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With an expected increase in population by 2050, the demand for high-quality protein for human consumption will increase. To improve the sustainability of US cattle production, producers and researchers have historically focused on improving the feed efficiency of steers through improvements in genetics, nutrition, and microbiome management, producing a more marketable beef product. However, without a successful pregnancy, there would be no marketable animal to feed. The objective of this study was to examine the impact of pregnancy on the rumen environment, microbial communities, and the correlation to feed efficiency status in Angus heifers. Utilizing 17 cannulated Angus heifers, feed efficiency status was determined using the GrowSafe 8000 feed intake monitoring system to monitor feed intake for a 70d trial period to calculate residual feed intake. Following the trial, heifers were bred and 40mL rumen fluid and content samples were collected every two weeks during gestation. Metagenomic DNA was extracted from the rumen samples, sequencing libraries were prepared targeting the bacterial V4 region of the 16S rRNA gene, and resultant libraries were sequenced on an Illumina MiSeq. All microbial analyses were conducted in the R environment v2.6.2 where alpha diversity, beta diversity, and differential abundances were determined. Metabolites were identified using ultra high-performance liquid chromatography high resolution mass spectrometry, and Metabolomic Analysis and Visualization Engine was used to select metabolite peaks. Differential abundance analyses using “ANCOMBC” indicated 10 amplicon sequence variants (ASVs), assigned to seven Prevotella and three Succiniclasticum, significantly varied among weeks of gestation (P

Once thought to be sterile, the reproductive tract microbiome has been characterized due to the transition from culture-dependent identification of bacteria to culture-independent sequencing methods. The urogenital microbiome was first identified in women through the Human Microbiome Project, which led to research in other species such as the bovine. Previous research focused on uterine bacteria associated with postpartum disease, but next generation sequencing methods identified a normal, healthy bacterial community of the reproductive tract of cows and heifers. Bacterial communities are now understood to differ between the uterus and vagina, and throughout the estrous cycle with changes in hormone dominance. In a healthy state, the bacterial communities largely interact with the uterine environment by assisting in maintaining the proper pH, providing and utilizing nutrients and metabolites, and influencing the immunological responses of the reproductive tract. If the bacterial communities become unbalanced due to an increase in potentially pathogenic bacteria, the health and fertility of the host may be affected. Although the presence of a reproductive tract microbiome has become widely accepted, the existence of a placental microbiome and in utero colonization of the fetus is still a popular debate due to conflicting study results. Currently, researchers are evaluating methods to manipulate the reproductive bacterial communities, such as diet changes and utilizing probiotics, to improve reproductive outcomes. The following review discusses the current understanding of the reproductive tract microbiome, how it differs between humans and cattle, and its relationship with the uterine environment.

IntroductionHigh quality semen is essential for reproductive efficiency. Nutrition and environmental factors impact the ejaculate components, like cytokines, that are essential for pregnancy establishment. We hypothesized that differing nutritional periods and body condition scores would affect seminal plasma cytokine concentrations of bulls.MethodsMature Angus bulls (n=11) were individually housed and randomly assigned to either over-fed (n=5) or restricted (n=6) treatment pathways. Bulls were fed different volumes of a single ration creating 8 individual treatment periods. Body weight and body condition scores were taken every 14 d to manage intake volumes. Ejaculates were collected every 84 d to determine seminal plasma cytokine profiles. A complete randomized design was used to evaluate seminal plasma cytokines after each nutritional treatment. Initial cytokine concentrations and volume of the ejaculate were included as covariates.ResultsAll cytokines returned to initial concentrations following maintenance treatments at an ideal body condition score of 6. Nutritional treatments affected (P ≤ 0.05) IFN-γ, IL-8, MIP-1α, MIP-1β, TNF-α, IL-1β, and VEGF-A. However, TNF-α, IFN-γ, and MIP-1α had the greatest impact on cytokine profile.DiscussionNutritional levels and adiposity altered seminal plasma cytokine concentrations which could potentially impact the inflammatory balance of the uterus and the immune responses necessary for pregnancy establishment.

Replacement heifer development is one of the most critical components in beef production. The composition of the ideal uterine environment could maximize fertility and reproductive efficiency. Our hypothesis was that protein supplementation would affect the uterine environment of beef heifers without inhibiting development or reproduction. To test the effects of dietary supplementation on these outcomes, a randomized complete block design with repeated measures was implemented. Angus heifers (n = 60) were blocked by body weight (BW) and randomly assigned to one of three supplemental protein treatment groups (10% (CON), 20% (P20), and 40% (P40)). Mixed model ANOVAs were used to determine whether protein supplementation treatments, time, and the interaction or protein supplementation, semen exposure, and the interaction influenced uterine luminal fluid (ULF) and pregnancy outcomes. Amino acids (AAs) were impacted (p < 0.001), specifically, the essential AAs: Arg, Iso, Leu, Val, His, Lys, Met, Phe, Trp. Protein supplementation influenced multiple AAs post-insemination: Arg (p = 0.03), CC (p = 0.05), 1-MH (p = 0.001), and Orn (p = 0.03). In conclusion, protein supplementation did not affect the reproductive development via puberty attainment or the timing of conception even with alterations in growth. However, uterine AA concentrations did change throughout development and protein supplementation influenced ULF d 14 post-insemination, which may affect the conception rates.

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.