Assessment of Welfare in Groups of Horses with Different Management, Environments and Activities by Measuring Cortisol in Horsehair, Using Liquid Chromatography Coupled to Hybrid Orbitrap High-Resolution Mass Spectrometry

SIMPLE SUMMARY: Identifying distressing practices in horse management can be challenging. There is little research available comparing the stress profile of horses with different management and activities. Currently, there is no full awareness of whether the stabling of the horse is more or less stressful than the free-ranging state. This study evaluated stress in horses with different management and activities by assessing the cortisol levels in the horsehair. All selected horses were from the same area (Central Italy) and in the same conditions according to the European Animal Welfare Indicators (AWIN) protocol. The horsehair was analyzed using liquid chromatography coupled to high-resolution mass spectrometry, which has not been previously used for this purpose. The free-range group of animals displayed much higher levels of cortisol than stabled horses, even higher than in the ones carrying out order service under the Italian State Police; these results demonstrated that horses leaving in the wild can be more subject to stress. Although further studies on this matter are desirable, this research adds an important piece to the stress associated with the management of horses. ABSTRACT: Horses have always been animals used for companionship, work, transportation, and performance purposes over the history of humanity; there are different ways of managing horses, but studies on how horse welfare is influenced by different activities and managements are scanty. Understanding how the management, the environment, and the different uses of horses can affect the level of stress and well-being is important not only for people associated with horses. Three groups of horses with different management, environments, and activities were selected: (1) stabled horses ridden frequently, (2) horses that perform public order service under the Italian state police, and (3) free-ranging horses. Cortisol analysis was carried out on horsehair samples using liquid chromatography coupled to hybrid orbitrap high-resolution mass spectrometry (LC-HRMS/MS), a laboratory technique used for the first time to quantify horsehair cortisol. The selection of horses to be included in the three groups was carried out by including only subjects with positive welfare assessment in accordance with the horse welfare assessment protocol (AWIN). These analyses demonstrated that the cortisol levels detected in the horsehair of free-ranging animals were significantly higher compared to those detected in stabled and working horses. These results may have been a consequence of complex environmental, managerial, and behavioral factors, which should be worth further investigation