Materials and Methods

4.1.3.1 Animals

Ten Beagle dogs participated in this study. Five Beagle dogs from the same litter, owned by the Small Animal Clinic (University of Veterinary Medicine Hannover, Foun-dation, Germany), represented the young group. The dogs were on average 2.0 ± 0.0 years of age (mean ± standard deviation), male, neutered and had a body weight of 18.3 ± 2.4 kg. Five more Beagle dogs, two from another institute of the uni-versity and three from private owners, represented the old group. They were 10.4 ± 0.9 years of age, three of the dogs were male and two female and all were neutered, except for one male dog. The old dogs had a body weight of 15.5 ± 2.4 kg.

The deviation of body weight between these two groups was not statistically significant.

All experiments were performed in accordance with the relevant statutory provisions.

The study was reported to the Lower Saxony State Office for Consumer Protection and Food Safety in Oldenburg, Germany (reference number 33.9-42502-05-13A369).

All participating dogs had to be free of orthopedic diseases. Therefore, detailed anam-nesis as well as general and orthopedic examinations were performed. There were no signs of general or orthopedic diseases in the participating dogs and none of the dogs

were receiving any medication. In the companion study by Willen et al. (manuscript submitted for publication) the vertical ground reaction force was recorded as an objec-tive measure of lameness. Kinetic and kinematic data were recorded parallel; except for one young dog, where the kinetic recordings had to be retaken and the later results were used for kinetic analysis. No statistically significant differences between the groups could be found in vertical impulse, peak vertical force or mean vertical force.

The body weight distribution, calculated according to Steiss et al. [41], was in both groups nearly 60 % for the forelimbs and 40 % for the hindlimbs, which is considered physiological in the Beagle dog [1]. The symmetry indices for the fore- and hindlimbs were calculated according to Herzog et al. [26] and a symmetry index of ˂ 6 % was considered physiological [11]. The symmetry indices of both groups confirmed an al-most symmetrical gait pattern without signs of lameness.

4.1.3.2 Data collection

Kinematic measurements were performed in the gait analysis laboratory of the Small Animal Clinic, similar to previous kinematic studies in this laboratory [10, 16, 21, 24, 25]. An instrumented four-belt treadmill (TM-07-B, Bertec Corporation, Columbus, OH, USA) was used. Six high-speed infrared cameras (MX3+, Vicon Motion Systems Ltd., Oxford, Great Britain; recording frequency 100 Hz) around the treadmill detected the three-dimensional movement of the markers during locomotion. A digital high-speed video camera (pilot piA 640-210gc, Basler AG, Ahrensburg, Germany) recorded the locomotion of the dogs from a lateral position. All devices were managed and controlled with the programs Vicon Nexus (version 1.8.5, Vicon Motion Systems Ltd., Oxford, Great Britain) and Treadmill Control Panel (version 1.7.12, Bertec Corporation, Colum-bus, OH, USA).

Prior to the recordings a training phase was carried out until the dogs were running at a relaxed and loose trot. The young dogs, which were used to the treadmill due to a previous study, had a predetermined training phase of 5.0 ± 0.0 minutes; the old dogs needed 18.2 ± 14.5 minutes. With double-sided adhesive tape and hair clips 35 retroreflective passive markers (16 mm diameter) were attached, by the same person,

to all four limbs and the back of each dog. The joint determining markers were attached above defined and well palpable anatomical landmarks, additional markers were attached to certain places on the intermediate segments (Fig. 1, above; [21, 24]). For each dog the treadmill speed was adjusted until the respective dog was able to trot smoothly and regularly. The young group trotted at 1.8 ± 0.0 m/sec, the old group at 1.7 ± 0.1 m/sec. In each case around ten recordings of about 30 seconds in length were made, therefore the complete duration of the recordings was about ten minutes.

4.1.3.3 Data analysis

For each dog a sequence of ten consecutive and representative strides was chosen in which the respective dog ran straight and uniformly. The marker points, recorded by the infrared cameras, were processed by the program Vicon Nexus with a deposited kinematic model of the four limbs and the back. Each marker point had to be linked to an anatomical location of this model. Thus, a linked stick model was created (Figure 1, below), in which the respective joint angles were defined by the location coordinates of three joint determining markers. The time points when the feet touched the ground and lifted off were determined manually. Therefore, the measured vertical ground re-action force, which indicates the beginning and end of the stance phase and as a con-sequence also the duration of the swing phase, and the parallel recorded video were used.

The angles of the shoulder, elbow, carpal, hip, stifle and tarsal joints were projected two-dimensionally in the sagittal plane and exported to the program Excel 2010 (Mi-crosoft Corporation, Redmond, WA, USA). For better comparability between the dogs, the data output was time normalized to a stride duration of 100 % and the same stance and swing phase duration of 50 % [21, 24]. The analysis of each joint angle included the joint angle progression curve in [°], the minimum joint angle in [°] (MIN, maximum flexion), the maximum joint angle in [°] (MAX, maximum extension) and the range of motion of the joint in [°] (ROM, difference between MIN and MAX) during each stride [10, 16, 21]. For each joint angle the data were averaged over the ten analyzed strides.

To compensate for possible differences between the marker positions, a standardiza-tion was performed, whereby the mean of each progression curve was subtracted from every value of this joint [7, 8, 31]. As all dogs were measured in trot, a symmetrical gait [23], and no statistically significant differences were found between the joint angles of the left and right side, the sides were averaged (Table 1).

4.1.3.4 Statistical analysis

Due to the small group size of n = 5, a normal distribution of data was not assumed.

Therefore, the non-parametric Wilcoxon rank-sum test for two independent samples was used to verify differences of body weight and standardized joint angles between the groups (differences with p < 0.05 were considered significant, differences with p < 0.10 were considered to be a tendency). To compare the joint angles between the left and right side the non-parametric Wilcoxon signed-rank test for two paired samples was used (differences with p < 0.05 were considered significant). The sides were com-pared within a group and across both groups. The statistical analysis was performed with the program SAS Enterprise Guide (version 7.1, SAS Institute Inc., Cary, NC, USA). To validate the results, the statistical power was analyzed retrospectively. For this purpose, a standard deviation of 10 % was assumed, which is an average value for kinematic analysis and in this study [2, 28]. Furthermore, a biologically relevant difference between the groups of 15 % was assumed. Bockstahler et al. found in sound dogs with borderline hip dysplasia alterations of hindlimb ROM of about 10 % [8]; in slightly lame dogs with bilateral degenerative joint disease of the hips they found alterations of hindlimb ROM of about 30 % [6]. Therefore, 15 % seemed to be an appropriate value that may be indicative of a clinically apparent orthopedic disease.

The α-risk was 0.05 and the test was one-tailed. For this the program PASS (version 14, NCSS LLC, Kaysville, UT, USA) was used.