mystery of yawning
Le bâillement, du réflexe à la pathologie
Le bâillement : de l'éthologie à la médecine clinique
Le bâillement : phylogenèse, éthologie, nosogénie
 Le bâillement : un comportement universel
La parakinésie brachiale oscitante
Yawning: its cycle, its role
Warum gähnen wir ?
Fetal yawning assessed by 3D and 4D sonography
Le bâillement foetal
Le bâillement, du réflexe à la pathologie
Le bâillement : de l'éthologie à la médecine clinique
Le bâillement : phylogenèse, éthologie, nosogénie
 Le bâillement : un comportement universel
La parakinésie brachiale oscitante
Yawning: its cycle, its role
Warum gähnen wir ?
Fetal yawning assessed by 3D and 4D sonography
Le bâillement foetal

mise à jour du
1 juin 2012
Assessment of Stress in Laboratory Beagle Dogs Constrained by a Pavlov Sling
Jenny Stracke, Bettina Bert, Heidrun Fink, Jörg Böhner
Freie Universität Berlin, Department of Veterinary Medicine, Institute of Pharmacology and Toxicology, Berlin, Germany;
University of Zielona Góra, Faculty of Biological Sciences, Zielona Góra, Poland


Le bâillement chez le chien, Canis lupus familiaris approches ethologique, pharmacologique et clinique
Nathalie Tomczyk
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.
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