The animal experiment for present microbiome study was described by Tench et al. (29 ). The protocol for the use of experimental animals was approved by the Institutional Animal Care and Use Committee at the University of Florida in Gainesville, FL (#201810324) under the Guide for the Care and Use of Agricultural Animals in Research and Teaching (30 ).
Briefly, 20 young and clinically healthy horses in training (mean ± SEM; initial age 22 ± 0.3 mo and BW 439 ± 3 kg) were paired by age and sex and randomly assigned to one of the two experimental treatments for 60 days. Treatments included supplementation with 0 g/d (Control; no treatment Control) or 21 g/d Diamond V TruEquine C (SCFP; Diamond V, Cedar Rapids, IA). A basal diet of 60% Coastal bermudagrass hay and 40% concentrate formulated to meet the nutrient requirements of horses at a moderate rate of growth (31 (link)) was offered to all horses. Treatment administration was done by top dressing SCFP on the concentrate ration. Horses were exercised 4 days per week for 30–45 min/d at light to moderate intensity. On day 57, horses were placed in individual stalls and tethered with their heads elevated 35 cm above wither height for 12 h to induce mild upper respiratory tract inflammation according to a previously established protocol to mimic long-distance transport stress (32 (link), 33 (link)). Induction of inflammation was confirmed by significantly elevated serum cortisol and blood leukocyte measurements performed after stress induction compared to pre-stress (34 (link), 35 (link)). The stress period was relieved after the 12 h timepoint by untethering of the horse heads. Fecal samples were collected into sterile containers at seven time points: days 0, 28, and 56 before induction of stress, and at 0, 12, 24, and 72 h post-stress, where 0 h is the time at which the horses were untethered. Samples were immediately placed on ice and transported to the laboratory where they were kept in a −80°C freezer until DNA extraction. A schematic of the experimental design and sample collection is given inFigure 1 .
Briefly, 20 young and clinically healthy horses in training (mean ± SEM; initial age 22 ± 0.3 mo and BW 439 ± 3 kg) were paired by age and sex and randomly assigned to one of the two experimental treatments for 60 days. Treatments included supplementation with 0 g/d (Control; no treatment Control) or 21 g/d Diamond V TruEquine C (SCFP; Diamond V, Cedar Rapids, IA). A basal diet of 60% Coastal bermudagrass hay and 40% concentrate formulated to meet the nutrient requirements of horses at a moderate rate of growth (31 (link)) was offered to all horses. Treatment administration was done by top dressing SCFP on the concentrate ration. Horses were exercised 4 days per week for 30–45 min/d at light to moderate intensity. On day 57, horses were placed in individual stalls and tethered with their heads elevated 35 cm above wither height for 12 h to induce mild upper respiratory tract inflammation according to a previously established protocol to mimic long-distance transport stress (32 (link), 33 (link)). Induction of inflammation was confirmed by significantly elevated serum cortisol and blood leukocyte measurements performed after stress induction compared to pre-stress (34 (link), 35 (link)). The stress period was relieved after the 12 h timepoint by untethering of the horse heads. Fecal samples were collected into sterile containers at seven time points: days 0, 28, and 56 before induction of stress, and at 0, 12, 24, and 72 h post-stress, where 0 h is the time at which the horses were untethered. Samples were immediately placed on ice and transported to the laboratory where they were kept in a −80°C freezer until DNA extraction. A schematic of the experimental design and sample collection is given in
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