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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.

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.

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.

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.

Extremes in body condition reduce fertility and overall productivity in beef cattle herds, due in part to altered systemic metabolic conditions that influence the intrafollicular and uterine environment. Follicular fluid and serum metabolome profiles are influenced by body composition in women and dairy cattle; however, such information is lacking in beef cattle. We hypothesized that body condition score (BCS)-related alterations in the metabolome of preovulatory follicular fluid and serum may influence oocyte maturation while impacting the oviductal or uterine environment. Therefore, we performed a study with the objective to determine the relationship between BCS and the metabolome of follicular fluid and serum in lactating beef cattle. We synchronized the development of a preovulatory follicle in 130 cows of varying BCS. We collected blood and performed transvaginal follicle aspirations to collect follicular fluid from the preovulatory follicle ~18 h after gonadotropin-releasing hormone administration to stimulate the preovulatory gonadotropin surge. We then selected follicular fluid and serum samples from cows with BCS 4 (Thin; n = 14), BCS 6 (Moderate; n = 18), or BCS8 (Obese; n = 14) for ultra-high performance liquid chromatography-high resolution mass spectrometry. We identified differences in the follicular fluid or serum of thin, moderate, and obese animals based on multiple linear regression. MetaboAnalyst 5.0 was used for enrichment analysis of significant metabolites. We identified 38 metabolites in follicular fluid and 49 metabolites in serum. There were no significant differences in follicular fluid metabolite content among BCS classifications. There were 5, 22, and 1 serum metabolites differentially abundant between thin-obese, moderate-thin, and moderate-obese classifications, respectively (false discovery rate [FDR]0.10). These metabolites were enriched in multiple processes including "arginine biosynthesis," "arginine/proline metabolism," and "D-glutamine/D-glutamate metabolism" (FDR0.04). Pathways enriched with serum metabolites associated with BCS indicate potentially increased reactive oxygen species (ROS) in serum of thin cows. ROS crossing the blood follicular barrier may negatively impact the oocyte during oocyte maturation and contribute to the reduced pregnancy rates observed in thin beef cows.Extremes in body condition affect fertility and pregnancy outcomes in beef cows. Much research has been done in women and dairy cows to evaluate body condition’s effect on oocyte and embryo quality, pregnancy rates, and pregnancy outcomes. However, little work of this type has been done in beef cows and most studies do not focus on the preovulatory time period. The preovulatory time period is an essential time for the oocyte, as final stages of prematuration and the completion of oocyte maturation take place in the peri-ovulatory follicle. The follicular fluid provides the microenvironment for oocyte maturation and exchanges substances with maternal circulation at the blood follicular barrier. Alterations in maternal circulation due to extremes in body condition may pass into the follicular fluid and affect the oocyte during the preovulatory time period. Such conditions may contribute to the reduced fertility seen in beef cows with extreme body condition.

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.

The rumen is a complex organ that is critical for its host to convert low-quality feedstuffs into energy. The conversion of lignocellulosic biomass to volatile fatty acids and other end products is primarily driven by the rumen microbiome and its interaction with the host. Importantly, the rumen is demarcated into five distinct rumen sacs as a result of anatomical structure, resulting in variable physiology among the sacs. However, rumen nutritional and microbiome studies have historically focused on the bulk content or fluids sampled from single regions within the rumen. Examining the rumen microbiome from only one or two biogeographical regions is likely not sufficient to provide a comprehensive analysis of the rumen microbiome and its fermentative capacity. Rumen biogeography, digesta fraction, and microbial rumen–tissue association all impact the diversity and function of the entirety of the rumen microbiome. Therefore, this review discusses the importance of the rumen biographical regions and their contribution to microbiome variation.

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.

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 rumen microbiome is critical in ruminant nutrition and contributes to nutrient utilization and feed efficiency in cattle. Therefore, the objective of this study was to interrogate microbial and biochemical factors affecting divergences in feed efficiency in Angus steers using 16S amplicon sequencing and untargeted metabolomics. Average residual feed intake (RFI) was calculated, and steers were divided into low- and high-RFI groups. Features were ranked in relation to RFI through supervised machine learning on microbial and metabolite compositions. Residual feed intake was associated with several attributes of the rumen bacterial community. Low-RFI steers were associated with decreased bacterial α- (P=0.03) and β-diversity (P

The milk microbiome in swine has been recently demonstrated to develop and alter the growing gut microbiome in offspring. Milk is the primary nutritional source for piglets and the established microbial communities from initial diet remain stable over time, thus being critical for long-term effects on the animal. Since little is known about the ontogeny of the milk microbiome, the objective of this study was to determine differences in the sow milk microbiome among multiple parities and days in lactation. A total of 32 sows were grouped by parity, 1–4, with 8 sows per parity. Sows were on the same diet and had ad libitum access to water. At sampling, teats were cleaned and stripped. Milk samples were manually collected on one side from a fore, mid, and hind teat, then combined for one whole milk sample per animal. Milk was sampled on days 2 and 16 to determine differences between days in lactation. Samples were sequenced using an Illumina MiSeq for V1-V3 regions of the bacterial 16S rRNA gene and downstream analyses were conducted in the R environment using ‘phyloseq’ and ‘DADA2’ packages. Statistical analyzes utilized a completely randomized design and a mixed model analysis of variance, with fixed effects of parity and days in lactation and examining a parity by day interaction. The genera Corynebacterium were greater (P < 0.05) abundance in parity 4. There was an increase (P < 0.05) in Staphylococcus at day 2 compared to day 16. Core microbial communities, Lactobacillus and Romboutsia increased (P < 0.05) by day 16. Identifying microbial communities present in the milk microbiome in sows can allow producers to understand how these microbes may influence the gut microbiome in growing piglets to augment gain. USDA is an equal opportunity provider and employer.

Objectives Extensive efforts have been made to characterize the rumen microbiome under various conditions. However, few studies have addressed the long-term impacts of ruminal microbiome dysbiosis and the extent of host control over microbiome stability. These data can also inform host-microbial symbioses. The objective was to develop preliminary data to measure the changes that occur in the rumen bacterial communities following a rumen content exchange to understand the effects major perturbations may impart upon the rumen microbiome, which may be host-driven. Data description We report here an initial rumen content exchange between two SimAngus (Simmental/Angus) non-pregnant, non-lactating cows of ~ 6 years of age weighing 603.4 ± 37.5 kg. To measure bacterial community succession and acclimation following the exchange, rumen content was collected via rumen cannula at the beginning of the study immediately prior to and following the rumen content exchange, and weekly for 12 weeks. The V4 hypervariable region of the 16S rRNA gene was targeted for DNA sequencing and bacterial analysis. Over 12 weeks, numerous genera and diversity varied, before partial return to pre-exchange metrics. These preliminary data help support potential host control for the rumen microbiome, aiding in efforts to define bovine host-microbe relationships.

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.

Feed accounts for as much as 70% of beef production costs, and improvement of the efficiency with which animals convert feed to product has the potential to have substantial financial impact on the beef industry. The rumen microbiome plays a key role in determining feed efficiency, however, previous studies of rumen microbiota have not focused on protozoal communities despite the estimation that these organisms represent approximately 50% of rumen content biomass. Protozoal communities participate in the regulation of bacterial populations and nitrogen cycling—key aspects of microbiome dynamics. The present study focused on identifying potential associations of protozoal community profiles with feed efficiency. Weaned steers (n = 50) 7 months of age weighing approximately 260 kg were adapted to a growing ration and GrowSafe for 2 weeks prior to a 70-day feed efficiency trial. The GrowSafe system is a feeding system that monitors feed intake in real time. Body weights were collected on the first day and then every 7 days of the feed efficiency trial, and on the final day, approximately 50 mL of rumen content were collected via orogastric tubing and frozen at −80 °C. Body weight and feed intake were used to calculate residual feed intake (RFI) as a measure of feed efficiency, and steers were categorized as high (n = 14) or low (n = 10) RFI based on ±0.5 standard deviations about the mean RFI. Microbial DNA was extracted, and the eukaryotic component profiled by amplification and sequencing of 18S genes using degenerate primers that can amplify this locus across a range of protists. The taxonomy of protozoal sequences was assigned using QIIME 1.9 and analyzed using QIIME and SAS 9.4 with significance determined at α ≤ 0.05. Greater abundances of unassigned taxa were associated with high-RFI steers (p = 0.03), indicating a need for further study to identify component protozoal species. Differences were observed between low- and high-RFI steers in protozoal community phylogenetic diversity, including weighted beta-diversity (p = 0.04), Faith’s phylogenetic diversity (p = 0.03), and observed Operational taxonomic unit (OUT) (p = 0.03). The unassigned taxa and differences in phylogenetic diversity of protozoal communities may contribute to divergences observed in feed efficiency phenotypes in beef steers.

The current study characterized the taxonomic composition of the uterine and vaginal bacterial communities during estrous synchronization up to timed artificial insemination (TAI). Postpartum beef cows (n = 68) were subjected to pre-synchronization step 21 d prior to TAI (day −21), followed by an industry standard 7 Day Co-Synch on day −9 and TAI on day 0. Uterine and vaginal flushes were collected on days −21, −9, and −2 of the protocol and pH was immediately recorded. Pregnancy was determined by transrectal ultrasound on day 30. Bacterial DNA was extracted and sequenced targeting the V1 to V3 hypervariable regions of the 16S rRNA bacterial gene. Results indicated 34 different phyla including 792 different genera present between the uterus and vagina. Many differences in the relative abundance of bacterial phyla and genera occurred between resulting pregnancy statuses and among protocol days (P < 0.05). At day −2, multiple genera were present in >1% abundance of nonpregnant cows but 0.05). Overall, our study indicates bacterial phyla and genera abundances shift over time and may potentially affect fertility by altering the reproductive tract environment.

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.

Overly elevated circulating non-esterified fatty acids (NEFAs) is an emerging health concern of obesity-associated energy disorders. However, methods to reduce circulating NEFAs remain elusive. The present study determined the effect of piceatannol, a naturally occurring stilbene, on adipocyte lipolysis and its underlying mechanism. Differentiated 3T3-L1 adipocytes, brown adipocytes and isolated white adipose tissue were treated with various concentrations of piceatannol for 1.5-hr both in the basal and stimulated lipolysis conditions. Piceatannol significantly inhibited NEFAs and glycerol release with a concomitant reduction of ATGL, CGI-58 and PLIN1 expression in adipocytes. Using a series of inhibitor assays, piceatannol-induced degradation of these proteins was found to be mediated by upregulation of the autophagy-lysosome pathway. Moreover, we demonstrated that piceatannol is capable of stimulating autophagy in vitro. Importantly, piceatannol administration tended to lower fasting-induced serum glycerol levels in healthy mice. Furthermore, piceatannol administration lowered lipolysis, central adiposity and hyperinsulinemia in diet-induced obese mice. Our study provides profound evidence of a novel inhibitory role of piceatannol in lipolysis through autophagy-lysosome-dependent degradation of the key lipolytic proteins in adipocytes. This study offers a mechanistic foundation for investigating the potential of piceatannol-containing foods in reducing lipolysis and its associated metabolic disorders.

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 (BW) and randomly assigned to 1 of 3 treatments based on protein supplementation level: control of 10% crude protein (CON), 20% crude protein (P20), or 40% crude protein (P40). BW, body condition score, and blood samples were taken every 2 wk for 140 d 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 1 mo 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.

Bacteria in the reproductive tract were previously thought to be “bad” and it was believed that they decreased the chances of successful pregnancies. However, healthy bacteria have been found in the reproductive tracts of humans and cattle! These “good” bacteria help maintain a healthy reproductive tract by reducing the ability of “bad” bacteria to grow, helping immune cells to function properly, and maintaining an appropriate pH in the environment. Researchers have found that differences in the bacterial communities of the uterus can influence whether cattle get pregnant and give birth to healthy calves. Future studies may evaluate how we can modify these bacterial communities to create the best environment in the reproductive tract for better reproductive success.

Local immune activity in the reproductive tract is crucial in the response to uterine diseases, normal reproductive functions, and establishing pregnancy. Few studies have evaluated the influence of the local immune environment of the reproductive tract on fertility outcomes. The objectives were to 1) evaluate reproductive cytokine concentrations in postpartum cows undergoing estrus synchronization followed by timed artificial insemination (TAI) and 2) correlate reproductive bacterial communities with cytokine concentrations. 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. Bacterial community profiling and analyses were conducted targeting the V1 to V3 hypervariable regions of the 16S rRNA gene. Cytokine concentrations for interleukin (IL)-1b, IL-6, IL-10, and transforming growth factor beta (TGF-β) were determined by commercial ELISA kits. Concentration data were analyzed using PROC MIXED and correlations using Pearson correlation in SAS. No differences were detected in vaginal samples (P >0.05). No differences in IL-10 or IL-1b concentrations were detected in uterine samples (P >0.05). Uterine TGF-β concentrations were greater in resulting pregnant than non-pregnant cows (44.0 ± 13.4 pg/mL vs 14.7 ± 4.9 pg/mL; P = 0.05). Uterine TGF-β was negatively correlated with the relative abundance of genera Treponema (r = -0.668; P = 0.05) in resulting non-pregnant cows on d -21. Uterine IL-6 concentrations were greater in resulting non-pregnant than pregnant cows (198.7 ± 21.8 pg/mL vs 144.3 ± 16.1 pg/mL; P = 0.05). Uterine IL-6 and the relative abundance of genera Butyrivibrio were positively correlated (r = 0.742; P = 0.02) in resulting non-pregnant cows on d -21. These results suggest possible relationships between uterine bacterial communities and cytokines prior to TAI that may ultimately affect fertility outcomes in beef cattle.

Fescue toxicosis causes substantial financial losses to the beef industry due to its negative effects on reproduction, growth and feed efficiency. Recent research has demonstrated that the negative effects of fescue toxicosis may be mitigated by supplementation of red clover isoflavones. To determine impacts on the ruminal environment, the objective of this study was to evaluate the effect of red clover isoflavones and endophyte-infected tall fescue on ruminal metabolites. Thirty-six Angus steers weighing 250±20 kg were randomly assigned to treatments in a 2×2 factorial arrangement, consisting of endophyte-free or endophyte-infected tall fescue seed with or without red clover isoflavones. For the 21d trial, steers were provided a basal diet supplemented with fescue seed targeting a minimum of 0.011 mg × kg of BW−1 × d−1 of total alkaloids. A total of 943 mg isoflavones were administered daily via bolus. Following the trial, 50mL of rumen fluid was collected via orogastric tubing. Untargeted metabolomic analysis was performed using an Exactive Plus Orbitrap MS, and 10µL sample was introduced using electrospray ionization into the Dionex UltiMate 3000 UPLC system. Peaks were identified in MAVEN, then normalized and analyzed in MetaboAnalyst 4.0, and 106 known metabolites were identified. Differences in seed type × isoflavone were analyzed by one-way ANOVA and main effects were analyzed by t-test. A total of 11 metabolites differed based on seed type × isoflavone treatment group (P ≤ 0.05), 11 differed by seed type (P ≤ 0.5), and seven differed by isoflavone treatment (P ≤ 0.05), namely amino acids or intermediates of amino acid metabolism. Methane (P = 0.03) and sulfur (P = 0.03) metabolism pathways were both impacted by isoflavone treatment, whereas 20 metabolic pathways were impacted as a result of differences in seed type (P ≤ 0.05). The rumen metabolome appears to be more affected by seed type, but a longer trial will likely result in more profound impacts.

Beef cattle is the most consumed red meat in the United States, and the United States is the largest producer and consumer of beef cattle globally. Feed is one of the largest input costs for the beef cattle industry, accounting for 40–60% of the total input costs. Identifying methods for improving feed efficiency in beef cattle herds could result in decreased cost to both producers and consumers, as well as increased animal protein available for global consumption. Identification of feed efficiency biomarkers could aid in the selection of more feed-efficient animals. To identify potential biomarkers of feed efficiency, rumen fluid was collected from low- (n = 14) and high-RFI (n = 15) Angus steers based on RFI ± 0.5 SD around the mean following a 70-d feed efficiency trial to determine rumen metabolites associated with variations in feed efficiency. Steers were 7 mo of age, weighing 264±2.7 kg. Rumen fluid was collected via gastric tubing, filtered through a 0.22 µM syringe filter, extracted for metabolomic analysis using 0.1% formic acid in acetonitrile:water:methanol (2:2:1), and injected into the Dionex UltiMate 3000 UHPLC system with an Exactive Plus Orbitrap MS. Peaks for known metabolites were identified using MAVEN and analyzed using MetaboAnalyst 4.0 and SAS. Significance was determined using an α ≤ 0.05. Based on fold change (high/low RFI), eight metabolites were greater in low-RFI steers compared to high-RFI steers, including 3,4-dihydroxyphenylacetate, 4-pyridoxate, citraconate, hypoxanthine, succinate/methylmalonate, thymine, uracil, and xylose (P ≤ 0.05), while 12 metabolites differed by ≥ 2-fold. These metabolites were predominantly involved in amino acid and lipid metabolism. Rumen fluid metabolomes differed in steers of varying feed efficiencies. These metabolites may be used as biomarkers of feed efficiency and may provide insight as to factors contributing to differences in feed efficiency that may be exploited to improve feed efficiency in beef cattle herds.

Fescue toxicosis impacts beef cattle production via reductions in weight gain and muscle development. Isoflavone supplementation has displayed potential for mitigating these effects. The objective of the current study was to evaluate isoflavone supplementation with fescue seed consumption on rumen and serum metabolomes. Angus steers (n = 36) were allocated randomly in a 2 &times, 2 factorial arrangement of treatments including endophyte-infected (E+) or endophyte-free (E&minus, ) tall fescue seed, with (P+) or without (P&minus, ) isoflavones. Steers were provided a basal diet with fescue seed for 21 days, while isoflavones were orally administered daily. Following the trial, blood and rumen fluid were collected for metabolite analysis. Metabolites were extracted and then analyzed by UPLC-MS. The MAVEN program was implemented to identify metabolites for MetaboAnalyst 4.0 and SAS 9.4 statistical analysis. Seven differentially abundant metabolites were identified in serum by isoflavone treatment, and eleven metabolites in the rumen due to seed type (p &lt, 0.05). Pathways affected by treatments were related to amino acid and nucleic acid metabolism in both rumen fluid and serum (p &lt, 0.05). Therefore, metabolism was altered by fescue seed in the rumen, however, isoflavones altered metabolism systemically to potentially mitigate detrimental effects of seed and improve animal performance.

Beef is the most consumed red meat in the United States, and the US is the largest producer and consumer of beef cattle globally. Feed is one of the largest input costs for the beef cattle industry, accounting for 40–60% of the total input costs. Identifying methods for improving feed efficiency in beef cattle herds could result in decreased cost to both producers and consumers, as well as increased animal protein available for global consumption. In this study, rumen fluid was collected from low- (n = 14) and high-RFI (n = 15) steers. Rumen fluid was filtered through a 0.22 µM syringe filter, extracted using 0.1% formic acid in acetonitrile:water:methanol (2:2:1) and injected into the Dionex UltiMate 3000 UHPLC system with an Exactive Plus Orbitrap MS. Peaks were identified using MAVEN and analyzed using MetaboAnalyst 4.0 and SAS. Significance was determined using an α ≤ 0.05. Eight metabolites were greater in low-RFI steers compared to high-RFI steers, including 3,4-dihydroxyphenylacetate, 4-pyridoxate, citraconate, hypoxanthine, succinate/methylmalonate, thymine, uracil, and xylose (P ≤ 0.05). These metabolites were predominantly involved in amino acid and lipid metabolism. Rumen fluid metabolomes differ in steers of varying feed efficiencies. These metabolites may be used as biomarkers of feed efficiency, and may provide insight as to factors contributing to differences in feed efficiency that may be exploited to improve feed efficiency in beef cattle herds.

Surveys of microbial populations in environmental niches of interest often utilize sequence variation in the gene encoding the ribosomal small subunit (the 16S rRNA gene). Generally, these surveys target the 16S genes using semi-degenerate primers to amplify portions of a subset of bacterial species, sequence the amplicons in bulk, and assign to putative taxonomic categories by comparison to databases purporting to connect specific sequences in the main variable regions of the gene to specific organisms. Due to sequence length constraints of the most popular bulk sequencing platforms, the primers selected amplify one to three of the nine variable regions, and taxonomic assignment is based on relatively short stretches of sequence (150-500 bases). We demonstrate that taxonomic assignment is improved through reduced unassigned reads by including a survey of near-full-length sequences specific to the target environment, using a niche of interest represented by the upper respiratory tract (URT) of cattle. We created a custom Bovine URT database from these longer sequences for assignment of shorter, less expensive reads in comparisons of the upper respiratory tract among individual animals. This process improves the ability to detect changes in the microbial populations of a given environment, and the accuracy of defining the content of that environment at increasingly higher taxonomic resolution.

The beef cattle industry represents a significant portion of the USA's agricultural sect, with beef cattle accounting for the most red meat consumed in the USA. Feed represents the largest input cost in the beef industry, accounting for approximately 70% of total input cost. Given that, novel methods need to be employed to optimize feed efficiency in cattle to reduce monetary cost as well as environmental cost associated with livestock industries, such as methane production and nitrogen release into the environment. The rumen microbiome contributes to feed efficiency by breaking down low-quality feedstuffs into energy substrates that can subsequently be utilized by the host animal. Attempts to manipulate the rumen microbiome have been met with mixed success, though persistent changes have not yet been achieved beyond changing diet. Recent technological advances have made analyzing host-wide effects of the rumen microbiome possible, as well as provided finer resolution of those effects. This manuscript reviews contributing factors to the rumen microbiome establishment or re-establishment following rumen microbiome perturbation, as well as host-microbiome interactions that may be responsible for possible host specificity of the rumen microbiome. Understanding and accounting for the variety of factors contributing to rumen microbiome establishment or re-establishment in cattle will ultimately lead to identification of biomarkers of feed efficiency that will result in improved selection criteria, as well as aid to determine methods for persistent microbiome manipulation to optimize production phenotypes.

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). 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.

This review highlights concerns with endophyte-infected tall fescue as a primary forage base in the southeastern United States and discusses specific physiological and ruminal effects caused by consumption of ergot alkaloids. Additionally, in an effort to promote various mitigation strategies to abate production limitations caused by fescue toxicosis, this review discusses the use of cool-season legumes, specifically red clover, with the already established forage base. Clovers are often mixed into pastures and help improve animal performance. Clovers contain phytoestrogenic compounds known as isoflavones that may be beneficial in reducing physiological limitations with consumption of endophyte-infected tall fescue.

Fescue toxicosis reduces animal performance, costing approximately $2 billion dollars to the beef industry annually. Emerging research has demonstrated effects of fescue toxicosis can be reduced by consumption of red clover isoflavones. The objective of the current study was to evaluate the effect of isoflavone supplementation with endophyte infected seed consumption on serum metabolites in beef steers. Angus steers (n = 36) were randomly allocated to treatments in a 2×2 factorial arrangement of endophyte-infected (E+) or endophyte-free (E-) tall fescue seed, with (P+) or without (P-) a red clover isoflavone supplement. For the 21d trial, steers were provided a basal diet supplemented with fescue seed head targeting a minimum of 0.011 mg×kg of body weight−1×d−1 of total alkaloids. A total of 943 mg isoflavones were administered daily via bolus. Following the 21d trial, blood samples were collected for metabolite analysis. Metabolites were filtered from serum and extracted using 0.1% formic acid in acetonitrile:water:methanol (2:2:1) for analysis on the Dionex UltiMate 3000 UHPLC system and Exactive Plus Orbitrap MS. The Metabolomic Analysis and Visualization Engine program was used to determine peaks and identify metabolites. Resulting metabolite data were analyzed in MetaboAnalyst 4.0 and SAS 9.4 with significance at P≤0.05. Principle component analysis indicated separation of metabolomes between E+P+ and E-P- steers. Orthogonal partial least squares discriminant analysis depicted distinct separation between P+ and P- steers and partial separation between E+ and E- steers. The variation between P+ and P- metabolomes were mainly due to differences in citrulline (r = 0.47, P = 0.003), and AMP (r = -0.35, P = 0.03) between E+ and E- metabolomes. A total of 13 and 8 metabolic pathways were impacted from differences in seed type and isoflavone treatment, respectively (P ≤ 0.05). Therefore, metabolism is altered by isoflavone supplementation that may improve animal performance during fescue toxicosis.

Numerous factors impact reproductive success in beef cattle; however, the presence and activity of bacteria in the reproductive tract and its effects on fertility is relatively unknown. The objective of this study was to evaluate the relationship between reproductive tract bacterial communities and cytokine profiles prior to timed-AI (TAI) and compare between resulting pregnancy status at d30. Sixty-eight beef cows, 80 ± 2.6 d postpartum at TAI, were synchronized and subjected to TAI on d0. Pregnancy was diagnosed via transrectal ultrasonography on d30. Uterine and vaginal flushes were collected on d -21 and -2 for bacterial DNA extraction to sequence the V1-V3 hypervariable regions of the 16S rRNA gene and to measure pro-inflammatory [interlukin-6 (IL-6)] and anti-inflammatory [IL-10 and transforming growth factor-beta (TGFβ)] cytokine concentrations using validated commercial ELISAs. Concentration data were analyzed using repeated measures in PROC MIXED of SAS and correlations were performed in SAS using Pearson correlation. There were no differences in cytokine concentrations by day or status x day. There were no differences in uterine IL-6 or IL-10 concentrations (P > 0.05). Uterine TGFβ concentrations were overall elevated in pregnant cows compared to open cows (72.6 vs. 13.7 ± 12.1 pg/mL, respectively; P < 0.05). On d -2, a positive correlation exists between TGFβ and the relative abundance of Actinobacteria in the uterus of pregnant cows (r=0.93, P = 0.02). There were no differences in vaginal IL-6 or TGFβ concentrations (P > 0.05). On d -2, vaginal IL-10 concentrations were elevated in open cows compared to pregnant cows (741.6 vs. 602.1 ± 47.0 pg/mL, respectively; P < 0.05) and a positive correlation exists between IL-10 and the relative abundance of Lentisphaerae in the vagina of pregnant cows (r=0.89, P = 0.04). These data suggest a possible relationship between bacterial communities and cytokines concentrations within the reproductive tract of beef cattle which may affect fertility.

Biochar, a byproduct from the biofuels industry, may be a potential feed additive in ruminant diets due to possible improvements in microbial fermentation. Therefore, the objective of this study was to determine the nutritive value, in vitro digestibility, volatile fatty acid (VFA) production, and gas production of biochar inclusion to an orchard grass (Dactylis glomerata) basal diet. The study was designed as a 3 × 2 factorial arrangement with 3 different biochar sources and 2 biochar processed sizes as the main effects factors. Experimental treatments were biochar from 3 different tree types: 1) Chestnut Oak (Quercus prinus L.; CO), 2) Yellow Poplar (Liriodendron tulipifera; YP), or 3) White Pine (Pinus strobus L.; WP), and processed at 2 different biochar particle sizes: a) 178 μm (Coarse). Biochar was added to the basal diet of orchard grass hay (872.35 g/kg of DM, 98.31 g/kg of CP, and 704.02 g/kg of aNDF, DM basis) at a rate of 81 g/kg DM. Biochar residual ash content was greater (P P P = 0.01) with lower aNDF content in both WP sizes compared with their respective biochar type and size. Gas production was not influenced (P = 0.23) by biochar tree type; however, gas production was increased (P = 0.05) by Fine particle size compared with Coarse biochar. The in vitro true digestibility (IVTD) of orchard grass hay was increased (P = 0.01) by the inclusion of Fine biochar particle size compared with Coarse particle size. Additionally, in vitro CP true digestibility (DCP) exhibited a type × size interaction (P = 0.01). Crude protein digestibility was lower for Fine particle-sized CO and WP biochar sources compared with Coarse particle-sized CO and WP (P ≤ 0.004). However, DCP was not different between Coarse and Fine particle-sized YP biochar (P = 0.70). Volatile fatty acids (acetate, propionate, and butyrate) were not altered by biochar type (P ≥ 0.66) or particle size (P ≥ 0.19). These results indicate that both tree type and particle size of biochar may need to be carefully considered before incorporating into a ruminant diet. Furthermore, Fine particle-sized biochar may be the most effective to incorporate as a feed additive in a ruminant diet based on digestibility parameters.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13059-019-1760-x) contains supplementary material, which is available to authorized users.

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 P = 0.005) and predictive of the rumen microbiome signature at week 10 (R2 = 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.

The mutualistic, commensal, and parasitic microorganisms that reside in the rumen and lower gastrointestinal tract of cattle and other ruminants exert enormous influence over animal physiology and performance. Because these microbial communities are critical for host nutrient utilization and contribute to the metabolic capacity of the rumen, past research has aimed to define host-microbe symbioses in cattle by examining the rumen and lower gut microbiomes with respect to production phenotypes, such as feed efficiency., The mutualistic, commensal, and parasitic microorganisms that reside in the rumen and lower gastrointestinal tract of cattle and other ruminants exert enormous influence over animal physiology and performance. Because these microbial communities are critical for host nutrient utilization and contribute to the metabolic capacity of the rumen, past research has aimed to define host-microbe symbioses in cattle by examining the rumen and lower gut microbiomes with respect to production phenotypes, such as feed efficiency. However, as the field of bovine gut microbial ecology progresses, multidisciplinary approaches must be employed, combining host genomics and other omics-based techniques to understand the complex host-microbe network. In this perspective, I discuss the direction of the field of bovine gut microbial ecology with regard to feed efficiency and explore how the grand challenge of such research will be to maintain host-efficient gut microbiomes in cattle production through manipulations of genome-microbiome interactions.

Studying abroad in agricultural biosciences can develop students' cultural, academic, and communication skills and enhance employability. However, in the United States, discussions of study abroad are limited to either one-way directionality (U.S. students to other countries) or to the experiences of international students who come to the United States for degree programs. We analyzed the perspectives and experiences of studying abroad by Zamorano University (Honduras) students who completed an agricultural bioscience pasantía (four-month internship) during the final year of their undergraduate program. We used mixed methods to collect data via focus group discussions and a survey with Zamorano students who had completed a pasantía in 2017, as well as key informant interviews with Zamorano pasantía coordinators. Study-abroad experiences were compared between students who completed their pasantía in the United States (37%) and those who completed their pasantía in any of 17 other countries worldwide. Significant relationships were identified (Estudiar en el extranjero puede desarrollar habilidades culturales, académicas y de comunicación en los estudiantes de biociencias agrícolas, sobre todo potencializar la empleabilidad. Sin embargo, las discusiones en los Estados Unidos sobre estudios en el extranjero son limitadas a vías unidireccionales (estudiantes de EE.UU. hacia otros países) o las experiencias de estudiantes internacionales durante sus estudios superiores en los Estados Unidos. Analizamos la perspectiva y experiencia de estudiar en el extranjero con estudiantes de la Universidad Zamorano (Honduras), quienes completaron una pasantía en biociencias agrícolas durante su último año del programa de pregrado. Utilizamos métodos mixtos para obtener datos a través de entrevistas con los coordinadores de las pasantías en Zamorano, discusión de grupos focales y encuestas a los estudiantes de Zamorano quienes habían completado su pasantía en el 2017. Se compararon estudios en el exterior entre estudiantes que completaron su pasantía en Estados Unidos (37%) contra aquellos que completaron su pasantía en cualquiera de los otros 17 países del mundo. Se identificaron relaciones significativas (P0.05) entre: realizar una pasantía en Estados (comparada a realizarla en cualquier otro lugar) y el aumento del conocimiento teórico y científico; teniendo experiencias prácticas y mejoras en la ciencia y la teoría; mejoras en la comunicación y la confianza en el uso de métodos científicos. El modelo de pasantía de Zamorano brinda información sobre el valor de estudios sobre biociencias agrícolas en el extranjero.

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.

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long "circular consensus" reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good's coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing.

Amplicon sequencing utilizing next-generation platforms has significantly transformed how research is conducted, specifically microbial ecology. However, primer and sequencing platform biases can confound or change the way scientists interpret these data. The Pacific Biosciences RSII instrument may also preferentially load smaller fragments, which may also be a function of PCR product exhaustion during sequencing. To further examine theses biases, data is provided from 16S rRNA rumen community analyses. Specifically, data from the relative phylum-level abundances for the ruminal bacterial community are provided to determine between-sample variability. Direct sequencing of metagenomic DNA was conducted to circumvent primer-associated biases in 16S rRNA reads and rarefaction curves were generated to demonstrate adequate coverage of each amplicon. PCR products were also subjected to reduced amplification and pooling to reduce the likelihood of PCR product exhaustion during sequencing on the Pacific Biosciences platform. The taxonomic profiles for the relative phylum-level and genus-level abundance of rumen microbiota as a function of PCR pooling for sequencing on the Pacific Biosciences RSII platform were provided. For more information, see “Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers” P.R. Myer, M. Kim, H.C. Freetly, T.P.L. Smith (2016) [1]. Keywords: 16S rRNA gene, MiSeq, Pacific Biosciences, Rumen microbiome

Research regarding the association between the microbial community and host feed efficiency in cattle has primarily focused on the rumen. However, the various microbial populations within the gastrointestinal tract as a whole are critical to the overall well-being of the host and need to be examined when determining the interplay between host and nonhost factors affecting feed efficiency. The objective of this study was to characterize the microbial communities of the jejunum among steers differing in feed efficiency. Within 2 contemporary groups of steers, individual ADFI and ADG were determined from animals fed the same diet. At the end of each feeding period, steers were ranked based on their standardized distance from the bivariate mean (ADG and ADFI). Four steers with the greatest deviation within each Cartesian quadrant were sampled ( = 16/group; 2 groups). Bacterial 16S rRNA gene amplicons were sequenced from the jejunum content using next-generation sequencing technology. The phylum Firmicutes accounted for up to 90% of the populations within all samples and was dominated by the families Clostridiaceae and Ruminococcaceae. UniFrac principal coordinate analyses did not indicate any separation of microbial communities within the jejunum based on feed efficiency phenotype, and no significant changes were indicated by bacterial diversity or richness metrics. The relative abundances of microbial populations and operational taxonomic units did reveal significant differences between feed efficiency groups ( < 0.05), including the phylum Proteobacteria ( = 0.030); the families Lachnospiraceae ( = 0.035), Coriobacteriaceae ( = 0.012), and Sphingomonadaceae ( = 0.035); and the genera ( = 0.019), ( = 0.018), and ( = 0.022). The study identified jejunal microbial associations with feed efficiency, ADG, and ADFI. This study suggests the association of the jejunum microbial community as a factor influencing feed efficiency at the 16S level.