Publications

With an expected increase in population by 2050, the demand for high-quality protein for human consumption will increase. To improve the sustainability of US cattle production, producers and researchers have historically focused on improving the feed efficiency of steers through improvements in genetics, nutrition, and microbiome management, producing a more marketable beef product. However, without a successful pregnancy, there would be no marketable animal to feed. The objective of this study was to examine the impact of pregnancy on the rumen environment, microbial communities, and the correlation to feed efficiency status in Angus heifers. Utilizing 17 cannulated Angus heifers, feed efficiency status was determined using the GrowSafe 8000 feed intake monitoring system to monitor feed intake for a 70d trial period to calculate residual feed intake. Following the trial, heifers were bred and 40mL rumen fluid and content samples were collected every two weeks during gestation. Metagenomic DNA was extracted from the rumen samples, sequencing libraries were prepared targeting the bacterial V4 region of the 16S rRNA gene, and resultant libraries were sequenced on an Illumina MiSeq. All microbial analyses were conducted in the R environment v2.6.2 where alpha diversity, beta diversity, and differential abundances were determined. Metabolites were identified using ultra high-performance liquid chromatography high resolution mass spectrometry, and Metabolomic Analysis and Visualization Engine was used to select metabolite peaks. Differential abundance analyses using “ANCOMBC” indicated 10 amplicon sequence variants (ASVs), assigned to seven Prevotella and three Succiniclasticum , significantly varied among weeks of gestation ( P <0.05). Based on a one-way ANOVA, 90 metabolites were determined significant throughout pregnancy ( P <0.05). The 10 metabolites with the highest variable importance of projection scores from a partial least squares discriminant analysis were used to calculate correlations between. Of the 10 ASVs and 10 metabolites, significant correlations with feed efficiency status were found at various time points ( P <0.05). The rumen microbiome and its fermentative profile were different at various time points during pregnancy with several correlations to feed efficiency status. Identifying pregnancy-related microbial and metabolite changes may aid producers to anticipate nutritional or management adjustments, ultimately contributing to the sustainability of beef production.

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.

Replacement heifer development is one of the most critical components of productive beef operations. By identifying the ideal uterine environment in pre-breeding heifers, fertility and reproductive efficiency can be maximized. Our hypothesis was that different levels of protein supplementation would affect the rate of development and uterine environment of beef heifers. To test the effects of dietary supplementation on these outcomes, a randomized complete block design with repeated measures was implemented. Commercial Angus heifers (n = 60) were blocked by BW into 4 weight classes. Within each weight class, three pens of 5 heifers each were randomly assigned to one of three treatment groups of supplemental protein [10% (control), 20% (P20), and 40% (P40)]. All heifers were allowed access to ad libitum grass hay. Bodyweight and BCS were collected every 2 weeks to monitor heifer development. Uterine fluid samples were collected monthly by flushing with sterile saline to evaluate components of the uterine environment. Relative uterine pH was calculated by subtracting saline pH from the uterine flush pH. Separate mixed model ANOVAs and mean separation via PROC GLIMMIX (SAS 9.4, Cary, NC) were used to determine if protein supplementation treatments, time and their interactions influenced BW, BCS, or uterine environment. Bodyweight of the control group was significantly lower (P < 0.05) than P20 or P40 groups from December to March. There was an interaction between treatment and time for relative uterine pH (P = 0.002). In November, the P40 group exhibited less change in pH compared to the control group (0.33±0.1 vs. 0.74±0.1; P < 0.05). Whereas in December the P40 (0.23±0.1) group showed more basic uterine environment compared to the P20 (-0.22±0.1) or control (-0.13±0.1) groups (P < 0.05). In conclusion, higher protein supplementation does improve heifer growth and development, as well as altering the pH of the uterine fluid from weaning to first breeding

Long-read assembly Prodigal ORF predictions. This file contains all Prodigal ORF predictions for the long-read MQ bins. (GZ 18904 kb)

Short-read unique rumen assembly sequence. A listing of the short-read contigs that did not have reciprocal alignments to the Hungate1000 or Stewart et al. rumen microbial assemblies. (GZ 17072 kb)

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 <1% abundance in pregnant cows (P < 0.05). Uterine pH increased in nonpregnant cows but decreased in pregnant cows (P > 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.

Feed efficiency is an important factor for improving the sustainability and economic value of beef production. Although feed efficiency is moderately heritable, it is a complex trait affected by numerous factors such as diet, the rumen microbiome, and energy metabolism. Recent work has examined the molecular control of cattle divergent in feed efficiency; however, limited work has focused on the transcriptomic regulation of variation in feed efficiency, specifically, the genes contributing to muscle growth and the variation in feed efficiency in beef production. The hypothesis of this study was that cattle with greater feed efficiency will have increased expression of genes that promote muscle growth and energy metabolism. A total of 100 Angus steers weighing approximately 400 kg were enrolled in 70 d feed efficiency trials to determine residual feed intake (RFI) using the Vytelle GrowSafe 8000 individual feed intake monitoring system. At mid-test, 500-mg of muscle tissue was collected from the wing of the ileum between the axis and transverse processes of the lumbar vertebrae and was punched with a biopsy probe tool. The collected biopsy samples were flash frozen in liquid nitrogen for storage until processing. RNA was extracted and 3’ RNA sequencing was conducted on the Novaseq 6000 to determine the differential skeletal muscle transcriptomes of steers differing in RFI. Differential expression analysis was performed with the DESeq2 R package. To determine connections between the differentially expressed genes identified via transcriptional profiling and RFI, gene expression results were examined for correlations. To visualize the correlation matrices, the Corrplot package in R (version 3.4) was utilized. Varying numbers of differentially expressed gene transcripts were detected between steers divergent in RFI (P &amp;lt; 0.05), and were identified with significant correlations to RFI (P &amp;lt; 0.05). Specifically, the genes identified were related to muscle growth and energy metabolism. These results further define the molecular control of muscle growth with regard to RFI, and the opportunity to improve feed efficiency and muscle growth while meeting the needs for food production and sustainability for the growing population.

To improve animal performance and modify growth by increasing lean tissue accretion, beef cattle production has relied on use of growth promoting technologies such as beta-adrenergic agonists. These synthetic catecholamines, combined with the variable inclusion of rumen degradable (RDP) and undegradable protein (RUP), may improve feed efficiency and rate of gain in finishing beef cattle. However, the impact of beta-adrenergic agonists and protein level and source on the ruminal microbiome is limited. The objective of this study was to determine the effect of different protein concentrations and beta-adrenergic agonist (ractopamine hydrochloride; RAC) on ruminal bacterial communities in finishing beef heifers. Heifers (n = 140) were ranked according to body weight and assigned to pens in a randomized complete design to 6 different treatments, containing 3 protein treatments (Control: 13.9% CP, 8.8% RDP, and 5.0% RUP; High RDP: 20.9% CP, 13.4% RDP, 6.1% RUP; or High RUP: 20.9% CP, 9.1% RDP, 10.4% RUP) and 2 RAC treatments (0 and 400 mg/day). Rumen samples were collected from heifers by oral lavage 7 days before harvest. The DNA from the samples were sequenced to identify bacteria based on the V1-V3 hypervariable regions of the 16S rRNA gene using the Illumina MiSeq. Sequences and data from the treatments were analyzed using the R environment and PROC MIXED in SAS 9.4 (SAS Inst.; Cary, NC). Beta diversity was analyzed using PERMANOVA based on PCoA Bray-Curtis distances and were significant among the treatments (P &amp;lt; 0.05). Alpha diversity metrics such as Chao1 and Shannon diversity indices were not significantly different (P &amp;gt; 0.05). Differences among treatments at variable taxonomic levels after analyses through DESeq2 were significantly different for the main effects of protein and RAC, as well as their interaction (P &amp;lt; 0.05). These results suggest possible effects on the microbial communities, potentially acting synergistically to improve performance.

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 BCS >8 (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" (FDR < 0.04). 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.

Feedlot cattle may be subjected to digestive disorders, including ruminal acidosis, due to high concentration of grain in their diet. Therefore, novel feeding strategies are required to maximize animal performance and mitigate economic losses in the operation. This study employed a two-period crossover design to assess the effect of direct ruminal administration of native rumen microorganisms (NRM) inoculation on cattle that underwent a high-grain challenge. The NRM inoculation consisted of six microorganisms (1.70 M CFU /day/animal) isolated from the rumen of healthy feedlot cattle: Succinivibrio dextrinosolvens ASCUSBF53, Prevotella albensis ASCUSBF41, Chordicoccus furentiruminis ASCUSBF65, Bacteroides xylanisolvens ASCUSBF52, Clostridium beijerinckii ASCUSBF26, and Syntrophococcus sp. ASCUSBF60. The trial consisted of 16 Angus heifers receiving NRM (n = 8) or a CON (CON = Carrier Buffer; n = 8) inoculation daily for 14-days as pre-challenge while on a high-grain diet and continued daily for a 21-day treatment period. The combined 35 days of microbial supplementation resulted in an improved average daily gain (ADG) of 29% (P = 0.037) and a tendency toward a 19% decrease in the feed efficiency metric, gain to feed ratio (G: F) (P = 0.055). Additionally, administration of NRM to animals on a high-grain diet, improved ruminal microbiome stability (P < 0.001), potentially encouraging the conversion of rumen lactate to propionate over time via the succinate pathway and alleviating metabolic stress.

ABSTRACT Effective science education relies on innovative and engaging teaching tools that enhance student learning and retention. This need is particularly important in agricultural science outreach, where understanding the role of microbiology in animal health and disease is essential for both livestock producers and the public. However, traditional teaching methods often fail to adequately convey complex concepts in an accessible, applicable, and memorable way. To address this gap, a memory-based card game was developed utilizing game-based learning (GBL) principles to improve comprehension of microbiology’s impact on animal health and disease within agricultural systems. This interactive tool balances educational content with gameplay, fostering an engaging learning experience that encourages knowledge retention and real-world application. Through the use of the game, pre- and post-self-assessments indicated learners perceived they were improved in their understanding of microbes in agriculture and noted their greater likelihood to use this information in real-world situations ( P &lt; 0.001). By integrating this game into extension and outreach programs, we aim to enhance public understanding of agricultural systems and microbiology’s role in animal health, ultimately improving informed decision-making and agricultural literacy. This approach not only advances microbiology education but also exemplifies the pedagogical benefits of incorporating GBL into agricultural science communication.

Long-read unique rumen assembly sequence. A listing of the long-read contigs that did not have reciprocal alignments to the Hungate1000 or Stewart et al. rumen microbial assemblies. (GZ 9 kb)

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.

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.