Effect of sample storage on pH-value in equine faeces

Measurements of pH-value in equine faeces are often performed in studies on nutrition and health in horses, but a standard protocol for it is lacking despite the knowledge that different sample handling may affect the pH-value. Sample handling prior to pH-measurement is often determined by practical settings and has been reported to vary in published studies within the area. The purpose of the current study was therefore to investigate the influence of different combinations of storage time and temperature on the pH-value in equine faecal samples in comparison to immediate pH-measurement at defecation. Faecal samples from 105 horses were collected, and each sample was divided into 14 sub-samples. The sub-samples were stored in room temperature (+20 to + 21°C), in a refrigerator (+4 to + 6°C) or in a freezer (-20°C) for different periods of time, after which pH was measured. Measurement of pH was performed immediately at defecation (reference value, RT0), after 2 h in room temperature (RT2), after 2, 4, 8, 12, 24 and 48 h in a refrigerator (RE2-RE48;), after 24, 48 and 72 h in a freezer (F24-F72), and after 1, 3 and 6 months in the same freezer (F1M-F6M). Measurements of pH were done on undiluted liquid pressed from whole faecal samples using a two-point calibrated pH-meter and was repeated three times on each sub-sample. Data were analysed using an ANOVA mixed model procedure with sample treatment (temperature and time) as a fixed effect and horse as a random effect, and with Dunnetts adjustment of P-values as all sample treatments were compared to the reference value at RT0. Results showed that mean pH ± SD at RT0 was 6.60 ± 0.04 and that it differed (P < 0.01) from all sample treatments except RT2, RE8 and RE48 (P > 0.34). All frozen samples had higher (P < 0.01) mean pH-values compared to RT0, and ranged from 6.65 ± 0.38 at F48 to 6.75 ± 0.40 at F72. All other frozen samples had pH-values between 6.65 and 6.75. Refrigerated samples RE2 and RE4 had lower (P < 0.001) mean pH-values (6.52 ± 0.36 for both) and RE12 and RE24 samples had higher (P < 0.001) mean pH-values (6.67 ± 0.34 and 6.67 ± 0.38, respectively) compared to RT0. The results of this study therefore showed that if pH cannot be measured immediately at defecation, storage of equine faecal samples in room temperature for 2 h or in a refrigerator at + 4 to + 6°C for 8 h or 48 h is another option, as pH at these timepoints did not differ from RT0.