## 2 Materials and Methods  
### Study Design  
This retrospective, observational, controlled study was conducted on privately owned, warm-blood older horses with a history of long-term under-saddle use. The horses were selected from a population of 72 horses kept in homogeneous environmental conditions for over 2 years housed in the Pegasus Sanctuary for Older Horses. In this large population of mainly older horses, the goal of screening was to find horses with tarsal joint OA. Considering that taking X-rays in healthy horses is not justified, the horses were pre-examined during a routine screening examination for hind limb lameness. The horses underwent a basic clinical examination and a detailed orthopedic examination following the protocol outlined by [PERSON] et al. [57]. Lameness was scored according to the guidelines and scale of the American Association of Equine Practitioners (AAEP) [58], the description of which is summarized here in Table 1. On this basis, 20 out of 72 horses (28%) were considered further. The sub-population of the 20 pre-selected horses underwent two selection criteria: they were at least 15 years of age and had at least 10 years of riding experience. The data for inclusion were verified based on the horses' histories and individual identification documents for age determination. Then, a conventional radiographic examination of the tarsal joint was conducted using an X-ray tube (Orange 9020 HF, Ecoray Co., Seoul, Republic of Korea), a radiographic cassette (Saturn 8000, Vieworks Co., Seoul, Republic of Korea), and a portable computer (HP Inc. UK Ltd., Reading, UK). The X-ray tube settings were 1.25 mAs and 60 kV, with an 80 cm distance between the X-ray tube and the radiographic cassette. Each horse underwent two radiographs of the right and two radiographs of the left tarsal joint in the dorsoplantar and lateral medial views. During the radiographic examination, the density standard-based CDA method was employed, following the protocol described for equine teeth radiography [48] and applied to normal tarsal joint radiography [59]. The radiographic signs of bone spawin were scored using a scale based on the findings of [PERSON] [60], [PERSON] et al. [28], and [PERSON] [61], as summarized in Table 2. Bone spawin was confirmed when the visible radiographic signs of OA involved the tarscorural joints (TCJs), distal intertalsal joint (DIJ), proximal intertalsal joint (PIJ), and/or tarscometatarsal joint (TMJ). The structural changes identified using the radiographs in the affected joints included subchondral cyst formation (lucency in the subchondral bone making the joint space appear wider), a local periosteal reaction, collapse of the central and third tarsal bones (osteolytic lesions), ossification of the central and third tarsal bones (sclerosis lesions), bone spur (exostosis, osteophyte, enthesophyte) formation, narrowing of the joint space, and ankylosis (bone fusion between the tarsal bones, which is possible with bony callus bridging the joint) [28; 60; 61; 62]. Exemplary radiographs of scores 0-3 are presented in Figure 1. \begin{table}
\begin{tabular}{c l} \hline \hline
**Grade** & **Clinical Symptoms** \\ \hline
0 & Lameness that is not perceptible \\ \hline
1 & Lameness that is difficult to observe regardless of the circumstances \\ \hline
2 &
\begin{tabular}{l} Lameness that is difficult to observe at a walk; lameness that is difficult to observe \\ when trotting in a straight line; lameness that is observable when trotting in a circle, \\ on an incline, on a hard surface, or under weight-carrying conditions \\ \hline
3 & Lameness that is observable at a trot regardless of the circumstances \\ \hline
4 & Lameness that is observable at a walk \\ \hline
5 &
\begin{tabular}{l} Lameness that is strongly observable at a walk; minimal weight bearing in motion \\ and/or at rest or a complete inability to move \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The scale used for grading the clinical symptoms of lameness summarized based on the AAEP guidelines [58].