Cortisol concentration of regrown hair and hair from a previously unshorn area in dairy cows

Accepted Manuscript Cortisol concentration of regrown hair and hair from a previously unshorn area in dairy cows

Ueli Braun, Nicolas Michel, Markus R. Baumgartner, Michael Hässig, Tina Maria Binz PII: DOI: Reference:

S0034-5288(17)30020-6 doi: 10.1016/j.rvsc.2017.07.005 YRVSC 3373

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

7 January 2017 2 July 2017 8 July 2017

Please cite this article as: Ueli Braun, Nicolas Michel, Markus R. Baumgartner, Michael Hässig, Tina Maria Binz , Cortisol concentration of regrown hair and hair from a previously unshorn area in dairy cows, Research in Veterinary Science (2017), doi: 10.1016/j.rvsc.2017.07.005

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ACCEPTED MANUSCRIPT Cortisol concentration of regrown hair and hair from a previously unshorn area in dairy cows

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Ueli Brauna,*, Nicolas Michela, Markus R. Baumgartnerb, Michael Hässiga, Tina Maria Binzb

Department of Farm Animals, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, CH-

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8057 Zurich, Switzerland

Center for Forensic Hair Analytics, Zurich Institute of Forensic Medicine, University of Zurich,

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Kurvenstrasse 17, 8006 Zurich, Switzerland

* Corresponding author: Tel.: +41-1-6358241; fax: +41-1-6358904 1

ACCEPTED MANUSCRIPT E-mail address: [email protected] ABSTRACT This study determined cortisol concentrations in hair that had grown for one month and in hair from a previously unshorn area and examined the effects of calendar month, pregnancy and illness on hair cortisol concentrations in dairy cows. The study was conducted over a one-year period using 27 cows.

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Electric clippers were used to collect two hair samples per cow each month. The first sample (A sample) consisted of hair that had grown for one month in a pre-clipped area and the second sample (B sample) comprised all hair from a previously unshorn area. Liquid chromatography tandem massspectrometry was used for cortisol measurement. The overall mean concentrations for A and B

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samples did not differ. Cortisol concentrations of A samples were significantly higher in the winter (0.86  0.37 pg/mg) than in the fall (0.67  0.33 pg/mg). The hair cortisol concentration in A samples

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increased during pregnancy and the maximum concentration of 1.40  1.08 pg/mg hair in the month

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of calving was significantly higher than the concentration measured in the first month (0.66  0.32 pg/mg). The findings show that the effect of short-term stressors such as parturition on hair cortisol

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previously unshorn area.

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concentrations are more easily detected in hair that has grown for one month than in hair from a

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Keywords: Stress; Cattle; Hair cortisol concentration; Pregnancy; Illness

1. Introduction

Hair cortisol concentration has evolved as a novel biomarker for chronic stress in humans (Meyer and Novak, 2012; Russel et al., 2012). Chronic stress caused by conditions such as chronic pain (Van Uum et al., 2008) in humans leads to increased cortisol concentrations in hair. During hair growth cortisol diffuses passively from the capillaries into the hair shaft (Pragst and Balikowa, 2006)

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ACCEPTED MANUSCRIPT where it is stored (Wosu et al., 2013). Alternatively, cortisol may be incorporated into hair during keratinisation, or may be taken up by cells in the sebaceous glands and secreted onto emerging fibres in lipids (Burnard et al. 2017). Strenuous physical exercise may increase hair cortisol concentration in humans, and thorough washing of human hair treated with a hydrocortisone solution for 60 minutes did not reduce its cortisol concentration (Russell et al. 2014). There are conflicting reports on cortisol

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concentrations in predominantly catagen- and telegon-phase hair: One study described decreasing concentrations in human hair (Kirschbaum et al., 2008), whereas other studies in humans (Sharpley et al., 2010), dogs (Bennett and Hayssen, 2010) and rhesus monkeys (Davenport et al., 2006) did not. Studies on hair cortisol concentration also have been carried out in cattle (González-de-la-Vara et al.,

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2011; Comin et al., 2011, 2012, 2013; Peric et al., 2013; Burnett et al., 2014, 2015) and showed that ill cattle have higher hair cortisol concentrations than healthy cattle (Comin et al., 2013; Burlett et al.,

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2015). An increase in hair cortisol concentration was seen on the day of parturition and three weeks postpartum compared with days 42 to 126 postpartum (Burnett et al., 2015). In another study, an

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increase occured 40 days after a change from winter indoor housing to summer grazing (Comin et al.,

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2011). Hair cortisol concentrations and the adrenal gland weights were recently investigated and compared in relation to the health status of slaughter cows. Chronically ill cows had significantly

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larger adrenal gland weights relative to body weight than acutely ill cows. Similarly, the hair cortisol

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concentration of chronically ill cows (1.37 pg/mg) was significantly greater than that of acutely ill cows (0.56 pg/mg). Furthermore, the total relative adrenal gland weight was positively and significantly correlated with hair cortisol concentration (Clavadetscher, 2016). It is not known whether cortisol concentration differs between hair that has grown for one month and hair that has grown for several months. The growth rate of hair in Holstein cows depends on the area of the body and is 0.30 mm/day at the shoulder and 0.40 mm/day at the hip (Burnett et al., 2014). Determining whether the cortisol concentration of hair varies among samples obtained at

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ACCEPTED MANUSCRIPT different periods of hair growth is relevant because the technique is an important tool for substantiating stress in an animal. The primary goal of this study was to compare the cortisol concentration of hair that has grown for one month with that of hair from a previously unshorn area. A secondary goal was to investigate the effect of calendar month, pregnancy and illness on hair

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cortisol concentration.

2. Materials and methods 2.1. Cows

The study was conducted during a one-year period (November 13, 2014 to November 12, 2015)

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and used 27 cows (16 Brown Swiss and 11 Swiss Fleckvieh). The cows were 3 to 17 years of age (mean ± SD = 6.6 ± 3.7 years) and produced 4,400 to 7,300 kg milk per lactation (5,950 ± 832 kg).

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The cows were kept in a tie stall during the day and on pasture at night from April 28 to November 11. During the remainder of the year, they were turned out in a 460 m2 exercise yard with a concrete

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floor for approximately four hours per day, five days per week. During the study period, the ambient

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temperature ranged from -6 °C (February 2015) to 34 °C (August 2015) and the relative humidity ranged from 25 to 100 %. The ration consisted of hay, grass and corn silage in the summer and of hay,

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haylage, sugar beet silage and high-moisture corn in the winter. Minerals and salt were fed year-

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round, and cows producing more than 20 kg milk/day received 1kg of concentrate (extracted soybean and canola meal, 24 % crude protein) for each 2.5 kg of extra milk produced. The study was approved by the veterinary authorities of the Canton of Zurich, Switzerland.

2.2. Hair samples Electric clippers (Aesculap® Favorita CL, Aesculap Suhl GmbH, Suhl, Germany) were used to collect two samples of pigmented hair from each cow every month for a total of 12 months. The first

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ACCEPTED MANUSCRIPT sample (A sample) was limited to anagen-phase hair that had grown for one month, and the second sample (B sample) comprised all hair from a previously unshorn area and included anagen-phase hair grown in the previous two to three months and catagen- and telogen-phase hair that had grown before that. - A samples: A 10 by 10 cm (100 cm2) area of the skin over the left shoulder was clipped to collect A

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samples. Approximately 3 g of hair was collected from the centre of this clipped area once per month, and then the entire area was re-clipped to ensure that the hair collected had grown for no more than one month.

- B samples: Each B sample was collected from a different area of the body, starting cranially on one

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side of the cow and then moving caudally before repeating the same sampling pattern on the other side. Samples 1 to 6 were collected from the left side of the body: sample 1 from the neck, sample 2

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from the shoulder, sample 3 from the cranial thorax, sample 4 from the caudal thorax, sample 5 from the flank and sample 6 from the pelvic region, and samples 7 to 12 were collected from analogous

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sites on the right side of the body. The hair samples were immediately wrapped in aluminium foil

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after collection and kept at room temperature until analysis.

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2.3. Measurement of hair cortisol concentration

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Liquid chromatography tandem mass-spectrometry (LC-MS/MS) was used as described in detail by Binz et al. (2016). Briefly, the hair was washed in 40 °C water for 1 h and then in acetone for 2 min and dried. Each sample (20 mg) of dried hair was cut into snippets measuring about 0.5 mm. 2 ng of D7-cortisone (CAS-Nr. 1261254-36-7, Cerilliant®) was added to 20 mg of hair as an internal standard. Hair snippets were extracted in 3 ml methanol (LC-MS CHROMASOLV®, Fluka®) for 16 h overnight in an ultrasonic bath at 55 °C. The supernatant was evaporated using nitrogen (N2) at 35 °C and reconstituted in 150 µl methanol and 350 µl reconstitution solution (1 M ammonium

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ACCEPTED MANUSCRIPT formate, 0.1 % formic acid). The samples were measured using a LC-MS/MS system that consisted of a Shimadzu Prominence XR high pressure liquid-chromatography (HPLC) system coupled to a Sciex QTRAP® 5500 linear ion trap quadrupole mass spectrometer (Sciex, Darmstadt, Germany).

2.4. Hair cortisol concentration over one year and during pregnancy

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The mean hair cortisol concentration for each calendar month was compared with the mean concentration of the other months. Comparisons were also made among seasons (winter, December to February; spring, March to May; summer, June to August; fall, September to November). Each hair sample was assigned to the current reproductive status of the cow, and the

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concentrations measured during pregnancy were compared with the concentrations measured in the

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other months.

2.5. Hair cortisol concentration in ill cows

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In cows that became ill during the study, the cortisol concentration of samples collected in the

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2.6. Statistical analysis

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month of illness and in the following month were compared using ANOVA.

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Data were recorded in Office Excel 2007 (Microsoft Inc.). Descriptive statistics were used to describe continuous data (StataCorp., 2011, Stata Statistical Software, Texas, USA) and normality was tested using the Wilk-Shapiro test. Data were transformed by . Means ± standard deviations were calculated for normally data and medians, minimum and maximum values for nonnormal data. The program STATA 12 (StataCorp., 2011, Stata Statistical Software, Texas, USA) was used for statistical analysis. A one-way ANOVA with Bonferroni post hoc test was carried out to analyse differences at each timepoint. An ANOVA with repeated measures was carried out to analyse

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ACCEPTED MANUSCRIPT time trends. The underlying Stata model for the one-way ANOVA was and for repeated measures ANOVA was , where varx1 is the independent variable, varx2 is the day variable and vary is sqrt_cortisol (square root of cortisol value). A P-value < 0.05 was considered significant.

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3. Results

3.1. Hair cortisol concentration over the course of a year

The monthly mean hair cortisol concentrations of A samples ranged from 0.43 to 1.25 pg/mg and those of B samples from 0.53 to 1.04 pg/mg (Table 1). The overall mean hair cortisol concentration

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(mean ± sd) for A samples was 0.73 ± 0.46 pg/mg and the overall mean for B samples was 0.69 ± 0.45 pg /mg; this difference was not significant. There were significant differences in hair cortisol

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concentrations over time (F value A samples = 6.83, P < 0.01; F value B samples = 4.94, P < 0.01; Fig. 1, details in Table 1). Cortisol concentrations of A samples were significantly higher in the winter

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(0.86 ± 0.37 pg/mg) than in the fall (0.67 ± 0.33 pg/mg) (P < 0.05) but did not differ between spring

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(0.78 ± 0.73 pg/mg) and summer (0.73 ± 0.30 pg/mg). Hair cortisol concentrations of B samples did

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not differ significantly between the 4 seasons.

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3.2. Hair cortisol concentration during pregnancy The hair cortisol concentrations (mean ± sd) measured in A and B samples in the first month of pregnancy were 0.67 ± 0.33 and 0.80 ± 0.31 pg/mg, respectively (Table 2). There were significant differences in hair cortisol concentrations over time (F-value A samples = 5.28, P < 0.01; F-value B samples = 2.23, P < 0.05; Table 2, Fig. 2). The concentration in A samples remained largely unchanged until the ninth month, after which time it increased to 1.41 ± 1.09 pg/mg in the month of parturition; this concentration was significantly higher than in the other months. The cortisol

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ACCEPTED MANUSCRIPT concentration decreased after parturition to 0.92 ± 0.69 mg/pg in the second month postpartum. The mean cortisol concentrations of B samples during pregnancy ranged from 0.52 (month 6) to 0.99 pg/mg hair (month 3).

3.3. Hair cortisol concentration in ill cows

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Twenty cows became ill during the study period with mastitis (n=15), lameness (n=11), abortion (n=3), colic (n=2), metritis/endometritis (n=3) or bronchopneumonia (n=1). All cows received immediate treatment, one cow with toxic mastitis was euthanased and another cow was slaughtered after an abortion. The mean hair cortisol concentration of A samples from cows that

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became ill was 0.80 ± 0.51 pg/mg in the month before illness and 0.83 ± 0.33 pg/mg in the month after illness. The respective concentrations of B samples were 0.80 ± 0.92 pg/mg and 0.79 ± 0.59

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pg/mg. The concentrations did not differ significantly before and after illness.

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4. Discussion

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The mean hair cortisol concentrations measured in A and B samples were considerably lower than values reported in other studies (2.5pg/mg, Comin et al., 2011; 2.35 pg/mg, Moya et al., 2013;

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5.7 pg/mg, Burnett et al., 2014; 12.15 pg/mg, González-de-la-Vara et al., 2011). Hair cortisol

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concentrations similar to those in the present study were recently measured in 142 slaughter cows, and possible reasons for the discrepancy among studies were discussed in detail (Clavadetscher, 2016). Methodical differences were the most likely reason for the discrepancies. We used a technique that combined liquid chromatography and mass spectrometry (LC-MS/MS) for cortisol measurement whereas others used a radioimmunoassay (González-de-la-Vara et al., 2011; Comin et al., 2011, 2012, 2013; Peric et al., 2013), an ELISA (Burnett et al., 2014) or enzyme immunoassay (Burnett et al., 2015). Mass spectrometry is considered the gold standard for hair analysis because it is more sensitive

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ACCEPTED MANUSCRIPT and more specific than other methods (Gow et al., 2010). Immunoassay techniques generally generate cortisol concentrations that are 2.5 to 10-fold higher than those measured by LC-MS/MS methods because of assay-dependent cross-reactivity with other steroid hormones (Russell et al., 2015). The method used in the present study had a recovery of about 85 %. We believe that a combination of factors including measurement technique (mass spectrometry versus ELISA or RIA), the use of

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pigmented hair rather than white hair and shredding of the hair using scissors instead of a milling cup and steel ball were responsible for differences in cortisol concentrations between the present study and other studies. Breed could be another factor because we used Brown Swiss and Swiss Fleckvieh cows, which had lower hair cortisol concentrations than Holstein Friesian cows (Clavadetscher,

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2016). Other studies used predominantly Holstein Friesian cows, one used Simmental cows (Comin et al., 2011) and another used Holstein Friesian-cross cows (Peric et al., 2013). Ideally clinicians and

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researchers should standardise the site of hair collection and the type of hair (pigmented or nonpigmented) to facilitate comparability among studies. We favour the use of pigmented hair because

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Brown Swiss cows are common in Switzerland.

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The cortisol concentration of A samples, but not B samples, reached a maximum concentration in the month of parturition and then decreased significantly in the following month. The difference

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between A and B samples most likely was attributable to differences in hair growth stages between

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samples. The actively growing, anagen-phase hair of the A samples is capable of capturing much more circulating cortisol than the predominantly catagen and telogen-phase of B samples. Therefore, the cortisol concentration of A samples is a much accurate reflection of the circulating cortisol concentration of the past 30 days than the concentration of the relatively static B samples. The variability in hair cortisol concentration of B samples, which all consisted of pigmented hair, most likely was not related to the different collection sites. Pigmented hair from the shoulder, top line and hip region of Holstein cows did not differ in cortisol concentration, whereas white hair from the top

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ACCEPTED MANUSCRIPT line and hip region had higher concentrations than white hair from the shoulder (Burnett et al., 2014). The concentrations measured in the A samples confirmed observations of higher levels around parturition and three weeks postpartum compared with later stages of lactation (Burnett et al., 2015). The numerical increase in hair cortisol concentrations observed from the eighth to the ninth month of gestation was in agreement with a study that showed an increase in late pregnancy (Alameen and

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Abdelatif, 2012). Apart from these fluctuations at the time of parturition, pregnancy per se was not associated with increasing hair cortisol concentrations, in agreement with an earlier study on peripheral cortisol levels during pregnancy in cattle (Patel et al., 1996).

An illness in the month preceding sampling did not lead to a significant increase in the cortisol

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concentrations in A and B samples, which contrasted with results of other studies (Comin et al., 2013; Burnett et al., 2015). A possible reason for this discrepancy is that all ill cows of the present study

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received immediate treatment.

The increased hair cortisol concentrations in April (and to a lesser extent in February) were not

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related to the time of year per se and were not caused by an incidental increase in the number of

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calvings in the preceding months; an increase in April was also seen when the cows that calved in March were excluded from the calculations. Rather, the increase in April was attributable to high hair

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cortisol concentrations (2.53 to 4.95 pg/mg) associated with parturition, fever of unknown origin and

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peritarsitis, respectively. Likewise, the increased hair cortisol concentration calculated for February was attributable to individual cows with high concentrations. Although there was no overall difference, the cortisol concentrations of A and B samples differed in the month of parturition.Thus, parturition results in increased cortisol deposition in hair and it can be concluded that stressful events manifest in changes in hair that has regrown in a short period but not in hair that has been present for a longer period. This implies that hair from a preclipped area is more suitable for detection of acute or subacute stressors such as parturition.

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5. Conclusions Parturition in the month preceding sampling increased the cortisol concentration in hair that had regrown within one month after clipping.

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6. Conflict of interest statement

The authors of this paper have no financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

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7. References

Alameen, A. O., Abdelatif, A. M., 2012. Metabolic and endocrine responses of crossbred dairy cows

Environ. Sci. 12, 1065-1074.

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in relation to pregnancy and season under tropical conditions. American-Eurasian J. Agric. &

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Bennett, A., Hayssen, V., 2010. Measuring cortisol in hair and saliva from dogs: coat color and

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pigment differences. Domest. Anim. Endocrinol. 39, 171-180. Binz T. M., Braun, U., Baumgartner, M. R., Kraemer, T., 2016. Development of an LC-MS/MS

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method for the determination of endogenous cortisol in hair using 13C3-labeled cortisol as

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surrogate analyte. J. Chromatogr. B 1033, 65-72. Burnard, C., Ralph, C., Hynd, P., Hocking Edwards, J., Tilbrook, A., 2017. Hair cortisol and its potential value as a physiological measure of stress response in human and non-human animals. Anim. Prod. Sci. 57, 401-414. Burnett, T. A., Madureira, A. M. L., Silper, B. F., Nadalin, A., Tahmasbi, A., Veira, D. M., Cerri, R. L. A., 2014. Factors affecting hair cortisol concentrations in lactating dairy cows. J. Dairy Sci. 97, 7685-7690.

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ACCEPTED MANUSCRIPT Burnett, T. A., Madureira, A. M. L., Silper, B. F., Tahmasbi, A., Nadalin, A., Veira, D. M., Cerri, R. L. A., 2015. Relationship of concentrations of cortisol in hair with health, biomarkers in blood and reproductive status in dairy cows. J. Dairy Sci. 98, 4414-4426. Clavadetscher, G., 2016. Vergleich der Cortisolkonzentrationen im Haar mit den Nebennierengewichten bei Kühen. Master Thesis, Vetsuisse Faculty, University of Zurich.

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Comin, A., Prandi, A., Peric, T., Corazzin, M., Dovier, S., Bovolenta, S., 2011. Hair cortisol levels in dairy cows from winter housing to summer highland grazing. Livest. Sci. 138, 69–73. Comin, A., Peric, T., Montillo, M., Faustini, M., Zufferli, V., Cappa, A., Cornacchia, G., Prandi, A., 2012. Hair cortisol levels to monitor hypothalamic-pituitary-adrenal axis activity in healthy dairy

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cows. J. Anim. Vet. Advances 11, 3623–3626.

Comin, A., Peric, T., Corazzin, M., Veronesi, M. C., Meloni, T., Zufferli, V., Cornacchia, G., Prandi,

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A., 2013. Hair cortisol as a marker of hypothalamic-pituitary-adrenal axis activation in Friesian dairy cows clinically or physiologically compromised. Livest. Sci.152, 36-41.

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Davenport, D. M., Tiefenbacher, S., Lutz, C. K., Novak, M. A., Meyer, J. S., 2006. Analysis of

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endogenous cortisol concentrations in the hair of rhesus macaques. Gen. Comp. Endocrinol. 147, 255-261.

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González-de-la-Vara M del, R., Valdez, R. A., Lemus-Ramirez, V., Vázquez-Chagoyán, J. C., Villa-

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Godoy, A., Romano, M. C., 2011. Effects of adrenocorticotropic hormone challenge and age on hair cortisol concentrations in dairy cattle. Can. J. Vet. Res. 75, 216-221. Gow, R., Thomson, S., Rieder, M., Van Uum, S., Koren, G., 2010: An assessment of cortisol analysis in hair and its clinical applications. Forensic Sci. Int. 196, 32-37. Kirschbaum, C., Tietze, A., Skoluda, N., Dettenborn, L., 2008. Hair as a retrospective calendar of cortisol production – Increased cortisol incorporation into hair in the third trimester of pregnancy. Psychoneuroendocrinology 34, 32-37.

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ACCEPTED MANUSCRIPT Meyer, J. S., Novak, M. A., 2012. Hair cortisol: a novel biomarker of hypothalamic-pituitaryadrenocortical activity. Endocrinology 153, 4120–4127. Moya, D., Schwartzkopf-Genswein, K. S., Veira, D. M., 2013. Standardization of a non-invasive methodology to measure cortisol in hair of beef cattle. Livest. Sci. 158, 138-144. Patel, O. V., Takahashi, T., Takenouchi, N., Hirako, M., Sasaki, N., Domeki, I., 1996. Peripheral

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cortisol levels throughout gestation in the cow: effect of stage of gestation and foetal number. Br. Vet. J. 152, 425-432.

Peric, T., Comin, A., Corazzin, M., Montillo, M., Cappa, A., Campanile, G., Prandi, A., 2013. Short communication: hair cortisol concentrations in Holstein-Friesian and crossbreed F1 heifers. J.

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Dairy Sci. 96, 3023-3027.

abuse. Clin. Chim. Acta 370, 17-49.

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Pragst, F., Balikova, M. A., 2006. State of the art in hair analysis for detection of drug and alcohol

Russell, E., Koren, G., Rieder, M., Van Uum, S., 2012. Hair cortisol as a biological marker of chronic

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stress: current status, future directions and unanswered questions. Psychoneuroendocrinology 37,

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589-601.

Russell, E., Koren, G., Rieder, M., Van Um, S. H. M., 2014. The detection of cortisol in human

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sweat: Implications for measurement of cortisol in hair. Ther. Drug. Mon. 36, 30-34.

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Russell, E., Kirschbaum, C., Laudenslager, M. L., Stalder, T., De Rijke, Y., Van Rossum E. F. C., Van Uum, S., Koren, G., 2015. Toward standardization of hair cortisol measurement: results of the first international interlaboratory round robin. Ther. Drug Monit. 37, 71-75. Sharpley, C. F., Kauter, K. G., McFarlane, J. R., 2010. An investigation of hair cortisol concentration across body sites and within hair shaft. Clin. Med. Insights Endocrinol. Diabetes 3, 17-23.

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ACCEPTED MANUSCRIPT Van Uum, S. H. M., Sauvé, B., Fraser, L. A., Morley-Forster, P., Paul, T. L., Koren, G., 2008. Elevated content of cortisol in hair of patients with severe chronic pain: a novel biomarker for stress. Stress 11: 483-488. Wosu, A. C., Valdimarsdóttir, U., Shields, A. E., Williams, D. R., Williams, M. A., 2013. Correlates of cortisol in human hair: implications for epidemiologic studies on health effects of chronic

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stress. Ann. Epidemiol. 23, 797-811.e2.

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Fig. 1

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Fig. 2

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ACCEPTED MANUSCRIPT Table 1 Hair cortisol concentration (pg/mg) in 25 dairy cows from December 2014 to November 2015. A samples

SD

0.32

0.53C

0.19

01 (January 15)

0.77A

0.29

0.65

0.24

02 (February 15)

0.97A

0.46

0.88D

0.49

03 (March 15)

0.64

0.35

0.58C

0.37

04 (April 15)

1.25A

0.99

1.04D

0.84

05 (May 15)

0.43

0.36

0.55C

0.35

06 (June 15)

0.69AB

0.26

0.71

0.32

07 (July 15)

0.79A

0.30

0.73

0.34

08 (August 15)

0.71B

0.33

0.66

0.23

09 (September 15)

0.67

0.30

0.84

0.62

10 (October 15)

0.63B

0.33

0.68

0.43

11 (November 15)

0.72AB

0.37

0.91

0.63

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0.86A

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Mean

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12 (December 14)

SD

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Mean

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Month

B samples

different from May : P < 0.05 (Nov.), and P < 0.01 (Dec., Jan., Febr., April, June, July)

B

different from April: P < 0.05 (Nov., Aug.), and P < 0.01 (June, Oct.)

C

different from April: P < 0.01 (March, May, Dec.)

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different from May: P < 0.05 (Febr., Nov.) and P < 0.01 (April)

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F value A samples = 6.83, P < 0.01 F value B samples = 4.94, P < 0.01

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ACCEPTED MANUSCRIPT Table 2 Hair cortisol concentration (pg/mg) in 25 dairy cows during pregnancy. B samples

Mean

SD

Mean

SD

Month 1 (P)a

0.67**

0.33

0.80

0.31

Month 2 (P)

0.70*

0.46

0.67

0.30

Month 3 (P)

0.67*

0.27

0.99

1.14

Month 4 (P)

0.60**

0.19

0.61

0.22

Month 5 (P)

0.65**

0.29

0.57

0.25

Month 6 (P)

0.55**

0.24

0.52

0.28

Month 7 (P)

0.60**

0.18

0.53

0.19

Month 8 (P)

0.55**

0.23

0.53

0.21

Month 9 (P)

0.79*

0.45

0.73

0.54

Month 0 (C)b

1.41

1.09

0.78

0.46

Month 1 (pp)c

0.94

0.33

0.75

0.22

Month 2 (pp)

0.92

0.69

0.75

0.37

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Stage

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A samples

P Pregnancy

b

Month 0 (C) Month of calving

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pp postpartum

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different from month 0 (Calving), P < 0.05

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a

**

different from month 0 (Calving), P < 0.01

F value A samples = 5.28, P < 0.01 F value B samples = 2.23, P < 0.01

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ACCEPTED MANUSCRIPT Highlights

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 We compared the cortisol concentration of hair that has grown for one month (A samples) with that of hair from a previously unshorn area in cows (B samples)  In the month of parturition, the hair cortisol concentration in A samples was significantly higher than in the preceding months  Hair cortisol concentrations of A samples were significantly higher in the winter than in the fall  Hair cortisol concentrations did not differ significantly before and after an illness

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