Toward standardized methods in canine vaginal microbiome research: evaluation of storage, host DNA depletion, and database selection.

Standardized methods for investigating the canine vaginal microbiome are not available yet. Data using next-generation sequencing (NGS) are still limited, and methodologies lack consistency. In theory, microbiome results can be significantly affected by factors like sampling technique, storage, DNA extraction methods, and reference databases, which can all introduce bias. To address these concerns, we compared two storage methods for the sample (no medium vs. medium), examined the effect of host DNA depletion, and tested two reference databases (Emu vs. SILVA) using samples from six bitches, totaling 26 samples. Host depletion showed no significant impact on bacterial composition, nor did the storage conditions. However, when comparing reference databases, we found significant differences in beta diversity, emphasizing the importance of database choice when comparing studies. The proposed protocol paves the way for future studies on the canine vaginal microbiome, setting the basis for more precise and comprehensive microbiome profiling in this field. IMPORTANCE: Understanding the vaginal microbiome in dogs could lead to new insights into reproductive health and fertility, but progress is limited by the lack of clear guidelines on how samples should be collected, stored, and analyzed. This study helps clarify which steps in the process truly matter and which have little impact, offering practical guidance for researchers entering this field. By highlighting where inconsistencies can influence outcomes and which methodological choices affect results, we take an important step toward more reliable and comparable research. These findings support future scientific studies and hold potential to improve veterinary care over time.