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

Abstract

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.

Fig 1

Box plots representing alpha diversity indices: (A) Shannon, (B) Pielou Evenness, (C) Observed features, and (D) Faith PD, under four different storage conditions: Immediate freezing at -80°C (0d), refrigeration for three (3d) and seven (7d) days at 4°C, and preservation in ethanol (E). For better visualization, the y-axis scale in plot B was adjusted. Significant differences (p ≤ 0.05) between methods are indicated by p-values shown on a solid line.

Fig 2

Box plots of beta diversity (A) Bray-Curtis (B) βw and (C) Weighted UniFrac for six pairs of four storage conditions: Immediate freezing at -80°C (0d), refrigeration for three (3d) and seven (7d) days at 4°C, and preservation in ethanol (E).

Fig 3. Clustering of samples due to storage conditions by PCoA.

Ordination plots of (A) Bray-Curtis, (B) βw and (C) Weighted UniFrac beta diversity measured for four storage conditions: Immediate freezing at -80°C (0d), refrigeration for three (3d) and seven (7d) days at 4°C, or preservation in ethanol (E). The ellipses indicate the clusters by groups, showing the 95% confidence intervals.

Fig 4. Bar plot of relative abundance at phylum level for samples storage under four different conditions.

Immediate freezing at -80°C (0d), refrigeration for three (3d) and seven (7d) days at 4°C, or preservation in ethanol (E).