Assessment of Commercially Available Immunoassays to Measure Glucocorticoid Metabolites in African Grey Parrot (Psittacus Erithacus) Droppings: A Ready Tool for Non-Invasive Monitoring of Stress
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Birds
2.2. Sample Collection
2.3. HLR Count
2.4. Methods Assessment and Selection
2.5. Analytical Validation of the Selected Procedure
2.6. Biological Assessment of the Procedure and Statistical Procedures
3. Results
3.1. Methods Assessment and Selection
3.1.1. Extraction Buffers Influence on EIA Key Parameters
3.1.2. Corticosterone Recovery in Extraction Buffers
3.1.3. Corticosterone “Virtual Recovery” in African Grey Parrots’ Droppings
3.1.4. Relative Accuracy of Each Methods Combination
3.2. Procedure Selection and Analytical Validation
3.3. Biological Assessment of the Selected Method
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Kit Provider | Enzo Life Sciences | Cayman Chemical | ImmunoDiagnostic Systems |
---|---|---|---|
Antibody | Sheep polyclonal corticosterone antibody | Corticosterone specific sheep antiserum | Polyclonal Corticosterone antibody |
Detection level | 26.99 pg/mL | 30 pg/mL | 0.55 ng/mL |
Compounds and % of cross-reactivity | Deoxycorticosterone 28.6% Progesterone 1.7% Testosterone 0.13% Tetrahydrocorticosterone 0.28% Aldosterone 0.18% Cortisol 0.046% Pregnenolone < 0.03% Estradiol < 0.03% Cortisone < 0.03% 11-dehydrocorticosterone acetate < 0.03% | 11-Dehydrocorticosterone 11% Progesterone 7% Cortisol 0.31% Aldosterone 0.17% Testosterone 0.06% Pregnenolone 0.3% 5α-DHT 0.02% Androstenedione 0.1% Cortisone < 0.1% DHEA < 0.1% DHEA-S < 0.1% | 11-Dehydrocorticosterone 6.60% 11-Deoxycorticosterone 5.93% Progesterone 1.39% Cortiso l 0.85% Prednisolone 0.60% 21-Deoxycortisol 0.34% 5α-Pregnan-3, 20-dione 0.21% Tetrahydrocortisone < 0.07% Dexamethasone 0.07% DHEA < 0.07% Prednisone < 0.07% Pregnantriol < 0.07% 20β-Hydroxyprogesterone < 0.07% 4-Pregnen-20α-ol-3-one < 0.06% Oestriol < 0.06% Oestradiol < 0.06% Oestrone < 0.06% Pregnenolone < 0.06% 17α-Hydroxypregnolone < 0.05% Cortisone 0.05% Testosterone 0.02% 11-Desoxycortisol 0.02% Aldosterone 0.02% 17α-Hydroxyprogesterone 0.01% Tetrahydrocortisol 0.01% |
Initial Sample (Mass in g) a | Sub-Sample Codes | Pre-Extraction Treatment | Dropping Mass (Fresh or Dry) before Extraction (g) | Extraction Buffer (Volume in mL) |
---|---|---|---|---|
S1 (13.78) | S1DE | Dry | 0.84 | 60% ethanol (8.4) |
S1DM | 0.82 | 60% methanol (8.2) | ||
S1FE | Fresh | 3.44 | 60% ethanol (34.4) | |
S1FM | 3.44 | 60% methanol (34.4) | ||
S2 (7.03) | S2DE | Dry | 0.45 | 60% ethanol (4.5) |
S2DM | 0.43 | 60% methanol (4.3) | ||
S2FE | Fresh | 1.74 | 60% ethanol (17.4) | |
S2FM | 1.74 | 60% methanol (17.4) | ||
S3 b (7.03) | S3DE | Dry | 0.42 | 60% ethanol (4.2) |
S3DM | 0.41 | 60% methanol (4.1) | ||
S3FE | Fresh | 1.74 | 60% ethanol (17.4) | |
S3FM | 1.74 | 60% methanol (17.4) |
Kit Provider | Enzo Life Sciences | Cayman Chemical | ImmunoDiagnostic Systems | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
OD of normal a Bo | 0.880 | 1.391 | 1.149 | |||||||||
OD of normal a Blk | 0.170 | 0.122 | 0.063 | |||||||||
Extraction buffer | 60% ethanol | 60% methanol | 60% ethanol | 60% methanol | 60% ethanol | 60% methanol | ||||||
Working dilution | 1:2 | 1:4 | 1:2 | 1:4 | 1:2 | 1:4 | 1:2 | 1:4 | 1:2 | 1:4 | 1:2 | 1:4 |
OD of Bo measured in extraction buffer | 0.799 | 0.936 | 0.982 | 0.978 | 0.747 | 0.923 | 1.054 | 1.178 | 0.888 | 0.873 | 1.052 | 1.046 |
OD of Blk measured in extraction buffer | 0.174 | 0.176 | 0.173 | 0.173 | 0.120 | 0.120 | 0.118 | 0.120 | 0.062 | 0.060 | 0.060 | 0.060 |
EIA Kit | Extraction Buffer | Working Dilution | Expected a Corticosterone Concentration (ng/mL) | Measured Corticosterone Concentration (ng/mL) | Recovery (%) |
---|---|---|---|---|---|
ELS | 60% ethanol | 1:2 | 0.80 | 0.09 | 11 |
1:4 | 0.10 | 13 | |||
60% methanol | 1:2 | <0.03 | ND | ||
1:4 | 0.11 | 13 | |||
CCC | 60% ethanol | 1:2 | 0.13 | 0.32 | 258 |
1:4 | 0.21 | 170 | |||
60% methanol | 1:2 | 0.13 | 101 | ||
1:4 | 0.11 | 88 | |||
IDS | 60% ethanol | 1:2 | 16.40 | 8.21 | 50 |
1:4 | 14.71 | 89 | |||
60% methanol | 1:2 | 4.16 | 25 | ||
1:4 | 11.68 | 71 |
EIA Kit | Sub-Sample Code (n = 2 or 3) | Working Dilution | Measured GCM Concentration in S2 (ng/g Droppings) | Expected GCM Concentration in S3 (ng/g Droppings) | Measured GCM Concentration in S3 (ng/g Droppings) | Recovery (%) |
---|---|---|---|---|---|---|
ELS | SnDE | 1:2 | 34.00 | 34.62 | 29.15 | 84 |
1:4 | 56.14 | 56.76 | 47.70 | 84 | ||
SnFE | 1:2 | 30.48 | 31.10 | 29.79 | 96 | |
1:4 | 47.93 | 48.55 | 47.13 | 97 | ||
SnDM | 1:2 | 47.17 | 47.79 | 58.73 | 123 a | |
1:4 | 79.53 | 80.15 | 99.59 | 124 a | ||
SnFM | 1:2 | 29.02 | 29.64 | 25.65 | 87 | |
1:4 | 64.04 | 64.66 | 50.55 | 78 a | ||
CCC | SnDE | 1:2 | ND | ND | 104.61 | ND |
1:4 | 76.67 | 77.29 | 64.9 | 84 | ||
SnFE | 1:2 | 143.55 | 144.17 | 138.77 | 96 | |
1:4 | 99.14 | 99.76 | 97.69 | 98 | ||
SnDM | 1:2 | 59.62 | 60.24 | 66.00 | 110 | |
1:4 | 52.25 | 52.87 | 65.12 | 123 a | ||
SnFM | 1:2 | 66.59 | 67.21 | 76.56 | 114 | |
1:4 | 71.78 | 72.40 | 72.65 | 100 | ||
IDS | SnDE | 1:2 | 582.23 | 582.85 | 488.73 | 84 |
1:4 | 485.71 | 486.33 | 485.30 | 100 | ||
SnFE | 1:2 | 898.81 | 899.43 | 1063.46 | 118 | |
1:4 | 684.06 | 684.68 | 702.36 | 103 | ||
SnDM | 1:2 | 368.91 | 369.53 | 408.95 | 111 | |
1:4 | 337.61 | 338.23 | 360.84 | 107 | ||
SnFM | 1:2 | 454.38 | 455.00 | 440.72 | 97 | |
1:4 | 185.02 | 185.64 | 219.13 | 118 |
EIA Kit | Sub-Sample Code (n = 1 or 2) | Working Dilution | Measured GCM Concentration in S1 (ng/g Droppings) | Measured GCM Concentration in S2 (ng/g Droppings) | Recovery (%) |
---|---|---|---|---|---|
ELS | SnDE | 1:2 | 34.76 | 34.00 | 102 |
1:4 | 52.83 | 56.14 | 94 | ||
SnFE | 1:2 | 28.77 | 30.48 | 94 | |
1:4 | 52.41 | 47.93 | 109 | ||
SnDM | 1:2 | 47.98 | 47.17 | 102 | |
1:4 | 79.71 | 79.53 | 100 | ||
SnFM | 1:2 | 34.96 | 29.02 | 120 | |
1:4 | 65.15 | 64.04 | 102 | ||
CCC | SnDE | 1:2 | ND | ND | ND |
1:4 | 67.76 | 76.67 | 88 | ||
SnFE | 1:2 | 125.74 | 143.55 | 88 | |
1:4 | 90.66 | 99.14 | 91 | ||
SnDM | 1:2 | 65.67 | 59.62 | 110 | |
1:4 | 57.09 | 52.25 | 109 | ||
SnFM | 1:2 | 69.35 | 66.59 | 104 | |
1:4 | 70.56 | 71.78 | 98 | ||
IDS | SnDE | 1:2 | 569.91 | 582.23 | 98 |
1:4 | 470.51 | 485.71 | 97 | ||
SnFE | 1:2 | 1048.88 | 898.81 | 117 | |
1:4 | 601.18 | 684.06 | 88 | ||
SnDM | 1:2 | 376.34 | 368.91 | 102 | |
1:4 | 334.55 | 337.61 | 99 | ||
SnFM | 1:2 | 430.53 | 454.38 | 95 | |
1:4 | 170.15 | 185.02 | 92 |
Parrot Code | Sex | HLR | Mass of Dry Droppings (g) | GCM Concentration (ng/g) |
---|---|---|---|---|
G1 | M | 0.99 | 0.22 | ND |
G2 | M | 1.12 | 0.27 | 44.25 |
G3 | M | 1.22 | NC | ND |
G4 | M | 1.53 | 0.11 | 40.80 |
G5 | M | 2.08 | 0.18 | 94.50 |
G6 | M | 0.82 | 0.12 | 48.79 |
G7 | M | 0.58 | 0.16 | 73.00 |
G8 | F | 1.49 | 0.38 | 81.63 |
G9 | M | 1.19 | 0.15 | 44.05 |
G10 | M | 1.19 | 0.40 | 36.53 |
G11 | F | 1.30 | 0.18 | 93.38 |
G12 | F | 1.11 | 0.03 | 66.43 |
G13 | M | 2.49 | 0.11 | 67.43 |
G14 | F | 1.23 | 0.20 | 69.64 |
G15 | M | 0.34 | 0.11 | 36.41 |
G16 | M | 0.86 | 0.12 | 54.37 |
G17 | M | 2.19 | 0.03 | ND |
G18 | M | 1.51 | 0.06 | 66.43 |
G19 | F | 1.06 | 0.33 | 36.78 |
G20 | F | 2.03 | NC | ND |
G21 | F | 0.85 | 0.19 | 33.08 |
G22 | M | 3.93 | 0.06 | 104.21 |
G23 | M | 0.90 | 0.22 | 104.29 |
G24 | F | 2.08 | 0.26 | 48.67 |
G25 | F | 0.81 | 0.24 | 26.73 |
G26 | F | 0.83 | 0.14 | 45.91 |
G27 | F | 1.58 | 0.07 | 107.27 |
G28 | F | 0.83 | 0.19 | 69.58 |
G29 | F | 1.69 | 0.14 | 88.09 |
G30 | M | 0.57 | 0.14 | 41.93 |
G31 | F | 1.99 | 0.09 | 97.64 |
G32 | F | 2.12 | 0.29 | 59.19 |
G33 | M | 0.77 | 0.15 | 75.33 |
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Bienboire-Frosini, C.; Alnot-Perronin, M.; Chabaud, C.; Asproni, P.; Lafont-Lecuelle, C.; Cozzi, A.; Pageat, P. Assessment of Commercially Available Immunoassays to Measure Glucocorticoid Metabolites in African Grey Parrot (Psittacus Erithacus) Droppings: A Ready Tool for Non-Invasive Monitoring of Stress. Animals 2018, 8, 105. https://doi.org/10.3390/ani8070105
Bienboire-Frosini C, Alnot-Perronin M, Chabaud C, Asproni P, Lafont-Lecuelle C, Cozzi A, Pageat P. Assessment of Commercially Available Immunoassays to Measure Glucocorticoid Metabolites in African Grey Parrot (Psittacus Erithacus) Droppings: A Ready Tool for Non-Invasive Monitoring of Stress. Animals. 2018; 8(7):105. https://doi.org/10.3390/ani8070105
Chicago/Turabian StyleBienboire-Frosini, Cécile, Muriel Alnot-Perronin, Camille Chabaud, Pietro Asproni, Céline Lafont-Lecuelle, Alessandro Cozzi, and Patrick Pageat. 2018. "Assessment of Commercially Available Immunoassays to Measure Glucocorticoid Metabolites in African Grey Parrot (Psittacus Erithacus) Droppings: A Ready Tool for Non-Invasive Monitoring of Stress" Animals 8, no. 7: 105. https://doi.org/10.3390/ani8070105
APA StyleBienboire-Frosini, C., Alnot-Perronin, M., Chabaud, C., Asproni, P., Lafont-Lecuelle, C., Cozzi, A., & Pageat, P. (2018). Assessment of Commercially Available Immunoassays to Measure Glucocorticoid Metabolites in African Grey Parrot (Psittacus Erithacus) Droppings: A Ready Tool for Non-Invasive Monitoring of Stress. Animals, 8(7), 105. https://doi.org/10.3390/ani8070105