## 4 Discussion
### Limitations  
The main limitation of this study is the lack of investigation into the relationship between hind limb lameness and the radiological signs of tarsal joint OA. It is important to note that intra-articular analgesia is utilized as a clinical tool to localize the source of lameness and remains the gold standard for diagnosing tarsal joint OA as a cause of lameness [53]. However, in this study, a lameness assessment was only a screening test performed as part of the Sanctuary for Older Horses' good practice in the routine veterinary examination of maintained horses. A lameness diagnosis using local anesthesia was not continued in these horses due to their free-range housing and the risk of injury after diagnostic analgesia. While radiography of the tarsus is routinely used to demonstrate and monitor changes in clinical cases of OA [53], its use in the current study is justified. Nevertheless, there was no evidence that lameness was related to the radiological signs of OA. The further limitation of this study primarily results from the small sample size. In previous studies on the equine tarsal joint, three radiographic rating systems were conducted on 54 horses [68], 15 horses [69], and 17 horses [70]. Even though the first studies were more than twice as large and the next two studies used fewer animals than ours, we would like to emphasize that the results of our research should be treated as preliminary. These findings are not intended to be generalized but rather to introduce progress in the quantification methodology. Hence, one of the next steps is to conduct this preliminary protocol on the larger, more diverse equine population covering different ages, housing systems, and geographic locations. Moreover, the current preliminary methodology requires expansion with subsequent steps of manual or computer-aided segmentation of the individual tarsal joint structures. In this preliminary study, the entire tarsal joint was designated as the ROI for pixel counting. Thus, manual image segmentation was not applied. However, the lack of manual segmentation in the proposed protocol limits its ability to identify the specific level of the tarsal joint and which bones are affected by radiological signs. The radiological signs of OA are primarily visible in the DJI, less frequently in the PIJ, and occasionally in the TCJ or TMJ [28; 29; 33; 36; 37; 38; 36; 38; 65]. Moreover, the subchondral bone (SCB) thickness pattern showed alterations in painful tarsi relative to the horses' sport and exercise history, with SCB pattern changes occurring at different locations throughout the tarsal joints as the disease progresses [41]. Thus, addressing bone remodeling in specific tarsal regions is strongly indicated. After demonstrating the feasibility of BMD quantification in the current study, further development of radiographic image processing techniques to quantify OA severity by segmenting individual joint structures is required to avoid oversimplification. Finally, one may postulate that the development of BMD and/or SCB patterns in bone spavin may differ depending on the primary cause of the disease. As bone spavin is a multifactorial disease [28; 29; 30; 32; 33; 36; 37; 38; 41; 53; 65; 70], it is important to investigate whether any primary cause of tarsal joint OA is related to specific bone patterns. Tarsal joint OA can originate from low-grade chronic inflammation, synovitis, subchondral bone remodeling, and/or articular cartilage degeneration [33]; however, the changes may be caused by trauma, developmental pathologies, infections, overload in the tarsus due to the incomplete ossification of the tarsal bones, overload in the tarsus due to pathologies in other limb regions, the instability of the tarsus caused by intra-articular or extra-articular ligament damage, and instability in other limb regions. Therefore, further research is needed on a much larger group of horses, with precisely diagnosed cases of bone spavin and long-term follow-up to identify the specific locations and patterns related to the disease.