Effects of storage conditions on the microbiota of fecal samples collected from dairy cattle

Microbiota analyses are key to understanding the bacterial communities within dairy cattle, but the impact of different storage conditions on these analyses remains unclear. This study sought to examine the effects of freezing at -80°C immediately after collection, refrigeration at 4°C for three days and seven days and absolute ethanol preservation on the microbiota diversity of pooled fecal samples from dairy cattle. Examining 16S rRNA gene sequences, alpha (Shannon, Pielou evenness, observed features and Faith PD indices) and beta (Bray-Curtis, βw and Weighted UniFrac) diversity were assessed. The effects of storage conditions on these metrics were evaluated using linear mixed models and PERMANOVA, incorporating the farm as a random effect. Our findings reveal that 7d and E significantly altered the Shannon index, suggesting a change in community composition. Changes in Pielou evenness for 3d and 7d storage when compared to 0d were found, indicating a shift in species evenness. Ethanol preservation impacted both observed features and Faith PD indices. Storage conditions significantly influenced Bray-Curtis, βw, and Weighted UniFrac metrics, indicating changes in community structure. PERMANOVA analysis showed that these storage conditions significantly contributed to microbiota differences compared to immediate freezing. In conclusion, our results demonstrate that while refrigeration for three days had minimal impact, seven days of refrigeration and ethanol preservation significantly altered microbiota analyses. These findings highlight the importance of sample storage considerations in microbiota research.