Applied Animal Behaviour Science 64 Ž1999. 81–90
A study of cortisol and beta-endorphin levels in
stereotypic and normal Thoroughbreds
S.M. Pell ) , P.D. McGreevy
Department of Animal Science, UniÕersity of Sydney, Sydney, NSW, 2006, Australia
Accepted 26 February 1999
Abstract
In recent studies of equine stereotypic behaviour, data on levels of cortisol and beta-endorphin
ŽBE. have been limited and sometimes contradictory. The current research aimed to investigate, in
a large number of horses, the relationships between these compounds and equine stereotypic
behaviour. Plasma and salivary cortisol levels were measured in stereotypic Ž n s 46. and normal
horses Ž n s 46. to determine whether a significant difference exists between these two groups. No
significant differences were found between the mean plasma or salivary cortisol concentrations of
stereotypic and normal horses, indicating that their arousal levels are similar. The correlation
between plasma and salivary cortisol concentrations of individual horses Ž n s 66. was also
examined. A significant correlation between plasma and salivary cortisol levels was found only in
horses with an oral stereotypy Ž r s 0.65; P s 0.01., which has implications for the use of salivary
cortisol as a cardinal indicator of equine acute physiological stress responses. Additionally, plasma
BE levels were compared between horses with an oral stereotypy Ž n s 17. and normal horses
Ž n s 17.. Mean plasma BE levels did not differ significantly between the two groups. Since
endogenous opioids are thought to facilitate stereotypic behaviour, and a hereditary component to
stereotypic behaviour has been observed, this may suggest that stereotypic horses have inherited
opioid receptors with a greater sensitivity than those of normal horses. q 1999 Elsevier Science
B.V. All rights reserved.
Keywords: Equine; Stereotypy; Cortisol; Beta-endorphin; Saliva; Cribbing
1. Introduction
Stereotypies are repetitive, invariant behaviours with no obvious goal or function
ŽMason, 1991.. In horses, stereotypies are primarily based on feeding and locomotory
)
Corresponding author. Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia. Tel.:
q61-02-9351-2810; fax: q61-02-9351-3957.
0168-1591r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 5 9 1 Ž 9 9 . 0 0 0 2 9 - 5
82
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
behaviours, and are associated with management practices that limit foraging behaviour
and social contact ŽHoupt, 1981; McGreevy et al., 1995.. Thwarted motivation to
perform these behaviours causes frustration which is thought to cause the development
of stereotypies ŽHoupt, 1991; Broom and Kennedy, 1993; Dantzer and Mittleman, 1993;
Houpt, 1993..
A hereditary component to stereotypy development is suggested by evidence that
certain Thoroughbred bloodlines are more likely than others to perform stereotypies
ŽHosoda, 1950; Vecchiotti and Galanti, 1986.. It is probable that these horses have
inherited a tendency to perform a particular stereotypy ŽVecchiotti and Galanti, 1986..
Furthermore, it has also been suggested that stereotypic animals may have a genetic
susceptibility to stress ŽVecchiotti and Galanti, 1986; Sambraus and Radtke, 1989;
Luescher et al., 1991..
The physiological concept of stress involves the interaction between external events
Ž‘stressors’. and individual predispositions Ždetermined by genetic factors and early
experience. giving rise to measurable ‘stress’ responses ŽLadewig et al., 1993.. Because
stressors consistently prompt cortisol production, plasma cortisol concentrations have
frequently been used to characterise the stress responses of horses ŽAlexander et al.,
1988; Martinez et al., 1988; Mal et al., 1991; Clark et al., 1993; Mills et al., 1997..
However, care must be taken when interpreting the results of cortisol assays, since
cortisol production is influenced by a number of factors ŽRushen, 1986.. Cortisol
secretion follows a circadian and ultradian rhythm similar to that of other pituitary
hormones, with peak secretion occurring in the early morning ŽIrvine and Alexander,
1994.. These rhythms may be disturbed by exercise, copulation, learning, excitement
and stressors such as venepuncture or removing an individual from its familiar environment ŽRushen, 1986; Alexander et al., 1991; Colborn et al., 1991; Ladewig et al., 1993;
Irvine and Alexander, 1994..
Cortisol is present in the plasma, saliva and urine of animals. It is transported in the
plasma primarily in association with binding proteins, although 10–15% remains
unbound and is able to cross into saliva ŽStabenfeldt, 1992; Beerda et al., 1996.. The
correlation between plasma and salivary cortisol levels has been examined in several
species ŽFell et al., 1985; Parrott et al., 1989; Vincent and Michell, 1992; Beerda et al.,
1996; Lebelt et al., 1996., and a recent study in four stallions found close correlation
Ž r s 0.83. between plasma and salivary cortisol levels ŽLebelt et al., 1996.. Because
saliva collection is generally less arousing than venepuncture, it has been suggested that
salivary cortisol concentrations are a suitable alternative to plasma cortisol concentrations as a measure of physiological stress responses ŽBeerda et al., 1996; Lebelt et al.,
1996..
Since it has been proposed that stereotypies arise in response to stress ŽMason, 1993.,
the relationship between plasma cortisol levels and stereotypic behaviour has been
investigated in several species, including horses ŽMcGreevy and Nicol, 1995.. McGreevy and Nicol Ž1995. found that mean baseline and response levels of plasma
cortisol were significantly higher in stabled crib-biters Ž n s 6. than in normal horses,
which supports the suggestion that stereotypies occur in animals with increased levels of
arousal. Furthermore, it has been suggested that stereotypies may form part of a coping
mechanism for stress ŽBroom, 1991; Mason, 1991.. McGreevy and Nicol Ž1995. found
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
83
that stereotypic horses deprived of both ad libitum hay and the opportunity to crib-bite
for 24 h showed an increase in plasma cortisol levels, while no rise in cortisol was
detected when crib-biting alone was prevented. This suggests that crib-biting may have a
function in satisfying foraging needs when these are thwarted by environmental conditions.
Because opioid antagonists can transiently eliminate stereotypic behaviour ŽDodman
et al., 1987, 1988., it has been proposed that endogenous opioids, such as beta-endorphin ŽBE., facilitate and reinforce stereotypies ŽDodman et al., 1987; Gillham et al.,
1994.. Therefore, one might expect stereotypic horses to have higher plasma BE levels
than normal horses. However, Gillham et al. Ž1994. found that mean baseline plasma BE
levels in crib-biters Ž n s 5. were half those of normal horses, while McGreevy and
Nicol Ž1995. found no significant difference between mean plasma BE levels of stabled
crib-biters Ž n s 6. and normal horses.
The current research aimed to investigate the relationships between cortisol and BE
levels and equine stereotypic behaviour. The correlation between equine plasma and
salivary cortisol concentrations was also examined.
2. Materials and methods
2.1. Animals and experimental design
Thoroughbreds in training were selected for both cortisol and BE studies. The study
of cortisol levels used horses with an age range of 1–6 years Žmean " SEM: 3.2 " 0.1
years.. Stereotypic horses were divided into two groups: those with oral stereotypies
Žcrib-biters and wind-suckers ŽOS.; n s 24. and those with locomotory stereotypies
Žweavers and box-walkers ŽLS.; n s 22., as defined by McGreevy et al. Ž1995.. Control
horses ŽOC; n s 24 and LC; n s 22. were matched for sex and age Žto within 1 year.
with stereotypic horses on the same yard. One LSrLC pair was an exception to this, in
that a 3-year old gelding was matched with a 1-year old colt on the same yard. The
plasma and salivary cortisol correlation study was performed on 33 pairs of stereotypic
and control horses. These were combined with an additional 13 pairs to study mean
plasma and salivary cortisol levels ŽTable 1..
The study of BE levels used horses with an age range of 1–5 years Žmean " SEM:
3.2 0.2.. Each crib-biterrwind-sucker ŽOS1; n s 17. was matched for sex and age Žto
within 1 year. with a normal horse ŽOC1; n s 17. on the same yard that acted as a
control ŽTable 1..
2.2. Sample collection
All samples were taken between 8:00 AM and 11:30 AM, with experimental pairs
sampled within 15 min of each other to minimise the effects of diurnal rhythms. Pairs
were matched as closely as possible for the amount and type of exercise performed prior
to sampling and for overall fitness, since these are factors known to affect cortisol
concentrations ŽRushen, 1986; Alexander et al., 1991.. Withdrawal of blood took no
84
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
Table 1
Gender distribution of horses in the study of plasma and salivary cortisol levels Žoral stereotypers, i.e.,
crib-biters and wind-suckers ŽOS., and locomotory stereotypers, i.e., weavers and box-walkers ŽLS.. and of
horses in the study of plasma BE levels Žoral stereotypers, i.e., crib-biters and wind-suckers ŽOS1 ... Equal
numbers of stereotypic and control horses were used
Study
Group
Geldings
Colts
Fillies
Mares
Total
Plasma and salivary cortisol
OS
LS
OS
LS
OS
LS
OS1
13
7
15
9
13
10
9
1
1
2
2
1
1
2
6
5
7
6
6
5
6
0
0
0
2
0
0
0
20
13
24
19
20
16
17
Plasma cortisol
Salivary cortisol
BE
more than 30 s and no medication was used during sampling. To minimise the effect of
any potential lag and to avoid operator effect, saliva was collected immediately after
blood samples were obtained from each horse.
Prior to sample collection the venepuncture site was prepared by wiping with cotton
wool soaked in 70% alcohol. Jugular blood samples Ž20 ml. were collected using a
20-ml plastic syringe and a 19-gauge 1.5 in. needle. Half of this sample, for use in the
cortisol studies, was decanted into ice-cold vacutainers containing potassium EDTA and
kept on ice prior to centrifugation at 3000 rpm for 15 min at 08C. The plasma was
pipetted off and distributed into 1.5 ml Eppendorf tubes. The remaining 10-ml blood
sample, for use in the study of BE concentrations, was decanted into ice-cold polystyrene
tubes containing potassium EDTA. These were stored on ice and centrifuged under the
same conditions, after which polyethylene pipettes were used to distribute the plasma
into polypropylene tubes. All plasma samples were stored at y708C until assayed.
Saliva samples were collected onto cotton surgical swabs which had previously been
soaked in 1% acetic acid then dried at 378C. Metal surgical clamps were used to hold the
swab while it was rubbed around the horse’s mouth until saturated. The swab was
returned to the container and stored on ice. Once in the laboratory, it was inserted into a
conical centrifuge tube containing a short length of drinking straw, then frozen and
thawed. This reduced the viscosity of the saliva prior to centrifugation ŽRaid-Fahmy et
al., 1983; Fell et al., 1985.. The tubes were centrifuged at 2500 rpm for 15 min at 08C.
Saliva ran down to the bottom of the tube and reabsorption was prevented by the straw
which kept the swab elevated. The swab was removed immediately after centrifugation
to prevent capillary action drawing the saliva back up the straw. The saliva was pipetted
into 1.5 ml Eppendorf tubes and stored at y708C until it was assayed.
2.3. Cortisol assays
These were performed using an 125 I-cortisol radioimmunoassay kit ŽOrion Diagnostica, Espoo, Finland; Cat. No. 68548.. This kit is designed for use with human samples
but is suitable for equine use ŽFell, 1997, personal communication. and has a detection
limit of 4–7 nmolrl. The intra-assay variation was 2% for plasma and 5% for saliva,
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
85
and the inter-assay variation was 8% and 6%, respectively. Mean recovery was 91% for
plasma and 94% for saliva. The mean binding percent of the tracer to the antibody for
plasma and saliva was 96% and 77%, respectively, while mean non-specific binding was
5% for both.
All samples were run as duplicates, with subject samples and control samples run in
the same assay. Six serum standards were provided with the kit, ranging from 0 to 2000
nmolrl. These were diluted with Tris–HCl buffer Ž0.1 M, pH 7.4, 0.2% BSA. for the
salivary cortisol assays, giving a range from 0 to 100 nmolrl, since the concentration of
cortisol in the saliva is 10–15% of plasma cortisol concentrations. The antiserum was
also diluted for these assays Žone part antiserum to four parts buffer.. A standard curve
was produced by calculating the binding of these samples as a percentage of the
maximum binding and plotting these on a semi-logarithmic scale.
In the assay, each sample Ž25 ml standard solution, 50 ml plasma or 100 ml saliva.
was mixed with 100 ml 125 I-labelled cortisol and 100 ml cortisol antiserum Žrabbit.. The
tubes were incubated in a waterbath at 378C for 1 h and allowed to equilibrate to room
temperature for 10 min. The free and antibody bound hormone were separated by
precipitating the antibody-bound portion with 1.0 ml polyethylene glycol and centrifuging at 3000 rpm for 15 min at room temperature. The supernatant was decanted and
the radioactivity of the precipitate measured using a gamma counter Ž1272 CliniGamma;
LKB Wallac.. The amount of cortisol in each sample was calculated using the standard
curve.
2.4. Beta-endorphin assay
This was performed using an 125 I-BE radioimmunoassay kit ŽPeninsula Laboratories,
Belmont, CA, USA; Cat. No. RIK 8616.. This kit has 100% cross-reactivity with equine
BE and is most sensitive to values greater than 9 pgr100 ml. The peptide was extracted
from the plasma before being assayed because the kit is designed to be used with the
buffer supplied, rather than with biological fluids. The recovery for the overall procedure was 99.8%. The binding percent of the tracer to the antibody was 32.9%, while the
non-specific binding was 3.2%. All samples were processed in one assay to avoid the
effect of inter-assay variation.
Each 1-ml sample was acidified with 1 ml of Buffer A Ž1% trifluoroacetic acid ŽTFA.
ŽHPLC grade; Cat: BUFF-A. to remove interfering proteins. The sample was centrifuged
at 4000 rpm for 40 min at 48C. The extraction columns Ž18 C Sep-Columns containing
200 mg of 18 C; Cat: RIK-SEPCOL1. were equilibrated in a vacuum apparatus ŽVacElut
SPS24; Sample Preparation Products, Varian. by washing them with 1 ml of 100%
acetonitrile followed by two flushings with 3 ml of Buffer A under vacuum Ž5 srdrop..
The plasma solution was loaded onto the pre-treated 18 C Sep-Column using plastic
pipettes and allowed to run through under gravity. The vacuum was reapplied as the
column was washed by two flushings with 3 ml of Buffer A and the wash discarded.
The peptide was eluted with 1 ml of Buffer B Ž60% acetonitrile ŽHPLC grade. in 1%
TFA; Cat: BUFF-B.. The tubes were maintained at 208C while the eluant was evaporated to dryness using nitrogen gas. The residue was then frozen at y708C overnight.
The following day, the residue was dissolved in 250 ml RIA buffer and the assay
procedure started. All samples were run as 100 ml duplicates. The standard peptide
86
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
Ž12.8 mg lyophilised powder. was reconstituted with 1 ml of RIA buffer and serially
diluted to provide eight standard solutions ranging from 1 to 128 pgr100 ml. The
binding of these samples as a percentage of the maximum binding was calculated. A
standard curve was produced by plotting these on a semi-logarithmic scale.
The assay involved two overnight incubations. In the first stage, each sample Ž100
ml. was mixed with 100 ml BE antiserum Žrabbit. and incubated overnight at 48C. The
following day 100 ml 125 I-BE was added and the tubes returned to 48C overnight. On the
third day, 100 ml goat anti-rabbit IgG serum and 100 ml normal rabbit serum were
added to each tube and the samples incubated at room temperature for 90 min. RIA
buffer Ž500 ml. was added to each tube and the samples centrifuged at 3000 rpm for 20
min at 48C. The supernatant was aspirated and the radioactivity of the pellets measured
using a gamma counter Ž1272 CliniGamma; LKB Wallac.. The amount of BE in each
sample was calculated using the standard curve.
2.5. Data analysis
Mean values Ž"SEM. were obtained for plasma and salivary cortisol levels of OS,
OC, LS and LC. Inspection of a plot of the residuals for each group indicated that log
transformation Žlog e . was necessary to approximate a normal distribution prior to
analysis. A two-tailed t-test was used to evaluate the significance of the difference
between the transformed mean values of each group. Pearson correlation coefficients
were calculated to investigate the significance of the relationship between plasma and
salivary cortisol levels.
Fig. 1. Mean plasma and salivary cortisol concentrations Žngrml. in oral stereotypers Žcrib-biters and
wind-suckers ŽOS.; plasma: ns 24, saliva: ns 20. and their controls ŽOC; plasma: ns 24, saliva: ns 20.,
and locomotory stereotypers Žweavers and box-walkers ŽLS.; plasma: ns19, saliva ns16. and their controls
ŽLC; plasma ns19, saliva ns16..
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
87
Table 2
The t-test of the differences between the log e-transformed mean values of plasma and salivary cortisol
concentrations in oral stereotypers Žcrib-biters and wind-suckers ŽOS.. and their controls ŽOC., and locomotory
stereotypers Žweavers and box-walkers ŽLS.. and their controls ŽLC.. An equal number of stereotypic and
control horses were used
log e Žplasma cortisol.
log e Žsalivary cortisol.
Group
n
Mean difference"SEM
df
t
P
OS vs. OC
LS vs. LC
OS vs. OC
LS vs. LC
24
19
20
16
y0.06"0.13
y0.80"0.65
y0.24"0.19
0.20"0.18
23
18
19
15
y0.44
y0.53
y1.26
1.10
0.66
0.60
0.22
0.29
Mean plasma BE levels were calculated for stereotypic and normal horses. Since the
data were not normally distributed, a sign test of the median plasma BE value was
performed. This is a quantitative measure that examines the sign of the difference
between paired OS1 and OC 1 values while the magnitude of the difference is ignored.
3. Results
Mean plasma and salivary cortisol levels for each group of horses are illustrated in
Fig. 1. When a two-tailed t-test was performed, no significant differences were found
between the transformed mean plasma and salivary cortisol values of either OS and OC,
or LS and LC ŽTable 2..
Fig. 2. Distribution of plasma and salivary cortisol levels Žngrml. in oral stereotypers Žcrib-biters and
wind-suckers ŽOS.., oral stereotyper control horses ŽOC., locomotory stereotypers Žweavers and box-walkers
ŽLS.. and locomotory stereotyper control horses ŽLC..
88
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
Pearson correlation coefficients indicate that a significant relationship exists between
the plasma and salivary cortisol levels of OS Ž r s 0.65; P s 0.01. but not between the
plasma and salivary cortisol levels of OC Ž r s 0.07., LS Ž r s y0.11. and LC Ž r s
y0.07.. The distribution of plasma and salivary cortisol levels in individual horses is
illustrated in Fig. 2.
No significant difference Žmedians y0.03; P s 1.00. was found between the mean
plasma BE levels of OS1 and OC 1 Žmean " SEM: 8.20 " 0.62 pgr100 ml and 8.99 "
0.52 pgr100 ml, respectively..
4. Discussion
No correlation was found between plasma and salivary cortisol levels in three of the
four groups of horses studied because high salivary cortisol values were found in
conjunction with low plasma cortisol values ŽFig. 2.. It is possible that these high values
were a response to a previous stressor. Although steroid concentrations appear to be
independent of flow rate in human saliva ŽRaid-Fahmy et al., 1983. and ovine saliva
ŽFell et al., 1985., this may not be the case in horses. Cortisol levels in the plasma
respond rapidly to increases in arousal levels and cortisol passes freely into saliva
ŽStabenfeldt, 1992; Beerda et al., 1996.. However, equine salivary cortisol may not be
able to cross back as readily into plasma, thus causing a build-up of cortisol in the
salivary gland. This suggestion is supported by the correlation found between plasma
and salivary cortisol concentrations in horses with oral stereotypies. It is possible that
the additional oral activity associated with these behaviours flushes cortisol from the
salivary glands and allows salivary cortisol levels to remain in equilibrium with plasma
cortisol levels.
In addition, it has been observed that the proportion of unbound cortisol in plasma,
and thus the amount of cortisol in saliva, varies between individuals ŽVincent and
Michell, 1992.. Furthermore, plasma cortisol binding proteins become saturated as
plasma cortisol concentrations increase, allowing a greater amount of cortisol to exist in
the unbound state and pass into saliva ŽRaid-Fahmy et al., 1983; Vincent and Michell,
1992.. Therefore, it is suggested that salivary cortisol levels may not always accurately
represent equine acute physiological responses to stressors.
In contrast to the results obtained by McGreevy and Nicol Ž1995., no significant
difference was found between mean plasma cortisol levels of stereotypic and normal
horses. Therefore, because the current study utilised focal samples, whereas repeated
sampling was used by McGreevy and Nicol Ž1995., it is possible that the differences
observed previously were experiential in origin. The present research suggests that
arousal levels of the two groups are similar. It is possible that stereotypic horses adapt to
cope with the stress that caused stereotypy development, and that the stereotypy itself
forms a part of that adaptive process. Longitudinal surveys of cohorts of young horses
would be useful in establishing whether a transient peak in stress levels occurs prior to
the emergence of stereotypic behaviour.
The current study demonstrates no significant difference between mean plasma BE
levels of horses with an oral stereotypy and normal horses. Since the motivation for
S.M. Pell, P.D. McGreeÕyr Applied Animal BehaÕiour Science 64 (1999) 81–90
89
stereotypic behaviour is thought to change with time ŽRushen et al., 1993., the results
obtained may have been affected by the unknown history of stereotypic behaviour of the
horses included in the study. However, the suggestion that endogenous opioids facilitate
stereotypic behaviour remains plausible. Rather than having an impaired release of BE,
it is possible that crib-biting horses have inherited an increased opioid receptor sensitivity ŽGillham et al., 1994.. Therefore, endogenous opioids could facilitate stereotypic
behaviour in the absence of abnormally high plasma BE levels. This may explain part of
the hereditary component to stereotypic behaviour. Further investigation of opioid
receptor sensitivity in stereotypic and normal horses would help to resolve this issue.
The current data demonstrate that arousal levels of stereotypic and normal horses are
similar, and it is suggested that salivary cortisol concentrations are an inappropriate
cardinal measure of equine acute physiological stress responses. Furthermore, it is
proposed that the hereditary component of stereotypic behaviour may be partly due to an
inherited upshift in opioid receptor sensitivity.
Acknowledgements
Trainers involved in this research are thanked for their generous cooperation. Kim
Heasman and Dot Lewis are thanked for their technical assistance, while Adrienne Kirby
is thanked for her statistical advice. David Evans and Jennifer Hodgson are thanked for
their helpful comments on this manuscript. This research was funded by a University of
Sydney Research Grant.
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