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Translational animal science2026; 10; txag131; doi: 10.1093/tas/txag131

Effect of sample storage on pH-value in equine faeces.

Abstract: 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 -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 (  0.34). All frozen samples had higher ( < 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 ( < 0.001) mean pH-values (6.52 ± 0.36 for both) and RE12 and RE24 samples had higher ( < 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.
© The Author(s) (2026). Published by Oxford University Press on behalf of the American Society of Animal Science.
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Publication Date: 2026-08-25 PubMed ID: 42707605PubMed Central: PMC13548788DOI: 10.1093/tas/txag131Google Scholar: Lookup
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  • Journal Article

Summary

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Overview

  • This study investigates how different storage times and temperatures affect the pH value of horse fecal samples compared to immediate measurement at the time of defecation.

Introduction

  • pH measurements in equine feces are commonly used in nutrition and health research in horses.
  • There is no standardized protocol for how samples should be handled before pH measurement.
  • Sample handling varies in published research and may influence pH values.
  • The study aims to understand how storage time and temperature impact fecal pH readings.

Methodology

  • Fecal samples were collected from 105 horses.
  • Each sample was divided into 14 subsamples.
  • Subsamples were stored under different conditions:
    • Room temperature (20-21°C): measured immediately (RT0) and after 2 hours (RT2).
    • Refrigerated (+4 to +6°C): measured after 2, 4, 8, 12, 24, and 48 hours (RE2-RE48).
    • Frozen (-20°C): measured after 24, 48, and 72 hours and after 1, 3, and 6 months (F24-F72, F1M-F6M).
  • pH was measured using a calibrated pH meter on liquid pressed from whole fecal material.
  • Measurements were repeated three times per subsample for accuracy.
  • Data analysis employed ANOVA mixed model with sample treatment as a fixed effect, horse as a random effect, and Dunnett’s adjustment for multiple comparisons to RT0 (immediate measurement).

Results

  • Baseline pH at RT0 (immediate measurement) was 6.60 ± 0.04.
  • Most storage conditions showed a statistically significant difference in pH compared to RT0 (< 0.01), except:
    • RT2 (2h at room temperature)
    • RE8 (8h refrigeration)
    • RE48 (48h refrigeration)
  • Frozen samples consistently had higher mean pH values (6.65 to 6.75) compared to RT0, all with significance (p < 0.01).
  • Shorter refrigerator storage (RE2 and RE4) showed lower mean pH (6.52 ± 0.36), significantly different from RT0.
  • Longer refrigeration times (RE12 and RE24) showed higher mean pH values (6.67 ± 0.34 and 6.67 ± 0.38), both significantly different from RT0.

Conclusions

  • Immediate pH measurement is optimal for accuracy.
  • If immediate measurement is not feasible:
    • Storing fecal samples at room temperature for up to 2 hours (RT2)
    • Storing in refrigerator for 8 to 48 hours (RE8 and RE48)

    are acceptable alternatives as pH values remain stable without significant change.

  • Freezing samples or other refrigeration timepoints can alter pH values significantly and should be interpreted with caution.
  • These findings provide guidance for standardizing fecal sample handling in equine pH studies to improve data reliability.

Cite This Article

APA
Lindroth KM, Müller CE. (2026). Effect of sample storage on pH-value in equine faeces. Transl Anim Sci, 10, txag131. https://doi.org/10.1093/tas/txag131

Publication

Translational animal science
ISSN: 2573-2102
NlmUniqueID: 101738705
Country: England
Language: English
Volume: 10
Pages: txag131
PII: txag131

Researcher Affiliations

Lindroth, Katrin Madelene
  • Department of Applied Animal Science and Welfare, Swedish University of Agricultural Sciences, Uppsala, 75007, Sweden.
Müller, Cecilia Elisabeth
  • Department of Applied Animal Science and Welfare, Swedish University of Agricultural Sciences, Uppsala, 75007, Sweden.

Conflict of Interest Statement

The authors declare no conflicts of interest.

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