The cattle rumen has a diverse microbial ecosystem that is essential for the host to digest plant material. Extremes in body weight (BW) gain in mice and humans have been associated with different intestinal microbial populations. The objective of this study was to characterize the microbiome of the cattle rumen among steers differing in feed efficiency. Two contemporary groups of steers (n=148 and n=197) were fed a ration (dry matter basis) of 57.35% dry-rolled corn, 30% wet distillers grain with solubles, 8% alfalfa hay, 4.25% supplement, and 0.4% urea for 63 days. Individual feed intake (FI) and BW gain were determined. Within contemporary group, the four steers within each Cartesian quadrant were sampled (n=16/group) from the bivariate distribution of average daily BW gain and average daily FI. Bacterial 16S rRNA gene amplicons were sequenced from the harvested bovine rumen fluid samples using next-generation sequencing technology. No significant changes in diversity or richness were indicated, and UniFrac principal coordinate analysis did not show any separation of microbial communities within the rumen. However, the abundances of relative microbial populations and operational taxonomic units did reveal significant differences with reference to feed efficiency groups. Bacteroidetes and Firmicutes were the dominant phyla in all ruminal groups, with significant population shifts in relevant ruminal taxa, including phyla Firmicutes and Lentisphaerae, as well as genera Succiniclasticum, Lactobacillus, Ruminococcus, and Prevotella. This study suggests the involvement of the rumen microbiome as a component influencing the efficiency of weight gain at the 16S level, which can be utilized to better understand variations in microbial ecology as well as host factors that will improve feed efficiency.

Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics—in addition to the traditional considerations when conducting an animal science study—makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

Reproductive inefficiency in cattle has major impacts on overall productivity of cattle operations, increasing cost of production, and impacting the sustainability of the cattle enterprise. Decreased reproductive success and associated disease states have been correlated with the presence of specific microbes and microbial community profiles, yet details of the relationship between microbial communities and host physiology are not well known. The present study profiles and compares the microbial communities in the bovine uterus and vagina using 16S rRNA sequencing of the V1-V3 hypervariable region at the time of artificial insemination. Significant differences (p<0.05) between the vaginal and uterine communities were observed at the level of α-diversity metrics, including Chao1, Shannon’s Diversity Index, and observed OTU. Greater clustering of vaginal OTU was apparent in principal coordinate analysis compared to uterine OTU, despite greater diversity in the vaginal community in both weighted and unweighted UniFrac distance matrices (p<0.05). There was a significantly greater relative abundance of unassigned taxa in the uterus (p=0.008), otherwise there were few differences between the overall community profiles. Both vaginal and uterine communities were dominated by Firmicutes, although the relative abundance of rRNA sequences corresponding to species in this phylum was significantly (p=0.007) lower in the uterine community. Additional differences were observed at the genus level, specifically in abundances within Clostridium (p=0.009), Anaerofustis (p=0.018), Atopobium (p=0.035), Oscillospira (p=0.035), 5-7N15 (p=0.035), Mycoplasma (p=0.035), Odoribacter (p=0.042), and within the families Clostridiaceae (p=0.006), Alcaligenaceae (p=0.021), and Ruminococcaceae (p=0.021). Overall, the comparison revealed differences and commonalities among bovine reproductive organs, which may be influenced by host physiology. The increased abundance of unassigned taxa found in the uterus may play a significant biological role in the reproductive status of the animal. The study represents an initial dataset for comparing bacterial communities prior to establishment of pregnancy.

The present study evaluated the bovine vaginal and uterine bacterial community diversity and its relationship to fertility. Postpartum beef cows (n = 68) were synchronized beginning on day −21 and ending with timed artificial insemination (TAI) on day 0. Pregnancy was diagnosed 30 d after TAI. Uterine and vaginal flushes were collected on day −21, −9, and −2 for bacterial DNA extraction to sequence the V1 to V3 hypervariable regions of the 16S rRNA gene. Results indicated a decrease in the number of bacterial species over time in the uterus of resulting pregnant and nonpregnant beef cows (P < 0.0001). Principal coordinate analyses (PCoA) depicted clustering of samples, indicating closely related bacterial communities, by day in the uterus and vagina (P < 0.0001). At day −2, uterine samples from nonpregnant and pregnant animals clustered separately (P < 0.0001), with nonpregnant animal samples clustering tightly together. Overall, the current study suggests the shift in the reproductive bacterial communities’ diversity and phylogenetic relationship leading up to the time of breeding may contribute to successful pregnancy establishment.