The 3Rs, Replacement, Reduction and
Refinement, have become increasingly important
in designing animal experiments. The Pavlov
sling is thought to be a non-invasive method to
restrain dogs for examinations. The aim of our
study was to investigate whether laboratory
Beagle dogs that had been trained to tolerate
restraint by a Pavlov sling are stressed by this
procedure and, furthermore, to analyze their
behavior during this period. Five male and five
female Beagle dogs were used, each three years
of age. Animals were restrained in the Pavlov
sling for 30 min on six days with an interval of
at least two days. The following behaviors were
recorded every minute for each session: postures
of body, head, and ears, as well as state of
eyes, tail, legs, and mouth. Additionally, the
animals were observed for the occurrence of
particular stress signs, including body shaking,
sweating of the paws, increased saliva
production, piloerection, blinking of eyes,
snout licking, yawning, and panting. As
an indicator for stress, salivary cortisol
levels were measured before, during, and after
each session. Our results show that for most
behavioral parameters, e.g., body, leg, head,
tail, and ear posture, the frequency of changes
between different behavior patterns, as well as
cortisol concentration, were not influenced by
restraint in the Pavlov sling. Therefore, the
Pavlov sling does not seem to be perceived as a
stressful situation by the Beagle dogs. Our
study demonstrates that under certain conditions
the use of the Pavlov sling in trained dogs can
substitute for more ordinary methods of
immobilization, e.g., the use of narcotics.
1 Introduction
A total of 3,832 dogs were used as
laboratory animals in Germany in 2009 (BMELV,
2011), mostly for the development of new
products or instruments used in human medicine
or dentistry, for toxicological studies, and for
fundamental biological research (BMELV, 2011;
Ritskes-Hoitinga et al., 2006). Many of the
experiments include direct measurements in the
conscious dog, such as taking blood samples,
intravenous dripping, imaging, or measuring
heart frequency, body temperature or other
physiological parameters. For imaging
techniques, especially, it is very important to
restrain the animal in its movements, sometimes
over a long period of time. Despite many efforts
to find alternatives for animal models in
science, it is unrealistic to expect a complete
end to laboratory animal use in the near future.
Therefore, the 3Rs &endash; Replacement,
Reduction and Refinement &endash; (Russell and
Burch, 1959) are still valid, and the third R
becomes increasingly important.
If neither replacement nor reduction is
possible, refinement is of utmost importance.
Refinement should minimize any pain and/or
distress for a laboratory animal in the
experiment and during housing. Over recent years
a lot of work has been done to improve
experimental conditions and to enrich the
environment, not only for the benefit of the
animals but also for the validity and
reliability of the experiment (European
Convention, 1986; TierSchHuV, 2001; Hubrecht,
2002; MUFV, 2003). One important issue in
refinement is the reduction of stress during the
experiment, and it can be very useful to develop
training programs for habituating dogs (as well
as other laboratory animals) to the experimental
situation (Adams et al., 2004; Hubrecht, 2002).
If possible, a lot of handling and training
should take place early, during the imprinting
period of puppies (Feddersen-Petersen, 1991). To
keep laboratory dogs calm and immobile during
long-term investigations, e.g., imaging
procedures, they usually are anaesthetized or
deeply sedated. One method to replace narcotics
or sedatives is for an assistant to restrain the
dog. Reduction of discomfort caused by this
procedure can be achieved by using the Pavlov
sling, in which the dogs are trained to stand
nearly motionless. The Pavlov sling is an
established alternative to restraint and is
expected to reduce the stress level caused by
the experiment. Dogs should first be carefully
trained by classical conditioning so they
associate restraint in the sling with a positive
experience. Nevertheless, in the Pavlov sling
dogs are restrained in their movements and
cannot escape from a situation that impedes
their normal resting positions, such as sitting
or lying down, and it is possible that the
Pavlov sling itself causes stress (Mikkelsen et
al., 2003). Avoiding stress during the
experimental situation is very important, and
not only for ethical reasons. The reaction of an
individual to stress is complex, integrating
responses of the central and the autonomic
system, the hypothalamic-pituitary-adrenal axis,
and the target organs. Each reaction can falsify
the experimental results in an interactive way
that is yet to be determined (Ritskes-Hoitinga
et al., 2006).
To evaluate the degree of stress of an
animal, the assessment of physiological
parameters, especially of stress hormones, are
diagnostically conclusive (Nagel and von
Reinhardt, 2003; Knies, 2005; Taylor, 2000). One
important hormone for the identification of a
stress reaction is the glucocorticoid cortisol.
The analysis of saliva provides a suitable
method to assess changes in cortisol
concentration. Traditionally, cortisol
concentration is measured in the plasma.
However, collecting blood samples is an invasive
method that may itself cause stress in the
animal (Beerda, et al., 1998), which can be a
confounding variable. The concentration of free
cortisol in saliva correlates significantly with
levels measured in the blood (Beerda et al.,
1996; Vincent and Michell, 1992). Therefore,
working with salivary cortisol provides a
non-invasive and currently very popular method
for measuring stress-related changes in cortisol
concentration (Kobelt et al., 2003). Stress also
can be measured by the occurrence of certain
behavioral patterns. Dogs learn submissive
behavior as puppies, and signals indicating
submission are normally used to avoid a threat
or prohibit aggressive behavior (Beaver, 1999).
Many of the behavioral patterns, e.g., calming
signals, are regarded as signs of stress
(Rugaas, 2002).
The first aim of our study was to quantify
and qualify the behavior shown by habituated
Beagle dogs during the Pavlov sling restraining.
Secondly, we investigated whether the animals
are stressed by this procedure and looked for
stress-related behavioral signs, such as body
shaking, sweating of the paws, increased saliva
production, piloerection, blinking of eyes,
snout licking, yawning, and panting.
Additionally, changes in the behavioral pattern
have been analyzed at the beginning, middle, and
end of restraining, since high levels of change
can be regarded as signs of restlessness.
Moreover, salivary cortisol concentrations were
measured before, during, and after the
experiment. Our results should help to evaluate
the Pavlov sling as an adequate instrument for
long-term restraining of dogs without causing
additional stress during experiments.
4 Discussion
This is the first study to reveal, review,
and discuss the behavior of trained Beagle dogs
that are restrained by a Pavlov sling. Overall,
the analysis of all behavioral patterns and the
physiological parameter cortisol clearly
indicate that the dogs did not suffer undue
stress in that situation.
The frequency of various behavioral states
and positions the animals displayed while
restrained in the Pavlov sling revealed no
marked differences across the five sessions and
confirms that the dogs were already familiarized
with this procedure and did not need further
habituation. This observation is underscored by
the finding that changes of state and positions
did not vary significantly between the
beginning, middle, and end of the sessions. Haug
(2004) describes a relaxed dog as sitting or
lying with muscles not tensed, body not shaking,
tail held low, and ears hanging. All of these
characteristics were frequently observed in our
dogs during the experiment. Additionally, most
of the time the animals had their paws touching
the ground, the mouth closed, and the eyes
totally or half closed. Although we cannot
quantify our observation by EEG, respiratory,
and heart rate recording, it appeared that most
dogs were at least dozing while their eyes were
half or fully closed. Further indications that
our dogs were relaxed during the experiments
are: head lying mainly on the hammock or on the
side metal rods and body either completely
hanging or, less often, just pelvis hanging in
the hammock.
However, it is sometimes difficult to decide
whether an immobilized dog is truly relaxed or
has simply "surrendered" to an unavoidable
situation. Therefore, we investigated additional
parameters, such as the frequency of changes of
behavioral patterns as well as unambiguous
stress signals. According to Beerda et al.
(1997) stress, i.e., unavoidable electrical
shock, results in an increase of restlessness in
dogs, such as increased walking, nosing, and
changing from one state of locomotion to
another. In our study we regarded the number of
changes from one behavioral category to another
as a measure of restlessness. We expected that
dogs should be more restless, i.e., show an
increased number of behavioral changes, at the
beginning and would calm down at the end of the
experiment, if they were stressed or agitated
during restraining. However, changes in body and
tail position, as well as in the state of the
mouth, occurred with only marginal frequency.
Most changes were observed in the position of
the ears and head and in the state of the eyes.
Since the experiment could not be carried out in
a soundproof room, it is quite likely that these
marginal changes in position/state of head,
ears, and eyes do not indicate stress or
discomfort but were caused by external, mainly
acoustic, stimuli from the adjacent rooms and
hallway, an assumption which we could confirm by
direct observation on several occasions.
Behavioral patterns that indicate some kind
of discomfort and that could be displayed by the
dogs restrained in the Pavlov sling can be
divided into distinct stress signals, such as
body shaking, sweating of the paws, an increased
saliva production and piloerection (Beerda et
al., 1997; Beerda et al., 2000; Nagel and von
Reinhardt, 2003), and more subtle submission
signals, such as eye blinking, snout licking,
yawning, and panting (Beerda et al.,
1998; Feddersen-Petersen, 2004; Haberland, 2002;
Rugaas, 2002). The quantitative analysis of
these behavioral patterns indicates a
non-stressed state of the dogs while standing in
the sling. Distinct stress signals were not
shown at all. Some submission signals occurred,
but in frequencies definitely below levels
indicating acute stress. Blinking of the eyes
occurred 2.7 times a minute on average, which is
far below a frequency given by Harmer and
Williams (2003) for relaxed dogs (13.7 times a
minute).
Beerda et al. (1997) measured stress of
freely moving dogs that were exposed to acoustic
signals (noise blasts of 3,000 Hz at levels of
70, 78, and 87 dB), and report a snout licking
frequency of approximately 26 times per 30
minutes, whereas control dogs showed such
behavior only three times per 30 minutes. With
9.8 instances of snout licking in the 30-minute
period, our dogs in the Pavlov sling displayed a
higher frequency than the control dogs used in
the study of Beerda et al. (1997) but did not
reach the frequency of the stressed dogs.
Furthermore, yawning and panting were not
observed in all dogs and occurred in less than
10% of the observation time. Therefore, there
was no indication of stress during the time the
dogs were restrained in the sling. In addition,
we have surveyed the behavior for any signs of
increased aggression and stress when the dogs
were reintegrated into their respective groups
after the sessions. Separating a dog from and
returning it to its group always leads to
agitation. Although we did not quantify our
observations, this agitation did not differ from
the behavior when a dog was taken out of the
group for examinations (e.g., control of body
weight and health status) or returning from a
walk on a leash outside. There were also no
differences in body weight before, during, and
after the experiments, indicating no additional
stress for the animals.
Our conclusions drawn from the behavioral
data are confirmed by the measurement of the
stress hormone cortisol. Firstly, salivary
cortisol concentrations of the experimental
samples ranged on average from 0.9 to 2.6 ng/ml.
This is in line with salivary cortisol
concentrations of unstressed Beagle dogs at a
similar age (one to four years of age, between
0.6 and 2.3 ng/ml) (Koyama et al., 2002; Vincent
and Michell, 1992). Secondly, there was no
difference in average cortisol levels between
the basal, pre-experimental, and experimental
samples, clearly indicating that the dogs were
neither stressed by the experimental preparation
nor by the experiment itself, i.e., spending 30
minutes standing or lying in the Pavlov
sling.
Hubrecht (2002) and Adams et al. (2004)
point out that an experimental situation should
be as comfortable as possible for the tested
animal. Therefore, the results of our study
could be helpful in improving future experiments
with laboratory dogs, because the Pavlov sling
obviously does not cause additional stress in
the trained animal. This advantage of the Pavlov
sling has implications not only for the ethics
concerning the use of laboratory animals but
also for the overall validity of the desired
data: The less stress an animal suffers, the
more reliable the experimental data are (Shuy et
al., 1987; Vogel, 1993). According to Hubrecht
(2002) and Adams et al. (2004), the animals
should be accustomed to an experimental
situation before the actual experiment is run, a
point of view certainly supported by the present
study. Our results show that a well-practiced
experimental procedure and habituation to the
special test situation can even lead to
relaxation of the animal, as indicated by a
completely hanging body, eyes closed for longer
periods of time. The Pavlov sling, therefore,
can substitute for more ordinary but often
discomfort-producing methods of immobilization
&endash; and even for the use of narcotics in
some cases. The setup of the Pavlov sling should
always be optimized in detail to make it as
comfortable as possible for the dogs (Mikkelsen
et al., 2003). In some cases it may be best, if
compatible with the aim of the experiment, to
have the dogs sitting instead of standing or
lying flat in the sling, so the hammock should
be constructed accordingly. The size of the
hammock and height of the Pavlov sling should be
fitted to the individual dog, as our experience
shows that even dogs of the same breed can
differ considerably in body height and length.
The possibility of raising or lowering the
position of the hammock, as well as using the
optimal hammock size, is very important for
enabling the animals to stand or lie down
comfortably. In summary, our study demonstrates
that, under certain conditions, the usage of a
Pavlov sling in trained animals can substitute
for other methods of immobilization, e.g., the
use of narcotics or sedating drugs, which result
in greater discomfort.
Le
bâillement chez le chien - Yawning in
dogs. Nathalie Tomczyk 2009
