in such cases, isopropanol may be used as an alternative.
This study demonstrated the applicability of the jawbone as a novel matrix for the reliable indication of stress in sturgeon. H. huso jawbone cortisol values were similar to those extracted from fins of the same species; future studies should confirm this result among different species, ages, and sexes via correlation analysis. A low number of fish was used in the current study due to the high cost of sturgeon; we attempted to overcome this limitation by testing each sample twice and also duplicating methanol extraction for the ELISA. Using quadruple multiplication could increase the power of the test to cover the low number of samples.
We conclude that the type of washing solvent moderately affected cortisol extraction from fins, but not jaws, of sturgeon. Before generalizing the conclusion of this study and to validate these results, further research using different species and solvents should be conducted. The present work provides evidence that the sturgeon jawbone can be applied as an alternative matrix in future studies using cortisol as an index of stress in sturgeon. The suitability of ELISA for fin and jawbone cortisol measurement was also demonstrated in the present study. Future research should
focus on two aspects: 1) determining the correlation between cortisol levels in jawbones and those in fins of sturgeon and 2) harvesting matrix samples for cortisol measurement from older fish and their caviar to determine long-term stress levels in different sturgeon species over the lifespan.
The authors have no conflicts of interest to disclose.
This work was conducted with the support of the Cooperative Research Program for Agriculture Science & 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 & 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 video component of this article can be found at https://www.jove.com/video/59961/
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