Journal of Equine Veterinary Science 34 (2014)

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Original Research

Electroencephalographic Responses to a Noxious Surgical Stimulus in

Mules, Horses, and Ponies
Nicola J. Grint BVSc, PhD a,*, Craig B. Johnson BVSc, PhD b, Silvia De Sa Lorena DVM, PhD c, Stelio
Luna DVM, PhD c, Carlos A. Hussni DVM, PhD c, Helen R. Whay BSc, PhD a, Joanna C. Murrell BVSc,
PhD a
a

Animal Welfare and Behaviour Group, School of Veterinary Science, University of Bristol, Langford, Bristol, UK b Institute of Veterinary,

Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand c Department of Veterinary Surgery and
Anaesthesiology, UNESPdUniversidade Estadual Paulista, Botucatu, Sao Paulo, Brazil

articleinfo
Article history:
Received 19 December 2013
Received in revised form 27 March 2014
Accepted 29 April 2014 Available
online 6 May 2014

Keywords:
Donkey
Mule
Pony
Electroencephalographic
Noxious

abstract

The aim of this study was to investigate the electroencephalographic (EEG) response of equidae to a
castration stimulus. Study 1 included 11 mules (28 years; 230315 kg) and 11 horses (13 years;
315480 kg); study 2 included four ponies (1517 months; 176229 kg). They were castrated under
halothane anesthesia after acepromazine premedication (IV [study 1] and intramuscular [study 2]) and
thiopental anesthetic induction. Animals were castrated using a semiclosed technique (study 1) and a
closed technique (study 2). Raw EEG data were analyzed and the EEG variables, median frequency (F50),
total power (Ptot), and spectral edge frequency (F95), were derived using standard techniques at skin
incision (skin) and emasculation (emasc) time points. Baseline values of F50, Ptot, and F95 for each
animal were used to calculate percentage change from baseline at skin incision and emasculation.
Differences were observed in Ptot and F50 data between hemispheres in horses but not mules (study 1)
and in one pony (study 2). A response to castration (>10% change relative to baseline) was observed in
eight horses (73% of animals) and four mules (36% of animals) for F50 and nine horses (82%) and four
mules (36%) for Ptot. No changes in F95 data were observed in any animal in study 1. Responses to
castration were observed in three ponies (75% of animals) for F50, one pony (25%) for F95, and all
ponies for Ptot. Alteration of acepromazine administration and castration technique produced a protocol
that identifed changes in EEG frequency and power in response to castration.
2014 Elsevier Inc. All rights reserved.

1. Introduction
The cerebral cortex is a region of the brain that is involved with
many functions in man, such as perception, learning, language, and
cognition [1]. There is acceptance that animals can perceive pain
rather than just experience nociception [2] and that the application
of a noxious stimulus to an animal activates regions of the
cerebral cortex including the cingulate, insular, and sensory
cortices, all associated with pain perception in man [3]. An animal
although unconscious under general anesthesia cannot perceive
* Corresponding author at: Nicola J. Grint, Cave Veterinary Specialists, Georges
Farm, West Buckland, Wellington, Somerset TA219LE, UK. E-mail address:
nicki.grint@yahoo.co.uk (N.J. Grint).
0737-0806/$ see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jevs.2014.04.008

veterinary species [57]. The minimal anesthesia model involves

maintaining a light plane of general anesthesia with halothane

pain per se; however, the nociceptive response of the cerebral

cortex to a noxious stimulus can be evaluated by recording the
electrical activity (the electroencephalography [EEG]) generated
by groups of pyramidal neurones of the cerebral cortex. In man,
the frequencies of EEG activity are categorized into bandwidths:
delta (04 Hz), theta (48 Hz), alpha (812 Hz), and beta (>12
Hz) [4].
A minimal anesthesia model has been used to identify the
response of the EEG to a noxious stimulus in various
during application of a noxious stimulus, with analgesia
administered after data collection are complete. Although the
clinical use of halothane in equine anesthesia has declined in
recent years, newer agents such as isofurane cause greater
depression of the cerebral cortex at surgical planes of anesthesia
[8]. The use of the minimal anesthesia model resulted in the
detection of signifcant changes in the frequency and power of the
EEG during noxious stimulation, and this response (i.e., change in
predominant bandwidth or shift in overall frequency) has been

N.J. Grint et al. / Journal of Equine Veterinary Science 34 (2014) 955962

reported in several species. Two patterns have been identifed. The

classical arousal pattern is a shift toward higher frequencies of the
EEG with lower amplitude waveforms and has been observed in
dogs [9,10], sheep [11], goats [12], cattle [13], red deer [6], and
horses [5]. The paradoxical arousal pattern is a shift toward lower
frequencies, higher amplitude waveforms, often with a
predominance of delta waves [14], and has been observed in
wallabies [15], rats [16], and sheep [17].
The original aim of this study was to repeat the minimal
anesthesia method [5] in a group of different equidae; horses
(Equus caballus) and mules (Equus asinus Equus caballus) to
compare the species response to a noxious surgical stimulus
(study 1). Study 2 describes the refnements made to the anesthesia
and surgical protocol of study 1 required to observe the EEG
markers of nociception previously reported in equidae under the
minimal anesthesia model [5]. As Murrell et al [5] recorded EEG
data from one cerebral hemisphere only, the third aim was to
record and compare EEG data between both cerebral hemispheres
(studies 1 and 2). Data from two studies that were carried out to
achieve these aims are reported here.

surgery before the animals were allowed to recover from

anesthesia.
2.1.3. Surgical Technique
One staff surgeon at UNESP performed all castrations using a
semiclosed castration technique. The left testicle was removed
frst, with the time point of skin incision recorded as T1skin on
the anesthetic record and the EEG recording. The incision was
deep enough to transect the tunica dartos and tunica vaginalis
comunalis. No surgical stimulation was then applied until a period
of two minutes after the frst incision had elapsed to standardize
the surgical approach. Two pairs of large hemostats were applied
across the spermatic cord, which was transected with scissors
between the hemostats. This time point was recorded as
T1emasc. Ligatures were applied across the tunica vaginalis to
close the structure. The right testicle was removed in an identical
manner through a separate skin incision. The time points of skin
incision and emasculation were recorded as T2skin and
T2emasc, respectively.
2.2. Study 2

2. Materials and Methods

The two studies received ethical committee approval from the
University of Bristol ethical review group (UB/ 09/029).
2.1. Study 1
2.1.1. Animals
Eleven mules aged 28 years, weighing 230315 kg, and 11
horses aged 13 years, weighing 315480 kg, were
anesthetized for castration at the University of So Paulo State
(UNESP), Brazil. They were deemed healthy based on routine
hematological and biochemical blood analyses. As they were
previously unhandled, only a brief clinical examination was
possible before anesthesia; but no abnormalities were identifed
which could cause a chronic pain state.
2.1.2. Anesthetic Protocol
Animals were starved of food but not water overnight before
general anesthesia. After skin infltration with lidocaine
(Xylocaina; Astrazeneca, Sao Paulo, Brazil), a 14-gauge catheter
was placed percutaneously in the jugular vein and secured in place.
Preanesthetic medication was acepromazine (Acepran; Univet, Sao
Paulo, Brazil) (0.03 mg kg1) injected through this catheter. Thirty
minutes later anesthesiawas induced with 10 mg kg1 thiopental
(Thiopentax; Cristalia, Itapira, Brazil) IV. After orotracheal
intubation, anesthesia was maintained with halothane (Tanohalo;
Cristalia) vaporized in 100% oxygen, delivered via a large animal
circle system. Intermittent positive pressure ventilation (2800
LAAV; Mallard Medical Inc, Redding, CA) was used to maintain
end-tidal carbon dioxide (CO2) between 35 and 50 mmHg.
Hartmann solution (Soluo de Ringer Lactato; Endomed
Laboratrio Farmacutico, Sao Paulo, Brazil) was infused IV (5
mL kg1 hr1) and dobutamine (Dobutamol; Hipolabor, Belo
Horizonte, Brazil) was added to this infusion to maintain mean
arterial blood pressure >60 mmHg. Flunixin (Flunixin injecta;
Chemitec, Campinas, Brazil) (1.1 mg kg 1 IV) and morphine
(Dimorf; Cristalia) (0.1 mg kg1 IV) were administered at the end of

2.2.1. Animals
Four Exmoor ponies, aged between 15 and 17 months,
weighing 176229 kg, were anesthetized for castration at the
University of Bristol School of Veterinary Sciences. They were
deemed healthy based on limited clinical examination.
2.2.2. Anesthetic Protocol
Animals were starved of food, but not water, overnight before
general anesthesia. In contrast to study 1, preanesthetic medication
was acepromazine (ACP; Novartis, Camberley, UK) (0.03 mg kg 1)
injected intramuscularly. A 14-g catheter was placed in the jugular
vein, after local anesthetic infltration with mepivacaine
(Intraepicaine; Dechra, Shrewsbury, UK), and secured in place.
Between 30 and 65 minutes after premedication, anesthesia was
induced with thiopental (Thiopental, Novartis) (10 mg kg 1),
administered through the jugular catheter.
After orotracheal intubation, anesthesia was maintained with
halothane (Halothane-Vet; Merial, Harlow, UK) vaporized in
oxygen and delivered via a large animal circle system. Intermittent
positive pressure ventilation was initiated once the animal had
been positioned in dorsal recumbency on the operating table.
Ventilator (Large Animal Ventilator; Drger, Telford, PA) settings
were adjusted to maintain end-tidal CO2 between 35 and 50
mmHg.
Hartmann solution (Hartmanns solution; Baxter, Newbury, UK)
was infused IV (5 mL kg1 hr1), and dobutamine (Dobutrex; Eli
Lilly Ltd, Basingstoke, UK) was added to this infusion to maintain
mean arterial blood pressure >60 mmHg. Flunixin (Mefosyl;
Pfzer, Sandwich UK) (1.1 mg kg 1 IV) and morphine (Morphine
sulphate; Martindale, Brentwood, UK) (0.1 mg kg 1 IV) were
administered at the end of surgery before the animals were allowed
to recover from anesthesia.
2.2.3. Surgical Technique
One staff surgeon at the University of Bristol School of
Veterinary Sciences performed all castrations using a closed
technique. The incision (marked at T1skin) was deep enough to
transect the tunica dartos but not the tunica vaginalis communalis.

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A transfxing suture was placed on the cord, and Serra

emasculators were then used to crush and transect the cord
(marked as T1emasc), and the emasculators were left in place
for three minutes. The right testicle was removed in an identical
manner through a separate skin incision, with skin incision and
emasculation time points recorded as T2skin and T2emasc,
respectively.
2.3. Anesthetic Monitoring (Studies 1 and 2)
Anesthetic monitoring (Capnomac v5; Datex-Ohmeda Ltd,
Stirling, UK) included pulse oximetry, electrocardiography,
capnography, end-tidal halothane concentration, and measurement
of arterial blood pressure directly via an arterial catheter placed in
the auricular artery (mules) or in the submandibular branch of the
facial artery (horses and ponies). Calibration of the gas module of
the anesthetic monitor was checked at the beginning of every study
day using calibration gas (Datascope Passport two calibration gas;
Datex-Ohmeda Ltd).
A European Specialist in Veterinary Anaesthesia and Analgesia
(N.G.) monitored the depth of anesthesia in all animals, using eye
position and palpebral refex, neck muscle tone, whether the
animal bucked the ventilator, and the tension the surgeon
reported in the cremaster muscle. The same person adjusted the
concentration of delivered halothane to achieve a light surgical
plane of anesthesia, that is, ventromedial eye rotation, a sluggish
palpebral refex, and relaxed muscle tone. End-tidal concentration
maintained between 0.9% and 1.1% (study 1) and 0.8% and 0.9%
(study 2). In each study, a staff anesthetist from the faculty
monitored the other aspects of the anesthetic.
Blood samples were drawn from the arterial catheter for blood
gas analysis (Chiron Diagnostics 348 analyser; Chiron
Diagnostics, Emeryville, CA) at three points during the study
period. A baseline sample was collected once the end-tidal
halothane and end-tidal CO2 concentrations had been stable for 30
minutes; T1 samples were drawn at the T1emasc time point and T2
at the T2emasc time point.
At baseline, T1 and T2 time points, heart rate, blood pressure,
and end-tidal halothane concentration were recorded.
2.4. EEG Monitoring (Studies 1 and 2)
Once the animals were anesthetized and positioned in dorsal
recumbency, 0.4mm diameter,12-mmlong27-gauge needle
electrodes (Needle electrodes; Carefusion Corporation,
Basingstoke, UK) were placed subcutaneously. Reference
electrodes (right and left) were placed at the midline of the
forehead and active electrodes (right and left) over the zygomatic
processes [7]. A common ground electrode was positioned caudal
to the poll on the left-hand side. The two channels of EEG signal
(channel 1 from the left side and channel 2 from the right side)
were amplifed through two bioamplifers (Iso Dam [study 1] and
DAM50 [study 2]; WPI Instruments, Hitchin, UK). The channel
recorded by each bioamplifer was kept constant throughout the
study. The EEG was recorded with a gain of 1,000 and a pass band
of 0.5500 Hz [7]. The EEG was digitized (MacLab; AD
instruments, Oxford, UK) at a rate of 1 kHz [7] and was recorded
continuously (Chart v5; AD instruments) using a personal
computer (Mac Power Book G4 laptop; Apple,
Cork, Ireland).

A 5-minute baseline epoch of EEG was recorded after the endtidal halothane concentration had been stable between 0.9% and
1.1% (study 1) or between 0.8% and 0.9% (study 2) for 30
minutes. Markers were inserted into the EEG recordings for
T1skin, T1emasc, T2skin, and T2emasc at the time
points detailed previously.
Noise was often observed during EEG recording, thought to be
because of electrical interference. Attempts were made to prevent
this by further grounding the electrodes using a connection to the
operating table. After data collection from six mules, the electrical
supply to the EEG monitoring equipment was changed from mains
to a 12V DC battery.
Two-minute sections of raw EEG data were analyzed from the
following time points for both left and right channels; the last 2
minutes of the baseline period (baseline), 2 minutes after skin
incision of testicles 1 and 2 (T1skin and T2skin), and 2 minutes
after emasculation of testicles 1 and 2 (T1emasc and T2emasc).
2.5. Data Analysis
Data generated from study 1 and study 2 were analyzed
separately.
The raw EEG data were inspected, and sections from the
epochs to be analyzed were manually rejected if noise (a random
fuctuation in electrical signal) was observed or when the signal
saturated the input amplifer resulting in a large offset. Fast Fourier
transformation was carried out on consecutive 1-second epochs of
data using purposewritten software (Spectral Analyser; CB
Johnson, Massey University, Palmerston North, New Zealand).
Median frequency (F50), spectral edge frequency (F95), and total
power (Ptot) were derived from the spectra using standard
techniques [13].
Datafromthetwo channelswere analyzedseparately.For each
two-minute section analyzed, 120 values were generated (one per
second) for each of F50, F95, and Ptot. These
datawerethensmoothed usinga10-pointforwardmoving average.
For each of F50, F95, and Ptot, mean smoothed values were
calculated for each two-minute section.
The mean baseline value for each animal was then used to
calculate the percentage change from baseline at the T1 and T2skin
and T1 and T2emasc time points. Animals were identifed as
responders if percentage change was >10% increase or
decrease relative to baseline [5]. For each group, a mean
percentage change from baseline for each time point was
calculated.
Normal distributions of all data were checked by plotting data
series as histograms. Median frequency, F95, and Ptot data were
found not to be normally distributed. Friedman two-way rank
analysis was used to compare the mean values for F50, F95, and
Ptot across time points. If results indicated that the distribution of
data were not similar over time points, then a Wilcoxon test was
used to indicate which time points yielded signifcant differences.
End-tidal halothane concentrations were compared between
baseline and T1 and T2 time points within each individual animal
in horses, mules, and ponies using repeated measures analysis of
variance.
If data were normally distributed, data were compared between
mule andhorse groups using a Student t test. If data were not
normally distributed, data were compared between horse and mule
groups using a MannWhitney test.

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N.J. Grint et al. / Journal of Equine Veterinary Science 34 (2014) 955962

All statistical analysis (SPSS v5.0 IBM corporation, Armonk,

NY) was carried out with signifcance set at P <.05. Results of
nonparametric analyses are presented as median (range) and results
of parametric analyses are presented as mean (standard deviation
[SD]).

species. In mules (channel 1), Ptot varied signifcantly (P .048)

across the fve time points. Mean values of Ptot in channel 2
(mules) and in both channels in horses did not vary signifcantly
over time points. Overall percentage changes (mean for the group)
for F50, F95, and Ptot are shown in Fig. 1A, B, and C (horse
group) and Figs. 2A, B, and C (mule group).

3. Results
3.2. Study 2
3.1. Study 1
Mean arterial blood pressure decreased <60 mmHg in three
mules and four horses at one time point and in one horse and one
mule at two time points but did not fall <50 mmHg at any time
point. Blood gas results for one mule demonstrated a PaO 2 of <60
mmHg over three time points (mean, 56.2 mmHg). Oxygen
tensions averaged across the three time points in both groups
demonstrated normoxia (>60 mmHg) in all other animals. Median
(range) PaO2 values were 176.7 (58.2391.6) mmHg for mules and
347.8 (199.3450.6) mmHg for horses. Median (range) PaCO2
were similar between groups (mules: 40.73 [32.9247.63] mmHg
and horses: 42.45 [34.6562.41] mmHg).
Averaged end-tidal halothane concentrations were signifcantly
(P .0375) lower in the horse group (mean [SD], 0.99 [0.05]%)
compared with mules (1.1 [0.07]%), but within each group did not
differ signifcantly over the course of castration.
Noise was often observed on EEG recordings; this often
coincided with frequent electrical storms at UNESP. Several
sections of the epochs intended for analysis were manually
rejected when the signal saturated the input amplifer resulting in a
large offset. In two horses and three mules, good quality data were
recorded for a time shorter than the two-minute epoch at some
time points before noise was observed; the good quality data only
were included in analysis.
3.1.1. Difference Between Channels 1 and 2
In the horse group, baseline F50 values for channels 1 and 2
were often different from each other. Frequencies in one channel
ranged from 1 to 3 Hz, and in the other channel ranged from 4 to 7
Hz, although the channel recording the higher frequencies was not
consistent. Median (range) baseline Ptot values were 49.3 (31.2
583.5) in the horse group and 44.7 (23.3218) in the mule group.
There was disparity between channel 1 and channel 2 Ptot data in
seven horses but no mules. No signifcant differences were
observed between channels in F95 data in either the horse or the
mule group.
3.1.2. Changes in EEG Across Time
Eight horses and four mules were responders for changes in
F50 at one or more time points across castration; however, this was
not consistent in both channels of EEG recording. Changesin F50
in mules were mainlyin a positive direction (indicating an increase
in F50 relative to the baseline period); however, in horses, a
combination of negative (decrease in F50 relative to baseline) and
positive changes was detected. Nine horses and four mules
responded with changes in Ptot that were >10% relative to
baseline, usually in a negative direction. There was a change in
F95 relative to baseline in one horse and none of the mules.
Overall, for the groups, mean values of F50 and F95 data did
not change signifcantly over time points in either channel in either

Mean arterial blood pressure was maintained >60 mmHg at all

time points in all ponies. PaCO2 and PaO2 were maintained at
clinically acceptable levels at all time points. Median (range)
PaCO2 was 48 (40.353.2) mmHg and median (range) PaO2 was
500.6 (291513) mmHg.
Averaged end-tidal halothane concentrations over the three
time points ranged between 0.8% and 0.9%. Mean end-tidal
halothane concentrations were 0.83% (baseline), 0.85% (T1), and
0.89% (T2); but this change was not statistically signifcant.
Less noise on the EEG recording was observed during data
collection and analysis in study 2 compared with study 1. In study
2, some sections of the epochs intended for analysis were manually
rejected when a noisy signal or a large offset was observed.
Suffcient good quality data were included in analysis in both
channels in all ponies, except for one pony where channel 1 data
for T1emasc, T2skin, and T2emasc could not be analyzed. In two
ponies, emasculation was carried out sooner than two minutes after
skin incision, and therefore, the sections analyzed were shorter.
Mean values for percentage change from baseline (F50, F95, and
Ptot) of the four ponies are shown in Fig. 3A, B, and C.
3.2.1. Differences Between EEG Channels
Channel 1 and Channel 2 data were similar in three ponies. In
the remaining, pony differences were observed in F50, F95, and
Ptot between channels. Median (range) baseline Ptot for the group
was 27.8 (14.538.7).

N.J. Grint et al. / Journal of Equine Veterinary Science 34 (2014) 955962

Fig. 1. (A, B, and C) Percentage change from baseline of (A) F50, (B) F95, and
(C) Ptot over four time points during castration. Data points represent the mean
percentage change from a group of 11 horses. A response is considered a 10%
increase or decrease relative to baseline. F50, median frequency; F95, spectral
edge frequency; Ptot, total power.

3.2.2. Changes in EEG Over Time

Three out of the four ponies responded with a change >10% in
F50 from baseline at T1skin and T1emasc time points. Statistically,
differences were found in channel 2 data where a signifcant
increase was observed between baseline and T1skin F50 values in
channel 2 (P .046).
All four ponies responded with changes in Ptot >10% at the
T1skin time point, with three of four ponies showing decreased
percentages relative to baseline for Ptot. At T1emasc, Ptot
increased to values above baseline, although only one pony
responded with a change >10%. Overall, for the group, signifcant
differences were found between baseline and T1skin Ptot values in
channel 1 (P .046). Total power values were not signifcantly
different from baseline at either T1emasc or T2 time points.
Spectral edge frequency in one pony increased >10% from
baseline in channel 1 at the T1skin time point. Otherwise, no pony
responded by increasing or decreasing F95 >10% at any time
point in any channel.

Fig. 2. (A, B, and C) Percentage change from baseline of (A) F50, (B) F95, and
(C) Ptot over four time points during castration. Data points represent the mean
percentage from a group of 11 mules. A response is considered a 10% increase
or decrease relative to baseline. F50, median frequency; F95, spectral edge
frequency; Ptot, total power.

4. Discussion
Here, we describe a two-stage study that repeated the minimal
anesthesia model already established in ponies [5]. We had
predicted that castration in horses and mules would be associated
with an increase in F50 and decrease in Ptot relative to an
unstimulated baseline recording period, as found by other authors
in ponies and other species [6,911,13]. However, this was not the
case in study 1, and therefore, achieving the original aim, to
compare EEG responses with a noxious stimulus by different
equidae, was not possible. Therefore, refnements to the anesthesia
protocol were made. For logistical reasons, it was not possible to
perform the study again at UNESP with a second cohort of horses
and mules. This was a limitation to the study. Instead, study 2 was
planned using a group of ponies, and EEG data collected during
castration repeated. As predicted, in study 2, EEG changes
mimicked those reported previously. Study 2 also served as a pilot
for a

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Fig. 3. (A, B, and C) Percentage change from baseline of (A) F50, (B) F95, and
(C) Ptot over four time points during castration. Data points represent the mean
percentage change from a group of four ponies. A response is considered a 10%
increase or decrease relative to baseline. F50, median frequency; F95, spectral
edge frequency; Ptot, total power.

study comparing the EEG changes with castration in ponies and

donkeys.
Notable disparity between EEG data collected from the left and
right hemispheres was found in study 1, which was unexpected.
Electroencephalographic data are often collected from one cerebral
hemisphere only; however, in studies in equidae where data from
both cerebral hemispheres have been recorded [1820], no
disparity between hemispheres has been reported previously.
Similarly in deer [6], cattle [13], and dogs [21], EEG data recorded
have not differed between recording channels. Murrell et al [22]
reported differences in EEG data recorded from right and left
primary
somatosensory
cortices
in
rats
undergoing
ovariohysterectomy, but concluded that the disparities were minor
and showed no consistent patterns.
In the horse group, baseline F50 values for channels 1 and 2
were often different from each other. Frequencies in one channel
ranged from 1 to 3 Hz and in the other channel ranged from 4 to 7
Hz, although the channel recording the higher frequencies was
inconsistent. Median frequencies in deer, calves, and rats

anesthetized with halothane before the application of a noxious

stimulus ranged from 2 to 4.5 Hz [6,13,22]. These F50 values were
therefore similar to ranges reported in other species.
The Ptot of the EEG was also often different between channels
in the horses. Comparison of Ptot between this study and other
models in other species is diffcult. Total power varies signifcantly
between studies [6,13,22] and is infuenced by anatomic factors
such as head size and amplifcation of the signal. Interestingly, the
channel producing the higher value was not consistent between
horses, although it was consistent within the data collected for
each horse. This makes it unlikely that differences in channels
were because of a lateralization (i.e., right or left sided) of the
changes in EEG frequency and power dependent on lateralization
of the surgical stimulus, as the left testicle was always removed
frst in the surgical protocol. In addition, the disparity between the
two channels was evident from the baseline time point before
castration started. The bioamplifers were labeled to ensure that a
particular bioamplifer was consistently used to amplify signal
from the left or right cerebral hemisphere, and so it is unlikely that
it was a fault with one of the bioamplifers. Electromyogenic
(EMG) interference could have been a cause, as a palpebral refex
was observed in some animals under anesthesia, which indicated
that they were maintained at a light surgical plane of anesthesia.
Spontaneous palpebral refex can occur unilaterally and so could
have affected a different channel in each animal. Electromyogenic
activity tends to occur as low as the alpha wave band (813 Hz)
[23].
Electrical interference, localized to one channel of recording,
was also considered as a cause of the disparity in EEG data
recorded from the left and right hemispheres. Electrical
contamination of the signal will generally produce activity at 50
Hz and so similar to EMG interference will tend to elevate F50,
although, to a greater extent. Spectral edge frequency would have
been more sensitive to outlying very high frequencies than F50,
but F95 was not infuenced by channel. A number of approaches
were adopted to try and increase the signal-to-noise ratio of EEG
data to reduce electrical interference. Strategies included additional
grounding of the electrodes, switching electrical supply from
mains to a DC battery, and avoiding data collection during the
frequent electrical storms.
The lack of change of the EEG frequencies and power during
castration in study 1 may have been for a variety of reasons
including premedication and induction protocols and surgical
technique. Because of the unhandled nature of the horses and
mules, it was decided to administer the acepromazine preanesthetic
medication by the IV route rather than the intramuscular route as
described by Murrell et al [5]. Animals appeared to be well sedated
after this preanesthetic medication alone. Acepromazine
administered at a dose rateof 0.05 mg kg1 IV reduced the minimum
alveolar concentration (MAC) of halothane in ponies by 37% from
a MAC of 0.91% to 0.58% end-tidal halothane concentration [24].
Although the end-tidal halothane concentrations maintained in the
study 1 (0.92%1.1%) were lower than the minimum end-tidal
concentration of 1.2% used previously [5] and the animals
appeared to be maintained under a light surgical plane of
anesthesia, it may be that the acepromazine depressed cortical
function and obtunded changes in response to nociception.
Sedation induced by acepromazine in horses can produce EEG
activity similar to slow-wave sleep [20], although acepromazine
administered to healthy horses and foals had little effect on the

N.J. Grint et al. / Journal of Equine Veterinary Science 34 (2014) 955962

EEG [25]. Although acepromazine is not inherently analgesic,

drugs with mechanisms other than analgesia such as muscle
relaxation can alter EEG changes to a noxious insult [26].
The surgical technique described in study 1 is an example of a
semiclosed castration where the tunica vaginalis was incised (open
castration) but then ligated at the end of surgery. This is in contrast
to the closed technique used for castration previously [5]. The
traction of the testis and stripping of the fascia in a closed
castration may represent a greater surgical stimulus than that of an
open technique, and this is another possible reason for the lack of
signifcant changes in EEG frequency or power during castrations
in study 1.
There was the opportunity to measure EEG changes during
castration in a group of ponies at the University of Bristol, and
therefore, it was decided to use these four ponies in a second study
to refne the minimal anesthesia model as applied in study 1 and
establish whether with an altered study protocol, the fndings of
Murrell et al [5] could be confrmed. Refnement to the
methodology between study 1 and study 2 included altering
surgical technique and anesthetic protocol. The acepromazine dose
was administered intramuscularly, and the target end-tidal agent
percentage was decreased to between 0.8% and 0.9% to ensure that
the ponies were not at a too deep a plane of anesthesia to mount
an EEG response to a noxious stimulus. The four ponies were also
castrated using a closed technique, with emasculation providing a
crushing and cutting stimulus to the spermatic cord.
In study 2, good quality EEG data was recorded with fewer
incidences of signal noise. Median frequency was similar between
channels in three of the four ponies studied. Total power tended to
be lower in study 2 compared with study 1, which suggests the
animals were at a lighter depth of anesthesia. Signifcant
differences were found between baseline and T1skin Ptot values in
channel 1 and between baseline and T1skin F50 values in channel
2. The changes in F50 were similar or greater in magnitude to
those reported previously (>10% change from baseline) as a
marker of nociception [5]. This could have been because of
lightening the plane of anesthesia, or changing the surgical
stimulus, or a combination of both.
Blood pressure, PaO2, and PaCO2 were recorded at baseline and
at T1emasc and T2emasc to ensure that aberrations in these
parameters did not affect cerebral perfusion or cerebral oxygen
delivery and thus infuence the EEG [27,28]. In man, cerebral
blood fow is maintained when mean arterial pressure is >50
mmHg [29], and in all the animals studied, mean arterial pressure
was >50 mmHg at all time points. Dobutamine was sometimes
used to increase arterial blood pressure; the effects of this drug on
the EEG are negligible [30]. There were no significant differences
in end-tidal halothane concentration across the time points. This
indicates that the comparison of EEG data between baseline and
both T1 and T2 is valid, as each animals baseline EEG acted as its
own control.
5. Conclusion

study 1, although differences between hemispheres were less

pronounced in study 2. This disparity has not been previously
reported in equidae.
Acknowledgments
This study was supported fnancially by UNESP, Universities
Federation for Animal Welfare, and the American Donkey and
Mule Society.
The authors thank Emma Love, Pamela Murison, Alan Jones,
Francesca Compostella, and Henry Tremaine for their assistance.
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