Volume 5 • 2017 10.1093/conphys/cox021
Toolbox
Compatibility of preparatory procedures for the
analysis of cortisol concentrations and stable
isotope (δ13C, δ15N) ratios: a test on brown bear
hair
Agnieszka Sergiel1, Keith A. Hobson2,3, David M. Janz4, Marc Cattet5,6, Nuria Selva1, Luciene Kapronczai7,
Chantel Gryba2 and Andreas Zedrosser8,9,*
1
Institute of Nature Conservation, Polish Academy of Sciences, 31120 Krakow, Poland
2
Environment Canada, 11 Innovation Blvd., Saskatoon, Saskatchewan, Canada S7N 3H5
3
Department of Biology, University of Western Ontario, London, Ontario, Canada N6A 5B7
4
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4
5
RGL Recovery Wildlife Health & Veterinary Services, Saskatoon, Saskatchewan, Canada S7H 4A6
6
Department of Veterinary Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaS7N 5B4
7
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3
8
Department of Natural Sciences and Environmental Health, University College of Southeast Norway, 3800 Bø, Norway
9
Institute for Wildlife Biology and Game Management, University for Natural Resources and Life Sciences, 1180 Vienna, Austria
*Corresponding author: Department of Natural Sciences and Environmental Health, University College of Southeast Norway, 3800 Bø, Norway.
Tel: +47-35-95-27-65. Email:
..............................................................................................................................................................
The measurement of naturally occurring glucocorticoids and stable isotopes of several elements has gained importance in
wildlife studies in recent decades and opened a myriad of ecological applications. Cortisol and stable isotopes equilibrate
in animal tissues over periods of integration related to the growth rate of the tissue, providing information reflecting sys-
temic cortisol secretion and dietary intake. Sample preparation shares the common step of first cleaning the sample of
external contamination. However, it is not well understood how different solvents used in sample preparation affect iso-
topic and cortisol values, and whether it is safe to follow the same procedures for both measures to optimize analyses of
the same sample. We conducted an experiment to compare different preparation protocols for the analysis of cortisol con-
centrations and stable carbon (δ13C) and nitrogen (δ15N) isotope ratios in hair. Hair samples from 12 brown bears (Ursus
arctos) were each divided into five aliquots; two aliquots were rinsed with a 2:1 chloroform:methanol (v/v) mixture with
one aliquot ground prior to cortisol analysis and the other left intact for stable isotope analyses; two aliquots were washed
with methanol with one aliquot ground prior to cortisol analysis and the other left intact for stable isotope analyses; and
one aliquot washed with methanol and ground prior to stable isotope analyses. The cortisol, δ13C and δ15N values
remained consistent following all treatments. Our results indicate that hair samples rinsed with a 2:1 chloroform:methanol
mixture or washed with methanol can be used for both types of analyses. Further, hair that has been ground in a standard
hair cortisol procedure can also be used for stable isotope analysis. This information is important for improving laboratory
efficiency and compatibility of procedures used for wildlife physiological ecology studies where concurrent measurements
of cortisol and stable isotopes in hair are required.
Key words: Dietary intake, laboratory procedures, stable isotopes, stress, Ursus arctos, wildlife physiological ecology
Editor: Steven Cooke
Received 13 December 2016; Revised 31 January 2017; Editorial Decision 26 February 2017; accepted 13 March 2017
..............................................................................................................................................................
© The Author 2017. Published by Oxford University Press and the Society for Experimental Biology. 1
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox021/3084405
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, Toolbox Conservation Physiology • Volume 5 2017
..............................................................................................................................................................
Cite as: Sergiel A, Hobson KA, Janz DM, Cattet M, Selva N, Kapronczai L, Gryba C, Zedrosser A (2017) Compatibility of preparatory procedures
for the analysis of cortisol concentrations and stable isotope (δ13C, δ15N) ratios: a test on brown bear hair. Conserv Physiol 5(1): cox021;
doi:10.1093/conphys/cox021.
..............................................................................................................................................................
samples (Cattet et al., 2014). Additionally, some studies have
Introduction made use of hair collected from museum and archeological
Levels of stress hormones and stable isotope ratios in animal specimens to determine cortisol levels (Webb et al., 2010;
tissues are increasingly recognized as important tools in stud- Bechshøft et al., 2012). However, the preparatory methods
ies on the physiology and ecology of wildlife (e.g. Kempster for the measurement of cortisol concentration differ from the
et al., 2007; Barger and Kitaysky, 2011; Deschner et al., methods used for the determination of stable isotope values,
2012; Bryan et al., 2013). Keratinous tissues, such as hair although both sets of measurements may be desired within
and feathers, offer the longest record of an animal’s circulat- the same study.
ing glucocorticoid (GC) concentrations (Bortolotti et al.,
For cortisol analysis, hair is commonly washed and
2009; Macbeth et al., 2010; Jenni-Eiermann et al., 2015).
extracted using methanol, and ground into a fine powder
Steroids are incorporated into the hair shaft and feather dur-
prior to extraction (Macbeth et al., 2010). For stable isotope
ing their growth, and therefore, GC levels in those matrices
analysis (SIA), hair samples are typically washed in a 2:1
are thought to reflect average systemic levels over the respect-
chloroform:methanol (v/v) mixture (e.g. Riofrío-Lazo and
ive growth phase by integrating baseline levels and elevated
Páez-Rosas, 2015), and then cut into small pieces (Newsome
adrenocortical secretion (Cattet et al., 2014; Jenni-Eiermann
et al., 2010) or powdered (Darimont et al., 2007; Riofrío-
et al., 2015). Hair or feather GC concentrations are increas-
Lazo and Páez-Rosas, 2015). Because cortisol concentrations
ingly applied to evaluate chronic exposure to various stres-
in blood and sweat are often far greater than those found in
sors or potentially stressful conditions (Koren et al., 2002;
hair, failure to adequately wash the external surface of hair
Davenport et al., 2006; Accorsi et al., 2008; Macbeth et al.,
shafts contaminated with these substances will falsely elevate
2010; Comin et al., 2011; Fairhurst et al., 2014).
measurement of the internal hair shaft (medullar) cortisol
Additionally, GCs in those sample matrices are stable over
concentration (e.g. Stalder and Kirschbaum, 2012). Similarly,
time and resistant to environmental exposure (Bortolotti
the single wash that is often used for stable isotope analyses
et al., 2009; Macbeth et al., 2010). Therefore, measuring GC
may not be sufficient to remove external contamination of
concentration provides a useful tool in understanding the
the hair shaft with blood and/or perspiration (Russel et al.,
role of stress in the life history, conservation physiology,
2014). On the other hand, if a solvent is too aggressive or if
health, and ecology of wildlife species (e.g. Wikelski and
hair is immersed in a solvent for prolonged time, hair shaft
Cooke, 2006; Macbeth et al., 2010; Cattet et al., 2014).
cortisol may be lost if the solvent fully penetrates the hair
Stable isotopes are also incorporated over time into hair cuticle (Eser et al., 1997). Stable isotope values may also be
and feathers (Wassenaar, 2008; Bontempo et al., 2014). affected by sample processing procedures (Font et al., 2007).
Stable nitrogen (δ15N) and carbon (δ13C) isotope ratios are Carbon composition of surface oils and waxes can be signifi-
widely employed to reconstruct and assess temporal and spa- cantly different from pure tissue composition and thus may
tial variation in diet using extant or archived materials influence stable isotope values. Therefore, in cleaning proto-
(Newsome et al., 2007; Crawford et al., 2008). These iso- cols for fixed tissue samples, volatile solvent mixtures are
topes are also used to characterize trophic niche and commu- used to remove surface lipids (Wassenaar, 2008). An import-
nity structure (Boecklen et al., 2011), elucidate animal ant question that remains is whether these preparatory proce-
migration and movements (Hobson and Wassenaar, 2008), dures used for washing and/or grinding hair can be merged
and determine individual specialization and habitat selection into a single protocol to increase the efficiency of sample pre-
(Newsome et al., 2009). Stable isotope analyses are also parations, and to reduce labor input and costs for research
employed in wildlife forensics and ecotoxicological studies projects.
(Bowen et al., 2005). Combining concurrent measures of
It is unknown whether the preparation process for δ13C
long-term stress and stable isotopes within the same study
and δ15N analysis affects the reliable measurement of cortisol
may open up a wide selection of testable ecological theories,
concentration in hair and vice versa. In this study, we com-
including relationships between diet and stress (e.g. Bryan
pared commonly used, but different, wash and sample pro-
et al., 2013; Fairhurst et al., 2014; Lafferty et al., 2015).
cessing procedures to determine their effect on cortisol and
Compared to blood, saliva, urine or feces, hair and feath- stable isotope values measured in hair. We used hair samples
ers are media that can be transported and stored at room collected from brown bears (Ursus arctos) as a model system.
temperature. The use of hair and feathers for quantifying Here, we evaluate whether different preparatory procedures
stress offers other desirable features, such as non-invasive yielded consistent results. Specifically, we test the null hypoth-
collection and retrospective analyses of stress using archived eses that (1) there are no differences in cortisol concentrations
..............................................................................................................................................................
2
Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox021/3084405
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on 07 March 2018
Toolbox
Compatibility of preparatory procedures for the
analysis of cortisol concentrations and stable
isotope (δ13C, δ15N) ratios: a test on brown bear
hair
Agnieszka Sergiel1, Keith A. Hobson2,3, David M. Janz4, Marc Cattet5,6, Nuria Selva1, Luciene Kapronczai7,
Chantel Gryba2 and Andreas Zedrosser8,9,*
1
Institute of Nature Conservation, Polish Academy of Sciences, 31120 Krakow, Poland
2
Environment Canada, 11 Innovation Blvd., Saskatoon, Saskatchewan, Canada S7N 3H5
3
Department of Biology, University of Western Ontario, London, Ontario, Canada N6A 5B7
4
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4
5
RGL Recovery Wildlife Health & Veterinary Services, Saskatoon, Saskatchewan, Canada S7H 4A6
6
Department of Veterinary Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaS7N 5B4
7
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B3
8
Department of Natural Sciences and Environmental Health, University College of Southeast Norway, 3800 Bø, Norway
9
Institute for Wildlife Biology and Game Management, University for Natural Resources and Life Sciences, 1180 Vienna, Austria
*Corresponding author: Department of Natural Sciences and Environmental Health, University College of Southeast Norway, 3800 Bø, Norway.
Tel: +47-35-95-27-65. Email:
..............................................................................................................................................................
The measurement of naturally occurring glucocorticoids and stable isotopes of several elements has gained importance in
wildlife studies in recent decades and opened a myriad of ecological applications. Cortisol and stable isotopes equilibrate
in animal tissues over periods of integration related to the growth rate of the tissue, providing information reflecting sys-
temic cortisol secretion and dietary intake. Sample preparation shares the common step of first cleaning the sample of
external contamination. However, it is not well understood how different solvents used in sample preparation affect iso-
topic and cortisol values, and whether it is safe to follow the same procedures for both measures to optimize analyses of
the same sample. We conducted an experiment to compare different preparation protocols for the analysis of cortisol con-
centrations and stable carbon (δ13C) and nitrogen (δ15N) isotope ratios in hair. Hair samples from 12 brown bears (Ursus
arctos) were each divided into five aliquots; two aliquots were rinsed with a 2:1 chloroform:methanol (v/v) mixture with
one aliquot ground prior to cortisol analysis and the other left intact for stable isotope analyses; two aliquots were washed
with methanol with one aliquot ground prior to cortisol analysis and the other left intact for stable isotope analyses; and
one aliquot washed with methanol and ground prior to stable isotope analyses. The cortisol, δ13C and δ15N values
remained consistent following all treatments. Our results indicate that hair samples rinsed with a 2:1 chloroform:methanol
mixture or washed with methanol can be used for both types of analyses. Further, hair that has been ground in a standard
hair cortisol procedure can also be used for stable isotope analysis. This information is important for improving laboratory
efficiency and compatibility of procedures used for wildlife physiological ecology studies where concurrent measurements
of cortisol and stable isotopes in hair are required.
Key words: Dietary intake, laboratory procedures, stable isotopes, stress, Ursus arctos, wildlife physiological ecology
Editor: Steven Cooke
Received 13 December 2016; Revised 31 January 2017; Editorial Decision 26 February 2017; accepted 13 March 2017
..............................................................................................................................................................
© The Author 2017. Published by Oxford University Press and the Society for Experimental Biology. 1
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox021/3084405
by Buskerud University colleg user
on 07 March 2018
, Toolbox Conservation Physiology • Volume 5 2017
..............................................................................................................................................................
Cite as: Sergiel A, Hobson KA, Janz DM, Cattet M, Selva N, Kapronczai L, Gryba C, Zedrosser A (2017) Compatibility of preparatory procedures
for the analysis of cortisol concentrations and stable isotope (δ13C, δ15N) ratios: a test on brown bear hair. Conserv Physiol 5(1): cox021;
doi:10.1093/conphys/cox021.
..............................................................................................................................................................
samples (Cattet et al., 2014). Additionally, some studies have
Introduction made use of hair collected from museum and archeological
Levels of stress hormones and stable isotope ratios in animal specimens to determine cortisol levels (Webb et al., 2010;
tissues are increasingly recognized as important tools in stud- Bechshøft et al., 2012). However, the preparatory methods
ies on the physiology and ecology of wildlife (e.g. Kempster for the measurement of cortisol concentration differ from the
et al., 2007; Barger and Kitaysky, 2011; Deschner et al., methods used for the determination of stable isotope values,
2012; Bryan et al., 2013). Keratinous tissues, such as hair although both sets of measurements may be desired within
and feathers, offer the longest record of an animal’s circulat- the same study.
ing glucocorticoid (GC) concentrations (Bortolotti et al.,
For cortisol analysis, hair is commonly washed and
2009; Macbeth et al., 2010; Jenni-Eiermann et al., 2015).
extracted using methanol, and ground into a fine powder
Steroids are incorporated into the hair shaft and feather dur-
prior to extraction (Macbeth et al., 2010). For stable isotope
ing their growth, and therefore, GC levels in those matrices
analysis (SIA), hair samples are typically washed in a 2:1
are thought to reflect average systemic levels over the respect-
chloroform:methanol (v/v) mixture (e.g. Riofrío-Lazo and
ive growth phase by integrating baseline levels and elevated
Páez-Rosas, 2015), and then cut into small pieces (Newsome
adrenocortical secretion (Cattet et al., 2014; Jenni-Eiermann
et al., 2010) or powdered (Darimont et al., 2007; Riofrío-
et al., 2015). Hair or feather GC concentrations are increas-
Lazo and Páez-Rosas, 2015). Because cortisol concentrations
ingly applied to evaluate chronic exposure to various stres-
in blood and sweat are often far greater than those found in
sors or potentially stressful conditions (Koren et al., 2002;
hair, failure to adequately wash the external surface of hair
Davenport et al., 2006; Accorsi et al., 2008; Macbeth et al.,
shafts contaminated with these substances will falsely elevate
2010; Comin et al., 2011; Fairhurst et al., 2014).
measurement of the internal hair shaft (medullar) cortisol
Additionally, GCs in those sample matrices are stable over
concentration (e.g. Stalder and Kirschbaum, 2012). Similarly,
time and resistant to environmental exposure (Bortolotti
the single wash that is often used for stable isotope analyses
et al., 2009; Macbeth et al., 2010). Therefore, measuring GC
may not be sufficient to remove external contamination of
concentration provides a useful tool in understanding the
the hair shaft with blood and/or perspiration (Russel et al.,
role of stress in the life history, conservation physiology,
2014). On the other hand, if a solvent is too aggressive or if
health, and ecology of wildlife species (e.g. Wikelski and
hair is immersed in a solvent for prolonged time, hair shaft
Cooke, 2006; Macbeth et al., 2010; Cattet et al., 2014).
cortisol may be lost if the solvent fully penetrates the hair
Stable isotopes are also incorporated over time into hair cuticle (Eser et al., 1997). Stable isotope values may also be
and feathers (Wassenaar, 2008; Bontempo et al., 2014). affected by sample processing procedures (Font et al., 2007).
Stable nitrogen (δ15N) and carbon (δ13C) isotope ratios are Carbon composition of surface oils and waxes can be signifi-
widely employed to reconstruct and assess temporal and spa- cantly different from pure tissue composition and thus may
tial variation in diet using extant or archived materials influence stable isotope values. Therefore, in cleaning proto-
(Newsome et al., 2007; Crawford et al., 2008). These iso- cols for fixed tissue samples, volatile solvent mixtures are
topes are also used to characterize trophic niche and commu- used to remove surface lipids (Wassenaar, 2008). An import-
nity structure (Boecklen et al., 2011), elucidate animal ant question that remains is whether these preparatory proce-
migration and movements (Hobson and Wassenaar, 2008), dures used for washing and/or grinding hair can be merged
and determine individual specialization and habitat selection into a single protocol to increase the efficiency of sample pre-
(Newsome et al., 2009). Stable isotope analyses are also parations, and to reduce labor input and costs for research
employed in wildlife forensics and ecotoxicological studies projects.
(Bowen et al., 2005). Combining concurrent measures of
It is unknown whether the preparation process for δ13C
long-term stress and stable isotopes within the same study
and δ15N analysis affects the reliable measurement of cortisol
may open up a wide selection of testable ecological theories,
concentration in hair and vice versa. In this study, we com-
including relationships between diet and stress (e.g. Bryan
pared commonly used, but different, wash and sample pro-
et al., 2013; Fairhurst et al., 2014; Lafferty et al., 2015).
cessing procedures to determine their effect on cortisol and
Compared to blood, saliva, urine or feces, hair and feath- stable isotope values measured in hair. We used hair samples
ers are media that can be transported and stored at room collected from brown bears (Ursus arctos) as a model system.
temperature. The use of hair and feathers for quantifying Here, we evaluate whether different preparatory procedures
stress offers other desirable features, such as non-invasive yielded consistent results. Specifically, we test the null hypoth-
collection and retrospective analyses of stress using archived eses that (1) there are no differences in cortisol concentrations
..............................................................................................................................................................
2
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