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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.

Uterine bacterial community abundances shift throughout the estrous cycle, potentially altering the immunological environment of the uterus and impacting subsequent fertility. The objective of the current study was to evaluate the immunological impact of lipopolysaccharide (LPS), as a model for potentially pathogenic bacteria, throughout the uterine endometrium between the luteal and follicular phase of the estrous cycle. Bovine uterine tracts were harvested in mid-luteal (n = 7) or follicular (n = 7) phase. Explants were collected from the contralateral and ipsilateral horn relative to the dominant follicle or corpus luteum, then subjected to one of three treatments: uncultured control, cultured control, or cultured with LPS (1 µg/mL). Explants underwent RNA extraction and targeted RNA sequencing for expression analyses of 40 immune response related genes. Sequencing reads were mapped to Bos taurus genome in CLC Genomics Workbench. Resulting total read counts were normalized by housekeeping gene GAPDH and analyzed for overall expression profile by Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and Variable Importance in Projection (VIP) analyses in Metaboanalyst. Individual gene expression differences were determined by GLIMMIX procedure in SAS with fixed effects of treatment, estrous phase, uterine horn, and their interaction, with random effect of individual uterus. Expression of 29 genes were affected among treatment groups, with seven genes increased in LPS treatment compared to other groups ( P < 0.05). Multiple genes were affected by estrous phase and uterine horn, independent of treatment ( P < 0.05). The OPLS-DA analyses indicated overall gene expression differences due to clustering by estrous cycle and treatment ( P < 0.001), with no effect of uterine horn ( P > 0.10). Similar clustering was observed between luteal and follicular phase explants of controls, but distinct separate clustering between phases with LPS treatment ( P = 0.001). According to VIP analyses, mucins were identified as contributing the most to differences observed between phase and treatment. In conclusion, estrous cycle phase resulted in differing overall endometrial gene expression profiles of immune response to LPS treatment. Therefore, altered immunological environment of the uterus in response to bacteria at different estrous cycle stages may lead to differences in reproductive success.

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.

Drylot beef cow-calf systems are viable alternative management systems to traditional forage-based systems. In such confinement, adding ionophores such as monensin to the diets of beef cattle is common, and has been shown to improve feed efficiency and increase average daily gain. The addition of monensin is also commonly utilized as a strategy for methane mitigation, as this ionophore class antimicrobial acts to interfere with ion flux primarily within Gram-positive cells through its action as an ion carrier. It is widely accepted that suppression of these ruminal organisms results in the reduction of substrates for rumen methanogenic archaea, reducing methane production. However, several studies have indicated that cattle may adapt to monensin, and within weeks of feeding, may return to prior levels of methane production. Our hypothesis is that feeding monensin to bred beef heifers in confinement will temporarily decrease methane production from shifts in the methanogenic archaeal and bacterial communities. Sixteen fall-born bred heifers were randomly assigned to 2 treatment groups (n = 8 per treatment) and were fed a control diet or a diet containing monensin for 70 days using headgates. In vivo gas exchange of oxygen consumption, carbon dioxide and methane production were measured for 24-h periods throughout the trial using individual calorimeter head boxes. Rumen content sampling was conducted on day 0, 18 and 53 of the trial through oral lavage. Upon completion of sampling, DNA was isolated for ruminal bacteriome composition utilizing deep, next-generation sequencing of the V1-V3 hypervariable regions of the 16S bacterial rRNA gene. Level of methanogen 16S rRNA was quantified using qPCR. There was a significant reduction in phylum SR1 (P < 0.05). The abundance of several OTUs was reduced between treatment with monensin, including Anaerofustis (P < 0.0001), Shuttleworthia (P < 0.0001) and Order Bacteroidales (P = 0.003). No significant shifts in key ruminal methanogenic archaeal groups as a percentage of total methanogen 16S rRNA occurred (P > 0.05). Heifers fed monensin did not produce significantly less methane than the control (P > 0.05) on a liters per day basis, which was consistent throughout the study. These data suggest methane production is not reduced long-term when animals are fed monensin in confinement. Additionally, the data suggest that monensin supplementation does not suppress all classical Gram-positive populations, rather it influences finer shifts in bacterial species that may be key in ruminant function. Determining the stability of the ruminal microbiome over time in heifers fed monensin may provide further insights to long-term methane mitigation in cow-calf systems.

The effects of growth-promoting implant strategies have been well-defined in research for their ability to impact growth performance in beef cattle. Production-relevant microbiomes in the rumen have also been associated with growth traits. However, the role of implant strategies on the rumen microbiome is not understood. The objective was to determine if varying doses of implant hormones cause gain-associated ruminal microbial community shifts. To assess this, a completely randomized design was used and 336 fall-born steers from the germplasm evaluation population between 450–470 days of age at the U.S. Meat Animal Research Center (Clay Center, NE) were divided into two treatment groups: 1) a moderate implant strategy of Revalor-IS (80 mg trenbolone acetate, 16 mg estradiol) followed by Revalor-S (120 mg trenbolone acetate, 24 mg estradiol) and 2) an aggressive implant strategy of Revalor-IS followed by Revalor-200 (200 mg trenbolone acetate, 20 mg estradiol). Steers were fed the same diet (57.0% dry-rolled corn, 30% wet distiller’s grains with solubles, 8.0% alfalfa hay, 4.25% supplement, and 0.75% urea, DM basis). Body weights were collected once per month with initial weights of 439.8 ± 43.1 kg. After implants were administered for 84 days, rumen content was collected via orogastric tubing. Samples were sequenced targeting bacterial V1-V3 16S rRNA gene regions, V3-V4 for archaea, and partial 18S rRNA gene of protozoa. Sequences were processed in R utilizing Phyloseq and analyzed with DESeq2 to test differential abundances. Production data between implant strategies were analyzed using a mixed model ANOVA (SASv9.4, Cary, NC). Alpha- and beta-diversity between strategies did not differ for bacteria, archaea, or protozoa (P &amp;gt; 0.05). Average daily gain was different (P = 0.01; 1.72 vs 1.66 ± 0.02 kg, aggressive vs moderate, respectively); however, large microbial community shifts were not associated implant strategy. USDA is an equal opportunity provider and employer.

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.

Virus-host associations. A listing of all associations (Hi-C linkage or long-read alignment) between predicted viral contigs and non-viral contigs. (XLSX 24 kb)

No abstract is provided for this article.

Overstocking can be a major issue in the dairy cattle industry, leading to negative changes in feeding and resting behavior. Additional stress imposed and alterations in feeding behavior may significantly impact the rumen microbiome. The rumen microbiome is responsible for the successful conversion of feed to usable energy for its host. Thus, understanding the effects of stocking density on the rumen microbiome is imperative for further elucidation of potentially negative consequences of overstocking in dairy cattle. This study implemented a Latin Square design accounting for four pens of cattle and four treatment periods so that all treatment combinations were assigned to every pen during one period of the study. Two treatment factors, including two levels of physically effective neutral detergent fiber, achieved with addition of chopped straw, and stocking density (100% vs. 142%) of freestalls and headlocks, were combined and tested within a factorial treatment design. Within each pen, three or four cannulated cows ( n = 15 total) were sampled for rumen content on the final day of each treatment period. Each treatment was randomly assigned to a single pen for a 14-day period. The V1–V3 hypervariable regions of the 16S rRNA gene were targeted for bacterial analyses. Variables with approximately normally-distributed residuals and a Shapiro–Wilk statistic of ≥0.85 were analyzed using a mixed model analysis of variance with the GLIMMIX procedure with fixed effects of feed (straw vs. no straw), stocking density (100% vs. 142%), and the interaction of feed × stocking density, and random effects of pen, period, feed × stocking × pen × period. Pen was included as the experimental unit in a given period and the sampling unit as cow. Variables included Shannon’s Diversity Index, Faith’s phylogenetic diversity index, chao1, observed OTU, and Simpson’s evenness E as well as most individual taxa. Data were analyzed in SAS 9.4 utilizing the GLIMMIX procedure to perform mixed model analysis of variance. If data were not normally distributed, a ranked analysis was performed. No differences were observed in α-diversity metrics by fiber or stocking density ( P &gt; 0.05). Beta diversity was assessed using weighted and unweighted Unifrac distances in QIIME 1.9.1 and analyzed using ANOSIM. No differences were observed in weighted ( P = 0.6660; R = −0.0121) nor unweighted ( P = 0.9190; R = −0.0261) metrics and R values suggested similar bacterial communities among treatments. At the phylum level, Tenericutes differed among treatments with an interaction of stocking density by feed ( P = 0.0066). At the genus level, several differences were observed by treatment, including Atopobium ( P = 0.0129), unidentified members of order RF39 ( P = 0.0139), and unidentified members of family Succinivibrionaceae ( P = 0.0480). Although no diversity differences were observed, taxa differences may indicate that specific taxa are affected by the treatments, which may, in turn, affect animal production.