###### Abstract  
This study aimed to validate eye temperature (ET) assessed using infrared thermography (IRT) as an indicator of welfare in horses. Moreover, this study aimed to determine the most accurate position for ET measured using IRT and to validate this approach as a gold-standard measurement method. As the quantitative data obtained by IRT have strongly influenced the ET results depending on the specific location of the measurement area, an accurate definition of the regions of interest (ROI) was established. A total of 176 horses (Thoroughbred, Warmblood, and Halla horses) were used at the racing course of the Korea Racing Authority and public horse-riding clubs in South Korea. The present study also compared temperatures among three ROIs of the eye--lacrimal sac, medial cannhus, and lateral cannhus--at rest. Correlations between ET, rectal temperature (RT), heart rate (HR), and respiratory rate (RR) were assessed. There were no significant correlations between HR, RR or RT; however, among the three ROIs, the temperature of the medial cannhus was positively correlated with RT (\(p<0.05\)). Furthermore, the size of ROI was negatively correlated with accuracy of temperature measurement. These results indicate that the most suitable area for ET measurement using IRT in horses is the medial cannhus, and it is recommended to use the average temperature of the smallest ROIs (\(2\times 2\) pixels) for temperature analysis. Therefore, this study offers a validated protocol in which ET measured using IRT in the horses is useful as an indicator of welfare. eye temperature; horse; infrared thermography; medial cannhus; rectal temperature +
Footnote †: journal: Medical  
management. Hence, the use of minimally invasive methodologies for stress assessment is advisable to assess animal welfare in this species. One of the ways to minimize stress in horses is IRT imaging. Infrared thermography (IRT) can detect infrared radiation emitted from an object, which is a measure of its surface temperature (ST), and can present it as a heat map [10]. IRT has been widely used as an auxiliary diagnostic tool for diagnosing musculoskeletal disorders in horses [11; 12; 13]. Moreover, IRT has emerged as a noninvasive and reliable method for assessing physiological stress in working [14; 15], companion [10; 16], and production animals [17; 18]. However, the quantitative data obtained from infrared thermography (IRT) show that even the slightest incorrect use can lead to substantial inaccuracies in the measurement of body temperature [19]. [PERSON] et al. [19] demonstrated that working distance, body-device angle, and light conditions can strongly impact temperature measurements, which could invalidate the screening results. In addition, [PERSON] et al. [20] reported that the collection of normal reference images is required, but it depends on the improvement of the reliability and cross calibration of camera systems. It is difficult to obtain reliable measurement values, especially in horse breeding environments, but studies that attempt quantitative measurements are increasing [21; 22; 23; 24]. Therefore, this study aimed to determine the most accurate position for eye temperature (ET) measured using IRT and to validate this approach as a gold-standard measurement method. Recent studies have used IRT to measure eye temperature (ET), which may reflect changes in body temperature in response to events and stimuli [10; 14; 25; 26]. Several IRT studies have shown that ET is significantly correlated with core body temperature compared with the ST of other anatomical regions [10; 14; 25; 26]. This association is attributed to the proximity of the orbit to the brain, as well as the rich blood supply it receives [27; 28]. A decrease and increase in ET may reflect a stress response dominated by the sympathetic nervous system [29] and the parasympathetic nervous system, respectively [30; 31]. An increase in ET may reflect general stress in a race meet, caused by transportation, race anticipation, and the race itself.