Sequencing and culture-based characterization of the vaginal and uterine microbiota in beef cattle that became pregnant or remained open following artificial insemination

Abstract

Emerging evidence suggests that microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. To develop such microbiome-based strategies, one of the first logical steps is to identify reproductive microbiome features related to fertility and to isolate the fertility-associated microbial species for developing a future bacterial consortium that could be administered before breeding to enhance pregnancy outcomes. Here, we characterized the vaginal and uterine microbiota in beef cattle that became pregnant or remained open via artificial insemination and identified microbiota features associated with fertility. We compared similarities between vaginal and uterine microbiota and between heifers and cows. Using culturing, we provided new insights into the culturable fraction of the vaginal and uterine microbiota and their antimicrobial resistance. Overall, our findings will serve as an important basis for future research aimed at harnessing the vaginal and uterine microbiome for improved cattle fertility.

Keywords: 16S rRNA gene sequencing; antimicrobial resistance; artificial insemination; bovine; culturing; pregnancy; uterine microbiota; vaginal microbiota.

Fig 1

Beta and alpha diversities and composition (phylum level) of the vaginal microbiota of heifers that remained open (open, n = 33) or became pregnant (n = 26) following artificial insemination. (A) Non-metric multidimensional scaling (NMDS) plots of the Bray-Curtis dissimilarities, (B) number of observed amplicon sequence variants, and Shannon and inverse Simpson diversity indices; (C) percentage of relative abundance of the eight most relatively abundant phyla.

Fig 2

Beta and alpha diversities and composition (phylum level) of the vaginal microbiota of cows that remained open (open, n = 31) or became pregnant (n = 29) following artificial insemination. (A) Non-metric multidimensional scaling plots of the Bray-Curtis dissimilarities, (B) number of observed amplicon sequence variants, and Shannon and inverse Simpson diversity indices; (C) percentage of relative abundance of the eight most relatively abundant phyla.

Fig 3

Beta and alpha diversities and composition (phylum level) of the uterine microbiota of cows that remained open (n = 31) or became pregnant (n = 29) following artificial insemination. (A) Non-metric multidimensional scaling plots of the Bray-Curtis dissimilarities, (B) number of observed amplicon sequence variants, and Shannon and inverse Simpson diversity indices; (C) percentage of relative abundance of the eight most relatively abundant phyla.

Fig 4

Differentially abundant (A) phyla and (B) genera in the uterine microbiota between cows that remained open or became pregnant following artificial insemination (P < 0.10). Displayed are the upper (75%) and lower (25%) quantiles and the median percent relative abundance for each taxon.

Fig 5

Ecological network of observed bacterial genera in the uterine microbiota of cows that remained open (n = 31) or became pregnant (n = 29) following artificial insemination.

Fig 6

Beta and alpha diversities and composition (phylum level) of the vaginal and uterine microbiota from cows (n = 60) that become pregnant or remained open following artificial insemination. (A) Non-metric multidimensional scaling plots of the Bray-Curtis dissimilarities, (B) number of observed amplicon sequence variants, and Shannon and inverse Simpson diversity indices; (C) percentage of relative abundance of the eight most relatively abundant phyla; (D) Venn diagram displaying the number of shared and unique ASVs between uterine and vaginal microbiota.

Fig 7

Heat map showing the 100 most abundant ASVs (log4) overall in the uterine and vaginal microbiota of cows (n = 60) that become pregnant or remained open following artificial insemination.

Fig 8

Beta and alpha diversities and composition (phylum level) of the vaginal microbiota between heifers (n = 59) and cows (n = 60). (A) Non-metric multidimensional scaling plots of the Bray-Curtis dissimilarities, (B) number of observed amplicon sequence variants, and Shannon and inverse Simpson’s diversity index. (C) Percentage of relative abundance of the eight most relatively abundant phyla.

Fig 9

Bacterial isolates from aerobic and anaerobic culturing of vaginal swab samples collected from yearling heifers and cows of various ages. Blue-highlighted genera were only present in heifers, and green-highlighted genera were only found in cows.

Fig 10

MICs of antibiotics against Gram-positive bacterial isolates (n = 10) isolated from the uterus (n = 3) and vagina (n = 7) of beef cows and heifers. MIC values (µg/mL) presented in the table were obtained from the Sensititre Gram Positive Companion panel (COMPGP1F). Green, no break points (isolate); blue, no/few break points (antibiotic); red, resistant; dark-red/white text, intrinsically resistant; yellow, highest test concentrations were lower than breakpoints; range, intermediate.

Fig 11

MICs of antibiotics against Gram-negative bacterial isolates (n = 19) isolated from the uterus (n = 4) and vagina (n = 15) of beef cows and heifers. MIC values (µg/mL) presented in the table were obtained from the Sensititre Gram Negative Companion panel (COMPGN1F). Green, no break points (isolate); blue, no/few break points (antibiotic); red, resistant; dark red/white text, intrinsically resistant; yellow, highest test concentrations were lower than breakpoints; orange intermediate.