Publications

Nutritional studies involving ruminants have traditionally relied on relatively short transition or wash-out periods between dietary treatments, typically two to four weeks. However, little is known about adequate adaptation periods required to reach stabilization of the rumen microbiome that could provide more accurate results from nutritional studies in ruminants. This study determined the rumen bacterial communities and rumen environment parameters over ten weeks following transition from a forage-based to concentrate-based diet. Several α-diversity metrics, including observed OTUs and Simpson’s Evenness fluctuated throughout the trial, but were typically either greatest (observed OTUs) or lowest (Simpson’s) at week 5 of the trial contrasted from weeks 1 and 10 ( P < 0.05). At week 4, several orders associated with the shift to the final bacterial community composition, including Pasteurellales, Aeromonadales, and Bacteroidales. At week 5, rumen pH was correlated with α-diversity ( P = 0.005) and predictive of the rumen microbiome signature at week 10 (R 2 = 0.48; P = 0.04). Rumen microbiome stability did not occur until approximately 9 weeks following adaptation to the diet and was associated with changes in specific bacterial populations and rumen environment. The results of this study suggest that adaptation and wash-out periods must be re-evaluated in order to accommodate necessary rumen microbiome acclimation.

Reproductive efficiency is largely impacted during heifer development, which generally requires nutrient supplementation for proper maturation. Nutritional status can also influence inflammation within the reproductive tract. Therefore, we hypothesized that cytokine concentrations in uterine luminal fluid (ULF) will be impacted by protein supplementation following exposure to semen via artificial insemination (AI). Commercial heifers (n = 60) were utilized to determine the effects of protein supplementation and AI on cytokine concentrations in ULF. Heifers were randomly assigned to one of three crude protein (CP) treatments (11%, 15%, and 19% overall CP) via supplementation: (1) CON (10% CP), (2) P20 (20% CP), and (3) P40 (40% CP). All heifers underwent estrus synchronization and ULF was collected 14 d after insemination. Cytokine profiles were constructed in MetaboAnalyst 5.0, and R Studio was used for individual cytokine analyses. Control heifers had increased (p = 0.05) MIP-1β concentrations (148.7 ± 123.9 pg) over P20 heifers (42.3 ± 123.9 pg), and P40 heifers (75.5 ± 123.9 pg) had intermediate concentrations. Semen exposure (1877 ± 550 pg) showed a trend (p = 0.06) to increase concentrations of IP-10 compared with heifers who were not inseminated (1556 ± 550 pg). In conclusion, although protein supplementation and semen exposure had minimal effects on overall cytokine profiles, MIP-1β, IP-10, and MIP-1α were identified as potential key regulators of uterine inflammation during early gestation.

The functional capacity of the rumen is central to ruminant feed efficiency, and therefore,differences in ruminal metabolism may contribute to cattle feed efficiency variations. The objective of this study was to determine differences in rumen metabolites associated withvariations in feed efficiency. Fifty steers were utilized and maintained on a backgrounding diet (11.57% CP; 76.93% TDN DM basis; 28 mg monensin/kg DM) for 70 days following a two-week diet adaptation. Feed intake was monitored using the GrowSafe system. Following the 70-day trial, steers were ranked by residual feed intake (RFI) and divided into low- (n = 14) and high-RFI (n = 15) based on RFI ±0.5 SD around the mean. On day 70, rumen fluid was collected viaorogastric tubing and filtered through a 0.22 μM syringe filter. Untargeted metabolomics wasconducted utilizing the Dionex UltiMate 3000 UPLC system and electrospray ionization used tointroduce samples into an Exactive Plus Orbitrap MS. Eighty-eight known metabolites wereidentified using MAVEN. Metabolites were median-centered, log-transformed, and pareto-scaled using Metaboanalyst 4.0. Normality was assessed using the SAS 9.4 PROC UNIVARIATE procedure. Normal distribution was considered based on a Shapiro-Wilk score of ≥ 0.85 andvisual analysis of histogram and plots of residuals. Normally distributed metabolites wereanalyzed using one-way ANOVA in SAS with Tukey’s HSD post-hoc test, whereas non-normally distributed variables were analyzed using Wilcoxon Rank Sum and Kruskal Wallis H test. Significance was set at P ≤ 0.05. Eight metabolites differed between high- and low-RFI steers. Twelve metabolites identified as having > two-fold change between low- and high-RFI steers, including 3,4-Dihydroxyphenylacetate and sulfolactate. Orthogonal PLSDA revealed overlap ofrumen fluid metabolites from high- and low-RFI steers. Understanding rumen metabolites in high- and low-RFI steers will provide researchers with information to identify more efficient cattle.

Negative impacts of endophyte-infected Lolium arundinaceum (Darbyshire) (tall fescue) are responsible for over $2 billion in losses to livestock producers annually. While the influence of endophyte-infected tall fescue has been studied for decades, mitigation methods have not been clearly elucidated. Isoflavones found in Trifolium pretense (red clover) have been the subject of recent research regarding tall fescue toxicosis mitigation. Therefore, the aim of this study was to determine the effect of ergovaline and red clover isoflavones on rumen microbial populations, fiber degradation, and volatile fatty acids (VFA) in an in vitro system. Using a dose of 1.10 mg × L −1 , endophyte-infected or endophyte-free tall fescue seed was added to ANKOM fiber bags with or without 2.19 mg of isoflavones in the form of a control, powder, or pulverized tablet, resulting in a 2 × 3 factorial arrangements of treatments. Measurements of pH, VFA, bacterial taxa, as well as the disappearance of neutral detergent fiber (aNDF), acid detergent fiber (ADF), and crude protein (CP) were taken after 48 h of incubation. aNDF disappearance values were significantly altered by seed type (P = 0.003) and isoflavone treatment ( P = 0.005), and ADF disappearance values were significantly different in a seed x isoflavone treatment interaction ( P ≤ 0.05). A seed x isoflavone treatment interaction was also observed with respect to CP disappearance ( P ≤ 0.05). Seventeen bacterial taxa were significantly altered by seed x isoflavone treatment interaction groups ( P ≤ 0.05), six bacterial taxa were increased by isoflavones ( P ≤ 0.05), and eleven bacterial taxa were altered by seed type ( P ≤ 0.05). Due to the beneficial effect of isoflavones on tall fescue seed fiber degradation, these compounds may be viable options for mitigating fescue toxicosis. Further research should be conducted to determine physiological implications as well as microbiological changes in vivo.

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 > 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.

The rumen microbiome is a complex microbial network critical to the health and nutrition of its host, due to their inherent ability to convert low-quality feedstuffs into energy. In rumen microbiome studies, samples from the ventral sac are most often collected because of the ease of access and repeatability. However, anatomical musculature demarcates the rumen into five sacs (biogeographical regions), which may support distinct microbial communities. The distinction among the microbes may generate functional variation among the rumen microbiome, thus, specialized tasks within different sacs. The objective of this study was to determine the rumen liquid metabolome and epimural, planktonic, and fiber-adherent bacterial communities among each rumen biogeographical region. It was hypothesized that differences in bacterial species and metabolome would occur due to differing anatomy and physiology associated with the respective regions. To assess this variation, epithelial and content microbial-associated communities were evaluated, as well as the metabolites among various rumen biogeographical regions. A total of 17 cannulated Angus cows were utilized to examine the fiber-adherent (solid fraction), planktonic (liquid fraction), and epimural microbial communities from the cranial, dorsal, caudodorsal blind, caudoventral blind, and ventral sacs. Metagenomic DNA was extracted and sequenced from the hypervariable V4 region of the 16S rRNA gene. Reads were processed using packages ‘phyloseq’ and ‘dada2’ in R. Untargeted metabolomics were conducted on rumen liquid from each sac using UHPLC-HRMS and analyzed in MetaboAnalyst 5.0. An analysis of variance (ANOVA) revealed 13 significant differentially abundant metabolites with pairwise comparisons against the five rumen sacs ( P < 0.05). Within the bacterial communities, neither alpha nor beta diversity determined significance against the rumen sacs ( P > 0.05), although there was significance against the fraction types ( P < 0.05). Utilizing multivariable association analysis with MaAslin2, there were significant differential abundances found in fraction type × location ( P < 0.05). Knowledge of similarities among fiber-adherent microbial communities provides evidence that single sac sampling is sufficient for this fraction. However, future projects focusing on either planktonic or epimural fractions may need to consider multiple rumen sac sampling to obtain the most comprehensive analysis of the rumen. Defining these variabilities, especially among the rumen epimural microbiome, are critical to define host-microbiome interactions.

Nutrition is a major component in successful heifer development, influencing age at puberty and the uterine environment, therefore, impacting reproductive efficiency. Different diets can influence systemic inflammation, however, the effect on uterine cytokines has not been determined. Our objective was to evaluate the effect of varied levels of dietary protein on inflammatory cytokines in the uterine environment of developing heifers. Commercial Angus heifers (n = 60) were blocked by body weight, then allocated to receive one of three dietary protein supplementation treatments (n = 20 per group, n = 5 per pen): 10% crude protein (CP; 100% corn), 20% CP [75% dried distillers’ grains (DDG) 25% corn], or 40% CP (75% soybean meal 25% DDG). Supplements were provided four times weekly for six months with ad libitum native grass hay, mineral supplement, and water. Body weight, BCS, and blood was taken every 14d. Uterine fluid was collected every 28d to determine presence of cytokines IL1α, IL1β, TNFα, IFNγ, IL10, IL4, and IL6. The GLIMMIX procedure (SAS 9.4) was used with fixed effects of supplementation, month, and their interaction; random effects of block, interaction of block and supplementation, and heifer within the interaction; and repeated measure of month. Concentrations of IL1β, TNFα, IFNγ, and IL10 shifted throughout development (P < 0.0002). Cytokine IFNγ was also affected by month of development (P = 0.002) and tended (P = 0.06) to decrease as CP increased. Presence of each cytokine was then recorded as binary and analyzed by the frequency procedure (SAS 9.4) to determine a uterine cytokine profile. Pro-inflammatory cytokines IL1α, IL1β, and TNFα occurred together more frequently following the beginning of supplementation. In conclusion, inflammatory cytokines shift throughout heifer development, potentially leading to differences in future reproductive success. Further research is needed to determine the dietary influence for optimal uterine environment development.

Protein supplementation to replacement heifers is utilized to reach targeted body weights, which may affect the animal’s gut and reproductive microbiomes and potentially impact breeding outcomes. The objective of the study was to evaluate the effects of protein supplementation on the ruminal and uterine bacterial communities of developing heifers. Thirty-nine pre-pubertal commercial Angus heifers were blocked by body weight (BW) and randomly assigned to one of three supplementation groups: 10% crude protein (CP), 20% CP, or 40% CP. Supplements were provided four times weekly for 140d. Blood and BW were taken every 14d to monitor development. Every 56d, uterine flushes and rumen content were collected for bacterial identification. Uterine flushes were collected via 20 mL sterile saline flushed through a Foley catheter, and rumen content by esophageal tubing. Bacterial DNA was extracted and amplified targeting the V1-V3 region of the 16S rRNA gene. Microbiome analyses were performed in R 4.1 with dada2 and phyloseq packages. Statistical analyses were performed in R and SAS 9.4. The GLIMMIX procedure was used including fixed effects of protein, month, and pubertal status. Random effects included BW, interaction of BW and protein, and heifer within the interaction, with repeated measures of month. Alpha diversity differed by month in the rumen, and by month and pubertal status in the uterus (P < 0.05). Bray Curtis analyses indicated clustering by month and pubertal status (P < 0.001) in the uterus, and by month in the rumen (P < 0.001). Multiple uterine and ruminal bacteria abundances differed over time and by protein supplementation and pubertal status (P < 0.05). The impact of pubertal status and month of development may indicate maturation of the uterine bacterial communities through development. Protein supplementation affecting the uterine bacterial communities may provide opportunity to manipulate the uterine microbiome, potentially affecting future reproductive success.

Short-read assembly bins. A tab-delimited text file containing contig ID names, contig lengths, bin assignment, and read depth coverage for the short-read assembly MQ bins. (GZ 13035 kb)

The rumen microbiome provides approximately 70% of the required energy for the host by converting low-quality feedstuffs into usable energy for ruminants. The energy produced by the microorganisms is subsequently absorbed through the rumen epithelium and used towards growth and energy maintenance. There is evidence that ruminal epimural microbes directly interact with the rumen epithelium, acting as an intermediary communicator between the rumen liquid fraction and the host. Epimural microbiota have been demonstrated to be distinct from the ruminal liquid microbiome and perform unique roles within the rumen environment. However, methods to sample epimural communities from the rumen wall are limited and typically invasive, requiring animal fistulation or harvesting. To characterize the epimural communities present on the rumen wall, a novel and minimally-invasive surgical method was developed to swab the epithelium of the ventral sac of the rumen. The objective of this study was to validate this sampling method by comparing epimural and liquid fraction bacterial communities. During a 70-day feeding trial, Angus steers ( n = 45) were sampled on day 35 using the novel surgery method and tubed on day 70 to sample rumen liquid content. Genomic DNA was used to generate amplicon libraries of the V4 region of the 16S rRNA gene. There were no differences between alpha diversity indices when comparing rumen versus epimural bacterial communities ( P > 0.05). The Bray-Curtis dissimilarity was used to ordinate ASV counts, and then tested for differences between rumen and epimural communities using a PERMANOVA with 999 permutations ( P < 0.05). Differential abundances of bacterial communities were tested using ANCOM-BC and MaAsLin2, where significance was determined by Q < 0.05 and overlap between both analysis methods. Within the 91 taxa that differed in abundance, 451 ASVs were found to be different between sample types ( Q < 0.05). Unique ASVs associated with Prevotella , Succinivibrio , family-level Eubacterium , and family-level Succinivibrio were in greater abundance for the rumen epithelial-associated bacterial communities ( Q < 0.05). The results demonstrate that the novel method of sampling from the rumen wall can capture differences between epimural and ruminal fluid bacterial communities, thus facilitating studies investigating the interactions between epimural bacteria with the host.

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 > 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.

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.

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.