ACTH trials were performed at the University of Alaska Fairbanks in February, 2008. Five adult male caribou and 5 adult female caribou were injected with 2 IU/kg ACTH, IM. One male and 1 female caribou were injected with saline and served as controls.

Hair was collected from three body regions (neck, shoulder and rump) before and one week after injection by shaving as close to the skin as possible with electric clippers. 100 hairs were also plucked from each body region in each animal for growth stage analyses (trichograms and special staining techniques)(12).

Intra assay coefficient of variation (CV) 6.04%, inter assay CV 18.39%, detection limit 0.32 pg cortisol/ mg hair and extraction efficiency (spike recovery) 102.16% +/- 5.20%.

Hair cortisol concentration in serially diluted caribou hair extracts and cortisol standards provided with the ELISA kit were highly correlated (r2=0.998, P<0.001).

HAIR CORTISOL CONCENTRATION: A POTENTIAL NON-INVASIVE BIOMARKER OF LONG-TERM STRESS IN FREE RANGING CARIBOU

Bryan J. Macbeth1, Marc R.L. Cattet 1, 2, Perry S. Barboza 3, Noah T. Ashley 4, Christine Cuyler 5, Mick Gauthier 6 and David M. Janz1

1Western College of Veterinary Medicine and 2Canadian Cooperative Wildlife Health Centre, University of Saskatchewan, Saskatoon, SK, Canada. 3 Department of Biology and Wildlife and Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, USA.4 Department of Biological Sciences, University of Alaska Anchorage, Anchorage, AK, USA. 5 Greenland Institute of Natural Resources, Nuuk, Greenland. 6 Ontario

Ministry of Natural Resources, Cochrane, ON, Canada.

Background Results Continued

Objectives

Materials and Methods

Results

Conclusions and Recommendations A minimum sample quantity of 100 mg hair (approximately a 2 cm by 2cm plucked or shaved patch of hair) is required for cortisol analysis. Assay performance in Rangifer was consistent with our previous work in other wildlife.

A wide range of HCC was determined in n=125 caribou from seven free ranging and one captive herds (mean 2.22 pg/mg, range 0.60-6.90 pg/mg).

HCC varied among body regions in caribou. Owing to its low intra-region variability, we recommend collecting only shoulder or rump hair for HCC analysis in this species.

HCC in quiescent hair was not influenced by adrenocorticotropic hormone (ACTH) challenge, a procedure that mimics short-term stress. HCC did not vary with sex class in captive animals. However, HCC varied with age class in some free ranging herds. HCC is a promising measure of long-term stress in Rangifer.

Further work to identify factors affecting HCC in caribou is warranted. HCC analysis should be expanded to include all age, sex and reproductive classes of free ranging caribou. Future studies should also explore the association of HCC with other measures of stress and health (e.g. parasite load, lactation, body condition, Brucella suis type 4).

References1. Wittmer, H.U., B.N. McLellan, R. Serrouya and C.D. Apps. 2007. Changes in landscape composition influence the decline of a threatened woodland caribou population. Journal of Animal Ecology 76:568-579. 2. Whitfield, P.H and D. Russell. 2005. Recent changes in seasonal variations of climate within the range of northern caribou populations. Rangifer Special Issue No. 16: 11-18.3. Habib, K. E., P.W. Gold and G.P. Chrousos. 2001. Neuroendocrinology of stress. Endocrinology and Metabolism Clinics of North America, 30:695-728.4. Sakkinen, H., J. Tornberg, P.J. Goddard, E. Eloranta, E. Ropstad and S. Saarela. 2004. The effect of blood sampling method on indicators of physiological stress in reindeer. Domestic Animal Endocrinology 26: 87-98.5. Keay, J.M., J. Singh, M.C. Gaunt and T. Kaur. 2006. Fecal glucocorticoids and their metabolites as indicators of stress in various mammalian species: a literature review. Journal of Zoo and Wildlife Medicine, 37:234-244. 6. Constable, S., A. Parslow, G. Dutton, T. Rogers and C. Hogg. 2006. Urinary cortisol sampling: A non-invasive technique for examining cortisol concentrations in the Weddell seal, Leptonychotes weddellii. Zoo Biology, 25:137-144.7. Millspaugh, J.J., B.E. Washburn, M.A. Milanick, J. Beringer, L.P. Hansen and T.M. Meyer. 2009. Non-invasive techniques for stress assessment in White-tailed deer. Wildlife Society Bulletin, 30:899-907.8. Pragst, F. and M.A. Balikova. 2006. State of the art in hair analysis for detection of drug and alcohol abuse. Clinica Chimica Acta, 370:17-49.9. Davenport, M.D., S. Tiefenbacher, C. K. Lutz, M.A. Novak and J.S. Meyer. 2006. Analysis of endogenous cortisol concentrations in the hair of rhesus macaques. General and Comparative Endocrinology, 147: 255-261.10. Accorsi, P.A., E. Carloni, P. Valsecchi, R. Viggiani, M. Gamberoni, C. Tamanini and E. Seren. 2008. Cortisol determination in hair and faeces from domestic cats and dogs. General and Comparative Endocrinology 155:398-402.11. Kirschbaum, C. A. Tietze, N. Skoluda and L. Dettenborn. 2009. Hair as a retrospective calendar of cortisol production: increased cortisol incorporation into hair in the third trimester of pregnancy. Psychoneuroendocrinology 34:32-37.12. Baden, H.P., J. Kubilus and L. Baden. 1979. A stain for plucked anagen hairs. Journal of the American Academy of Dermatology 1: 121-122.

Acknowledgements: This project was funded by the CircumArctic Rangifer Monitoring and Assessment Network (CARMA) and the Natural Sciences and Engineering Research Council of Canada. Photo of caribou © J. Eastcott and Y. Momatiuk.

Hair was obtained from fresh caribou hides (n=2) collected as part of a research hunt on Southhampton Island, Canada, from captive caribou (n=12) maintained at the University of Alaska Fairbanks, Fairbanks, AK, USA, from caribou killed during two research collections in Greenland (n=97) and caribou captured as part of ongoing research programs in Ontario, Canada (n=15).

Human caused environmental change is believed to negatively affect the sustainability of many wildlife populations. Increasingly, caribou (Rangifer tarandus) are threatened by anthropogenic landscape change and the effects of global warming (1,2).

Long-term physiological stress in individual animals may be the predominant mechanism linking environmental change with impaired wildlife population health. While the physiological response to short-term stress is adaptive, long-term stress may lead to a pathological syndrome of immunosuppression, decreased reproduction and diminished growth which may eventually manifest at the population level as reduced rates of survival and reproduction (3).

Biological markers of long-term physiological stress should be measurable in individuals before health effects are apparent at the population level.

Cortisol, the primary glucocorticoid of most mammals, is a key component of the physiological stress response and has been used extensively to measure stress in the plasma, urine, feces and saliva of many wild species (4,5,6,7).

Plasma collection requires capture, immobilization and invasive sampling. Urine and fecal samples are especially prone to contamination and degradation. The collection of saliva may be impractical in free ranging wildlife. More importantly, all four matrices can only assess stress in the time frame of a few hours to a few days (4,5,6,7).

Hair is a relatively stable medium which can be collected non-invasively and is known to incorporate blood borne hormones and xenobiotics during its active growth phase lasting weeks to months (8).

Hair cortisol concentration (HCC) has recently been validated as a biomarker of long-term stress in a variety of species (9,10,11) and building on techniques we have developed for use in free ranging grizzly bears (Ursus arctos) and polar bears (Ursus maritimus), we are investigating HCC as a sensitive, reliable and non-invasive measure of long-term stress in caribou.

To develop, validate and apply a technique for the determination of HCC in free ranging caribou.

To examine the effect of acute stress (adrenocorticotropic hormone (ACTH) challenge) on HCC in a controlled experiment.

Caribou Herd Designation Median Hair Cortisol (pg/mg)

Hair Cortisol Range

(pg/mg)

Sample Size

Akia-Manittisoq 2.14 0.60-6.90 n=48

Kangerlussuaq-Sisimut 2.30 1.21-3.98 n=49

3. ACTH Challenge and Growth Stage Analyses:

1. Measures of Accuracy, Precision, Sensitivity and Specificity:

2. Body Regions and Sex Classes in Captive Adult Caribou:

1. Sources of Hair :

Samples were washed three times (3 minutes per wash with 10.0 ml methanol /100 mg hair) and dried hair was ground to a fine powder using a Retsch MM301 ball mill (3 minutes /100 mg hair at 30HZ).

Steroids were extracted by spinning samples for 24 hours on a slow rotator with 1.0 ml methanol / 50 mg powdered hair. Extracted samples were centrifuged, supernatant was collected, and solvent evaporated under a gentle stream of Nitrogen gas at 38 oC.

Samples were reconstituted with 0.4 ml of phosphate buffer for 12 hours at 4 oC in the dark and HCC assayed with a commercial enzyme linked immunoassay kit (Oxford EA-65 Cortisol EIA kit, Oxford Biomedical, MI, USA).

Figure 1. Hair cortisol concentration among body regions (neck, shoulder and rump) in captive caribou. Data are presented as mean +/- SD of hair cortisol determinations in n=12 caribou. Hair cortisol concentration varied among body regions (One-way repeated measures ANOVA, P<0.001). Data marked with different letters are significantly different from each other (Tukey-Kramer, P≤0.05).

Figure 4. Growth stage analysis in hair collected post ACTH (or saline) administration from 12 caribou. Club shaped hair bulbs (e.g. #) are indicative of non-growing hair. Most hairs from all body regions in all animals were club shaped. These findings are consistent with known patterns of hair growth in caribou as samples were collected in late winter when all animals should have mature hair coats. Cortisol from the systemic circulation can only enter hair during periods of active growth suggesting that changes in shoulder HCC observed post ACTH were not due to ACTH administration.

Figure 3. Hair cortisol concentration pre and post ACTH administration in neck, shoulder and rump hair of caribou. Data are presented as mean +/- SD of hair cortisol determinations in n=10 caribou (black). There was no significant difference in the cortisol concentration of caribou neck hair (Paired samples t-test, t (9)=2.213, P=0.055) or rump hair (Paired samples t-test, t (9)=2.195, P=0.056) pre or post ACTH administration. *There was a significant increase in hair cortisol concentration in shoulder hair post ACTH administration (Paired Samples t-test, t (9)=10.581, P<0.001). In all body regions the pattern of HCC pre and post ACTH administration followed that in n=2 control animals (red) injected with saline. Variability in HCC within body regions may best explain the differences observed in shoulder hair.

Figure 2. Hair cortisol concentration in adult male and adult female captive caribou. There was no difference (Independent samples t-test, t (10) =0.287, P=0.780) in hair cortisol concentration among male and female caribou. Data are presented as the mean +/- SD of cortisol determinations in n=6 caribou of each sex class.

5. Free Ranging Caribou: Ontario

Figure 5. Hair cortisol concentration in free ranging caribou from the Akia-Manittisoq and Kangerlussuaq-Sisimut herds in Greenland. Data are presented as the mean +/- SD of hair cortisol determinations in female caribou and calves. Adult females were > 3 years of age, subadult females were 1-3 years old and calves were 9-10 months old. No adult or sudadult males were harvested during these research collections.

In both herds, hair cortisol concentration varied among age classes (Akia-Manittisoq: One-way ANOVA, P=0.010 [n=34 adults, n=7 subadults and n=7 calves] and Kangerlussuaq-Sisimut: One-way ANOVA, P=0.021 [n=36 adults, n=4 subadults, n=9 calves]). Significant differences (Tukey-Kramer, P≤0.05) in hair cortisol are designated by a>b. There is no difference (Tukey-Kramer, P>0.05) in data designated ab from that designated a or b. In both herds, hair cortisol was highest in calves of the year.

Figure 6. Hair cortisol concentration determined in 4 herds of free ranging woodland caribou in Ontario, Canada. Herd ranges are outlined in violet and designated with highlighted letters. Animals from the Fraserdale and Nagagami regions inhabit heavily disturbed landscape owing to the presence of forestry and significant infrastructure development (e.g. roads, railways and transmission lines). Onakawana animals are only impacted by a railway line on the eastern aspect of their range and have access across large rivers to untouched habitat to the north and west. Detour animals have been exposed to mines, mining exploration, forestry operations, paved highways and many seasonal roads.

4. Free Ranging Caribou: Greenland

2. Sample Processing , Steroid Extraction and Analysis:

3. ACTH Challenge:

Body RegionMean % Club Shaped

Hair BulbsMean% Broken Hairs

Number Hairs

Examined

Neck 97 3 n=1100

Shoulder 99 1 n=1200

Rump 95 6 n=1200

Caribou Herd Designation Mean Hair Cortisol (pg/mg) Hair Cortisol Range (pg/mg)

Sample Size

Fraserdale (A) 2.08 NA n=1

Nagagami (B) 2.94 1.20-4.67 n=2

Onakawana (C) 1.91 0.82-2.61 n=5

Detour (D) 1.84 0.89-3.87 n=7

#

Key:

Red: paved roads

Black: unpaved roads and railways*

Dark green: provincial parks

Lighter green: conservation reserves.

*Forestry roads not included.

D

A

B

C

Ontario