Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota

Abstract

Sample storage conditions are an important factor in fecal microbiota analyses in general. The objective of this study was to investigate the effect of sample storage at room temperature on the equine fecal microbiota composition. Fecal samples were collected from 11 healthy horses. Each sample was divided into 7 sealed aliquots. One aliquot was immediately frozen at −80 °C; the remaining aliquots were stored at room temperature (21 to 22 °C) with one transferred to the freezer at each of the following time points: 6, 12, 24, 48, 72 and 96 h. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. Fibrobacteraceae (Fibrobacter) and Ruminococcaceae (Ruminococcus) were enriched in samples from 0 h and 6 h, whereas taxa from the families Bacillaceae, Planococcaceae, Enterobacteriaceae and Moraxellaceae were enriched in samples stored at room temperature for 24 h or greater. Samples frozen within the first 12 h after collection shared similar community membership. The community structure was similar for samples collected at 0 h and 6 h, but it was significantly different between samples frozen at 0 h and 12 h or greater. In conclusion, storage of equine fecal samples at ambient temperature for up to 6 h before freezing following sample collection had minimal effect on the microbial composition. Longer-term storage at ambient temperature resulted in alterations in alpha-diversity, community membership and structure and the enrichment of different taxa when compared to fecal samples immediately frozen at −80 °C.

Impact of Storage Conditions on Equine Fecal Inoculum for Estimating In Vitro Digestibility

“…In contrast, a recent publication by de Bustamante et al found that aliquots of 2–3 g of feces stored in closed 118 mL containers for up to 6 h at room temperature prior to freezing did not affect the composition of the microbial population in equine feces, as compared to samples frozen immediately after collection. The same study also found that microbial populations were significantly changed by storage at ambient temperatures for periods longer than 6 h prior to freezing [ 12 ]. Samples from this de Bustamante et al study were not used to form inoculum to digest forages.…”

Evaluation of Bacterial Composition and Viability of Equine Feces after Processing for Transplantation

“…However, this means that the T 0 samples were kept for a longer period (45–60 min), refrigerated at 4 °C, in regard to the T 3 samples, which could have altered the bacterial viability. Bustamante et al, 2021 revealed that storage of equine fecal samples at room temperature for up to 6 h before freezing had a minimal effect on the fecal microbiota [ 41 ]. Furthermore, Burz et al, 2019 showed that refrigeration at 4 °C can be a safe solution to avoid changes in bacterial populations during fecal sample storage for 24 h, which supports the result that the differences in the storage times in our study seem negligible [ 9 ].…”

Identification of a Sub-Clinical Salmonella spp. Infection in a Dairy Cow Using a Commercially Available Stool Storage Kit

“…For the purposes of this study, samples were transported back to the laboratory where the stool could be weighed and aliquoted inside a Class II biological cabinet. As the total processing time was under 6 h, it was assumed that any changes to the microbiota would have been insignificant [10,11]. However, future studies where samples are placed directly into commercial stool storage kits on the farm would be useful for replicating what would be done in a clinical setting.…”

“…However, it must be considered that, in a farm setting, there may be limited access to cold storage. Therefore, it is not unusual for samples to remain at ambient temperature for prolonged periods before they reach the laboratory for analysis, resulting in the growth of minor species in the samples and a distortion of the true composition of the microbiota [7,[10][11][12][13][14]. Better methods of preserving bovine faecal microbiota are, therefore, required, particularly one that does not involve freezing the sample and is not reliant on immediate refrigeration.…”

“…Better methods of preserving bovine faecal microbiota are, therefore, required, particularly one that does not involve freezing the sample and is not reliant on immediate refrigeration. Limited research on the preservation of the faecal microbiota in cow stool samples exists, and, due to the clear variation between results in other species, findings from these studies cannot be extrapolated to the ruminant gut [10][11][12][13][14][15].…”