The vaginal and fecal microbiomes are related to pregnancy status in beef heifers

Abstract

Background: The greatest impact on profitability of a commercial beef operation is reproduction. However, in beef heifers, little is known about the vaginal and fecal microbiota with respect to their relationship with fertility. To this end, we followed heifers through gestation to examine the dynamics of vaginal and fecal microbial composition throughout pregnancy.

Results: Heifers were exposed to an estrus synchronization protocol, observed over a 12-day period, artificially inseminated 12 h to 18 h after observed estrus, and subsequently exposed to bulls for a 50-day breeding season. Vaginal samples were taken at pre-breeding (n = 72), during the first (n = 72), and second trimester (n = 72) for all individuals, and third trimester for individuals with confirmed pregnancies (n = 56). Fecal samples were taken at pre-breeding (n = 32) and during the first trimester (n = 32), including bred and open individuals. Next generation sequencing of the V4 region of the16S rRNA gene via the Illumina MiSeq platform was applied to all samples. Shannon indices and the number of observed bacterial features were the same in fecal samples. However, significant differences in vaginal microbiome diversity between gestation stages were observed. No differences in beta-diversity were detected in vaginal or fecal samples regarding pregnancy status, but such differences were seen with fecal microbiome over time. Random Forest was developed to identify predictors of pregnancy status in vaginal (e.g., Histophilus, Clostridiaceae, Campylobacter) and fecal (e.g., Bacteroidales, Dorea) samples.

Conclusions: Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction. Further experiments are needed to validate these biomarkers and to examine their roles in a female's ability to establish pregnancy.

Keywords: Beef cattle; Pregnancy; Random forest; Reproduction; Vaginal microbiome.

Fig. 1

Vaginal and fecal microbial community alpha diversity measures between bred and open female cattle by stage. Diversity in the vaginal and fecal community was measured using Shannon index (a, c) and observed OTUs (b, d). The bottom and top of each box are the first and third quartiles, respectively, and the band inside the box is the median. Bred: cattle that were pregnant after the breeding season; Open: cattle that never established pregnancy. Vaginal and fecal swabs were collected at the sampling points (e.g. 1st trimester) from both pregnant and open cattle. The labels were defined based on the status of the pregnant cattle. Open cattle stayed open throughout the whole experiment

Fig. 2

Beta diversity measures in vaginal (a, b) and fecal (c, d) samples across gestation stages and between open and bred cattle. a, c show the Principal Coordinate Analysis (PCoA) plot based on community membership as measured by the Jaccard distances. b, d show the PCoA based on community structure based on Bray-Curtis dissimilarity matrices. Triangles and circle represent bred and open females, respectively. Stages are indicated by color: red, blue, green and purple represent pre breeding, and gestational trimesters 1 through 3 respectively. These stages were defined based on the status of the pregnant cattle. Open cattle stayed open throughout the whole experiment. Samples were collected prospectively but pregnancy was defined retrospectively. The ellipses were calculated and drawn with 0.95 of confidence level. Bred: cattle that were pregnant after the breeding season; Open: cattle that never established pregnancy

Fig. 3

Relative abundance of bacterial features of different pregnancy status and stages in the vaginal microbiota of beef heifers. Multi-colored stacked bar graphs represent the relative abundance of the top 15 bacterial features. These features were classified against the Greengenes database and were shown at the deepest known classification. Each panel represents a stage (a: Pre-breeding, b: first trimester, c: second trimester, d: third trimester) and each bar represents a sample. These stages were defined based on the status of the pregnant cattle. Open cattle stayed open throughout the whole experiment

Fig. 4

Relative abundance of bacterial features of different pregnancy status and stages in the fecal microbiota of beef heifers. Multi-colored stacked bar graphs represent the relative abundance of the top 15 bacterial features. Each panel represents a stage (a: Pre-breeding, b: first trimester) and each bar represents a sample. These stages were defined based on the status of the pregnant cattle. Open cattle stayed open throughout the whole experiment

Fig. 5

Predicting pregnancy outcome using random forest model with vaginal microbiota dataset of the pre-breeding stage. a ROC curve of the optimal random forest model. b Selected features in repeated cross-validation of the optimal random forest model. c-e Relative abundance of the top three features with the highest probability of selection

Fig. 6

Predicting pregnancy outcome using Random forest model with fecal microbiota dataset of the pre-breeding stage. a ROC curve of the optimal random forest model with selected 94 features. b Top 15 features with the highest probability of selection in repeated cross-validation of the optimal random forest model. c-e Relative abundance of the three features with the highest probability of selection