Effects of bedding materials on equine airway inflammation

Equine asthma (EA) is a chronic lower airway disease that affects 10–20% of adult horses in the Northern Hemisphere, making it a major welfare issue. Inhaled particles, such as moulds and pollens, play a crucial role in its pathogenesis. As many horses spend a considerable portion of their lives indoors, the factors that affect stable air quality come into play. Although the effects of hay quality on EA have been long acknowledged, the effects of bedding materials have been studied less. Peat, which has traditionally been a popular bedding material for horses in Nordic and Baltic countries, has been investigated minimally from this perspective.  

The main diagnostic tool for EA in practice is bronchoalveolar lavage fluid (BALF) cytology. The technique has some weaknesses, and the cut-off values for inflammatory cell proportions have been debated. More sensitive diagnostic methods are thus desired. The field of lipidomics is still rather an unexplored territory in equine medicine. Studying the lipidome offers an in-depth view into inflammation, as many proinflammatory and proresolving inflammatory mediators are lipids.

The three studies in this thesis are based on one clinical experiment. The objective of study I was to investigate whether bedding material (peat vs. wood shavings/WS) influences clinical respiratory variables, tracheal mucus score and lower respiratory tract cytology of healthy horses. The objectives for studies II and III were formulated based on the results from study I. The objective of study II was to compare different bedding materials to the peat bedding that was used in study I. The objectives of study III were to explore the BALF cells’ lipidome in horses for the first time, and to identify lipids associated with progression or resolution of airway inflammation.

The experiment was a prospective controlled study in which 32 healthy adult horses were exposed to different bedding materials over a period of one in-housing season. Each bedding material was used for 35 consecutive days in the following order: baled peat (Peat 1), WS, baled peat (Peat 2), wood pellet (WP), a mixture of baled and loosely stored peat (Peat 3), straw pellet, loosely stored peat (Peat 4). During the experiment, horses were stabled in a single stable and fed haylage with some concentrate feed. Horses spent part of the day on an outdoor paddock and were used as riding school teaching horses. During the experiment horses acted as their own controls. Clinical signs of respiratory disease were monitored daily. The cytology of lower airway samples (tracheal wash [TW], BALF) was examined after each bedding period and the relative proportions of inflammatory cells were

compared. Lipid profiling of the BALF cells were then conducted comparing BALF cells that were collected after Peat 1, WS, and Peat 2.  

Study I compared WS to peat. The hypothesis was that peat induces less airway inflammation than WS. No difference between bedding periods was observed in the respiratory rate (RR) or tracheal mucus score. TW neutrophil percentage (neutr%) with WS was higher than Peat 1 (P = 0.040) and Peat 2 period (P = 0.0045. BALF neutr% with WS was higher than that of Peat 2 period (P < 0.001). Between the two bedding materials used in this study, peat was associated with lower BALF and TW neutr%.  

In study II, results from study I were further explored. WP, straw pellet, and loosely stored peat (Peat 3) were compared to Peat 2 from study I. The hypothesis was that peat induces less airway inflammation than straw pellets or WP. The tracheal mucus scores (P = 0.014) and RR (P = 0.026) were higher during the straw pellet period than during Peat 2 period. RR was lower during WP than during Peat 2 period (P = 0.004). TW neutr% during straw pellet period was higher than during Peat 2 period (P = 0.0003). BALF neutr% was higher during the straw pellet period (P = 0.005) and during Peat 3 period than during Peat 2 period (P = 0.04). In conclusion of this study, baled peat (Peat 2) resulted in lower neutr% in the airway samples than with straw pellet and loosely stored peat (Peat 3). No difference was observed between Peat 2 and WP. The information gained from studies I and II may assist veterinarians and horse owners in selecting bedding materials, especially for horses suffering from EA. 

In study III, lipidomic analysis of the BALF cells was performed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. Lipid species profiles of equine BALF cells indicated changes in airway inflammation between the bedding periods. Differences between the bedding periods were found in the molar percentage (mol%) of 60 individual lipid species. Phosphatidylcholine (PC) mol% was higher after Peat 2 period than after WS period (P < 0.001). PC 32:0 (main molecular species 16:0_16:0) was higher after Peat 2 period than after WS period (P < 0.001). Phosphatidylinositol (PI) 38:4 (main molecular species 18:0_20:4) was higher after WS period than after both peat periods (P = 0.004). BALF cell count (cells/µ l) correlated positively with PI 38:4. The results of this study indicate that numerous specific lipids may contribute to the process of airway inflammation in horses. Mass spectrometry-based lipidomics was used for the first time on equine BALF cells. Based on these results, this technique may be a sensitive tool to detect and monitor resolution of inflammation in equine airways. On top of these three studies, a pilot study exploring a possible link between obesity/insulin dysregulation and airway inflammation was conducted. The objective of this fourth study was to investigate the association between airway neutrophilia and obesity and/or insulin dysregulation (ID) in healthy adult horses. No associations between these phenomena were found in this study population (unpublished material).

Altogether, these studies provide new information about the effects of common bedding materials on equine airway inflammation. Studies I and II indicate that peat is a suitable bedding material when horse airway health is considered. Study III describes for the first time the lipidome of equine BALF cells.