Ameliorating the adverse cardiorespiratory effects of chemical immobilization by inducing general anaesthesia in sheep and goats: implications for physiological studies of large wild animals

Abstract

Chemical immobilization is necessary for the physiological study of large wild animals. However, the immobilizing drugs can adversely affect the cardiovascular and respiratory systems, yielding data that do not accurately represent the normal, resting state. We hypothesise that these adverse effects can be ameliorated by reversing the immobilizing agent while holding the animal under general anaesthesia. We used habituated sheep Ovis aries (N = 5, 46.9 ± 5.3 kg body mass, mean ± SEM) and goats Capra hircus (N = 4, 27.7 ± 2.8 kg) as ungulate models for large wild animals, and measured their cardiorespiratory function under three conditions: (1) mild sedation (midazolam), as a proxy for the normal resting state, (2) immobilization (etorphine and azaperone), and (3) general anaesthesia (propofol) following etorphine antagonism (naltrexone). Cardiac output for both sheep and goats remained unchanged across the three conditions (overall means of 6.2 ± 0.9 L min^-1 and 3.3 ± 0.3 L min^-1, respectively). For both sheep and goats, systemic and pulmonary mean arterial pressures were significantly altered from initial midazolam levels when administered etorphine + azaperone, but those arterial pressures were restored upon transition to propofol anaesthesia and antagonism of the etorphine. Under etorphine + azaperone, minute ventilation decreased in the sheep, though this decrease was corrected under propofol, while the minute ventilation in the goats remained unchanged throughout. Under etorphine + azaperone, both sheep and goats displayed arterial blood hypoxia and hypercapnia (relative to midazolam levels), which failed to completely recover under propofol, indicating that more time might be needed for the blood gases to be adequately restored. Nonetheless, many of the confounding cardiorespiratory effects of etorphine were ameliorated when it was antagonised with naltrexone while the animal was held under propofol, indicating that this procedure can largely restore the cardiovascular and respiratory systems closer to a normal, resting state.

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Vol.:(0123456789)
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Journal of Comparative Physiology B
https://doi.org/10.1007/s00360-018-1184-z
ORIGINAL PAPER
Ameliorating theadverse cardiorespiratory effects ofchemical
immobilization byinducing general anaesthesia insheep andgoats:
implications forphysiological studies oflarge wild mammals
AdianIzwan1 · EdwardP.Snelling2 · RogerS.Seymour3 · LeithC.R.Meyer2,4 · AndreaFuller2,4 ·
AnnaHaw2· DuncanMitchell1,2 · AnthonyP.Farrell5,6· Mary‑AnnCostello7· ShaneK.Maloney1,2
Received: 11 May 2018 / Revised: 28 August 2018 / Accepted: 11 September 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Chemical immobilization is necessary for the physiological study of large wild animals. However, the immobilizing drugs can
adversely affect the cardiovascular and respiratory systems, yielding data that do not accurately represent the normal, resting
state. We hypothesize that these adverse effects can be ameliorated by reversing the immobilizing agent while holding the
animal under general anaesthesia. We used habituated sheep Ovis aries (N = 5, 46.9 ± 5.3kg body mass, mean ± SEM) and
goats Capra hircus (N = 4, 27.7 ± 2.8kg) as ungulate models for large wild animals, and measured their cardiorespiratory
function under three conditions: (1) mild sedation (midazolam), as a proxy for the normal resting state, (2) immobilization
(etorphine and azaperone), and (3) general anaesthesia (propofol) followed by etorphine antagonism (naltrexone). Cardiac
output for both sheep and goats remained unchanged across the three conditions (overall means of 6.2 ± 0.9 and 3.3 ± 0.3L
min−1, respectively). For both sheep and goats, systemic and pulmonary mean arterial pressures were significantly altered
from initial midazolam levels when administered etorphine + azaperone, but those arterial pressures were restored upon transi-
tion to propofol anaesthesia and antagonism of the etorphine. Under etorphine + azaperone, minute ventilation decreased in
the sheep, though this decrease was corrected under propofol, while the minute ventilation in the goats remained unchanged
throughout. Under etorphine + azaperone, both sheep and goats displayed arterial blood hypoxia and hypercapnia (relative
to midazolam levels), which failed to completely recover under propofol, indicating that more time might be needed for the
blood gases to be adequately restored. Nonetheless, many of the confounding cardiorespiratory effects of etorphine were
ameliorated when it was antagonized with naltrexone while the animal was held under propofol, indicating that this procedure
can largely restore the cardiovascular and respiratory systems closer to a normal, resting state.
Keywords Cardiac output· Etorphine· Mammal· Opioid· Propofol· Ventilation
Introduction
Little is known about normal physiological function in large
wild animals because measurements cannot be made without
either habituating the animals, physically restraining them,
Communicated by I. D. Hume.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0036 0-018-1184-z) contains
supplementary material, which is available to authorized users.
* Adian Izwan
Adian.izwan@research.uwa.edu.au
1 School ofHuman Sciences, University ofWestern Australia,
35 Stirling Hwy, Crawley, WesternAustralia, Australia
2 Brain Function Research Group, School ofPhysiology,
University oftheWitwatersrand, Johannesburg, SouthAfrica
3 School ofBiological Sciences, University ofAdelaide,
Adelaide, SouthAustralia, Australia
4 Department ofParaclinical Sciences, University ofPretoria,
Pretoria, SouthAfrica
5 Department ofZoology, University ofBritish Columbia,
Vancouver, BritishColumbia, Canada
6 Faculty ofLand andFood Systems, University ofBritish
Columbia, Vancouver, BritishColumbia, Canada
7 Central Animal Service, University oftheWitwatersrand,
Johannesburg, SouthAfrica

Journal of Comparative Physiology B
1 3
or chemically immobilizing them. Because the habituation
of large wild animals is generally impractical, and physi-
cal restraint introduces additional physiological stresses,
many researchers have adopted chemical immobilization as
a means to obtain measurements that would otherwise be
extremely challenging in the fully awake state (Boesch etal.
2011; Brøndum etal. 2009; Buss etal. 2016; Ozeki etal.
2014; Rey etal. 2016; Springer etal. 2015). However, the
chemical agents used for immobilization can have profound
effects that compromise normal physiological function.
One of the most widely used drugs for immobilizing large
mammals is etorphine hydrochloride, a potent synthetic opi-
oid popular due to its rapid and reversible immobilization
(Woolf etal. 1973).
While etorphine is recognized as an effective immobi-
lizing agent, it has various species-dependent and dose-
dependent side effects, varying from bradycardia and
hypotension to tachycardia and hypertension, as well as
respiratory and central nervous system depression (Cabral
etal. 1980; Marano etal. 1996; Alford etal. 1974; Buss and
Meltzer 2001; Meyer etal. 2010; Perrin etal. 2015; Woolf
1970; Radcliffe etal. 2014). The cardiovascular effects can
be mediated by vagal feedback from the lungs (Willette
etal. 1982; Willette and Sapru 1982), via direct effects on
the heart itself (Gautret and Schmitt 1984, 1985), and also
through central autonomic stimulation (Alford etal. 1974;
Heard etal. 1990; McQueen 1983). Additionally, the etor-
phine-induced respiratory depression presents an increased
risk of asphyxiation for the immobilized animal. Etorphine
acts on opioid receptors within the respiratory centres of the
brainstem, inhibiting both the rate and depth of ventilation,
and reduces the ventilatory response to carbon dioxide (Buss
and Meltzer 2001; Shook etal. 1990). Animals immobilized
with etorphine are therefore more likely to become hypoxic
and hypercapnic (Boom etal. 2012). To mitigate this prob-
lem, etorphine is commonly given in combination with
other chemical agents, both to lower the dose of etorphine
and to help alleviate some of its side effects. For example,
etorphine is often used in conjunction with low doses of
azaperone, a neuroleptic butyrophenone that enhances the
opioid’s sedative effects (Wenger etal. 2007) while appar-
ently ameliorating its depressive respiratory effects (Greth
etal. 1993).
Nonetheless, cardiorespiratory data from animals under
etorphine, even when used in conjunction with azaperone,
are generally unreliable for assessment of normal physiolog-
ical function of the cardiovascular and respiratory systems,
as they are highly unlikely to reflect normal resting values.
Therefore, there is a need to identify and assess alternative
immobilizing agents with less impact on the cardiovascular
and respiratory systems. Unfortunately, many of the current
alternatives are impractical for darting because of the large
and continuous volumes required to effectively immobilize
large wild animals. A potential solution is to persist with
etorphine as the initial immobilizing agent, but once immo-
bilization is achieved, induce a general anaesthesia with an
alternative chemical agent that has milder cardiorespiratory
side effects, and then provide naltrexone, an opioid antago-
nist, to reverse the effects of etorphine. A candidate for an
alternative chemical agent that has anaesthetic effects but
impacts less on the cardiovascular and respiratory systems
is propofol, commonly used in human and veterinary sur-
gical procedures. Although propofol’s exact mechanism
of action is unclear, it is thought to bind to and activate
GABA(A) and GABA(B) receptors of neurons within the
central nervous system, inhibiting their activity and leading
to a loss of consciousness (Trapani etal. 2000). As with
most anaesthetics, propofol does have side effects, namely
hypotension, as propofol can have negative chronotropic
effects on heart rate, secondary to increased venous capaci-
tance, which may reduce overall cardiac output, as well as
reduced systemic vascular resistance, possibly mediated by
the drug’s inhibitory effect on sympathetic action within the
vessel walls (Hoka etal. 1998; Muzi etal. 1992). A reduced
cardiac output and systemic vascular resistance, and a possi-
bly blunted baroreflex, could result in a persistently reduced
mean arterial pressure (Sinclair 2003). Propofol can also
have similar dose-dependent respiratory depressant effects to
those of etorphine, such as reduced minute ventilation, and
an attenuated ventilatory response to hypoxia and hypercap-
nia (Mirenda and Broyles 1995; Madan etal. 2010; Nieu-
wenhuijs etal. 2001; Goodman etal. 1987). However, the
severity of these side effects appears to be generally much
milder under propofol compared to etorphine (Rosa etal.
1992; Madan etal. 2010; Nieuwenhuijs etal. 2001; Good-
man etal. 1987).
Although no chemical agent is without side effects, if
a balance can be achieved between the adequate chemical
restraint of large wild animals and the integrity of acute
physiological measurements of the cardiovascular and res-
piratory systems, then cardiorespiratory data can be obtained
that more closely represent the normal, resting state. In this
study, we assess whether or not the adverse cardiorespira-
tory effects of etorphine can be ameliorated by reversing it
with an opioid receptor antagonist, while holding animals
under general anaesthesia using propofol. We used habitu-
ated sheep and goats as a two-species ungulate model,
because previous research suggests that species can differ
in their physiological response and sensitivity to opioids
(Alford etal. 1974; Woolf etal. 1973; Mir etal. 2000). Fur-
thermore, both species are routinely used as animal models
and both are known to provide reliable cardiorespiratory
measurements (Egermann etal. 2008; Lenaerts etal. 2013;
Vahle-Hinz and Detsch 2002). We measured cardiovascular
variables (heart rate, stroke volume, cardiac output, and sys-
temic and pulmonary arterial blood pressures), respiratory

Journal of Comparative Physiology B
1 3
variables (respiratory rate, tidal volume, minute ventilation)
and arterial blood gas tensions (partial pressures of oxygen
and carbon dioxide), under (1) mild sedation using mida-
zolam as a proxy for the normal resting state, (2) immo-
bilization using etorphine and azaperone, and (3) general
anaesthesia using propofol after administration of the opi-
oid receptor antagonist, naltrexone. We hypothesized that
propofol anaesthesia could largely return cardiovascular and
respiratory function to normal in domestic ungulates ini-
tially immobilized with etorphine, thus enabling the future
assessment of normal cardiorespiratory function in large
wild ungulates.
Materials andmethods
Animals
The procedures used in this study were approved by the Ani-
mal Ethics Committees of the University of Western Aus-
tralia (UWA AEC RA/3/100/1241) and of the University of
the Witwatersrand (AESC 2013/25/2B). Adult sheep Ovis
aries (N = 5, 46.9 ± 5.3 kg body mass, mean ± SEM) and
adult goats Capra hircus (N = 4, 27.7 ± 2.8kg), purchased
from farm stock, were housed in large indoor enclosures,
under a controlled temperature (23 ± 1°C) and lighting
regime (12:12h day-night), at the Central Animal Services,
University of the Witwatersrand, South Africa (~ 1750m
elevation). Animals were fed once daily on a mix of teff
(Eragrostis tef), lucerne (Medicago sativa), vegetables, and
commercial pellet. Water was provided adlibitum. Animal
health was assessed daily, and body mass measured twice a
week. During the 3-week period leading up to day of experi-
mentation, each animal was habituated to stand or lie quietly
while being handled and wearing a respiratory face mask.
The domestic nature of the sheep and goats mean they are
predisposed to being more readily approached by humans
and less stressed in a captive environment (Price 1999).
Furthermore, the additional training during the habituation
period helped to familiarize the animals to their surround-
ings and prepare them for handling on the day of experi-
mentation, with the ultimate aim of minimizing stress and
thus providing physiological measurements more reflective
of the resting state.
Cardiovascular function
Measurements were made in a large-animal surgery main-
tained at a constant temperature (23 ± 1°C). Initially, each
animal was mildly sedated with an intramuscular injec-
tion of midazolam (0.3mg kg− 1; Dormicum; Roche Prod-
ucts, Johannesburg, South Africa) and was in a state that
human medicine terms “minimal sedation”, which allows
the individual to tolerate uncomfortable procedures while
maintaining cardiorespiratory function (American Society of
Anesthesiologists 2004). Individuals exhibited intact corneal
reflex, partial loss of muscle tone, and were nonresponsive
to innocuous stimuli. The skin and underlying tissue of the
left mid-neck region was locally anesthetized with lidocaine
(5ml; Lignocaine Hydrochloride Fresenius 2%; Fresenius
Kabi, Midrand, South Africa), then a fluid-filled Swan-
Ganz continuous cardiac output thermodilution catheter (7
French gauge; 139HF75P; Edwards Life Sciences, Irene,
South Africa) was introduced into the left jugular vein at
the mid-neck region, and advanced into the superior vena
cava, through the right atrium and right ventricle, before ter-
minating in the pulmonary artery. The port at the tip of the
thermodilution catheter was connected via a fluid-filled line
to a pre-calibrated Deltran II pressure transducer (DPT-200;
Utah Medical Products, Midvale, UT, USA), which allowed
for real-time monitoring of pressure waveforms at the tip,
ensuring its correct placement, and providing continuous
measurement of pulmonary arterial pressures (mmHg). In
addition, the auricular artery of each animal was catheter-
ized with an intravenous catheter (22 gauge; Introcan; B/
Braun, Melsungen, Germany) connected via a fluid-filled
line to another pre-calibrated Deltran II pressure transducer,
which allowed for continuous measurement of systemic
arterial pressures (mmHg). The pressure transducers were
placed at the level of the scapulohumeral joint (level with
the base of the heart) and zeroed to atmospheric pressure to
standardize pressure readings. The pressure transducers were
connected via blood pressure amplifiers (FE117; ADInstru-
ments, Castle Hill, Australia) to a PowerLab Exercise Physi-
ology System (ML870B80; ADInstruments), which captured
and displayed real-time data through LabChart software
(Chart5; ADInstruments). Heart rate (beats min−1) was also
captured and displayed in real time via the same software.
The thermodilution catheter’s heating coil lay in the right
ventricle and the thermistor at the catheter tip lay in the
pulmonary artery, allowing measurement of cardiac output
following thermodilution principles (Fegler 1954). The ther-
modilution catheter was connected to a Vigilance Monitor
(450070-R11; Edwards Life Sciences), which continuously
measured cardiac output (L min−1). Stroke volume (ml) was
calculated as the quotient of cardiac output and heart rate.
Pulmonary artery temperature was recorded and used as a
measure of body temperature.
Respiratory function
A respiratory face mask was placed around the muzzle of
each animal, connected through a respiratory flow head
(MLT1000L; ADInstruments) and gas mixing chamber
(MLA246; ADInstruments) to the spirometer component
(FE141, ADInstruments) of the same PowerLab system.

Journal of Comparative Physiology B
1 3
A Powerlab 8/30 amplifier (ML870; ADInstruments) col-
lected the data from these modules before integrating with
the system’s metabolic module software to determine the
tidal volume (L), respiratory rate (breaths min−1) and minute
ventilation (L min−1). A 3-L precision volume syringe was
used to calibrate the spirometer prior to each use.
Arterial blood gases
Blood samples were drawn from the auricular artery via
its respective catheter and the partial pressures of arterial
oxygen (PaO2; mmHg) and carbon dioxide (PaCO2; mmHg)
were measured with a blood gas analyzer using pre-cali-
brated blood gas cassettes (Roche OPTI CCA Analyzer and
OPTI cassette B; Kat Medical, Johannesburg, South Africa)
and corrected to body temperature. Barometric pressures
were measured by the built-in barometer of the blood gas
analyzer, which was calibrated against a Fortin-type pre-
cision mercury barometer (On; F D & Co. Ltd., Watford,
UK). The partial pressure of alveolar oxygen (PAO2; mmHg)
was calculated from the alveolar gas equation, PAO2 = FiO2
(Pb–PAH2O) – PACO2/RER, where FiO2 is the fraction of
oxygen in the inspired air (= 0.209), Pb is the barometric
pressure (mmHg), PAH2O is the water vapour pressure of
saturated alveolar air (mmHg) corrected to temperature
and pressure (Barenbrug 1974), PACO2 is the partial pres-
sure of alveolar CO2 (mmHg) which we assumed to equal
the measured PaCO2, and RER is the respiratory exchange
ratio (i.e. ratio of CO2 production to O2 consumption rates)
(Curran-Everett 2006) for which we assumed a value of 1,
as previously measured in healthy sheep and goats (Beker
etal. 2010). The alveolar–arterial oxygen partial pressure
gradient (P(A–a)O2; mmHg) could then be calculated as the
difference in oxygen partial pressure between the calculated
value for the alveoli and the measured value for the arterial
circulation.
Experimental procedure
The timeline of the experimental procedure is summarized
in Fig.1. Following instrumentation, each animal was given
approximately 5min for conditions to stabilize, before car-
diorespiratory variables under midazolam (described ear-
lier) were measured for 15min. Animals were then injected
intramuscularly with etorphine (0.05mg kg−1; Captivon;
Wildlife Pharmaceuticals, White River, South Africa) and
azaperone (0.5mg kg−1; Stressnil; Janssen Pharmaceuticals
Ltd, Halfway House, South Africa), and the cardiorespira-
tory variables were measured for a further 15min following
a 5min drug stabilization period. The animals then received
a bolus of propofol (3–5mg kg−1; Diprivan; Fresenius Kabi,
Bad Homburg, Germany) followed by propofol infusion at a
constant rate (6–12mg kg−1 h−1 depending on the animal),
and an intravenous injection of naltrexone (1mg kg−1; Trex-
onil; Wildlife Pharmaceuticals, White River, South Africa)
to reverse the etorphine. Once again, the same cardiorespira-
tory variables were measured for a further 15min following
a 5min drug stabilization period. After the physiological
measurements were complete, each animal was euthanized
with an injection of sodium pentobarbitone (200mg kg− 1;
Euthapent; Kyron Laboratories, Johannesburg, South Africa)
and the hearts were harvested for use in another study (Snel-
ling etal. 2016).
Statistical analyses
All values are reported as mean ± standard error (SEM).
Mean values were calculated as the average of the last
15min of each drug phase. The effects of the different drugs
on the measured cardiorespiratory variables were analysed
using a one-way ANOVA. Where the ANOVA indicated
significance, a Student–Newman–Keuls post hoc test was
used to compare individual means. Statistical significance
was set at P < 0.05 a priori. All statistical analyses were per-
formed using STATISTICA (Version 4.1; Statsoft, Tulsa,
OK, USA).
Fig. 1 Timeline of the experimental procedure. Midazolam was given
prior to instrumentation. Each experimental phase lasted approxi-
mately 20min with approximately 5min for stabilization (S) before
commencing approximately 15 min of data collection. Naltrexone
was given after propofol infusion commenced

Journal of Comparative Physiology B
1 3
Results
Cardiovascular function
In the sheep, heart rate increased from 110 ± 14 beats
min−1 under midazolam to 180 ± 7 beats min−1 under
etorphine + azaperone (P < 0.001; Fig.2A). When etor-
phine was reversed and the sheep placed under propofol,
there was a significant decrease in heart rate to 147 ± 6
beats min−1, although it nonetheless remained signifi-
cantly higher than the initial midazolam phase (P < 0.05).
In the goats, the heart rate was 86 ± 5 beats min−1 under
midazolam and was unaffected by the introduction of
etorphine + azaperone (86 ± 2 beats min−1; P = 0.96),
while etorphine antagonism and introduction of propofol
increased heart rate to 110 ± 8 beats min−1 (P < 0.05). In
general, stroke volume showed an inverse response to that
of heart rate across the three drug phases, particularly in
the sheep. In sheep, stroke volume decreased significantly
from 53 ± 4ml under midazolam to 35 ± 6ml under etor-
phine + azaperone (P < 0.05; Fig.2B), before increasing
again during the propofol phase to 42 ± 6ml, although it
nonetheless remained significantly reduced compared to
the initial midazolam phase (P < 0.05). In goats, stroke
volume did not change across all three drug phases with an
overall mean of 37 ± 2ml. Because changes in heart rate
tended to cancel changes in stroke volume, particularly in
the sheep, cardiac output in both sets of animals did not
vary significantly between the three drug phases (Fig.2C).
Thus, the overall average cardiac output was 6.2 ± 0.9L
min−1 for the sheep and 3.3 ± 0.3L min−1 for the goats. Or,
expressed in mass-independent units, cardiac output was
0.29 ± 0.02L min−1 kg−0.79 for the sheep and 0.24 ± 0.04L
min−1 kg−0.79 for the goats (Holt etal. 1968).
Systemic mean arterial pressure decreased signifi-
cantly in sheep and goats from initial levels of 120 ± 8 and
96 ± 4mmHg under midazolam, respectively, to 95 ± 8
and 71 ± 3mmHg under etorphine + azaperone (P < 0.05;
Fig.3A). However, in both sheep and goats, systemic
mean arterial pressure recovered, following thepropo-
fol administration andetorphine antagonism, to 108 ± 12
and 101 ± 6mmHg, respectively, which were both statis-
tically indistinguishable from their respective midazolam
levels (P > 0.05). In the transition from midazolam to
etorphine + azaperone, the systemic vascular resistance
decreased significantly from 21 ± 2 to 16 ± 2mmHg min
L−1 in the sheep (P < 0.05) and from 31 ± 2 to 23 ± 2mmHg
Fig. 2 Mean ± SEM A heart rate, B stroke volume and C cardiac out-
put of sheep (unfilled circles, N = 5) and goats (filled circles, N = 4)
under the different drug regimes. The drug regimes comprised mida-
zolam (mild sedation), followed by etorphine + azaperone (immobili-
zation), and then followed by the antagonism of etorphine with nal-
trexone while held under propofol (general anaesthesia). Statistical
differences between the mean values of the different drug regimes are
identified for sheep (a, b, c) and goats (x, y). Statistical significance
was set at P < 0.05. In the different drug phases, asterisk and dou-
ble asterisk signify potential residual effects of the drug(s) from the
previous treatment (midazolam only, and midazolam and azaperone,
respectively)

Journal of Comparative Physiology B
1 3
min L−1 in the goats (P < 0.05; Fig.3B). After thetransi-
tion onto propofol and antagonism of etorphine, systemic
vascular resistance returned to levels that were statistically
indistinguishable from the initial midazolam levels in both
the sheep, 18 ± 3mmHg min L−1 (P = 0.54), and the goats,
30 ± 4mmHg min L−1 (P = 0.94).
Pulmonary mean arterial pressure increased from
14 ± 1mmHg under midazolam in both groups of animals,
to 19 ± 1 and 20 ± 2 mmHg under etorphine + azaperone
in the sheep and goats, respectively (P < 0.05; Fig.4A).
These pressures then decreased again following thepropo-
fol administrationandetorphine antagonism, to 14 ± 1 and
Fig. 3 Mean ± SEM A systemic
mean arterial blood pressure
and B systemic vascular resist-
ance of sheep (unfilled circles)
and goats (filled circles) under
the three drug regimes. See
Fig.2 for explanation of statis-
tics and asterisks
Fig. 4 Mean ± SEM A pulmo-
nary mean arterial blood pres-
sure and B pulmonary vascular
resistance of sheep (unfilled
circles) and goats (filled circles)
under the three drug regimes.
See Fig.2 for explanation of
statistics and asterisks

Journal of Comparative Physiology B
1 3
16 ± 1mmHg in the sheep and goats, respectively, which
was not significantly different from their respective mida-
zolam levels (P > 0.05). In the transition from midazolam
to etorphine + azaperone, the pulmonary vascular resistance
increased from 1.7 ± 0.4 to 2.4 ± 0.6mmHg min L−1 in the
sheep (P < 0.05) and from 3.2 ± 0.2 to 4.6 ± 0.6mmHg min
L−1 in the goats (P < 0.05; Fig.4B). Following the propo-
fol administration andantagonism of etorphine, pulmonary
vascular resistance returned to levels that were statistically
indistinguishable from the initial midazolam levels in both
the sheep, 1.3 ± 0.5mmHg min L−1 (P = 0.07), and the goats,
2.6 ± 0.6mmHg min L−1 (P = 0.23).
Respiratory function
Both species showed a significant decrease in respiratory
rate after the administration of etorphine + azaperone,
decreasing from 54 ± 17 to 26 ± 4 breaths min−1 in the
sheep and from 31 ± 3 to 22 ± 1 breaths min−1 in the goats
(P < 0.05 for both species; Fig.5A). However, following the
propofol administrationandantagonism of etorphine, respir-
atory rate recovered to 68 ± 7 breaths min−1 in the sheep and
42 ± 6 breaths min−1 in the goats, which were statistically
indistinguishable from their respective frequencies recorded
under midazolam (P > 0.05 for both species). Tidal volume
of the sheep did not change across the three drug phases,
with an overall mean of 0.14 ± 0.02L. In the goats, how-
ever, tidal volume increased significantly from 0.14 ± 0.03L
under midazolam to 0.25 ± 0.05L under etorphine + azaper-
one (P < 0.05; Fig.5B), before decreasing again, following
the propofol administrationandetorphine antagonism, to
0.12 ± 0.02L, which was not significantly different from
the initial midazolam volumes (P = 0.62). Minute ventila-
tion in the sheep decreased significantly from 7.4 ± 1.8L
min−1 under midazolam to 3.1 ± 0.3L min−1 under etor-
phine + azaperone (P < 0.05; Fig. 5C), before recovering
to 9.1 ± 1.8L min−1 following thepropofol administration
andetorphine antagonism, and wasnot significantly different
from the initial midazolam levels (P = 0.23). In the goats,
minute ventilation was not significantly different between
the three phases, with an overall mean of 4.8 ± 0.7L min−1.
Arterial blood gases
The arterial partial pressure of oxygen (PaO2) in the sheep
decreased significantly from 69 ± 5mmHg under midazolam
to 35 ± 5 mmHg under etorphine + azaperone (P < 0.05),
and then partially recovered to 57 ± 4mmHg following
thepropofol administration andetorphine antagonism,
though it was still lower than the initial midazolam levels
(P < 0.05; Fig. 6A). For the goats, PaO2 also decreased
significantly from 68 ± 1mmHg under midazolam to
48 ± 5 mmHg under etorphine + azaperone (P < 0.05),
but did not change following the propofol administra-
tionandetorphine antagonism, 51 ± 7mmHg, and therefore
remained below the initial midazolam level (P < 0.05).
The arterial partial pressure of carbon dioxide (PaCO2) in
the sheep increased significantly from 32 ± 2mmHg under
midazolam, to 53 ± 2 mmHg under etorphine + azaperone
(P < 0.05), and then partially recovered to 38 ± 1mmHg
Fig. 5 Mean ± SEM A respira-
tory rate, B tidal volume and
C minute ventilation of sheep
(unfilled circles) and goats
(filled circles) under the three
drug regimes. See Fig.2 for
explanation of statistics and
asterisks

Journal of Comparative Physiology B
1 3
following the propofol administration andetorphine antago-
nism, although it was still higher than the initial midazolam
level (P < 0.05; Fig.6B). For the goats, PaCO2 also increased,
from 32 ± 1 under midazolam to 39 ± 3mmHg under etor-
phine + azaperone (P < 0.05), but then did not change signifi-
cantly following the propofol administration andetorphine
antagonism, 38 ± 3mmHg, and thus remained significantly
higher than the initial midazolam level (P < 0.05).
Lastly, the alveolar–arterial oxygen partial pressure differ-
ence (P(A–a)O2) for the sheep increased from 21 ± 5mmHg
under midazolam, to 32 ± 4mmHg under etorphine + azaper-
one (P < 0.05), and then recovered to 25 ± 3mmHg follow-
ing the propofol administration andetorphine antagonism,
which was not significantly different from the initial mida-
zolam level (P = 0.07; Fig.6C). In the goats, the P(A–a)O2 also
increased significantly, from 21 ± 1mmHg under midazolam,
to 32 ± 3 mmHg under etorphine + azaperone (P < 0.05),
although unlike the sheep, P(A–a)O2 remained elevated at
30 ± 4mmHg following the propofol administration andetor-
phine antagonism, and was thus significantly higher than the
initial midazolam level (P < 0.05).
Discussion
The main problem with collecting physiological data from
chemically immobilized animals is that it is generally unrep-
resentative of the normal resting state, owing to the adverse
cardiorespiratory effects of the immobilizing drugs. In this
study, etorphine had significant adverse cardiorespiratory
effects, with the sheep appearing relatively more sensitive than
the goats. However, when etorphine was antagonized while the
animals were held under a general anaesthesia using propo-
fol, many of the negative cardiorespiratory effects partly—or
completely—improved. Thus, we believe that the protocol
used in this study, albeit with some modification to extend the
recovery period, could be adopted to allow for safe and reli-
able acquisition of acute physiological data from large wild
animals.
Fig. 6 Mean ± SEM A arterial
partial pressure of oxygen
(PaO2), B carbon dioxide
(PaCO2) and C alveolar–arte-
rial oxygen partial pressure
difference (P(A–a)O2) of sheep
(unfilled circles) and goats
(filled circles) under the three
drug regimes. See Fig.2 for
explanation of statistics and
asterisks

Journal of Comparative Physiology B
1 3
Validation ofthedrug phases
Sedation under midazolam appeared to be mild in our ani-
mals, as evidenced by the fact that their heads remained
mostly upright with only slight additional support from the
handler, they maintained an intact corneal reflex, and they
exhibited only partial loss of muscle tone (American Soci-
ety of Anesthesiologists 2004). The anxiolytic use of mida-
zolam was necessary to obtain our initial cardiorespiratory
measurements as it assisted in keeping the animals calm and
closer to a resting state. It has been demonstrated in goats
that clinical levels of sedation require intramuscular doses
of at least 0.6mg kg−1 of midazolam, with the severity being
dose dependent, and that cardiorespiratory variables such as
heart rate and ventilation rate were not significantly affected
under those doses, which are notably higher than those used
in this study (Stegmann and Bester 2001).
Midazolam is presumed to work by binding to glycine
and enhancing the affinity of GABA(A) to its receptor, thus
promoting its inhibitory effects (Splinter etal. 1995). While
midazolam itself can have some effect on cardiorespiratory
function, the severity of the effect is proportional to the
level of sedation (Upton etal. 2009), which in our study
was mild. Furthermore, the cardiorespiratory variables for
our animals under midazolam were similar to those reported
in the literature for non-anesthetized sheep and goats (Chahl
1996; Greene 2002; Holt etal. 1968; Jackson and Cock-
croft 2008; Seymour and Blaylock 2000) as documented in
Table1. Therefore, we believe that midazolam, at the mild
dose used in the present study, had little effect on our initial
and subsequent cardiorespiratory measurements, and indeed
helped maintain a physiological state closer to that experi-
enced under resting conditions.
Etorphine immobilization was reversed by the opioid
receptor antagonist, naltrexone. It is possible that there could
have been some lingering effects of etorphine, especially in
the initial stages of the propofol drug phase. However, the
effect was likely minimal because we analysed data only
from the final 15min of each drug phase and because the
antagonism of etorphine, using intravenous naltrexone, is
generally a very rapid process (Presnell etal. 1973; Alford
etal. 1974; Roussel and Patenaude 1975). Previous reports
of recovery range from almost instantaneous (Lynch and
Hanson 1981) to within a few minutes (Atkinson etal.
2002).
Azaperone may also affect cardiorespiratory function.
However, previous studies show that it has only a slight
stimulatory effect on respiration (Clarke 1969), and little or
no effect on cardiac output and heart rate (MacKenzie and
Snow 1977). Nonetheless, azaperone has been reported to
decrease mean arterial pressure (Lees and Serrano 1976),
and because it cannot be antagonized in the same way that
etorphine was in the present study, it is possible that its
effects may have persisted into the propofol phase. However,
our data shows that systemic mean arterial pressure only
improved under propofol after the etorphine was reversed,
which suggests that azaperone likely did not have substantial
effects.
Cardiovascular function
Compared to the initial midazolam phase, we found no
change in heart rate, stroke volume or cardiac output of the
goats when placed under etorphine + azaperone (Fig.2).
This differs from the results of Meyer etal. (2015), who
showed a significant decrease in cardiac output after admin-
istration of etorphine, although in that study etorphine was
given at twice the dose as in the present study and without
azaperone. We also found no change in cardiac output of the
sheep between the midazolam and the etorphine + azaper-
one phases; however, there was a decrease in stroke volume
which was compensated for by an increase in heart rate.
This reduction in stroke volume could have resulted from
the direct vasoactive effects of opioids, which can induce
mast cell histamine release, binding with H1 and H2 recep-
tors on vascular smooth muscle, causing relaxation and
Table 1 Mean ± SEM absolute,
body-mass-specific, and body-
mass-independent cardiac
output and minute ventilation
of sheep (N = 5) and goats
(N = 4) recorded under mild
sedation (midazolam) in the
present study compared to
values recorded from non-
anaesthetized individuals in the
literature
Literature source: Non-anaesthetized cardiac output data and scaling exponent (Holt etal. 1968), minute
ventilation data (Jackson and Cockcroft 2008; Greene 2002) and scaling exponent (Stahl 1967)
Sheep Goats
(midazolam) (non-
anaesthe-
tized)
(midazolam) (non-
anaesthe-
tized)
Cardiac output (L min−1)6.0 ± 1.0 7.1 3.2 ± 0.2 3.9
Mass-specific cardiac output (L min−1 kg−1)0.13 ± 0.01 0.15 0.12 ± 0.01 0.13
Mass-independent cardiac output (L min−1 kg−0.79)0.28 ± 0.03 0.34 0.23 ± 0.02 0.26
Minute ventilation (L min−1)7.4 ± 1.8 6.7 4.2 ± 0.6 4.3
Mass-specific minute ventilation (L min−1 kg−1)0.15 ± 0.02 0.14 0.15 ± 0.02 0.14
Mass-independent minute ventilation (L min−1 kg−0.8)0.33 ± 0.06 0.30 0.29 ± 0.03 0.28

Journal of Comparative Physiology B
1 3
venodilation (Grossmann etal. 1996). The exact mechanism
behind this histamine release is currently unknown, although
it is speculated to occur via the interaction of the opioid with
G-proteins on mast cells (Barke and Hough 1993). Opioids
can also act centrally to reduce venous tone, similar to their
effects on the rest of the systemic circulation (Lowenstein
etal. 1972; O’Keefe etal. 1987; Mansour etal. 1970), and
can have negative chronotropic effects by direct stimulation
of opioid receptors in the heart itself (Gautret and Schmitt
1984, 1985).
The administration of etorphine and azaperone led to a
decrease in the systemic mean arterial pressure in both sheep
and goats, consistent with etorphine’s reported effects of
systemic hypotension (McQueen 1983) (Fig.3a). In addi-
tion to causing systemic hypotension, etorphine also resulted
in pulmonary hypertension (Fig.4a). In both instances, the
observed changes in blood pressure were almost entirely due
to changes in vascular resistance, because cardiac output
remained unchanged between the different phases.
The observed pulmonary hypertension occurred concur-
rently with a decrease in PaO2 (Fig.6b) and, consequently,
an increase in the P(A–a)O2 in both species (Fig.6c), and
may have led to pulmonary congestion and oedema (Hat-
tingh etal. 1994; Shaw etal. 1995). This would have
increased the oxygen diffusion distance and, assuming
oxygen flow rate across the surface was unaffected and in
the steady state, would increase the oxygen partial pressure
difference between the alveoli and the arterial circulation
(Meyer etal. 2015). It is likely that the inadequate oxygen
diffusion resulting from the oedema persisted into the propo-
fol phase, anddid not clear immediately following etorphine
antagonism, explaining the incomplete recovery of P(A–a)
O2, especially in the goats. Although an RER of 1 is consid-
ered normal for sheep and goats (Beker etal. 2010; Fernán-
dez etal. 2012), calculating the P(A–a)O2 for the animals
assuming an RER of 0.7 showed that the observed trends
were still apparent, with the P(A–a)O2 widening under
etorphine and remaining slightly higher until the end of
the experiment. Further studies utilizing this method might
benefit from extending the recovery time under propofol to
account for the slower resolution of potential pulmonary
congestion and oedema.
The simultaneous systemic hypotension and pulmonary
hypertension might be explained by the numerous mecha-
nisms by which opioids are known to alter blood pressure.
Opioid stimulation of pulmonary J receptors in the periph-
ery transmits a chemoreceptor reflex via vagal afferents to
the central vasomotor regions (Paintal 1969; Willette and
Sapru 1982), which can inhibit splanchnic neurotransmis-
sion to systemic vascular beds (Willette etal. 1982), leading
to a reduction in vascular tone and hence reduced resist-
ance through these vessels. Given that we found cardiac
output to be virtually unaffected by etorphine, the decrease
in systemic mean arterial pressure is likely due to the com-
mensurate decrease in systemic vascular resistance observed
in both species under the opioid (Fig.3b). Activation of
opioid receptors within the lung can also cause the release
of histamine into the rest of the circulation that leads to
systemic hypotension. However, the local effects of his-
tamine release have the opposite effect, leading to potent
vasoconstriction within the lung itself, and thus pulmonary
hypertension and subsequent oedema (Hakim etal. 1992).
Opioids can also directly influence vasomotor centres within
the central nervous system (Laubie etal. 1974; Daskalopou-
los etal. 1975). It is believed that the medulla region of the
brainstem possesses several distinct types of opioid receptors
that elicit either constrictive or dilatory vasoactive responses
(McQueen 1983). Therefore, it is possible that activation of
different receptors within the same area may elicit different
responses in different areas of the body, like those seen in the
medulla, or that activation of the same receptors may cause
different downstream responses in different areas, as in the
case of the lungs, thus resulting in systemic hypotension but
pulmonary hypertension.
While under propofol, and with etorphine reversed,
systemic vascular tone was restored in both species, with
systemic vascular resistance and systemic mean arterial
pressure returning to the same levels observed under the
initial midazolam phase (Fig.3). The opioid antagonism was
accompanied by increased stroke volume and reduced heart
rate in the sheep (Fig.2). However, heart rate in both sheep
and goats was still higher during the propofol phase than
during the midazolam phase. Propofol has been reported to
affect the cardiovascular system by inhibiting sympathetic
activity to vascular smooth muscle, leading to decreases in
systemic vascular resistance and increased venous capaci-
tance (Hoka etal. 1998; Muzi etal. 1992), as well as having
a direct chronotropic effect on the heart (Runciman etal.
1990), possibly explaining the elevated heart rate seen in
the propofol phase. Importantly, for both sheep and goats,
despite the significant effect of etorphine on systemic and
pulmonary pressures, and on systemic and pulmonary vascu-
lar resistances, these variables returned to their initial mida-
zolam levels once etorphine was reversed and the animals
were held under propofol. Similarly, despite the effect of
etorphine on sheep heart rate and stroke volume, and despite
the apparent effect of propofol on goat heart rate, the overall
cardiac output remained unchanged for both sets of animals
across the three drug phases.
Respiratory function
Under etorphine + azaperone, the respiratory rates of both
species were significantly reduced compared to the initial
levels under midazolam (Fig.5A), likely due to etorphine’s
interaction with pons and medulla opioid receptors, which is

Journal of Comparative Physiology B
1 3
known to alter breathing patterns (Boom etal. 2012; Pattin-
son 2008). Opioids also have been shown to reduce the sen-
sitivity of central and peripheral chemoreceptors, depressing
the respiratory system and reducing the response to hypox-
aemia and hypercapnia (Boom etal. 2012; Pattinson 2008).
While both sheep and goats had a reduced respiratory rate
under etorphine + azaperone, the goats displayed an increase
in tidal volume (Fig.5b) such that overall minute ventila-
tion was maintained under the opioid (Fig.5c). This might
explain why there was less deviation in PaO2 and PaCO2 in
the goats between the midazolam and the etorphine + azap-
erone drug phases as they were taking deeper, albeit fewer,
breaths.
After the animals were placed under propofol and the
etorphine was antagonized, breathing patterns were restored
to those observed under midazolam. However, PaO2 and
PaCO2 levels for both species did not return to the initial
partial pressures recorded under midazolam, with the sheep
showing significant, albeit incomplete, recovery and the
goats showing no significant recovery under propofol. That
the blood gases did not return completely to the levels meas-
ured in the midazolam phase could be explained by the slow
resolution of potential pulmonary congestion and oedema;
given enough time these could possibly have corrected. Per-
haps most importantly, all the animals were kept under a
sufficient level of chemical restraint, evidenced by a lack of
movement and response to external stimuli (no palpebral
reflex), throughout the experiment.
Conclusion
The overall purpose of this study was to develop a chemi-
cal-restraint drug protocol that allows for the safe and reli-
able acquisition of acute physiological data from large wild
animals. While darting with etorphine remains a crucial
tool to provide access to species in the wild, physiological
data collected under this drug do not reflect normal rest-
ing values. Therefore, subsequent antagonism of etorphine
and transition onto a replacement agent, such as propofol,
is essential to obtain more accurate physiological data. In
this study, we showed that the negative cardiorespiratory
effects associated with etorphine can largely be reversed
by transitioning the animals to propofol and restoring car-
diovascular and respiratory function to a rest-like state. We
expect that etorphine-induced pulmonary hypertension may
have resulted in pulmonary congestion and oedema, and so,
despite the rapid action of the drugs used in this study, a
longer recovery period under propofol may be required to
normalize gas exchange and blood gases.
Acknowledgements The authors acknowledge the expertise and con-
tribution made by the academics, technicians, and volunteers at the
School of Physiology, and the Central Animal Service, at the Uni-
versity of the Witwatersrand. We thank especially David Gray, Zipho
Zwane, Robyn Hetem, Benjamin Rey, Nico Douths, Peter Kamer-
man, Richard McFarland, Hilary Lease, Peter Buss, Michelle Miller,
Tapiwa Chinaka, and W. Maartin Strauss. We also thank Tobias Wang
of Aarhus University, and one anonymous reviewer, for providing valu-
able feedback on the manuscript. This research was supported by an
Australian Research Council Discovery Project Award to R. S. Sey-
mour, S. K. Maloney, and A. P. Farrell (DP-120102081). E. P. Snelling
holds a South African Claude Leon Foundation Postdoctoral Fellow-
ship. A. P. Farrell holds a Canada Research Chair and is supported by a
Discovery Grant from the Natural Sciences and Engineering Research
Council of Canada.
Author contributions EPS, RSS, LCRM, AF, AH, DM, APF, M-AC,
and SKM: conception and design of research; EPS, RSS, LCRM, AF,
AH, DM, APF, M-AC, and SKM: performed experiments; AI: ana-
lysed data; AI, EPS, RSS, LCRM, and SKM: interpreted results of
experiments; AI: prepared figures; AI, EPS, RSS, and SKM: drafted
the manuscript; AI, EPS, RSS, AF, LCRM, APF, and SKM: edited and
revised the manuscript; AI, EPS, RSS, LCRM, AF, AH, DM, APF,
M-AC, and SKM: approved the final version of the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All applicable international, national, and/or insti-
tutional guidelines for the care and use of animals were followed. All
procedures performed in studies involving animals were in accordance
with the ethical standards of the institution or practice at which the
studies were conducted.
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