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
2. Fin preparation for cortisol extraction
3. Fin cortisol analysis
4. Fin cortisol detection
5.Statistical analysis
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 analysis5,6 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.
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 |
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.
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.
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.