Ovine model
This investigation was approved by the Institutional Animal Care and Use Committee of the University of Trás-os-Montes e Alto Douro (IACUC Approval No. 6/2015). All procedures were performed with the approval of the Portuguese Veterinary Authorities, in accordance with the EU Directive 2010/63/EU for animal experiments. The experimental sheep were female Portuguese Churra-da-Terra Quente breed (age: ≈ 2 years; weight: ≈ 40 kg) belonging to a breeding colony based at University of Trás-os-Montes e Alto Douro. Upon delivery, all animals were examined by a veterinarian and deemed non-pregnant and free of disease. The sheep were judged to be healthy on account of results of complete physical, orthopaedic and neurologic examinations. The animals were fed a diet of hay supplemented with concentrate according to their requirements and had free access to fresh water. The present experimental design did not induce any pain or discomfort to the included ovine, and by the end of the studied timepoint, all the animals returned to their normal activities without any disturbance nor pathology.
Training Procedure
The general procedure for training was similar to that in previous work [14]. Two weeks before the collection of kinematic data, sheep were trained daily for 30 minutes to walk over ground at a natural speed on a 2 m by 0.6 m walkway and to step over a rectangular obstacle (6 cm high × 6 cm wide).
Kinematic Recording
Hemispherical markers with a diameter of 2 cm were placed on the skin over six anatomic landmarks on the lateral side of the left hindlimb: the iliac crest, the greater trochanter, the knee joint, the lateral malleolus, the distal end of the metatarsal bone and distal end of the middle phalanx, as in the previous study [14].
Three CMOS cameras (PhotonFocus MV-D640C, Lachen, Switzerland) were strategically placed around the left hindlimb to minimize marker occlusion, maximize resolution and to improve the accuracy of the 3D reconstruction process. The camera in the middle, which was placed perpendicular to the direction of the movement, was used for 2D analysis. Kinematic data were collected at a sampling rate of 144 Hz. The cameras’ field of view was calibrated to cover 2 meters in length of the walkway and allowed the recording of a complete gait cycle (Fig. 1). The images were acquired using the software Video Savant 4 (IO Industries Inc, Ontario, Canada). The colour image had a resolution of 640 × 480 pixels. The camera calibration and the 3D reconstruction process were similar to a previously described procedure [21].
During each trial, sheep walked at a natural speed over the walkway, led by a handler using a halter. The present work limited walking velocity to between 1.1–1.5 m/s [14]. A total of ten trials for each sheep were analysed.
Joint flexion-extension angles were measured at the flexor side for hip, knee, ankle and metatarsophalangeal (MTP) joints. The 2D approach was described in detail previously [14, 22]. Briefly, the knee position was computed indirectly by superimposing two circles (centred on hip and ankle pivots) with a radius of the femur and tibia length, respectively. The knee position was determined as the intersection of the two circles. For the 3D biomechanical model, we applied a slightly different approach, as previously described (Fig. 2) [24]. The two circles were replaced by two spheres centred on the greater trochanter and the lateral malleolus with a radius equal to the length of the femur and tibia and segments, respectively. We presumed that the true position of the knee marker would lie on the plane defined by the greater trochanter, the knee and the lateral malleolus markers.
Numerical Analysis
The general procedure for numerical analysis was similar to our recent paper [14].
The marker-based angular kinematic curves were filtered using a fourth-order Butterworth filter (cut-off frequency at 10 Hz). For each stride, the duration of the stance and swing phases was normalized. Cubic spline interpolation was applied to the original data regarding the angular position of the pelvis, hip, knee, ankle, and MTP joints to obtain 101 samples per gait cycle regardless of their duration. We recorded the maximal and minimal joint flexion-extension angles during both the stance and swing phases of the gait, as well as the angles at the point of initial hoof-ground contact (IC, start of the stance phase) and immediately when the hoof is lifted from the ground (TO, start of the swing phase). This numerical analyse was performed using Matlab computational software (The MathWorks Inc., Natick, MA, USA).
In addition, the following gait parameters were included: gait cycle duration, stance duration, swing duration, stride length and the maximal vertical displacement (MVD). Stride length was defined by the distance between the middle phalanx markers of the hindlimb in two consecutive steps. Maximal vertical displacement was quantified by measuring the maximum height reached by the middle phalanx marker when stepping over the obstacle (Fig. 3).
Statistical analysis
Differences in joint kinematics data collected in 2D and 3D were tested using paired Student’s t-test. Mean standard deviation (SD) values for all the measured variables are reported. The two-way mixed model intra-class correlation coefficient (ICC) for absolute agreement was calculated as a preliminary measurement of intra-trial, inter-step reliability of joint kinematics data. The statistical significance was set at the level of P < 0.05. All statistical tests were performed using SPSS v. 22 software (Statistical Package for the Social Sciences Inc., Chicago, USA).