Evaluation of thermal antinociceptive effects

and pharmacokinetics after intramuscular

administration of butorphanol tartrate
to American kestrels (Falco sparverius)
David Sanchez-Migallon Guzman, LV, MS; Tracy L. Drazenovich, DVM;
Butch KuKanich, DVM, PhD; Glenn H. Olsen, DVM, PhD; Neil H. Willits, PhD;
Joanne R. Paul-Murphy, DVM

ObjectiveTo evaluate antinociceptive effects and pharmacokinetics of butorphanol tartrate after IM administration to American kestrels (Falco sparverius).
AnimalsFifteen 2- to 3-year-old American kestrels (6 males and 9 females).
ProceduresButorphanol (1, 3, and 6 mg/kg) and saline (0.9% NaCl) solution were administered IM to birds in a crossover experimental design. Agitation-sedation scores and
foot withdrawal response to a thermal stimulus were determined 30 to 60 minutes before
(baseline) and 0.5, 1.5, 3, and 6 hours after treatment. For the pharmacokinetic analysis,
butorphanol (6 mg/kg, IM) was administered in the pectoral muscles of each of 12 birds.
ResultsIn male kestrels, butorphanol did not significantly increase thermal thresholds
for foot withdrawal, compared with results for saline solution administration. However, at
1.5 hours after administration of 6 mg of butorphanol/kg, the thermal threshold was significantly decreased, compared with the baseline value. Foot withdrawal threshold for female
kestrels after butorphanol administration did not differ significantly from that after saline
solution administration. However, compared with the baseline value, withdrawal threshold
was significantly increased for 1 mg/kg at 0.5 and 6 hours, 3 mg/kg at 6 hours, and 6 mg/kg
at 3 hours. There were no significant differences in mean sedation-agitation scores, except
for males at 1.5 hours after administration of 6 mg/kg.
Conclusion and Clinical RelevanceButorphanol did not cause thermal antinociception
suggestive of analgesia in American kestrels. Sex-dependent responses were identified.
Further studies are needed to evaluate the analgesic effects of butorphanol in raptors. (Am
J Vet Res 2014;75:1118)

aptors are frequently brought to veterinarians at

wildlife rehabilitation centers, zoological institutions, or private clinical practices because of conditions
that require analgesia. Traumatic injury is the most
common reason that raptors are brought to wildlife
centers, and 58.2% to 82% of these raptors are affected by collisions or traumatic wounds and fractures.16
However, despite advances in analgesia for birds, there
is limited information regarding dose response and dos-

Received June 13, 2013.

Accepted September 4, 2013.
From the Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine (Sanchez-Migallon Guzman,
Drazenovich, Paul-Murphy), and the Department of Statistics, College of Letters and Science (Willits), University of California-Davis,
Davis, CA, 95616; the Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS
66506 (KuKanich); and the United States Geological Survey, Patuxent Wildlife Research Center, 12100 Beech Forest Rd, Ste 4039,
Laurel, MD 20708 (Olsen).
Supported by the Morris Animal Foundation (grant No. D10ZO-305).
The authors thank Dr. Bruno H. Pypendop for technical assistance.
Address correspondence to Dr. Sanchez-Migallon Guzman (guzman@
ucdavis.edu)
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13-06-0175r.indd 11

ing interval for analgesic drugs in raptors, and veterinarians have been compelled to extrapolate from studies conducted in other species.
Opioids are a diverse group of drugs that bind reversibly to specific receptors in the CNS and peripheral nervous
system and modify the transmission and perception of a
noxious stimulus in numerous vertebrate species.7 Opioid
drugs are used for their analgesic properties, acting on the
-, - and -opioid receptors as well as the orphan opioidlike receptor.8 The action of opioid drugs on these receptors activates a G-protein, which leads to a reduction in the
transmission of nerve impulses and inhibition of neurotransmitter release.9 Research on the distribution, quantity, and functionality of each opioid receptor type in birds
has been limited.1012 Butorphanol tartrate and nalbuphine
hydrochloride, a -opioid receptor agonist and -opioid
receptor antagonist, are the opioid drugs currently recommended for the management of acute pain and to provide
preemptive analgesia in psittacine birds,1316 but their
analgesic properties have not been evaluated in raptors.
Fentanyl, a -opioid receptor agonist, administered as a
continuous rate infusion in red-tailed hawks (Buteo jamaicensis) decreases isoflurane minimum anesthetic concen11

12/20/2013 12:56:44 PM

tration, which suggests that fentanyl could have analgesic

properties in this species.17 Hydromorphone, a -opioid
receptor agonist, has been investigated in American kestrels (Falco sparverius), and it was found that it provides
dose-responsive thermal antinociception, which suggests
that hydromorphone would provide analgesia in this species.18 Other investigators evaluated the pharmacokinetics
of butorphanol in red-tailed hawks and great-horned owls
(Bubo virginianus )19 and tramadol in bald eagles (Haliaeetus leucocephalus)20 and red-tailed hawks,21 but the analgesic efficacy of these drugs was not investigated. The
pharmacokinetics of butorphanol has also been evaluated
in Hispaniolan Amazon parrots (Amazona ventralis)22 and
domestic chickens (Gallus domesticus).23
Antinociceptive analgesimetry is one of the simplest and least invasive methods that can be used to
quantify analgesic effects.24,25 The use of a thermal
noxious stimulus for evaluating analgesia has been
validated for several domesticated mammalian species,
including cats, dogs, rabbits, and rats.25 This method
has also been used in chickens,25 several species of parrots,1416,26,27 and American kestrels.18 To our knowledge,
there have been no studies conducted to investigate the
antinociceptive effects of butorphanol in raptors. The
purpose of the study reported here was to determine
the antinociceptive effects, sedation-agitation effects,
and pharmacokinetics of butorphanol tartrate after
IM administration to American kestrels. Our hypothesis was that butorphanol tartrate would cause significant dose-dependent increases in thermal thresholds
and sedative effects in American kestrels and have a
pharmacokinetic profile similar to that in other species
of birds.
Materials and Methods
The study comprised 2 phases. The first phase involved evaluation of thermal antinociception after administration of butorphanol tartrate, and the second
phase involved a pharmacokinetic analysis after butorphanol administration.
THERMAL ANTINOCICEPTION
AnimalsFifteen 2- to 3-year-old American kestrels (9 females and 6 males) were used in the experiment. Mean SD body weight was 108.4 6.4 g. All
kestrels were captive-bred birds and were healthy as
determined by results of physical examinations performed before and during the experiment. Eleven of the
15 kestrels had been included in a study18 conducted to
evaluate the thermal antinociceptive effects of hydromorphone hydrochloride; that study was completed 3
weeks before the start of the experiments reported here.
Kestrels were maintained in three 2.5 X 2.5 X 3.2-m
rooms; perches were spaced throughout each room.
Kestrels were maintained on a cycle of 12 hours of light
and 12 hours of darkness. They were fed frozen-thawed,
medium-sized mice and provided water ad libitum. The
experimental protocol was approved by the Institutional Animal Care and Use Committee of the University
of California-Davis. Because the experiments involved
a species for which the effects (eg, antinociceptive effects, duration of action, and interindividual variabil12

13-06-0175r.indd 12

ity) of other common analgesics were not definitively

known, the use of a positive control group in place of a
negative control group28 was not feasible for the evaluation of antinociceptive effects and duration of action
of butorphanol.
Experimental designA within-subject, complete
crossover experimental design was used. Each kestrel
received 4 treatments, which were administered IM in
the left pectoral muscle. Birds received butorphanol
tartratea (1, 3, and 6 mg/kg; 10 mg/mL of solution)
and saline (0.9% NaCl) solution (0.33 mL/kg; control treatment). Randomization for the order of treatments for each kestrel was determined by an integer
set generator.b There was a washout period of 14 days
between subsequent treatments (ie, periods).
Antinociception testing proceduresThermal
foot withdrawal threshold was determined for all kestrels by use of a test box equipped with a test perch. The
test box was 52.1 cm high, 10.2 cm wide, and 34.3 cm
deep. The perch was placed 7 cm from the front of the
box and 18.4 cm from the bottom of the box. The bottom of the box had a V shape to discourage birds from
standing on it. There were small holes on top of the box
for ventilation. The test box had dark sides to inhibit a
bird from viewing its surroundings or observers and a
clear front that allowed observers to monitor real-time
behavioral responses by use of a remote video camera.
Two weeks prior to the experiment, each kestrel was
allowed to acclimate to the test chamber for a full day.
The test perch was designed to deliver a thermal
stimulus to the right plantar surface of a birds foot;
thermal microchips rapidly changed the temperature
of the perch.26 The thermal stimulus generated by the
thermoelectric modules ranged from 29 to 55C and
resulted in a rapid increase in perch temperature (rate
of temperature increase, 0.3C/s). The cutoff temperature was 55C to avoid tissue damage to the plantar
surface of a birds foot. A bird could escape the brief
noxious thermal stimulus by lifting the foot, and the
foot could then be placed back on the perch within 2
or 3 seconds after the withdrawal response because the
temperature of the perch decreased rapidly (rate of temperature decrease, 0.3C/s).
A thermal threshold withdrawal response was defined as the perch temperature at the time of a foot
withdrawal response. A separate baseline thermal withdrawal threshold value was determined for each period
by a single measurement obtained 30 to 60 minutes
before treatment administration. Time of treatment
administration was designated as time 0. Thermal foot
withdrawal threshold was determined by a single measurement at 0.5, 1.5, 3, and 6 hours after IM administration of butorphanol or saline solution. All thermal thresholds were determined by a single observer
(TLD); the observer was not aware of the treatment
administered to each kestrel. The observer initiated the
thermoelectric module and then observed and recorded
the kestrels with the video camera.
Agitation-sedation score and adverse effectsAll
birds were placed in the test box 5 minutes prior to
thermal testing and observed to determine their behavior. An agitation-sedation score was assigned by use of
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a scoring system based on a parrot agitation-sedation

scoring system29 modified for kestrel behavior (Appendix). During the 7 hours of data collection on each test
day, kestrels remained in the same test room. Between
testing times, birds were placed in transport carriers
(23 X 30.5 X 43 cm); each carrier contained a perching brick. Thus, the observer was able to continuously
monitor the birds for any adverse effects, including
vomiting and diarrhea, throughout the testing period.
Statistical analysisThe difference between
withdrawal temperature at any given time point after
treatment administration and the baseline withdrawal
threshold temperature for each bird in each period was
calculated. A repeated-measures ANOVA was used,
with fixed effects of dose, time, sex, period, and all
associated interactions. Correlation within birds over
time within a period was modeled with a spatial power
structure. Analysis of residuals resulting from the fitted
model revealed that they were acceptably normally distributed and had no evidence of heteroscedasticity. The
least squares mean of changes in withdrawal temperature
was obtained from values generated by the fitted model.
Pairwise comparisons of the least squares mean for the
various treatments, both within each time point and over
all time points, were performed with the post hoc least
significant difference method. Values were considered significant at P < 0.05. The sedation-agitation scores were
analyzed in the same manner. All data were analyzed with
commercially available software.c
PHARMACOKINETIC ANALYSIS
AnimalsTwelve 3-year-old American kestrels
(6 females and 6 males) were used. Mean SD body
weight was 114.2 6.0 g. The kestrels were a subgroup
of the birds used for the thermal antinociception testing
and were healthy as determined on the basis of results
of physical examinations performed before and during
the experiments. Kestrels were maintained in the same
conditions as described previously. The experimental
protocol for this portion of the study was approved by
the Institutional Animal Care and Use Committee of
the University of California-Davis.
Experimental designKestrels were assigned to 3
groups (A, B, and C). Each group comprised 4 birds (2
males and 2 females). Each kestrel was manually restrained and received 6 mg of butorphanol tartrate/kg,
IM, in the left pectoral muscle. Birds were manually restrained for collection of blood samples. Samples were
collected at predetermined time points after butorphanol administration (group A, 5 minutes, 1 hour, and 3
hours; group B; 0.25, 1.5, and 9 hours; and group C,
0.5, 2, and 6 hours). The birds were housed in transport
carriers throughout the sample collection period.
Blood samples (0.3 mL/sample) were collected
from a jugular vein or medial metatarsal vein into heparin-lithium microtainer tubes and placed in ice-packed
containers. Within 1 hour after collection, samples
were centrifuged at 3,500 X g for 6 minutes. Plasma was
harvested and stored at 80C until analysis.
Measurement of plasma butorphanol concentrationsPlasma concentrations of butorphanol were
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13-06-0175r.indd 13

determined with liquid chromatographyd and triple

quadrupole mass spectrometry.e Qualifying ions monitored had an m/z of 328.27 for butorphanol and 337.14
for the internal standard (ie, fentanyl). Quantifying
ions had an m/z of 157.20 for butorphanol and 105.3
for fentanyl. The mobile phase consisted of acetonitrile
and 0.1% formic acid. The mobile phase gradient was
95% formic acid from 0 to 0.5 minutes, 95% formic acid
to 65% formic acid from 0.5 to 1.5 minutes, 65% formic
acid from 1.5 to 6.5 minutes, and 65% formic acid to
95% formic acid from 6.5 to 7 minutes. Separation was
achieved with a C18 columnf at 40C.
Sample processing consisted of liquid extraction.
Plasma (0.05 mL) was mixed with 100 L of the internal standard (fentanyl; 250 ng/mL in 2% ammonium
hydroxide) and 1 mL of methyl tertiary-butyl ether. The
solution was mixed in a vortexer for 10 seconds and
then centrifuged at 10,000 X g for 5 minutes. The upper organic layer was transferred to a culture tube and
evaporated in a 40C water bath under an air stream for
10 minutes. Samples and standards were reconstituted
with 0.2 mL of 50% methanol, mixed in a vortexer for 5
seconds, and transferred to an injection vial. Injection
volume was 25 L. Plasma standard curves were created in canine plasma and were linear from 5 to 1,000 ng/
mL. Plasma standard curves were accepted if the correlation coefficient was 0.99 and the predicted values
were within 15% of the actual values. Accuracy of the
assay was determined in kestrel plasma. Accuracy was
97.3%, and the coefficient of variation was 13.7% for 3
replicates at each of 3 concentrations (5, 50, and 500
ng/mL).
Pharmacokinetic analysisPlasma concentrations
were analyzed with nave pooling of data and a 2-compartment open model with an absorption phase and biphasic elimination phase. Weighting (1/actual plasma
concentration) was used.g The pharmacokinetic model was chosen on the basis of visual inspection of the
goodness of fit and residuals.
Results
Thermal antinociceptionBaseline temperature for thermal foot withdrawal ranged from 35.9 to
47.3C. The within-bird SD for the 6-hour period after
administration of saline solution ranged from 0.65 to
1.65C. There were no significant (P = 0.815) changes
in thermal threshold over time after administration of
saline solution. There was no significant effect attributable to dose (P = 0.366), time (P = 0.082), or period
(P = 0.097); however, there was a significant effect of
sex (P = 0.002) and the dose-by-time interaction (P =
0.025). Because of the significant effect of sex, results
were analyzed separately for males and females.
At all doses of butorphanol administered to female
kestrels, the thermal foot withdrawal threshold did not
change significantly (P = 0.392), compared with the value for the saline solution, but withdrawal threshold was
significantly increased after butorphanol administration,
compared with baseline values, for 1 mg/kg at 0.5 hours
(P = 0.027) and 6 hours (P = 0.011), 3 mg/kg at 6 hours (P
= 0.031), and 6 mg/kg at 3 hours (P = 0.034). For all doses
of butorphanol administered to male kestrels, the thermal
13

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Figure 2Plasma concentrations of butorphanol after IM administration of 6 mg/kg in the pectoral muscles of 12 American kestrels (6 females [black circles] and 6 males [white circles]). The
birds were assigned to 3 groups (A, B, and C); each group comprised 4 birds (2 males and 2 females). Blood samples were collected at predetermined times after drug administration (group A,
5 minutes, 1 hour, and 3 hours; group B, 0.25, 1.5, and 9 hours;
and group C, 0.5, 2, and 6 hours).
Table 1Least squares mean changes in thermal thresholds
from baseline values in 15 American kestrels (Falco sparverius)
after IM administration of saline (0.9% NaCl) solution (control
treatment) or butorphanol tartrate at 1, 3, and 6 mg/kg.

Time

1 mg/kg 3 mg/kg 6 mg/kg

Male (n = 6)

0.5
1.5
3.0
6.0

1.362
0.002
0.222
0.922

0.568
0.552
0.452
0.652

Female (n = 9)

0.5
1.5
3.0
6.0

Sex
Figure 1Estimated mean change in thermal threshold from
baseline values in 6 male (A) and 9 female (B) American kestrels (Falco sparverius) after IM administration of saline (0.9%
NaCl) solution (diamonds with solid line; control treatment) and
butorphanol tartrate at 1 mg/kg (squares with dashed line), 3 mg/
kg (triangles with dashed-and-dotted line), and 6 mg/kg (squares
with dotted line). Baseline values were obtained 30 to 60 minutes before injections; time of treatment administration was
designated as time 0. There was a washout period of 14 days
between subsequent treatments. *Value differs significantly (P
< 0.05) from the baseline value for 6 mg/kg in panel A and for 1
mg/kg at 0.5 and 6 hours, 3 mg/kg at 6 hours, and 6 mg/kg at 3
hours in panel B. Within a time point, value for 6 mg/kg differs
significantly (P < 0.05) from the value for the control treatment.

foot withdrawal threshold did not change significantly,

compared with the threshold after administration of saline solution; however, compared with the baseline value,
the withdrawal threshold was significantly (P = 0.008) decreased 1.5 hours after administration of butorphanol at 6
mg/kg (Figures 1 and 2; Table 1).
We did not detect significant effects on sedationagitation for either sex at any dose of butorphanol, except male kestrels had a significantly higher sedationagitation score 1.5 hours after administration of butorphanol at 6 mg/kg (Table 2).
Pharmacokinetic analysisPlasma concentrations
over time and pharmacokinetic parameters of butorphanol tartrate administered to American kestrels were
determined (Figure 2; Table 3). Plasma butorphanol
concentrations were below the limit of detection (5 ng/
mL) in 1 of 4 birds at 6 hours and in all 4 birds at 9
hours. Mean plasma concentrations were > 100 ng/mL
for approximately 2 hours after IM administration of
butorphanol.
14

13-06-0175r.indd 14

Butorphanol

Saline
solution

0.889*
0.145
0.189
0.489

0.860*
0.260
0.693
0.993*

0.098
0.258
0.282
0.122
0.105
0.583
0.194
0.850*

0.328
2.328*
0.348
1.568
0.134
0.390
0.812*
0.112

Baseline values were obtained 30 to 60 minutes before injections; time of treatment administration was designated as time 0.
There was a washout period of 14 days between subsequent treatments. The SE for males was 0.749 for saline solution, 0.798 for 1 mg/
kg, 0.750 for 3 mg/kg, and 0.774 for 6 mg/kg. The SE for females was
0.384 for saline solution, 0.384 for 1 mg/kg, 0.389 for 3 mg/kg, and
0.378 for 6 mg/kg.
*Within a sex within a column, value differs significantly (P <
0.05) from the baseline value. Within a sex within a row, value differs significantly (P < 0.05) from the value for the control treatment.

Discussion
Butorphanol tartrate administered IM at 1, 3, and
6 mg/kg caused sex-dependent responses in kestrels,
which resulted in increased thermal thresholds in females and decreased thermal thresholds in males. To
the authors knowledge, this is the first report of sex differences for opioid drugs in birds. Changes in thermal
nociception in females were suggestive of mild analgesia at specific time points, whereas the changes in males
suggested mild hyperesthesia to the thermal stimulus
or mild hyperalgesia as well as mild agitation at higher
doses. An explanation that must be considered for the
fact there was not a significant difference from results
for the saline solution is a type 1 error. The significant
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Table 2Least squares mean changes in sedation-agitation

scores in 15 American kestrels after IM administration of saline
solution (control treatment) or butorphanol tartrate at 1, 3, and
6 mg/kg.

Sex

Time

Saline
solution

Male (n = 6)

0.5
1.5
3.0
6.0
0.5
1.5
3.0
6.0

0.987
0.987
0.987
0.987
1.060
0.782
1.060
1.004

Female (n = 9)

Butorphanol
1 mg/kg 3 mg/kg 6 mg/kg
1.171
1.088
1.088
1.005
1.011
0.844
0.788
1.066

0.994
0.994
1.161
1.077
1.033
1.089
0.978
0.978

1.010
1.224
1.010
1.010
1.063
0.952
1.063
1.174

For males, the SE was 0.089 for saline solution, 0.083 for 1 mg/
kg, 0.082 for 3 mg/kg, and 0.076 for 6 mg/kg. For females, the SE was
0.117 for saline solution, 0.117 for 1 mg/kg, 0.119 for 3 mg/kg, and
0.116 for 6 mg/kg.
See Table 1 for remainder of key.
Table 3Pharmacokinetic parameters derived from nave pooling of data in a 2-compartment pharmacokinetic analysis after IM
administration of butorphanol tartrate at 6 mg/kg to 12 American
kestrels.
Parameter
A (ng/mL)
B (ng/mL)
K01 (/h)
(/h)
(/h)
AUC0 (hng/mL)
t1/2 (h)
t1/2 (h)
Volcc/F (L/kg)
CL/F (mL/min/kg)
Volpc/F (L/kg)
Tmax (h)
Cmax (ng/mL)

Value
3,460
155
3.27
2.46
0.47
631
0.28
1.48
6.06
159
5.18
0.38
445

A = intercept. = Rate constant for the distribution phase.

t1/2 = Half-life of the distribution phase. AUC0 = Area under the
concentration-time
curve extrapolated to infinity. B = intercept. = Rate constant for
the elimination phase. t1/2 = Half-life of the terminal phase. CL/F
= Clearance per fraction of the dose absorbed. Cmax = Maximum
plasma concentration. K01 = Absorption rate constant. Tmax = Time
to maximum plasma concentration. Volcc/F = Volume of distribution of the central compartment per fraction of the dose absorbed.
Volpc/F = Volume of distribution of the peripheral compartment per

time-by-dose interaction and small magnitude of the

change in thermal thresholds preclude broad clinical
application. On the basis of results of the present study,
butorphanol tartrate at the doses used may not provide
effective analgesia in American kestrels. Alternatively,
the method used may not have been sensitive enough
to assess the effects of butorphanol.
The thermal nociceptive response has been used
to study several opioids and doses in psittacine species14,15,23,26,2933 and raptors.18 Nociception is caused by
a thermal stimulus activating thermal receptors. Thermal receptors in birds are associated with afferent A
and C fibers, which transmit nociceptive information to
different areas of the midbrain and forebrain by ascending spinal pathways.3436 The use of a thermal stimulus to affect the natural perching behavior of raptors
is a noninvasive method for evaluation of nociceptive
thresholds and modulation of nociceptive thresholds
by analgesic treatments. The within-kestrel SD over the
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13-06-0175r.indd 15

6-hour period after the administration of saline solution

ranged from 0.65 to 1.65C. This was similar to results
of another study18 in American kestrels and substantially smaller than the within-parrot SD over a 6-hour
period after IM administration of saline solution.16 The
sample size (n = 15) was larger than that in a similar
study18 in American kestrels,0 which was conducted to
evaluate the effectiveness of hydromorphone, similar
to or larger than that in other nociception studies with
psittacine species performed by our research group,37
and larger than that in several studies in dogs38,39 and
cats.4043 Lack of a significant period effect and lack of
significant changes in thermal threshold during the 6
hours after the administration of saline solution (except in the females at 0.5 hours) validated the use of
the control group and baseline values for the analysis.
Results of the present study differ from those in
other studies1315,4446 with psittacine species, in which
butorphanol and nalbuphine were recommended as the
opioid drugs for the management of acute pain. The butorphanol doses used in the present study were based
on doses recommended for psittacine species.14,15,22 In
the experience of one of the authors (JRPM), electrical stimulus thresholds in Hispaniolan Amazon parrots
were significantly increased throughout the 90-minute
study period after IM administration of 3 mg of butorphanol/kg. However, it is unclear whether the results of
thermal and electrical stimuli can be directly compared.
All methods for assessment of nociception have their
limitations. Thermal stimuli also activate temperaturesensitive neurons; thus, the response may be to warmth
rather than pain, and we would not expect this to be
affected by an opioid. Electrical stimuli activate all neurons (touch, motor, temperature, autonomic, and pain).
Other factors can also affect results of studies.
The distribution of nociceptors on the feet may differ
among species of birds; thus, different responses may
be obtained to a stimulus. The sensitivity of the nociceptors may also differ, which could result in different responses independent of the drug administered.
Differences in the thickness of the sole of a birds foot
could also influence the results. The difference in response to butorphanol for psittacine birds, compared
with the response for kestrels, might be attributable to
a variety of factors such as species differences in drug
metabolism, protein binding, or peripheral thermal receptors or dissimilarities in the quantity, distribution,
or function of - and -opioid receptors in the limbic
system (subcortical and spinal levels).
Individual variation in the antinociceptive effects of
opioids has been detected in many species and appears
to be multifactorial, including genotype, sex, age, type
of noxious stimulus, receptor, and relative efficacy of
the agent.47,48 In particular, there are marked differences
between sexes in rodents and nonhuman primates after
injection of low-efficacy opioids such as butorphanol,
with the drug being more potent and effective in males
than females.49 Despite the relatively small number of
kestrels of each sex in the present study, there was a
significant difference between males and females in
the response to the thermal stimulus and treatments.
Males generally had a decrease in the thermal threshold
for both the control and butorphanol treatments, with
15

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hyperalgesia in the males for 6 mg/kg at 1.5 hours, whereas there was an increase in the thermal threshold in the
females. The difference between males and females for a
low-efficacy opioid such as butorphanol suggests a fundamental difference in signal transduction mechanisms or
receptor density between males and females.49
The pharmacokinetic method used in the present
study was a nave pooling of drug concentrations from
multiple birds, which was necessitated because the small
size of the birds precluded blood collection from each kestrel at all time points. A limitation of this method is that
it does not allow measurement of variation in calculated
pharmacokinetic parameters because pooled concentrations are analyzed as though they have been derived from
a single bird.50 However, strong interindividual variation
in the pharmacokinetics and duration of the effects of butorphanol has been reported in other species.51,52
Butorphanol tartrate administered IM at 6 mg/kg
rapidly resulted in high plasma concentrations (maximum concentration, 444.68 ng/mL at 0.38 hours) in
American kestrels. Plasma concentrations of butorphanol after IM administration decreased rapidly over time.
The termination half-life for butorphanol in American
kestrels that we calculated for the present study (1.48
hours) was comparable to that reported for red-tailed
hawks and great horned owls19 injected IM with a
dose of 0.5 mg/kg (0.93 and 1.78 hours, respectively)
or chickens23 injected IV with a dose of 2 mg/kg (1.19
hours), but it was longer than that in Hispaniolan Amazon parrots22 injected IM with a dose of 5 mg/kg (0.51
hours). Clearance per fraction of the dose absorbed was
also higher for the present study (158.59 mL/min/kg)
than that reported after IV administration in red-tailed
hawks and great horned owls19 (58.03 and 26.1 mL/
min/kg, respectively). The higher clearance per fraction
of the dose absorbed in American kestrels may have
been attributable to greater metabolism, which occurs
by hepatic hydroxylation in other species,53 or to a
lower bioavailability after IM administration. It may be
more likely to truly be a slower clearance because the
terminal half-life was much longer in the kestrels, but
evaluation after IV administration is needed to confirm
the true clearance.
In kestrels, IM administration of 6 mg of butorphanol/kg resulted in plasma concentrations 100 ng/
mL for 2 hours in 3 of 4 birds. Plasma concentrations
of butorphanol and metabolites > 80 ng/mL reportedly
provide analgesia in Hispaniolan Amazon parrots.15
However, a direct relationship between plasma drug
concentrations and antinociceptive effects could not be
inferred from the study15 of Hispaniolan Amazon parrots because the assay measured concentrations of butorphanol and its metabolites together, and some of the
metabolites may not be effective analgesics. The plasma
concentrations in the present study would be sufficient
to provide analgesia in other species, so caution should
be used when plasma concentration is used alone to
predict analgesia. Antinociceptive effects are likely determined by the concentration at the receptor, which
lags behind the plasma concentration.54 Furthermore,
the affinity of a drug for the receptors, the quantity and
distribution of the receptors, and the interactions with
other receptors might also determine the effect.
16

13-06-0175r.indd 16

Adverse effects observed in the present study

were hyperesthesia or hyperalgesia and agitation.
Opioid-induced hyperesthesia or hyperalgesia
was seen in male kestrels (6 mg/kg at 1.5 hours).
Hyperesthesia is an increased sensitivity to sensation. Therefore, the response may not necessarily indicate an increase in pain sensitivity. Opioid-induced
hyperalgesia is defined as a state of nociceptive sensitization caused by exposure to opioids.55 The condition is characterized by a paradoxical response
whereby a patient receiving opioids for the treatment
of pain could actually become more sensitive to certain painful stimuli.55 Opioid-induced hyperalgesia
is associated with upregulation of the compensatory
pronociceptive pathways.56 Acute receptor desensitization via uncoupling of the receptor from G-proteins, upregulation of the cAMP pathway, activation
of the N-methyl-D-aspartate receptor system, and descending facilitation have been proposed as potential
mechanisms underlying opioid-induced hyperalgesia.57 Opioid-induced hyperalgesia occurs in several
distinct settings and is characterized on the basis of
the opioid dose administered and the pattern of administration. Most of the studies on opioid-induced
hyperalgesia have been performed during ongoing
(maintenance) therapy or withdrawal from opioids,
but another study56 has revealed opioid-induced
hyperalgesia during administration of extremely
high or extremely low doses of opioids.56 Factors
determining the net effect after administration of
-opioid receptor agonists are complex and incompletely understood, and they likely include the site of
drug administration and the nociceptive method used
for testing. Contrary to the biphasic, analgesic-hyperalgesic temporal response observed after administration
of -opioid receptor agonists, the response elicited by
-opioid receptor agonists appears to be monophasic
(ie, it is either analgesic or hyperalgesic).56
Sedation was not observed in individual birds in
the present study, but the male kestrels appeared agitated at 1.5 hours after administration of 6 mg of butorphanol/kg. The sedation-agitation scoring in the study
involved evaluation of the birds after they were placed
in the perching box and may not have fully reflected the
sedative effects of this drug in another setting.
The cardiorespiratory effects of butorphanol were
not evaluated in the study reported here. However, in
red-tailed hawks and great horned owls administered
0.5 mg/kg, IV and IM, the decreases in heart rate and
respiratory rate were not considered clinically relevant.19 In that study,19 only minor sedative effects of
short duration were detected in some birds. Butorphanol at a dose of 2 mg/kg administered IM did not cause
significant changes in anesthetic and cardiopulmonary
variables in Hispaniolan Amazon parrots anesthetized
with sevoflurane.58 There were no other adverse effects
detected during that study. Results of the present study
supported the contention that butorphanol appears to
be safe for use in American kestrels, but further studies to evaluate the antinociceptive, sedation-agitation,
cardiorespiratory, and thermoregulatory effects of butorphanol in American kestrels and other raptor species
are needed.
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Butorphanol tartrate administered IM at 1, 3, and

6 mg/kg did not cause significant increases in thermal
thresholds or sedative effects in American kestrels.
Instead, it caused hyperesthesia or hyperalgesia and
agitation in males receiving 6 mg/kg. These findings
suggested that butorphanol may not provide effective
analgesia in American kestrels. Additional studies with
other types of stimulation, formulations, doses, routes
of administration, and testing times are needed to fully
evaluate the antinociceptive and adverse effects of butorphanol in American kestrels and other species of
birds and their relevance for clinical application.
a.
b.
c.
d.
e.
f.
g.

Fort Dodge Animal Health, Overland Park, Kan.

RANDOM.ORG random integer generator, Randomness and Integrity Services Ltd, Dublin, Ireland. Available at: www.random.
org. Accessed Aug 20, 2011.
Mixed Procedure, SAS, version 9.1.3, SAS Institute Inc, Cary,
NC.
Shimadzu Prominence, Shimadzu Scientific Instruments, Columbia, Md.
API 2000, Applied Biosystems Inc, Foster City, Calif.
ACE-C18AR, 150 X 3 mm, 5M, Mac-Mod Analytical Inc,
Chadds Ford, Pa.
WinNonlin, Pharsight Corp, Mountain View, Calif.

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Appendix
Agitation-sedation scores used to assess behavioral effects of butorphanol tartrate administered to American kestrels (Falco
sparverius).
Score

Description

+3
+2
+1
0
1
2
3
4

Bird does not stay on the perch and constantly flies off.
Bird intermittently flies off the perch but returns to the perch on its own.
Bird stays on the perch but constantly looks around.
Bird stays on the perch, is calm, and does not look around but is reactive to movement in front of the test box.
Bird stays on the perch, is calm, and has only sluggish response to movement in front of the test box.
Bird does not react to movement in front of the test box and only reacts if the back of the box is opened and a hand is inserted into the box.
Bird is only responsive when touched.
Bird is nonresponsive to a visual or tactile stimulus.

Adapted from Geelen S, Sanchez-Migallon Guzman D, Souza MJ, et al. Antinociceptive effects of tramadol hydrochloride after intravenous
administration to Hispaniolan Amazon parrots (Amazona ventralis). Am J Vet Res 2013;74:201206. Reprinted with permission.

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