Research Note Feather corticosterone evaluated by ELISA in broilers: A potential tool to evaluate broiler welfare A. Carbajal, o. Tallo-Parra, M. Sabes-Alsina, I. Mular, and M. Lopez-Bejar Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain feathers were collected from each bird, and sex, weight, and morphological aspects of feather status were recorded. We tested the feasibility for detecting CoRT in broiler feathers by ELISA, which had never been done before, and an assay validation test was performed. No significant relationships were found between feather CoRT concentrations and physiological variables such as sex, weight, and fault bars in broilers. To our knowledge, this is the first study that uses broiler feathers as a matrix that provides a retrospective record of their hypothalamic-pituitary-adrenal activity. Results indicate that ELISA is a valid tool to detect feather CoRT levels in broilers.
Key words: corticosterone, feather, broiler, welfare, ELISA 2014 Poultry Science 93:2884–2886 http://dx.doi.org/10.3382/ps.2014-04092
INTRODUCTION At present, much effort is being directed toward the development of an objective and precise tool for animal welfare assessment (Hõrak et al., 2013). The measure of corticosterone (CORT) in feathers has been introduced as a valid and easily obtainable measure of prolonged glucocorticoid secretion (Bortolotti et al., 2008, 2009a). This measure provides integrated information of the past hypothalamic-pituitary-adrenal (HPA) axis activity (Bortolotti et al., 2008, 2009a), and thus, meaningful interpretations of bird welfare state. Since the usefulness of feather CoRT (FCORT) was described, this new matrix has been used with different goals in conservation studies of wild birds. It has been used as a tool to associate physiology with feather coloration, feather quality, and social signals (Bortolotti et al., 2009b; Lattin et al., 2011; Kennedy et al., 2012; Martínez-Padilla et al., 2013). It has also been applied to assess health condition (Harms et al., 2010; Koren et al., 2012) and to comprehend relationships between ©2014 Poultry Science Association Inc. Received April 7, 2014. Accepted July 11, 2014. 1 Corresponding author: [email protected]
habitat and animal physiology (Fairhurst et al., 2011, 2013), among other applications. Nonetheless, research in the possibilities and potential offered by this matrix is still in its beginnings, with important gaps hindering proper interpretation (Hõrak et al., 2013). Glucocorticoids in feathers have never been used before as a valid tool to assess welfare in farm broilers. The current study attempts to validate a tool for broiler welfare assessments by measuring CoRT levels in feathers, providing a stable and objective physiological record. The development of an ELISA to detect FCoRT would be advisable because, to date, RIA has been the common method used (Bortolotti et al., 2008). The assay validation will allow us to test the hypothesis that ELISA is a useful implement to detect CoRT deposited in feathers.
MATERIALS AND METHODS Fieldwork was conducted at Xert (40°31′2″ North, 0°9′37″ East, Valencia, Spain) from May 1 to June 15, 2013. The group of animals consisted of 8,000 broilers located in a 1,000 m2 industrial area and was kept under conditions regulated by the national legislation (RD 692/2010). Twenty-two commercial broilers ran-
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Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
ABSTRACT The measure of corticosterone (CoRT) in feathers has been recently recognized as a valid and easily obtainable measure of chronic glucocorticoids secretion in avian species. This measure provides meaningful interpretations of how individuals respond to environmental perturbations. The growing interest of the public toward animal-food production welfare shows the need for improving and expanding objective tools to evaluate this issue. The present study evaluates whether it is possible to detect CoRT in broiler feathers, and thus, assess if it would be a useful measure to study broiler welfare. Twenty-two broilers were randomly selected from an intensive farm. Four to 6 dorsal
2885
RESEARCH NOTE
domly selected were evaluated at the age of 44 d, when samples were collected. Four to 6 feathers from each individual were pulled from the interscapular area. All the feathers were collected the same day and individually stored at room temperature until analysis.
Feather CORT Measurement
Assay Validation The assay was validated following the essential criteria for the immunological validation: specificity, accuracy, precision, and sensitivity. Specificity was given by the dilution and parallelism test. Accuracy was calculated with the spike recovery. Precision was measured from intra- and interassay variability. Sensitivity was given by the smallest amount of unlabeled hormone distinguished and measured. Extracts from 20 birds were pooled for assay validation. Intraassay CV was calculated by running ELISA samples by duplicate. Interassay precision was assessed running the same samples in different ELISA kits by duplicate. The dilution set ratios created using a single pool and the EIA buffer were 1:1, 1:2, 1:5, and 1:10. The dilution curve, and consequently, the linearity test were calculated by using the set ratios performed. Parallelism test was assessed by comparing 2 different calibration curves. One of them was the curve created through the ELISA kit standards. The other, was performed by adding known amounts of CORT to the pool created with feather samples (modified standard). The spike recovery was assessed measuring the final recovery of CORT added to the sample pools.
Figure 1. Correlation line acquired in the linearity test of the corticosterone (CORT) ELISA validation.
the relation of the number of fault bars and values of FCORT was studied using an ANOVA.
RESULTS Assay Validation Intraassay CV was 4.4%. The CV for interassay precision was 17.2%. Figure 1 shows the correlation line acquired in the linearity test, with an R2 = 0.99. Hormone standards spiked with feather extracts produced a mean recovery percentage of 129.2 ± 49.7%. Results of the parallelism test are represented in the Figure 2. To compare the parallelism of the correlation lines, the logarithm (log10) of the FCORT concentrations was performed. The slope of the line was 0.29 for the standard curve and 0.19 for the modified one, with an R2 of 98 and 93%, respectively.
FCORT Concentrations Mean FCORT concentrations were 0.71 ± 0.55 for males and 0.76 ± 0.52 for females. All values were used to execute the statistical analysis. Sex did not affect significantly FCORT concentrations (P > 0.05; Figure 3). No significant correlation
Statistical Analyses Data obtained were analyzed using SAS software (version V.8, SAS Institute Inc., Cary, NC). A P-value lower than 0.05 was considered statistically significant. To determine if the concentration of CORT deposited on feathers differed between sexes, a Student t-test was performed. After that, a Pearson correlation was calculated between FCORT and weight variables. Finally,
Figure 2. Correlation lines performed in the parallelism test of the feather corticosterone (CORT) ELISA validation.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
The CORT was extracted from feathers using a methanol-based technique modified from that previously described by Bortolotti et al. (2008). Bortolotti et al. (2009a) previously showed that feathers with fault bars contain significantly more CORT than the adjacent sections lacking these deformities. Consequently, before feathers were minced, the number of fault bars was macroscopically recorded for each sample. Defects found consisted in the partial absence of barbs forming conspicuously marked transverse lines extending across the entire feather vane. Between 0 and 3 fault bars were recorded for each sample. The ELISA kits (Corticosterone ELISA kit; Neogen Corporation, Ayr, UK) were used to asses and quantify the CORT concentration of feather samples following manufacturer instructions.
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tive record of their HPA activity. These encouraging results beg the question as to whether this methodology can be applied in detecting FCORT differences between healthy and unhealthy animals, and thus improve their welfare state.
ACKNOWLEDGMENTS We thank Simon Bel Marzà for allowing us to perform this work at his farm and for all his help with the sample collection and management of the birds. Figure 3. Boxplot displaying differences in feather corticosterone (CORT) concentrations between sexes.
DISCUSSION Results indicate that the methodology developed in this study, that includes the hormone extraction procedure and the EIA is an efficient tool to detect the amounts of CORT deposited in broiler feathers. The FCORT can be reliably measured by using RIA, the usual method used to measure CORT in avian feathers (Bortolotti et al., 2008, 2009a,b). The use of ELISA would reduce health hazards, costs of handling radioisotopes, radioisotope license requirements, and the short stability time associated with RIA (Sink et al., 2008). Validation tests for FCORT determination were assessed because no proper ELISA validation had been done before. Intra- and interassay CV were below 10 and 20%, respectively, displaying good assay precision. Linearity and parallelism tests yielded acceptable results. This is the first time that validation tests are performed to assess the capacity of ELISA in the detection of FCORT concentrations in broiler feathers. Results obtained are promising, revealing that ELISA is feasible for FCORT analysis. No significant relationships were found between feather CORT concentrations and physiological variables such as sex, weight, and fault bars in broilers. Because fault bars can be a useful trace of stressful events, more work has to be performed to check if this feature can be a valuable indicator in broilers. To our knowledge, this is the first study that uses broiler feathers as a matrix that provides a retrospec-
Bortolotti, G. R., T. Marchant, J. Blas, and S. Cabezas. 2009a. Tracking stress: Localisation, deposition and stability of corticosterone in feathers. J. Exp. Biol. 212:1477–1482. Bortolotti, G. R., T. A. Marchant, J. Blas, and T. German. 2008. Corticosterone in feathers is a long-term, integrated measure of avian stress physiology. Funct. Ecol. 22:494–500. Bortolotti, G. R., F. Mougeot, J. Martinez-Padilla, L. M. I. Webster, and S. B. Piertney. 2009b. Physiological stress mediates the honesty of social signals. PLoS ONE 4:e4983. Fairhurst, G. D., M. D. Frey, J. F. Reichert, I. Szelest, D. M. Kelly, and G. R. Bortolotti. 2011. Does environmental enrichment reduce stress? An integrated measure of corticosterone from feathers provides a novel perspective. PLoS ONE 6:e17663. Fairhurst, G. D., M. Vögeli, D. Serrano, A. Delgado, J. L. Tella, and G. R. Bortolotti. 2013. Can synchronizing feather-based measures of corticosterone and stable isotopes help us better understand habitat-physiology relationships? Oecologia 173:731–743. Harms, N. J., G. D. Fairhurst, G. R. Bortolotti, and J. E. G. Smits. 2010. Variation in immune function, body condition, and feather corticosterone in nestling tree swallows (Tachycineta bicolor) on reclaimed wetlands in the Athabasca oil sands, Alberta, Canada. Environ. Pollut. 158:841–848. Hõrak, P., M. Männiste, R. Meitern, E. Sild, L. Saks, and T. Sepp. 2013. Dexamethasone inhibits corticosterone deposition in feathers of greenfinches. Gen. Comp. Endocrinol. 191:210–214. Kennedy, E. A., C. R. Lattin, L. M. Romero, and D. C. Dearborn. 2012. Feather coloration in museum specimens is related to feather corticosterone. Behav. Ecol. Sociobiol. 67:341–348. Koren, L., S. Nakagawa, T. Burke, K. K. Soma, K. E. Wynne-Edwards, and E. Geffen. 2012. Non-breeding feather concentrations of testosterone, corticosterone and cortisol are associated with subsequent survival in wild house sparrows. Proc. Biol. Sci. 279:1560–1566. Lattin, C. R., J. M. Reed, D. W. DesRochers, and L. M. Romero. 2011. Elevated corticosterone in feathers correlates with corticosterone-induced decreased feather quality: A validation study. J. Avian Biol. 42:247–252. Martínez-Padilla, J., F. Mougeot, J. T. García, B. Arroyo, and G. R. Bortolotti. 2013. Feather corticosterone levels and carotenoidbased coloration in common buzzard (Buteo buteo) nestlings. J. Raptor Res. 47:161–173. Sink, T. D., R. T. Lochmann, and K. A. Fecteau. 2008. Validation, use, and disadvantages of enzyme-linked immunosorbent assay kits for detection of cortisol in channel catfish, largemouth bass, red pacu, and golden shiners. Fish Physiol. Biochem. 34:95–101.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
was found between FCORT and the remainder variables, weight, and fault bars.
REFERENCES
Key words: corticosterone, feather, broiler, welfare, ELISA 2014 Poultry Science 93:2884–2886 http://dx.doi.org/10.3382/ps.2014-04092
INTRODUCTION At present, much effort is being directed toward the development of an objective and precise tool for animal welfare assessment (Hõrak et al., 2013). The measure of corticosterone (CORT) in feathers has been introduced as a valid and easily obtainable measure of prolonged glucocorticoid secretion (Bortolotti et al., 2008, 2009a). This measure provides integrated information of the past hypothalamic-pituitary-adrenal (HPA) axis activity (Bortolotti et al., 2008, 2009a), and thus, meaningful interpretations of bird welfare state. Since the usefulness of feather CoRT (FCORT) was described, this new matrix has been used with different goals in conservation studies of wild birds. It has been used as a tool to associate physiology with feather coloration, feather quality, and social signals (Bortolotti et al., 2009b; Lattin et al., 2011; Kennedy et al., 2012; Martínez-Padilla et al., 2013). It has also been applied to assess health condition (Harms et al., 2010; Koren et al., 2012) and to comprehend relationships between ©2014 Poultry Science Association Inc. Received April 7, 2014. Accepted July 11, 2014. 1 Corresponding author: [email protected]
habitat and animal physiology (Fairhurst et al., 2011, 2013), among other applications. Nonetheless, research in the possibilities and potential offered by this matrix is still in its beginnings, with important gaps hindering proper interpretation (Hõrak et al., 2013). Glucocorticoids in feathers have never been used before as a valid tool to assess welfare in farm broilers. The current study attempts to validate a tool for broiler welfare assessments by measuring CoRT levels in feathers, providing a stable and objective physiological record. The development of an ELISA to detect FCoRT would be advisable because, to date, RIA has been the common method used (Bortolotti et al., 2008). The assay validation will allow us to test the hypothesis that ELISA is a useful implement to detect CoRT deposited in feathers.
MATERIALS AND METHODS Fieldwork was conducted at Xert (40°31′2″ North, 0°9′37″ East, Valencia, Spain) from May 1 to June 15, 2013. The group of animals consisted of 8,000 broilers located in a 1,000 m2 industrial area and was kept under conditions regulated by the national legislation (RD 692/2010). Twenty-two commercial broilers ran-
2884
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
ABSTRACT The measure of corticosterone (CoRT) in feathers has been recently recognized as a valid and easily obtainable measure of chronic glucocorticoids secretion in avian species. This measure provides meaningful interpretations of how individuals respond to environmental perturbations. The growing interest of the public toward animal-food production welfare shows the need for improving and expanding objective tools to evaluate this issue. The present study evaluates whether it is possible to detect CoRT in broiler feathers, and thus, assess if it would be a useful measure to study broiler welfare. Twenty-two broilers were randomly selected from an intensive farm. Four to 6 dorsal
2885
RESEARCH NOTE
domly selected were evaluated at the age of 44 d, when samples were collected. Four to 6 feathers from each individual were pulled from the interscapular area. All the feathers were collected the same day and individually stored at room temperature until analysis.
Feather CORT Measurement
Assay Validation The assay was validated following the essential criteria for the immunological validation: specificity, accuracy, precision, and sensitivity. Specificity was given by the dilution and parallelism test. Accuracy was calculated with the spike recovery. Precision was measured from intra- and interassay variability. Sensitivity was given by the smallest amount of unlabeled hormone distinguished and measured. Extracts from 20 birds were pooled for assay validation. Intraassay CV was calculated by running ELISA samples by duplicate. Interassay precision was assessed running the same samples in different ELISA kits by duplicate. The dilution set ratios created using a single pool and the EIA buffer were 1:1, 1:2, 1:5, and 1:10. The dilution curve, and consequently, the linearity test were calculated by using the set ratios performed. Parallelism test was assessed by comparing 2 different calibration curves. One of them was the curve created through the ELISA kit standards. The other, was performed by adding known amounts of CORT to the pool created with feather samples (modified standard). The spike recovery was assessed measuring the final recovery of CORT added to the sample pools.
Figure 1. Correlation line acquired in the linearity test of the corticosterone (CORT) ELISA validation.
the relation of the number of fault bars and values of FCORT was studied using an ANOVA.
RESULTS Assay Validation Intraassay CV was 4.4%. The CV for interassay precision was 17.2%. Figure 1 shows the correlation line acquired in the linearity test, with an R2 = 0.99. Hormone standards spiked with feather extracts produced a mean recovery percentage of 129.2 ± 49.7%. Results of the parallelism test are represented in the Figure 2. To compare the parallelism of the correlation lines, the logarithm (log10) of the FCORT concentrations was performed. The slope of the line was 0.29 for the standard curve and 0.19 for the modified one, with an R2 of 98 and 93%, respectively.
FCORT Concentrations Mean FCORT concentrations were 0.71 ± 0.55 for males and 0.76 ± 0.52 for females. All values were used to execute the statistical analysis. Sex did not affect significantly FCORT concentrations (P > 0.05; Figure 3). No significant correlation
Statistical Analyses Data obtained were analyzed using SAS software (version V.8, SAS Institute Inc., Cary, NC). A P-value lower than 0.05 was considered statistically significant. To determine if the concentration of CORT deposited on feathers differed between sexes, a Student t-test was performed. After that, a Pearson correlation was calculated between FCORT and weight variables. Finally,
Figure 2. Correlation lines performed in the parallelism test of the feather corticosterone (CORT) ELISA validation.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
The CORT was extracted from feathers using a methanol-based technique modified from that previously described by Bortolotti et al. (2008). Bortolotti et al. (2009a) previously showed that feathers with fault bars contain significantly more CORT than the adjacent sections lacking these deformities. Consequently, before feathers were minced, the number of fault bars was macroscopically recorded for each sample. Defects found consisted in the partial absence of barbs forming conspicuously marked transverse lines extending across the entire feather vane. Between 0 and 3 fault bars were recorded for each sample. The ELISA kits (Corticosterone ELISA kit; Neogen Corporation, Ayr, UK) were used to asses and quantify the CORT concentration of feather samples following manufacturer instructions.
2886
Carbajal et al.
tive record of their HPA activity. These encouraging results beg the question as to whether this methodology can be applied in detecting FCORT differences between healthy and unhealthy animals, and thus improve their welfare state.
ACKNOWLEDGMENTS We thank Simon Bel Marzà for allowing us to perform this work at his farm and for all his help with the sample collection and management of the birds. Figure 3. Boxplot displaying differences in feather corticosterone (CORT) concentrations between sexes.
DISCUSSION Results indicate that the methodology developed in this study, that includes the hormone extraction procedure and the EIA is an efficient tool to detect the amounts of CORT deposited in broiler feathers. The FCORT can be reliably measured by using RIA, the usual method used to measure CORT in avian feathers (Bortolotti et al., 2008, 2009a,b). The use of ELISA would reduce health hazards, costs of handling radioisotopes, radioisotope license requirements, and the short stability time associated with RIA (Sink et al., 2008). Validation tests for FCORT determination were assessed because no proper ELISA validation had been done before. Intra- and interassay CV were below 10 and 20%, respectively, displaying good assay precision. Linearity and parallelism tests yielded acceptable results. This is the first time that validation tests are performed to assess the capacity of ELISA in the detection of FCORT concentrations in broiler feathers. Results obtained are promising, revealing that ELISA is feasible for FCORT analysis. No significant relationships were found between feather CORT concentrations and physiological variables such as sex, weight, and fault bars in broilers. Because fault bars can be a useful trace of stressful events, more work has to be performed to check if this feature can be a valuable indicator in broilers. To our knowledge, this is the first study that uses broiler feathers as a matrix that provides a retrospec-
Bortolotti, G. R., T. Marchant, J. Blas, and S. Cabezas. 2009a. Tracking stress: Localisation, deposition and stability of corticosterone in feathers. J. Exp. Biol. 212:1477–1482. Bortolotti, G. R., T. A. Marchant, J. Blas, and T. German. 2008. Corticosterone in feathers is a long-term, integrated measure of avian stress physiology. Funct. Ecol. 22:494–500. Bortolotti, G. R., F. Mougeot, J. Martinez-Padilla, L. M. I. Webster, and S. B. Piertney. 2009b. Physiological stress mediates the honesty of social signals. PLoS ONE 4:e4983. Fairhurst, G. D., M. D. Frey, J. F. Reichert, I. Szelest, D. M. Kelly, and G. R. Bortolotti. 2011. Does environmental enrichment reduce stress? An integrated measure of corticosterone from feathers provides a novel perspective. PLoS ONE 6:e17663. Fairhurst, G. D., M. Vögeli, D. Serrano, A. Delgado, J. L. Tella, and G. R. Bortolotti. 2013. Can synchronizing feather-based measures of corticosterone and stable isotopes help us better understand habitat-physiology relationships? Oecologia 173:731–743. Harms, N. J., G. D. Fairhurst, G. R. Bortolotti, and J. E. G. Smits. 2010. Variation in immune function, body condition, and feather corticosterone in nestling tree swallows (Tachycineta bicolor) on reclaimed wetlands in the Athabasca oil sands, Alberta, Canada. Environ. Pollut. 158:841–848. Hõrak, P., M. Männiste, R. Meitern, E. Sild, L. Saks, and T. Sepp. 2013. Dexamethasone inhibits corticosterone deposition in feathers of greenfinches. Gen. Comp. Endocrinol. 191:210–214. Kennedy, E. A., C. R. Lattin, L. M. Romero, and D. C. Dearborn. 2012. Feather coloration in museum specimens is related to feather corticosterone. Behav. Ecol. Sociobiol. 67:341–348. Koren, L., S. Nakagawa, T. Burke, K. K. Soma, K. E. Wynne-Edwards, and E. Geffen. 2012. Non-breeding feather concentrations of testosterone, corticosterone and cortisol are associated with subsequent survival in wild house sparrows. Proc. Biol. Sci. 279:1560–1566. Lattin, C. R., J. M. Reed, D. W. DesRochers, and L. M. Romero. 2011. Elevated corticosterone in feathers correlates with corticosterone-induced decreased feather quality: A validation study. J. Avian Biol. 42:247–252. Martínez-Padilla, J., F. Mougeot, J. T. García, B. Arroyo, and G. R. Bortolotti. 2013. Feather corticosterone levels and carotenoidbased coloration in common buzzard (Buteo buteo) nestlings. J. Raptor Res. 47:161–173. Sink, T. D., R. T. Lochmann, and K. A. Fecteau. 2008. Validation, use, and disadvantages of enzyme-linked immunosorbent assay kits for detection of cortisol in channel catfish, largemouth bass, red pacu, and golden shiners. Fish Physiol. Biochem. 34:95–101.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 8, 2015
was found between FCORT and the remainder variables, weight, and fault bars.
REFERENCES
