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

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.

Determination of hair cortisol in horses: comparison of immunoassay vs LC-HRMS/MS

“…HCC was also related to somatic cell count (SSC), body condition score (BCS), and milk yield and results evidenced a beneficial impact of grazing on the health of animals although associated to a worsening BCS, increased SCC, a reduction of milk yield during the grazing, and a slight rise of HCC during the transition followed by its stabilization. The HCC comparison between different management has been described in our previous paper [4]; briefly, lower level of cortisol was detected in horses that carried out public order activity and had access to the paddock, followed by animals belonging to other two groups. The free-ranging horses provided higher HCC, probably because they lived in more stressful conditions (e.g., fear of being predated, long distance covered in the wild).…”

“…In a few, other cases, the HCCs were only reported in the graphs or figures [13,16]. It is noteworthy that the detection/quantitation of horsehair cortisol has been carried out almost exclusively with immunoassay techniques (i.e., EIA and RIA) except for the previous published paper [4].…”

“…The presented method was based on our previous paper [4]. Particularly, chromatography was performed on a Thermo Ultimate 3000 High-Performance Liquid Chromatography system (San Jose, CA, USA) using an Acquity UPLC BEH C18 column (50 × 2.1 mm; 1.7 μm, 130 Å) (Waters, Milford, MA, USA), connected to an Acquity guard column (5 × 2.1 mm).…”

“…Hair samples were selected and collected as described by Cerasoli et al ( 2022) [4]. Briefly, forty-seven horses belonged to three groups: group 1 (n = 16) carried out training activities and flatwork for the Italian State Police of Lazio region 3-4 times a week (the animals stand alone in the boxes with daily access to the paddock), while group 2 (n = 16) exercised daily public order activities for the…”

“…The comparison between the two cited techniques was only investigated by Russell and collaborators in human hair [3]. To the best of our knowledge, only our previous study has analyzed hair cortisol in horses with LC-HRMS/MS [4], and no study has so far compared it with immunoassays. The aims of this work were to compare validation method performances and the equine HCCs of real samples, obtained by means of LC-HRMS/MS and an immunoassay (i.e., ELISA) and validation data of the two approaches.…”