107 Equine fecal pH measurement methods and storage protocols

Abstract

The pH of the equine gastrointestinal lumen influences digestion, the microbiome, and enterocyte physiology, each having important impacts on horses. Fecal pH is a simple and noninvasive variable that reflects changes in hindgut pH. The objectives were to evaluate whether there is agreement between fecal pH measured by a meter and pH strips and to determine the effect of storage temperature and time on pH. Fecal samples were collected from 5 broodmares and 8 yearlings on one farm and 12 school horses on another. Feces were collected within 1 min of defecation from an uncontaminated region. A portable pH meter (Hanna Instruments model #9812) and 3 pH strips were used to measure the pH of 4 sub-samples: fresh, 24 h refrigerated at 4°C, 6- and 16-weeks frozen at −20°C. Samples were thawed in a water bath to 20–23°C before measurement. Fecal liquid without the addition of distilled water was used for meter readings. The ranges of the 3 pH strips were 4.5–10 (S1), 5.1–7.2 (S2), and 6.0–7.7 (S3). A 1:2 ratio of feces to distilled water was used for pH strip measurements to ensure readability related to color staining. All pH strips had a strong positive correlation (>0.8) with pH meter. Linear regressions between pH measured by strips and meter resulted in r2 between 0.76 (S1) and 0.86 (S2). Bland-Altman plots quantified agreement between pH meter and S1 (bias 0.005, 95% CI −0.448 to 0.458), S2 (bias −0.033, 95% CI −0.408 to 0.342), and S3(bias −0.044, 95% CI −0.426 to 0.338). Therefore, S2 best reflects the meter pH, with the highest r2 and the narrowest Bland-Altman CI. The influence of storage temperature and time were analyzed with a mixed ANOVA. Significance was set at P < 0.05 and data are presented as means ± sem. The pH of fresh and 6-weeks frozen samples was higher than 24 h refrigerated samples (6.49 ± 0.31, 6.47 ± 0.31, 6.39 ± 0.31, P < 0.001, respectively). No difference was detected comparing the pH of 16-week frozen (6.43 ± 0.31) to other sub-samples. There was a trend for it to be lower than fresh (P = 0.082). This may indicate relevant change due to long-term frozen storage. A difference in the fresh fecal pH (P < 0.001) was detected using all techniques between the 2 farms (pH meter results: 6.78 ± 0.11, 6.20 ± 0.09; respectively). The reason for the difference is beyond the scope of this study. The ability to detect pH differences could be valuable to those interested in management strategies influencing gastrointestinal health. The results of this study indicate that pH strips differ in their ability to reflect pH meter readings, storage temperature and time of storage may influence pH, and pH strips may be useful in detecting relevant differences in fecal pH.