The gastrointestinal microbiome is a diverse colony of microorganisms that has a synergistic and specifically adapted relationship to the digestive function of the host and may be altered by changes in the diet or environment, administration of medications (including antimicrobials), stress, or disease.1,2 Large-scale studies3–5 involving humans have been conducted to investigate the relationship between the microbiome and diseases such as obesity and inflammatory bowel disease. This area of research has since expanded to include veterinary species including equids. Results of a pilot study6 indicate that the fecal microbiome of ponies maintained with similar diets and housing conditions is fairly stable, but the fecal microbiome of individual ponies tends to have unique characteristics. In a study7 of Thoroughbred racehorses, a dietary change had a significant effect on the fecal microbial community structure and intestinal metabolites. Alterations in the fecal microbiome are associated with postpartum colic,8 chronic laminitis,9 and colitis,10 conditions that have high morbidity and mortality rates in horses.
Equine fecal samples used for DNA extraction and analysis in previous studies7–10 were obtained from the ground or stall floor, and sample collection following defecation ranged from immediately to within 24 hours after hospital admission. Those inconsistencies indicate lack of a standardized protocol for collection and handling of fecal samples for microbiome analysis. Although research on the equine microbiome is ever increasing, to our knowledge, the effect of sample collection protocol, including the portion (surface vs center) of the fecal ball from which the sample is obtained and the duration between defecation and sample collection, on the composition of the fecal microbiome has not been investigated. Protocols of fecal microbiome studies11,12 involving dogs and humans also vary in terms of sample collection, handling, and processing, but the various techniques used in those studies have been more comprehensively studied and validated than those used in studies involving horses.13 In regard to horses, because the fecal surface is generally exposed to air and the stall or pasture environment, it seems reasonable to postulate that environmental exposure may produce temporal differences in the absolute number or relative proportions of certain aerobic and anaerobic bacteria between the surface and center of fecal balls, which may occur within minutes or hours after defecation. Moreover, inconsistencies in fecal sample collection, processing, and handling, such as freezing and thawing, may contribute to variation in the microbial composition of the microbiome among horses.6
The purpose of the study reported here was to characterize the fecal microbiota of healthy horses with access to pasture and fed a diet of free-choice timothy hay and to investigate alterations in that microbiota on the basis of sample collection site (rectum vs stall floor), sample location in the fecal ball (surface vs center), and duration of environmental exposure (collection time). Our hypotheses were that the microbiota composition would differ significantly between fecal samples collected per rectum and those collected from the stall floor and between samples extracted from the surface and those extracted from the center of a fecal ball and that the fecal microbiota would change rapidly over time owing to environmental exposure.
All research was performed at New Bolton Center, University of Pennsylvania, Kennett Square, Pa.
Supported by the Raymond Firestone Trust Research Grant. The authors declare that there were no conflicts of interest.
Operational taxonomic unit
Ag-Tek, Neogen, Lansing, Mich.
Amsino International Inc, Pomona, Calif.
PSP Spin Stool DNA Plus Kit, Invitek, Berlin, Germany.
Accuprime Taq DNA Polymerase System, Invitrogen, Carlsbad, Calif.
Agencourt AMPure XP Beads, Beckman-Coulter, Fort Collins, Colo.
Ion Torrent, Thermo Fisher Scientific, Waltham, Mass.
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