Genetic differences in temperament determine

Physiology & Behavior 105 (2012) 1117–1123

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Genetic differences in temperament determine whether lavender oil alleviates or exacerbates anxiety in sheep P.A.R. Hawken a,⁎, C. Fiol b, D. Blache a a b

UWA Institute of Agriculture (Animal Production), The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia Departamento de Bovinos, Facultad de Veterinaria, Montevideo, Uruguay

a r t i c l e

i n f o

Article history: Received 21 March 2011 Received in revised form 23 November 2011 Accepted 7 December 2011 Keywords: Anxiety Depression Temperament Isolation Stress Alternative therapies

a b s t r a c t Growing concerns about the risk of addiction to benzodiazepines have led to increasing interest in alternative therapies to treat anxiety and depression. Lavender oil (Lavendula augustifolia) is reportedly anxiolytic in a number of species but little is known about how it affects individuals that are more or less anxious when faced with a stressor. In this study, we used changes in locomotor activity and the plasma concentrations of cortisol to test whether lavender oil would reduce behavioral and endocrine correlates of anxiety in calm and nervous sheep exposed to an isolation stressor. During the non-breeding season, ‘calm’ or ‘nervous’ female sheep from the UWA temperament flock were exposed to a mask containing either 1 mL of 10% lavender oil (calm: n = 8; nervous: n = 8) or peanut oil (calm: n = 8; nervous: n = 8). After 30 min, each sheep was isolated for 5 min and then returned to the group. Blood was sampled prior to the mask, prior to isolation, 1 min and 30 min after isolation to profile changes in the plasma concentrations of cortisol. Agitation score, locomotor activity and vocalizations were recorded as correlates of anxiety associated with the isolation stressor. Irrespective of whether they were exposed to lavender oil, calm sheep had a lower agitation score (Pb 0.001), crossed the central lines of the isolation box less frequently (Pb 0.001), expressed fewer vocalizations (Pb 0.001) and had lower plasma concentrations of cortisol immediately after isolation (Pb 0.001) than nervous sheep. Exposure of calm sheep to lavender oil decreased the agitation score (Pb 0.001), frequency of vocalizations (Pb 0.05), decreased the number of crosses of the central lines of the isolation box (Pb 0.05), and the plasma concentrations of cortisol prior to isolation (Pb 0.05) (after mask application) compared to calm control sheep. Exposure of nervous sheep to lavender oil increased the frequency of vocalizations (Pb 0.05), the number of sheep attempting to escape (Pb 0.05) and the plasma concentrations of cortisol 30 min after isolation (Pb 0.05) compared to nervous control sheep. We conclude that genetic differences in temperament determine whether lavender oil alleviates or exacerbates the behavioral and/or endocrine correlates of anxiety in sheep. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Anxiety can be interpreted as the emotional capacity of an individual to anticipate an aversive situation that is unpredictable and outside of their control [1]. Abnormally high levels of anxiety are associated with dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis, and, in humans, affective disorders such as pathological anxiety and depression [1]. Anxiety disorders affect 40 million people annually in the USA, and are often co-morbidly expressed with depression [2]. Pathological anxiety and depression are frequently treated with benzodiazepines which act on the gamma-aminobutyric acid complex (GABA) to suppress neural activity in regions of the brain that mediate

⁎ Corresponding author at: M085, School of Animal Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: + 61 8 6488 3588; fax: + 61 8 6488 1029. E-mail addresses: [email protected] (P.A.R. Hawken), cfi[email protected] (C. Fiol), [email protected] (D. Blache). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.12.005

anxiety [3]. In the short term, benzodiazepines are effective at reducing anxiety but long term use can be associated with a number of adverse side effects and a relatively high risk of abuse and addiction (review; [4]). Furthermore, polymorphisms in the enzymes and receptors responsible for mediating the effects of anti-depressant or antipsychotic drugs reduce the reliability and predictability of the drug response [5]. In recent years, there has been increasing interest in the use of alternative therapies to treat a number of medical conditions, including anxiety disorders and depression [6]. Lavender oil (Lavendula augustifolia) is commonly used in aromatherapy and allegedly has anxiolytic effects in a number of species including rats [7], mice [8], Mongolian gerbils [9], dogs [10] and humans [11]. Both the mechanism of action [12] and anxiolytic effect of lavender oil are reputedly similar to the benzodiazepines, making lavender oil a potentially viable alternative to benzodiazepines for the treatment of anxiety [9,13,14]. In humans, lavender oil appears to be calmative for young infants [15] and reduces the anxiety associated with visiting the dentist [16]

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or taking examinations [17]. However, interpretation of human studies can be difficult because lavender oil is often combined with other relaxation strategies such as massage [18], foot-baths [19] or baths [15,20]. A recent study also showed that humans expect lavender oil to be calmative and that this expectation, rather than the aroma per se, was responsible for mediating the relaxing effects of lavender oil [21]. These issues, combined with the difficulty in sourcing an adequate placebo, make it difficult to differentiate between the real and perceived effects of lavender oil on anxiety in humans [21]. Animal models provide a viable alternative to test the anxiolytic effects of lavender oil because we can directly measure behavioral and endocrine correlates of anxiety and expectation is unlikely to be an issue. We can also use natural variation in anxiety within a population to select animals for extremes in this trait, thus allowing the anxiolytic effects of lavender oil to be tested in individuals that are more or less anxious when faced with a stressor. HAB/LAB rats have been selected for decades for extremes in anxiety related behavior when faced with a mild stressor [1,22,23]. Specifically, HAB rats have a lower propensity to enter the open arms of the elevated plus maze than LAB rats and secrete more corticosterone when forced to do so [24]. The repeatability and heritability of anxiety related behavior of the two lines within and between generations thus provides predictability over the level of anxiety associated with a stressor. Sheep at the University of Western Australia have been divergently selected for 17 generations on the basis of their behavioral responses to social isolation and human presence. Isolation and human presence elicit a strong fear response in sheep [26], that can be quantified by increased locomotor activity and vocalization frequency [26,27]. The scores from the behavioral tests used to select the sheep are repeatable over time (r= 0.40–0.76) [28] and moderately heritable (h = 0.45) [28]. A recent cross-fostering study found the phenotype of calm and nervous sheep to be evident at 1 week of age and minimally affected by non-genetic factors [29]. Fear and anxiety are closely related emotions that describe the reaction of an individual to an actual or potential threat [30]. The stressors used to select the UWA temperament flock pose no actual, physical danger to the sheep, so one could argue that nervous sheep are predisposed to view an ambiguous situation as more threatening than calm sheep. This type of negative bias is characteristic of high anxiety in rats, which combined with the divergence in the behavioral [29,31,32] and endocrine [32] responses of calm and nervous sheep to social isolation supports the notion that nervous sheep are more ‘anxious’ than calm sheep when faced with an isolation stressor. The calm and nervous sheep of the UWA temperament flock thus draw many parallels to the HAB/LAB rats (strong phenotypic differences of genetic origin that are evident early in life) [25] and as such may provide an opportunity to study anxiety in a non-rodent species. Genetic factors play an integral role in determining the level of anxiety associated with a stressor [22] and the phenotypic expression of the drug response [33] so may, in turn, affect the anxiolytic properties of lavender oil. In this study, we tested the effect of lavender oil on the behavioral and endocrine correlates of anxiety in calm and nervous sheep exposed to an isolation stressor. The nervous sheep have been genetically selected for high expression of locomotor activity [29,31] when faced with an isolation stressor that is associated with significantly higher plasma concentrations of cortisol than those observed in calm sheep [32]. Therefore, we hypothesized that exposure to lavender oil would reduce locomotor activity and plasma concentrations of cortisol in sheep selected for nervous temperament during exposure to an isolation stressor. Calm sheep express low levels of locomotor activity when faced with an isolation stressor [29,31] that are associated with significantly lower plasma concentrations of cortisol than those observed in nervous sheep [32]. Therefore, we hypothesized that exposure to lavender oil would be unlikely to further reduce the behavioral and endocrine correlates of anxiety in calm sheep during exposure to an isolation stressor.

2. Materials and methods 2.1. Animals These experiments were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th Edition, 2004) and were approved by the Animal Ethics Committee of The University of Western Australia (RA3/100/947). 2.1.1. The UWA temperament flock The UWA temperament flock are Merino sheep kept at the University of Western Australia (UWA) research farm in Wundowie (latitude 31°46′S, longitude 116°29′E; 330 m in altitude) that have been divergently selected for ‘calm’ or ‘nervous’ temperament for 17 generations [29,31,34]. In this context, we define ‘temperament’ as the behavioral reactivity of sheep to the psychosocial stress of social isolation in the presence or absence of humans [31,34]. For the purpose of genetic selection, behavioral reactivity to stress is measured at 14–16 weeks of age using a series of tests that assess the animal's relative ability to cope with stress. The first test is a 3 min arena test that quantifies the expression of locomotor activity and vocalizations when faced with the conflict of approaching a human to gain contact with a small group of con-specifics [31,34]. In the second test, the animal is placed in visual isolation in a solid plywood box (1.5 m3) for 1 min. Physical activity inside the box (agitation score) is recorded using a digital agitation meter that is calibrated prior to the test for low, medium and high levels of activity [31,34]. The scores from the two tests are then used in a selection index to formulate a score for the temperament of the sheep as described in detail by Beausoleil et al. [35]. Males with the most extreme scores within the calm and nervous lines are used for breeding the next generation of sheep when they reach sexual maturity. Calm and nervous sheep are maintained as one flock at pasture and subject to conventional Australian farm practice. We have screened over 10,000 sheep across Australia using the isolation box test (test 2) and found that the progeny bred from unselected or ‘wild type’ sheep that were ‘more or less reactive’ to the isolation stressor, were also ‘more or less’ reactive to the isolation stressor, as indicated by differences in their agitation score. These data indicate that the genes underlying calm and nervous temperament in this model are likely to be common in the general population, thus supporting the heritability, relevance and validity of our animal model [28]. 2.1.2. Experimental animals During August 2010 (non-breeding season; southern hemisphere), females from the calm (n = 16) or nervous (n = 16) lines of the UWA temperament flock were allocated to one of four treatments; calm and nervous sheep exposed to 10% lavender oil (Calm Lav; n = 8; Nerv Lav; n = 8) or exposed to peanut oil as a control (Calm Con; n = 8; Nerv Con; n = 8). The groups were balanced for age (14–16 months) and live weight within temperament (Table 1). The sheep were initially selected based on their parental temperament (i.e. calm or nervous) and their agitation score in the standard isolation box at weaning (3–4 months). Before they were finally allocated to their treatment groups, the sheep were screened during a 1 min test in a smaller (0.5 m × 1.2 m × 1.2 m), portable version of the isolation box (screening agitation score) to ensure that the screening agitation scores were balanced within temperament across the lavender oil and control groups (Table 1). 2.2. Experimental procedures The isolation box was set up in an enclosed shed under natural light with access to yards for holding the sheep before and after the isolation stressor. The isolation box was stood on a base of four tires of equal size and pressure to provide suspension and stability for movement within the box. On the day of the test, a mechanical device


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Table 1 Mean ± SEM live weight and screening agitation score in calm and nervous sheep exposed to wool scented with lavender oil (Calm Lav, Nerv Lav) or peanut oil (Calm Con, Nerv Con). Different superscripts (a,b) indicate significant differences between treatment groups (P b 0.05).

Calm Con Calm Lav Nerv Con Nerv Lav

Live weight (kg)

Screening agitation score

29.4 ± 2.0 31.7 ± 2.5 35.4 ± 1.1 35.4 ± 1.0

38.4 ± 5.2a 44.3 ± 8.1a 187.1 ± 14.8b 193.9 ± 13.4b

fitted with four piston driven legs was placed inside the box that mimicked a low, medium and high level of activity inside the box. The sensitivity of the agitation meter was then adjusted so that low, medium and high levels of activity during 1 min resulted in scores of approximately 60, 90 and 120 arbitrary units respectively (agitation score). The calibration was repeated at the end of the day to confirm the consistency of the agitation meter over the experiment. Calm and nervous sheep were assigned to one of two treatment groups (control and lavender oil) that were held in pens at least 50 m apart and managed by two teams of people to minimize the risk of cross contamination of odor between the lavender oil and control groups. The same, standard size isolation box was used for both groups to minimize variation in the isolation stimulus and because the risk of odor transmission from inside the mask to the box was minimal. Approximately 30 min prior to the isolation stressor, each test animal was moved from the holding pen into the test pen (approximately 2 m × 2 m) with companion sheep (n = 4/5). The test sheep was then fitted with a mask containing wool scented with either lavender oil or peanut oil that remained on the sheep for the duration of the experiment. The timing of initial mask application was coordinated to ensure that control and lavender exposed sheep from each temperament were subjected alternatively to the isolation stressor, approximately 30 min after initial exposure to the mask. During the isolation stressor, the animal was isolated for 5 min the standard isolation box that prevented visual communication with other sheep, although auditory communication was possible. During the test, the behavior of the animal was recorded using a video camera fixed to the top of the isolation box. After 5 min, the test agitation score was recorded and the animal was released and returned to the companion sheep. The mask remained on the sheep for another 30 min after exiting the isolation box. 2.2.1. Exposure to lavender/peanut oil Sheep exposed to lavender oil were fitted with a mask containing cotton wool infused with 1 mL of 100% lavender oil (L. augustifolia; Oil Garden Aromatherapy, Victoria, Australia) diluted in peanut oil at a ratio of 1:9. This concentration of lavender oil was selected based on the work of Diego et al., [36] in humans. Peanut oil was used as the diluent because it has a neutral aroma and was thus unlikely to affect any of the behavioral or endocrine correlates of anxiety. Control sheep were fitted with an identical mask containing a hand sized amount of cotton wool (Fig. 1) infused with 1 mL of peanut oil. The masks were made of fabric with a mesh cover at the end to allow airflow through the mask. A mesh bag containing the cotton wool infused with oil was attached to the mesh-covered end of the mask to optimize inhalation of the aroma and keep the wool at a fixed distance (1–2 in.) from the nose of the sheep (Fig. 1). Neither the mask, wool or peanut oil were expected to affect the behavioral or endocrine responses of the sheep to the isolation stressor, so an empty mask or no mask control were not included in this study. 2.2.2. Quantification of data from the video footage The video footage was watched twice by an observer who was unaware of the temperament or treatment (i.e. control or lavender) of the test animal; firstly to quantify the frequency of all vocalizations

Fig. 1. Top panel: Example of a mask fitted to a sheep (1a) and unzipped (1b), showing the placement of the mesh bag containing the cotton wool infused with either lavender oil or peanut oil. Bottom panel: Example of a sheep in the isolation box (1c) and a schematic diagram of the floor of the isolation box — the bold lines represent those used to quantify the crosses of the central line of the box (1d).

and secondly to quantify locomotor activity within the isolation box. The observer recorded the number of times that the front feet of the animal crossed the central lines of the floor of the isolation box shown in Fig. 1. The observer also recorded the frequency of urinations, pawing and whether the animal tried to escape from the box. An escape attempt was defined as any jumping movement towards the wall with a minimum of two feet off the ground.

2.2.3. Blood collection To profile the effects of treatment (i.e. lavender or control) and temperament on the plasma concentration of cortisol, blood was sampled by jugular venepuncture using 1″ 18 G needles and 4 mL vacutainers coated with lithium heparin (Interpath, WA, Australia) at 4 time points; prior to fitting the animal with the mask; prior to entering the isolation box (30 min after fitting the mask): immediately (1 min) post isolation, and 30 min post isolation. All blood samples were taken in the test pen with the sheep held in a standing position by an experienced handler with the head and neck slightly elevated for ease of access to the jugular vein. The necks of the sheep were clipped prior to the experiment and the blood sample taken within 1 min of initial restraint of the animal, thus minimizing the impact of the procedure on the plasma concentrations of cortisol in the sample collected. Blood was stored immediately on ice and centrifuged within 30 min of collection for 10 min at 3000 rpm. Plasma was then harvested and frozen in plastic tubes at − 20 °C until immunoassay.

2.3. Immunoassay Plasma concentrations of cortisol were quantified using commercial radioimmunoassay kits (Diasorin Australia Ltd. NSW) modified and validated for sheep as described by Beausoleil et al. [35]. The sensitivity of the assay was 1.1 ng/mL. All samples were processed within one assay. The mean intra assay variation for quality control samples of plasma cortisol (low:14.0 ng/mL; medium: 29.1 ng/mL) were 9% and 7.9% respectively.

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2.4. Statistical analysis All the tests used in the data analysis were run in Genstat 5 (Second Edition, Lawes Agricultural Trust, Rothamsted Experimental Station, Hertfordshire UK) or Minitab ® 14.1 (Minitab Inc, State College, Pennsylvania, USA). The distributions of all data were initially assessed for normality using the Shapiro Wilk test. Where the data were not from a normal distribution, they were transformed (see below) and the tests were repeated to ensure the validity of the data for parametric analysis. Agitation score, crosses of the central line and vocalizations were subject to rank transformation prior to analysis with two-way ANOVA to determine the effect of treatment (i.e. +/− lavender) and temperament and any interaction between the two factors. Live weight directly affects the agitation score so was included as a covariate in analysis of this parameter. Where a significant effect of treatment, temperament or interaction was detected, data were compared between each combination of treatment groups by Students t-test (e.g. Calm Lav versus Calm Con; Calm Lav versus Nerv Con; Calm Lav versus Nerv Lav). Nontransformed data are presented in the tables for ease of interpretation. The numbers of sheep attempting to escape from the box were compared between treatment groups using a chi-squared test. The limited numbers of sheep observed pawing or urinating prevented statistical analysis of these parameters relative to temperament or treatment (i.e. +/− lavender). Cortisol data were subject to square root transformation prior to analysis with two way repeated measures ANOVA with time, temperament and treatment (i.e. +/− lavender) as factors and live weight as a covariate. Where a significant effect of time (pre-mask, preisolation, 1 min and 30 min post isolation), treatment (i.e. +/− lavender), temperament (calm, nervous) or interaction was detected, data were compared between time points within each treatment group by paired t-test (e.g. pre-mask sample versus pre-isolation sample within Calm Lav sheep) or between each combination of treatment groups by Students t-test (e.g. Calm Lav versus Calm Con at each time point). Non-transformed data are presented in the tables for ease of interpretation. 3. Results 3.1. Behavior 3.1.1. Test agitation score Two-way ANOVA revealed an effect of temperament (F (1, 31) = 1017.95; P b 0.001), no effect of treatment (i.e. +/− lavender oil; F (1, 31) = 51.61; P > 0.1) and an interaction between on temperament and treatment (F (1, 31) = 114.43; P b 0.05) on the test agitation score. Further analysis revealed that both nervous control sheep and nervous sheep exposed to lavender oil had a higher mean agitation score than calm control sheep (Nerv Con: t14 = 5.18; P b 0.001; Nerv Lav: t14 = 4.45; P b 0.01; Table 2) and calm sheep exposed to lavender oil (Nerv Con: t14 = 8.46; P b 0.001; Nerv Lav: t14 = 7.21; P b 0.001; Table 2). Calm sheep exposed to lavender oil had a lower agitation score than control sheep of the same temperament (t14 = 4.81; P b 0.001; Table 2). 3.1.2. Crosses over the central lines of the isolation box Two-way ANOVA detected an effect of temperament (F (1, 31) = 10,507.6; P b 0.001) and an interaction between temperament and treatment (i.e. +/− lavender oil; F (1, 31) = 687.7; P b 0.05) but no effect of treatment (i.e. +/− lavender oil; F (1, 31) = 214.4; P > 0.1) on the number of crosses of the central lines of the isolation box. Subsequent analysis revealed that both nervous control sheep and nervous sheep exposed to lavender oil crossed the central lines of the box more frequently than calm control sheep (Nerv Con: t14= 4.93;

Table 2 Mean ± SEM test agitation score, vocalization frequency and crosses of the central lines in calm and nervous sheep exposed to wool scented with lavender oil (Calm Lav, Nerv Lav) or peanut oil (Calm Con, Nerv Con). Different superscripts (a,b,c) indicate significant differences between treatment groups (at least P b 0.05).

Test agitation score Crosses of central line of isolation box Vocalizations (bleats per min)

Calm Con (n = 8)

Calm Lav (n = 8)

Nerv Con (n = 8)

Nerv Lav (n = 8)

67.8 ± 20.03a 16.6 ± 4.23a

28.1 ± 7.46b 3.42 ± 0.81b

299 ± 43.84c 53.8 ± 4.38c

374 ± 71.98c 57.2 ± 6.18c

1.95 ± 0.86a

0.34 ± 0.19b

5.60 ± 1.01c

9.2 ± 1.13d

0 (0) 1 (13) 0a (0)

1 (13) 2 (25) 0a (0)

2 (25) 3 (38) 4b (50)

Numbers of animals observed: Urinating 0 (%) (0) Pawing 1 (%) (13) Escape attempts 1ab (%) (13)

P b 0.001; Nerv Lav: t14 = 4.60; P b 0.001; Table 2) and calm sheep exposed to lavender oil (Nerv Con: t14 = 6.58; P b 0.001; Nerv Lav: t14 = 5.86; P b 0.001; Table 2). Calm sheep exposed to lavender oil crossed the central lines of the box less frequently than calm control sheep (t14 = −2.26; P b 0.05; Table 2). There were no differences in this variable between nervous sheep exposed to lavender oil and control sheep of the same temperament (t14 = −0.68; P > 0.1; Table 2). 3.1.3. Vocalizations Two-way ANOVA detected an effect of temperament (F (1, 31) = 3349.4; P b 0.001) and an interaction between temperament and treatment (i.e. +/− lavender oil; F (1, 31) = 825.4; P b 0.001) but no effect of treatment (i.e. +/− lavender oil; F (1, 31) = 93.4; P > 0.1) on the frequency of vocalizations during the isolation stressor. Subsequent analysis revealed that nervous control sheep and nervous sheep exposed to lavender oil vocalized more frequently than calm control sheep (Nerv Con: t14 = 2.49; P b 0.05; Nerv Lav: t14 = 5.08; P b 0.001; Table 2) and calm sheep exposed to lavender oil (Nerv Con: t14 = 5.46; P b 0.001; Nerv Lav: t14 = 8.93; P b 0.001; Table 2). Nervous sheep exposed to lavender oil vocalized more frequently than nervous control sheep (t14 = 2.46; P b 0.05; Table 2). Conversely, calm sheep exposed to lavender oil vocalized less frequently than calm control sheep (t14 = −2.26; P b 0.05; Table 2). 3.1.4. Other behaviors The number of sheep attempting to escape from the box was greater among nervous sheep exposed to lavender oil than nervous control sheep or calm sheep exposed to lavender oil (Chi square value = 10.19; P b 0.05; Table 2). The limited numbers of sheep observed pawing or urinating prevented any meaningful analysis of these parameters relative to temperament or treatment (Table 2). 3.2. Cortisol secretion Two way, repeated measures ANOVA detected significant effects of time (F (3, 81) = 50.76; P b 0.001) and temperament (F (1, 27) = 46.76; P b 0.001) but no effect of treatment (i.e. +/− lavender oil; F (1, 27) = 0.35; P > 0.1) on the mean plasma concentrations of cortisol. There were also interactions between temperament and time (F (3, 81) =2.93; P b 0.01), treatment (i.e. +/− lavender oil) and time (F (3, 81) = 2.68; P b 0.05) and treatment (i.e. +/− lavender oil), temperament and time (F (3, 81) = 7.53; P b 0.001) on the mean plasma concentrations of cortisol over the experimental period.


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3.2.1.1. Pre-mask sample. Mean plasma concentrations of cortisol prior to the mask being fitted did not differ among any of the treatment groups (P > 0.05; Table 3). Nervous control sheep tended to have higher mean plasma concentrations of cortisol than calm control sheep but this difference failed to reach significance (t14 = − 1.86; P b 0.1; Table 3).

3.2.2.2. Calm lavender. There was no change in the mean plasma concentrations of cortisol between the pre-isolation and pre-mask sample in calm sheep exposed to lavender oil (t7 = 1.61; P > 0.1; Table 3). Exposure to the isolation stressor increased the mean plasma concentrations of cortisol in the post isolation sample (1 min) above those reported in the pre-mask sample (t7 = 6.23; P b 0.001; Table 3). Mean plasma concentrations of cortisol 30 min post isolation were lower than those reported at 1 min post isolation (t7 = −5.75; P b 0.001; Table 3) and in the pre-mask sample (t7 = −3.27; P b 0.05; Table 3).

3.2.1.2. Pre-isolation sample. Mean plasma concentrations of cortisol were lower in calm sheep exposed to lavender oil than calm control sheep (t14 = − 2.73; P b 0.05; Table 3), nervous control sheep (t14 = −5.79; P b 0.05; Table 3) and nervous sheep exposed to lavender oil (t14 = −3.93; P b 0.01; Table 3). Mean plasma concentrations of cortisol did not differ between calm control sheep and nervous control sheep (t14 = 0.155; P > 0.1; Table 3) or nervous sheep exposed to lavender oil (t14= −0.88; P > 0.1; Table 3).

3.2.2.3. Nervous control. Mean plasma concentrations of cortisol did not increase significantly between the pre-isolation sample and the pre-mask sample (t7 = 2.22; P b 0.1; Table 3). However, the mean plasma concentrations of cortisol did increase between the premask sample and the post isolation (1 min) (t7 = 6.65; P b 0.001; Table 3). Mean plasma concentrations of cortisol 30 min post isolation were lower than those reported at 1 min post isolation (t7 = −8.30; P b 0.001; Table 3) and did not differ from the pre-mask sample (t7 = 0.90; P > 0.1; Table 3).

3.2.1. Differences in cortisol secretion between temperaments and treatments

3.2.1.3. Post-isolation (1 min) sample. Both calm sheep exposed to lavender oil and calm control sheep had lower mean plasma concentrations of cortisol than nervous control sheep (Calm Con: t14 = −10.61; P b 0.001; Calm Lav: t14 = −5.55; P b 0.001; Table 3) and nervous sheep exposed to lavender oil (Calm Con: t14 = −5.99; P b 0.001; Calm Lav: t14 = −5.03; P b 0.001; Table 3). The plasma concentrations of cortisol did not differ between calm control sheep and calm sheep exposed to lavender oil (t14 = 0.90; P > 0.1; Table 3) or between nervous control sheep and nervous sheep exposed to lavender oil (t14 = −1.08; P > 0.1; Table 3). 3.2.1.4. Post-isolation (30 min) sample. Mean plasma concentrations of cortisol were greater in nervous sheep exposed to lavender oil than nervous control sheep (t14 = 3.53; P b 0.01; Table 3), calm control sheep (t14 = 4.82; P b 0.001; Table 3) and calm sheep exposed to lavender oil (t14 = 5.67; P b 0.001; Table 3). Mean plasma concentrations of cortisol 30 min post isolation in calm sheep exposed to lavender oil did not differ from nervous control sheep (t14 = 1.35; P > 0.1; Table 3) and were only numerically lower than calm control sheep (t14 = − 2.10; P b 0.1; Table 3). 3.2.2. Differences in cortisol secretion over time 3.2.2.1. Calm control. Mean plasma concentrations of cortisol increased between the pre-isolation sample and the pre-mask sample (t7 = 4.51; P b 0.01; Table 3) and between the pre-mask sample and the post isolation sample taken 1 min after the isolation stressor (t7 = 2.96; P b 0.05; Table 3). Mean plasma concentrations of cortisol 30 min post isolation were lower than those reported at 1 min post isolation (t7 = −11.59; P b 0.001; Table 3) and did not differ significantly from the pre-mask sample (t7 = −1.53; P > 0.1; Table 3). Table 3 Mean ± SEM plasma concentrations of cortisol (ng/mL) in calm and nervous sheep exposed to wool scented with lavender oil (Calm Lav, Nerv Lav) or peanut oil (Calm Con, Nerv Con). Different superscripts (a,b,c,d) indicate differences within rows (i.e. within treatment group over time as indicated by paired t-test; at least P b 0.05). Different superscripts x,y indicate differences within columns (i.e. between treatment groups at a specific time point as indicated by Student's t-test; at least P b 0.05).

Calm Con Calm Lav Nerv Con Nerv Lav

Pre-mask sample

Pre-isolation sample

Post-isolation (1 min) sample

Post isolation (30 min) sample

11.38 ± 2.31a,x 18.10 ± 2.21a,x 19.61 ± 3.72a,x 15.73 ± 3.11a,x

24.36 ± 3.41b,x 14.55 ± 0.90a,c,y 31.10 ± 2.87a,x 28.88 ± 3.97b,x

34.59 ± 1.01c,x 32.04 ± 2.79b,x 53.29 ± 1.45b,y 58.52 ± 4.23c,y

13.48 ± 1.55a,x 8.41 ± 1.94c,x 14.76 ± 2.99a,x 37.11 ± 5.82d,y

3.2.2.4. Nervous lavender. Mean plasma concentrations of cortisol increased between the pre-isolation sample and the pre-mask sample (t7 = 2.35; P b 0.05; Table 3) and between the pre-mask sample and the post isolation (1 min) sample (t7 = 6.98; P b 0.05; Table 3). Mean plasma concentrations of cortisol 30 min post isolation were lower than those reported at 1 min post isolation (t7 = − 5.87; P b 0.001; Table 3) but were higher than those reported during the pre-mask sample (t7 = 4.02; P b 0.01; Table 3). 4. Discussion Lavender oil (L. augustifolia) is reportedly anxiolytic in a number of species [7,16,37,38] but this is the first study to investigate the effects of lavender oil on animals that differ genetically in their behavioral reactivity to a specific stressor. We originally hypothesized that exposure to lavender oil would reduce the behavioral and endocrine correlates of anxiety in nervous sheep exposed to an isolation stressor. However, exposure of nervous sheep to lavender oil appeared to increase rather than decrease the anxiety associated with the stressor, reflected by a higher frequency of vocalizations, more animals attempting to escape and higher plasma concentrations of cortisol 30 min after isolation compared to control sheep of the same temperament. In contrast, exposure of genetically ‘calm’ sheep to lavender oil appeared to reduce the anxiety associated with the stressor, reflected by reduced locomotor activity and frequency of vocalizations compared to control sheep of the same temperament. Polymorphisms in genes involved in the processing of anti-depressant and anti-psychotic drugs directly affect the proportion of individuals showing positive or adverse reactions to these drugs [5]. It is therefore possible that the divergent effects of lavender oil on the level of anxiety associated with the isolation stressor are an artifact of co-selection for polymorphisms in the enzymes and/or receptors responsible for mediating the effects of lavender oil on anxiety in sheep. Further studies are required to accept or reject this hypothesis, including direct comparisons with sheep of the same breed not selected for calm or nervous temperament. Exposure of calm sheep to lavender oil appeared to reduce the plasma cortisol response of calm sheep to mask application and was associated with significantly lower plasma concentrations of cortisol 30 min after isolation than pre-treatment values. These observations indicate that lavender oil had an anxiolytic effect on calm sheep, at least in terms of cortisol secretion. However, the absence of an effect of lavender oil on the plasma concentrations of cortisol immediately after the isolation stressor indicates that, at least in calm sheep, there may be a threshold concentration of cortisol above which the anxiolytic effects of lavender oil are ineffective. This concept is supported by Bradley et al. [39] who found that an oral dose of lavender

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oil reduced anxiety in humans exposed to neutral film clips but failed to reduce anxiety when the same individuals were exposed to film clips expected to induce anxiety in humans. However, lavender oil reduced locomotor activity and vocalization frequency in calm sheep during exposure to the isolation stressor, indicating a disconnection between the behavioral and endocrine correlates of anxiety. A similar disconnection between the behavioral and endocrine response of animals to a stressor was reported in pigs, where exposure to lavender straw reduced the behavioral expression of travel sickness but not the effects of transport on cortisol secretion [40]. We therefore propose that 10% lavender oil was sufficient to reduce the behavioral correlates of anxiety in calm sheep exposed to an isolation stressor but that its impact on cortisol secretion was dependent on the plasma concentrations of cortisol. The ‘nervous’ sheep were more active than ‘calm’ sheep, both in terms of their behavior and HPA activity when faced with an isolation stressor, indicating that they have been selected for a pro-active strategy to cope with social stressors [41]. This type of coping strategy in rodents is associated with higher levels of aggression and activation of the HPA axis when faced with a social stressor than those with a more reactive coping strategy [42]. Reports of positive, neutral or negative effects of drugs are common in the literature, especially with respect to the expression of aggressive behavior [43,44]. For example, benzodiazepine treatment of an aggressive species of mice (Omychomys leucogaster) increased the expression of aggressive behavior in response to electric shock but had no effect on less aggressive, albino white mice [43]. We propose that the divergent effects of lavender oil on the behavioral and endocrine correlates of anxiety in calm and nervous sheep may be at least in part due to differences in the strategies that they use to cope with stress. An alternative explanation for the greater expression of the behavioral and endocrine correlates of anxiety in nervous sheep exposed to lavender oil is the novelty of the aroma. Sheep are fearful of novel environments and objects, and these stimuli are frequently used to induce a fear response in sheep [26,45]. Little work to date has studied the impact of novel odors on fear and anxiety in sheep, but their responses to unfamiliar feeds may be mediated, at least in part, by the novelty of the odor [46]. However, the concentrations of plasma cortisol during the pre-isolation sample (i.e. after 30 min of exposure to lavender oil) did not differ between nervous control sheep or nervous sheep exposed to lavender oil, indicating that the novelty of the aroma was unlikely to be responsible for the elevated secretion of cortisol in nervous sheep. Parallels are frequently drawn between lavender oil and benzodiazepines because they have similar effects on anxiety in Mongolian gerbils and humans [9,13] that are mediated, at least in part, via the gammaaminobutyric acid complex [12]. There is currently no information on the effects of benzodiazepines on anxiety in sheep selected for calm or nervous temperament. We propose that further studies are warranted to directly compare the effects of anxiolytic drugs, such as benzodiazepines, on the behavioral and endocrine correlates of anxiety in sheep. The elevation in plasma concentrations of cortisol 30 min after initial exposure to the mask in all treatment groups, except calm sheep exposed to lavender oil, indicates a degree of stress associated with the mask and/or experimental procedure. We did not include a non-mask or empty mask control because masks have been successfully used in sheep with no apparent effect on hormone secretion [47,48]. However, changes in plasma concentrations of LH were the end point of these studies [47,48] rather than plasma concentrations of cortisol, as was the case in this study. In spite of this issue, calm and nervous sheep clearly differed in their cortisol response to the isolation stressor but we acknowledge that the mask itself may have influenced the magnitude of the changes in cortisol secretion. Lavender oil appears to have bi-directional effects on anxiety in calm and nervous sheep exposed to an isolation stressor. This outcome raises the question of whether this pattern is likely to be mirrored during exposure to other stressors? The two lines of sheep have been selected for

their behavioral reactivity to social isolation and human presence but there are limited studies into their reactivity to other stressors and these have yielded variable results. For example, Beausoleil et al. [35] found that nervous sheep were more active when faced with the conflict of approaching a human to access a pen of companion sheep but that the cortisol responses of the two lines to this stressor did not differ. Bickell [32] found that calm and nervous sheep initially had a similar increase in plasma concentrations of cortisol when faced with isolation combined with a novel stressor (white, flapping plastic). However, they found that the magnitude of this cortisol response decreased during subsequent exposures to the same stressor in calm sheep but not in nervous sheep, even when the novel stressor was removed [32]. The latter study demonstrated an important divergence between calm and nervous sheep in their propensity to interpret an ambiguous situation as negative or threatening, similar to that purported for rodent models of anxiety [25]. Social isolation clearly appears to be the dominant stressor in the index used to select calm and nervous sheep but further studies are required to determine whether the behavioral and endocrine correlates of anxiety differ between the two lines during initial and repeated exposure to different stressors. Only once this baseline information has been established can we conclusively determine whether lavender oil has a bi-directional effect on anxiety in calm and nervous sheep. 5. Conclusions Exposure to lavender oil had divergent effects on the behavioral and endocrine correlates of anxiety in calm and nervous sheep. Specifically, lavender oil reduced locomotor activity and vocalization frequency in calm sheep during exposure to an isolation stressor but only appeared to affect cortisol secretion when plasma concentrations were relatively low. In nervous sheep, lavender oil increased locomotor activity, vocalization frequency and plasma concentrations of cortisol 30 min after the isolation stressor relative to control sheep. We conclude that genetic differences in temperament determine whether lavender oil alleviates or exacerbates anxiety in sheep. It may therefore be important to consider the emotional state of an individual prior to using lavender oil to treat symptoms of anxiety. Acknowledgments This research was supported by operating funds from the UWA Lefroy fellowship. We thank S Gray for his assistance in the care and management of the animals, M Blackberry for her assistance with the cortisol assays, and S Plug and M Shikh Maidin for their assistance in data collection. References [1] Landgraf R. HAB/LAB rats: an animal model of extremes in trait anxiety and depression. Clin Neurosci Res 2003;3:239–44. [2] Kessler RC, Chiu WT, Demler O, Walters EE. Prevalence, severity, and comorbidity of twelve-month DSM-IV disorders in the National Comorbidity Survey Replication (NCS-R). Arch Gen Psychiatry 2005;62:617–27. [3] Roy-Byrne PP. The GABA-benzodiazepine receptor complex: structure, function, and role in anxiety. J Clin Psychiatry 2005;66(Suppl 2):14–20. [4] Lader MH. Limitations on the use of benzodiazepines in anxiety and insomnia: are they justified? Eur Neuropsychopharmacol 1999;9(Suppl 6):S399–405. [5] Kirchheiner J, Nickchen K, Bauer M, Wong M-L, Licino J, Roots I, et al. Pharmacogenetics of antidepressants and antipsychotics: the contribution of allelic variations to the phenotype of drug response. Mol Psychiatry 2004:442–73. [6] Cline M, Taylor JE, Flores J, Bracken S, McCall S, Ceremuga TE. Investigation of the anxiolytic effects of linalool, a lavender extract, in the male Sprague–Dawley rat. AANA J 2008;76:47–52. [7] Shaw D, Annett JM, Doherty B, Leslle JC. Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats. Phytomedicine 2007;14:613–20. [8] Kim Y, Kim M, Kim H, Kim K. Effect of lavender oil on motor function and dopamine receptor expression in the olfactory bulb of mice. J Ethnopharmacol 2009;125:31–5. [9] Bradley BF, Starkey NJ, Brown SL, Lea RW. Anxiolytic effects of Lavendula augustifolia odour on the Mongolian gerbil elevated plus maze. J Ethnopharmacol 2007;111:517–25.

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