Stabilility of bacterial communities in horse dung samples tested

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How long can samples of horse manure be kept at room temperature before the bacterial composition starts to change significantly?

It is an important factor in field studies, where access to freezing or refrigeration may be limited or non-existent, resulting in samples remaining at room temperature until being taken to the laboratory.

Michelle Martin de Bustamante and her fellow researchers devised an experiment to determine how storage techniques may affect the collective community composition of bacteria present in horse feces (the fecal bacterial microbiota).

Eleven healthy adult horses from the University of Florida’s College of Veterinary Medicine equine research program were enrolled in the study.

Fecal samples were collected from their rectums.

Each sample was divided into seven sealed samples. One was immediately frozen at −80 degrees Celsius.

The remaining samples were stored at room temperature (21 to 22 °C), with one transferred to the freezer after 6, 12, 24, 48, 72 and 96 hours.

The study team used molecular-based sequencing technology to identify the bacteria present.

Fibrobacteraceae and Ruminococcaceae were found to be enriched in the sample that was frozen immediately, and the sample frozen after six hours, whereas taxa from the families Bacillaceae, Planococcaceae, Enterobacteriaceae and Moraxellaceae were enriched in samples stored at room temperature for 24 hours or longer.

The community structure was similar for the samples frozen straight away and after 6 hours.

Samples frozen within the first 12 hours after collection also shared similar community membership, the study team reported in the journal Animals.

However, there were significant differences between the sample frozen fresh and those left at room temperature for 12 hours or more.

“In conclusion, storage of equine fecal samples at ambient temperature for up to 6 hours before freezing following sample collection had minimal effect on the microbial composition,” the study team reported.

However, 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.

“When ultra-low temperature storage conditions are unavailable for immediate freezing, equine fecal samples should be frozen within 6 hours after collection to minimize storage-induced alterations in bacterial composition.”

In order to better establish appropriate storage protocols (more than 6 hours after sample collection) for equine fecal microbiota field studies, further investigations into commercial DNA preservation solutions or short-term refrigeration are warranted, they said.

The authors noted that the results were in contrast to studies investigating the effect of room temperature fecal sample storage on the human and feline fecal microbiota.

No significant differences were noted in alpha and beta-diversity indices or the relative abundance of different taxa over time between feline fecal samples stored at room temperature for up to 96 hours before freezing and sample processing.

In humans, some studies showed that fecal sample storage at ambient temperature for up to 24 hours did not lead to significant changes in fecal microbial composition when compared to direct freezing. In another human study, room temperature storage for up to 14 days had minimal effect on community structure and the relative abundance of different taxa in fecal samples.

“While the underlying cause of these inter-species inconsistencies in the fecal microbial community stability over time is unknown, it can be in part attributed to dietary differences altering the fecal bacterial and metabolic composition,” they said.

The full study team comprised Michelle Martin de Bustamante and Caryn Plummer, with the University of Florida; and Jennifer MacNicol and Diego Gomez, with the University of Guelph in Canada.

Martin de Bustamante, M.; Plummer, C.; MacNicol, J.; Gomez, D. Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota. Animals 2021, 11, 819. https://doi.org/10.3390/ani11030819

The study, published under a Creative Commons License, can be read here

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