According to the Hungarian regulations of animal experimentation our non-invasive polysomnography research does not qualify as an animal experiment. The Hungarian Scientific Ethical Committee of Animal Experiments issued permission (under the number PE/EA/853-2/2016) approving our non-invasive protocol. All owners volunteered to participate in the study and were informed about the procedure before beginning.
Family dogs (N=50) participated in the research, but 15 of them did not enter REM sleep (we did not find any apparent characteristic differences that would justify the dissimilarity between the dogs that did not fall into REM sleep and dogs that did). In four cases there were some missing data (e.g., owners did not provide the age of the dog), thus the final sample consisted of N=31 dogs (16 males and 15 females). Their age ranged between 6 months and 15 years (median ± SD=4.17 ± 4.08), their height varied between 20 cm and 68 cm (mean ± SD=51.89 ± 10.71), and they weighed between 5 and 38 kg (mean ± SD=21.77 ± 8.47).
Our study was conducted within the confines of the Family Dog Project (Abdai & Miklósi, 2015). Participation was voluntary, the owners signed up through the internet. There were no requirements regarding what kind of dogs could participate, except that dogs needed to tolerate a stranger touching their head during electrode placement. There was no need for any kind of pre-training of the dogs. Additionally, we asked the owners to come to the recording session after an ordinary day (without any unusual occurrences that could possibly have an effect on the dogs’ sleep).
The study consisted of a single 3-h polysomnography recording (parallel monitoring of EEG, EOG, ECG, respiration, and EMG), which took place between 12:00 and 18:00 – since similar to humans, dogs are most prone to fall asleep during this time of the day (Tobler & Sigg, 1986). The recordings took place in a darkened room, where the owner could lay down on a mattress with his/her dog, read, or watch movies on a computer (with headphones) (Fig. 1).
After arrival, the owner allowed his/her dog to explore the environment and then the experimenter placed the respiratory belt and the electrodes on the dog according to a previously validated canine polysomnography procedure (Fig. 2) (Kis et al., 2014). During this time the dogs received social reinforcements from both the owner and the experimenter. They also received food treats when required. As per our protocol, no force was applied during the placement of the measuring equipment in any case. There were also no tranquillizers or soporifics used – the dogs fell asleep due to the absence of stimuli and movement.
Before attaching each electrode, we used PARKER® SIGNA® spray to clean the surface of the skin. Gold (Au) coated silver-chloride (Ag|AgCl) scalp electrodes were used (with a diameter of 10 mm), which were attached with NATUS® EC2® GENUINE GRASS® electrode cream and gauze.
For the present analysis data from the eye movement electrode was used (marked as EOG(+) on Fig. 2), which was placed on the left zygomatic arch and referenced to the central posterior electrode (marked as EOG(-) on Fig. 2). The recording and monitoring of the signals were done with SYSTEMPLUS EVOLUTION® (MICROMED®) software.
Sleep stage scoring (identification of the stages of awake, drowsiness, NREM sleep, REM sleep) was carried out with FERCIO’S EEG 0.8 (© Ferenc Gombos 2012) software in 20-s epochs manually according to standardized criteria for dogs (Kis et al., 2014) based on a conventional scheme for humans (Rechtschaffen & Kales, 1968).
Rapid eye movements (excursions on the EOG with a minimum of 50 μV) were manually detected in all the 2-s epochs initially marked as REM. REMD was calculated as the number of 2-s epochs that contained rapid eye movements divided by the total number of 2-s epochs of REM sleep (Table 1 shows REM data and basic demographic information for the sample).
Data analysis was done using R 3.3.1 statistical software (R Core Team, 2016). The effect of REM duration and different individual characteristics (age, sex, and body mass) on REMD was investigated by general linear models (GLMs). Since there was a strong correlation between the dogs’ height and body mass (Pearson’s product-moment correlation, R=0.72, t25=5.23, p<0.001) only the effect of body mass was used as an explanatory variable. First a model that included the main effects and all two-way interactions was built. REM duration was included in the model as a second-order orthogonal polynomial due to the fact that when initially plotting the data before statistical analysis a strong quadratic relationship was apparent between REM duration and REMD (Fig. 3). Second, non-significant terms were removed by backward elimination. Third, the effect of the quadratic term of REM duration was tested by comparing the model with the polynomial term with a model containing a linear term. Fourth, since the quadratic term was significant (see Results), subjects were divided into two groups using the median of the REM sleep duration (15.33 min) as a cut-off. The short REM sleep duration group included the dogs that spent 15.33 min or less in REM sleep (n=16), whereas the long REM sleep duration group included the dogs that spent more than 15.33 min in REM sleep (n=15). We repeated the above GLM analysis separately for the two groups; however, in these models REM duration was included as a linear term.