Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

Summary

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.

Abstract

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 ± 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.

Introduction

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^1,2, monkeys^3,4, cattle⁵, sheep⁶, and goldfish^7,8. 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 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 stage^2,7,12, 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 conditions^13,14. 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. Heimbürge 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 output^1,6,8,17; the technique is applicable to fish species without scales such as sturgeon. The technique requires slaughter of the fish, selection of appropriate washing solvents^2,4, proper grinding of samples^3,5, professional enzyme-linked immunosorbent assay (ELISA) application^5,7, 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.

Protocol

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

1. Capture the sturgeon gently using a net to minimize injury and stress.
2. Rinse the fish carefully with fresh water and then wipe the body surface with an absorbent towel prior to euthanasia.
3. 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.
4. Measure the body weight (g) and length (cm).
5. 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 ± 2.1 months, body weight = 2,700 ± 300 g, and body length = 55 ± 5 cm; Siberian sturgeon (A. baerii): age = 9.6 ± 2.4 months, body weight = 1,750 ± 250 g, and body length = 45 ± 5 cm; sevruga sturgeon (A. stellatus): age = 14 ± 1.3 months, body weight = 1,000 ± 100 g, and body length = 65 ± 5 cm.

2. Fin preparation for cortisol extraction

1. Place the fin samples (one sample per tissue: ~3 g) on laboratory weighing paper (107 mm × 210 mm) and dry at room temperature for a few days until dry.
2. Wrap samples in sheets of aluminum foil, place in labeled plastic bags, and transfer to the laboratory.
3. Store samples in a refrigerator for further use, including washing, cortisol extraction, drying, and ELISA analysis (Figure 2).

3. Fin cortisol analysis

1. Calibrate the digital analytical scale (accuracy: 0.0001) and weigh out 300 ± 10 mg samples with weighing paper on the scale pan.
2. Wash the samples.
   1. Transfer each sample into a 15 L conical polypropylene tube. Add 3 mL of isopropanol to each tube using a 5,000 µL single-channel pipette.
   2. Rotate the tubes at 80 rpm for 2.5 min to wash out cortisol and remove any potential external contamination. Repeat this procedure twice.
   3. Air-dry the washed samples at room temperature (22-28 °C) for 7 days.
   4. Repeat the washing procedure using ultrapure water as the washing agent.
3. Extract the jawbone from the body tissue using bone-cutting forceps. Apply steps 1.5-3.2.4 to the jawbone samples.
4. Weigh out (75 ± 5 mg) dried fin or jawbone samples and grind using a bead beater at 50 Hz for 32 min.
   1. Deliver 1.5 mL of methanol into each tube containing powdered fin or jawbone using a 1000 µL 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.
5. 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.
   1. Dry the samples by incubation at 38 °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.

4. Fin cortisol detection

1. Thaw the dried fin or jawbone samples at room temperature for 1.5 h prior to using the ELISA kit.
2. Add 400 µL of phosphate buffer, vortex, and centrifuge at 1,500 x g for 15 min.
3. Run each sample (25 µL) in duplicate to improve assay accuracy and reliability. Remove any data outside the standard curve as outliers.
4. Set a microplate reader to 450 nm, then set to µg dL^-1 and read the optical density of the plate.
   1. 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 (µg 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.

5.Statistical analysis

1. Divide each sample into two sub-samples prior to the washing procedure and then run in duplicate during the ELISA kit assay (2 × 2 = 4 observations per sample) to improve the power of the test and reliability of the results.
2. 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.
3. Test differences between means using Tukey's test at a significance level of p < 0.05. Accept 0.05 < p < 0.10 as evidence of a tendency rather than as a significant difference.

Representative Results

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. Please click here to view a larger version of this figure.

Figure 2. Infographic of fin cortisol analysis^5,6 performed in the laboratory. All photographs presented in the infographic abstract were taken in the laboratory. Please click here to view a larger version of this figure.

Sturgeon species (SS)					Washing solvent (WS)			P-value
	Huso huso	Acipenser baerii	Acipenser stellatus	SEM	Water	Isopropanol	SEM	SS	WS	SS×WS
Cortisol (pg mg-1)
	3.46	2.85	3.34	0.41	2.86	3.69	0.33	0.52	0.08	0.95

Table 1. Fin cortisol levels in three sturgeon species obtained using two different washing solvents.

	Washing solvent (WS)		SEM	P-value
	Water	Isopropanol
Cortisol (pg mg-1)	1.11	1.43	0.31	0.45

Table 2. Jawbone cortisol levels in beluga sturgeon (Huso huso) using two different washing solvents.

Discussion

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 life¹⁹. 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.

The role of a cortisol wash solvent is to remove external cortisol sources from the skin mucus⁹. Aerts et al.¹⁴ used distilled water to remove external cortisol contamination from fish skin; in previous studies^2,5,12,17, 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 eggs¹³, skin¹⁵, fins, and jawbones. Brossa⁷ 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.²⁰ 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^4,5,6, 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.¹⁷ 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 study⁷. Isopropanol is known to penetrate hair follicles and fish scales^4,7. 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.

Materials

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