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.

The development of replacement heifers is crucial for breeding success and herd efficiency. Nutritional management can affect not only reproductive development, but also the inflammatory status of the uterine environment, which may impact reproductive functions such as pregnancy establishment and development. The study herein evaluated the concentration of cytokines and chemokines in the uterus of heifers supplemented with different levels of protein. Angus heifers (n = 60) were blocked by body weight and randomly assigned to one of three treatments based on protein supplementation level: control of 10% crude protein (CON), 20% crude protein (P20), or 40% crude protein (P40). Body weight, body condition score, and blood samples were taken every two weeks for 140 days to monitor development. Uterine flushes were performed monthly and concentrations of cytokines (IL-1α, IL-1β, TNF-α, IFN-γ, IL-10, VEGF-α, IL-17A, and IL-36RA) and chemokines (IL-8, MCP-1, MIP-1α, and MIP-1β) were quantified via ELISA multiplex. To test if there were mean differences in cytokines between the treatment groups or over time, PROC GLIMMIX (SAS v 9.4) was utilized. Concentrations of all cytokines and chemokines, except IL-1α, changed throughout heifer development (P < 0.05). Heifers in the P40 treatment group displayed reduced concentrations of MCP-1 (P = 0.007) and tended to have decreased concentrations of IFN-γ (P = 0.06). Cytokine IL-36RA tended (P = 0.06) to be affected by protein level, with the lowest concentrations observed in CON heifers. Most cytokines and chemokines increased following the initial month of supplementation (P < 0.05). The increase in concentrations after one month may indicate an adaptive response in the uterus to diet change. Cytokines and chemokines fluctuated due to physiological changes occurring during development. Further research is needed to determine the influence of nutrition on uterine inflammation and long-term impacts on reproductive function.

The activity of the immune system in the reproductive tract has been proven to be crucial in the response to uterine diseases, normal reproductive functions, and tolerance to the allogeneic fetus during pregnancy. The objectives of the current study were to (1) evaluate uterine and vaginal cytokine concentrations in postpartum cows undergoing estrus synchronization followed by timed artificial insemination (TAI) and (2) correlate bacterial communities with cytokine concentrations. Postpartum Angus cows (n = 20) were subjected to a 7-Day Co-Synch protocol with pre-synchronization beginning 21 days prior (d −21) to TAI (d 0). Uterine and vaginal flushes were collected on d −21 and −2. Pregnancy was determined by transrectal ultrasound on d 30. Cytokines include interleukin (IL)-1b, IL-6, IL-10, transforming growth factor beta (TGF-β), and immunoglobin A (IgA) and concentrations were determined by commercial ELISA kits. No differences by day or pregnancy status in cytokine concentrations were detected in vaginal samples. No differences by day or pregnancy status in IgA, IL-10, or IL-1b concentrations were detected in uterine samples. Overall TGF-β concentrations in the uterus were greater in resulting pregnant than non-pregnant cows (44.0 ± 13.4 pg/mL vs. 14.7 ± 4.9 pg/mL; P = 0.047). Uterine TGF-β was correlated with the relative abundance of genera Treponema (r = −0.668; P = 0.049) in resulting non-pregnant cows on d −21 and with the relative abundance of genera Ureaplasma (r = 0.901; P = 0.0004) in resulting pregnant cows on d −2. In resulting pregnant animals, a tendency for a strong correlation was detected between d −2 progesterone concentrations and uterine TGF-β concentrations (r = 0.591, P = 0.07). Overall IL-6 concentrations in the uterus were greater in resulting non-pregnant than pregnant cows (198.7 ± 21.8 pg/mL vs. 144.3 ± 16.1 pg/mL; P = 0.045). A correlation was also detected between uterine IL-6 concentrations and the relative abundance of genera Butyrivibrio (r = 0.742; P = 0.022) in resulting non-pregnant cows on d −21. These results suggest possible relationships between different bacterial communities and cytokine concentrations within the uterus of beef cattle prior to TAI that may ultimately affect fertility outcomes.

Bacterial communities play major roles in rumen and uterine function toward optimal animal performance and may be affected by changes occurring during heifer development such as nutritional supplementation for optimal growth and the attainment of puberty. The effect of different levels of protein supplementation on ruminal and uterine bacterial communities following weaning was examined through first breeding of heifers. Angus heifers (n = 39) were blocked by initial body weight (BW) and randomly assigned to one of three 163-day (d) crude protein (CP) supplementation diets including control (10% CP, n = 14), 20% CP (n = 11), or 40% CP (n = 14) treatment groups. Growth and development were monitored by body weight, with blood progesterone concentration determined every 14 d to determine pubertal status. Uterine flush and rumen fluid were collected on d 56, 112, and 163 relative to the start of supplementation. Bacterial DNA was extracted from fluid samples, the V1–V3 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced then processed in R 4.1. Statistical analyses were performed in SAS 9.4 with a GLIMMIX procedure utilizing fixed effects of protein, month, pubertal status, and interactions, with random effects including BW, interaction of BW and protein, and heifer within the interaction, and repeated measures of day. In the uterus, pubertal status and day of supplementation affected the observed amplicon sequence variants (ASVs) and led to clustering of samples in a principal coordinate analysis (PCoA; P < 0.05), but no effect of protein supplementation was observed. Ruminal samples clustered in PCoA (P = 0.001), and observed ASVs were impacted over time (P < 0.0001), but no effect of protein supplementation was detected. In contrast, protein supplementation, pubertal status, and day of supplementation affected the abundance of multiple phyla and genera in the uterus and rumen (P < 0.05). Temporal and pubertal status effects on the heifer’s uterine bacterial communities potentially indicate a maturing uterine microbiome. Protein supplementation did not impact microbial diversity measures but did affect the abundance of individual bacterial phyla and genera that may provide future opportunities to manipulate bacterial community composition and maximize productivity.

This study determined potential microbial and metabolic biomarkers of feed efficiency in Angus heifers. Seventeen ruminal cannulated Angus heifers underwent a 70-day feed efficiency trial. Residual feed intake was used to determine high and low feed efficient heifers. On day 70 of the trial, rumen content and blood were collected and used for microbial and metabolomic analyses, respectively. Bacterial populations were examined by targeting the V4 region of the 16S rRNA gene and analyzed using QIIME and SAS. Rumen fluid and serum metabolites were analyzed using MetaboAnalyst. No microbial taxa differed after false discovery rate correction, but seven did differ (p ≤ 0.05) prior to correction, including Lachnospiraceae (Other), Desulfobulbaceae, Neisseriaceae, Shuttleworthia, Corynebacterium, p-75-a5, and L7A-E11. No differences were observed in alpha diversity metrics. Beta diversity utilizing unweighted UniFrac distances analyzed via PERMANOVA was significant (p = 0.03). Several metabolites in rumen fluid metabolites were correlated with bacteria that differed by feed efficiency phenotype. The metabolites correlated with bacteria were primarily involved in nutrient signaling and microbial crude protein availability. These data suggest variation in the availability of nutrients, primarily amino acids, as well as a relationship among microbiota, metabolome, and host feed efficiency phenotypes in heifers.