Investigation of the vaginal microbiota of dairy cows through genetic sequencing of short (Illumina) and long (PacBio) reads and associations with gestational status

Abstract

The vaginal microbiota has been shown to be important in local immune regulation and may play a role in reproduction and fertility. Next-generation sequencing (NGS) technologies have been used to characterize the bovine vaginal microbiota, mainly using short-read sequencing (Illumina). However, the main limitation of this technique is its inability to classify bacteria at the species level. The objective of this study was to characterize the bovine vaginal microbiota at the species level using long-read sequencing (PacBio) and to compare it with the results of short-read sequencing. In addition, the vaginal microbiota of cows that became pregnant after artificial insemination (AI) was compared with that of infertile animals. Thirteen Holstein cows had vaginal swabs collected prior to AI. DNA was extracted and subjected to Illumina and PacBio sequencing to characterize the V4 region and the entire 16S rRNA gene, respectively. PacBio sequencing yielded 366,509 reads that were assigned to 476 species from 27 phyla. However, none of the most abundant reads (>1%) could be classified at the species level. Illumina sequencing yielded more reads and consequently was able to detect a more observed species, but PacBio sequencing was able to detect more unique and rare species. The composition of the vaginal microbiota varies according to the sequencing method used, which might complicate the interpretation of results obtained in the majority of the current studies. The present study expands on the current knowledge of bovine microbiota, highlighting the need for further efforts to improve the current databanks.