Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
(N/A)
Non Technical Summary
Beef cattle vary in how efficiently they convert feed into energy, which affects the viability and competitiveness of US beef production. While microbes in the rumen, the stomach of cattle, play a key role in this process through fermentation of feed, little is known about the specific microbes attached to the rumen wall and how they interact with the animal's tissue to influence its nutrient absorption. Understanding this relationship is essential for improving feed efficiency and supporting US beef production. The overarching hypothesis of this project is that the genetics of cattle affect both the cattle cells at the rumen wall and the microbes that live there. In turn, the microbes and the cattle cells influence each other's gene activity, which helps control how nutrients are used at the rumen wall. To test the overarching hypothesis of this project, the specific objectives include, 1) Identify the different cell types in the rumen wall and examine how their gene activity is related to feed efficiency in beef cattle, 2) Determine the types and functions of active microbes attached to the rumen wall and how they are connected to gene activity in the rumen tissue, and 3) Identify genetic markers in cattle that are linked to both gene activity in the rumen wall and the makeup of the microbial community. At the completion of this project, we expect to have a comprehensive understanding of how different rumen wall cell types and their gene activity interact with wall-associated microbes and cattle genetics to influence feed efficiency. This project will provide critical new knowledge that can be used to develop targeted breeding strategies and microbial interventions aimed at improving feed efficiency in beef cattle. The findings will support enhanced cattle management practices, advance genetic improvement efforts, and ultimately help to strengthen US beef production and US agriculture.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The overall goal in this application is to determine the genetic and microbial factors influencing nutrient absorption and utilization at the rumen wall to enhance feed efficiency and sustainability in beef cattle. We will pursue the following objectives:1: Identify cell populations, cell type-specific gene expression in the rumen epithelium, and their relation to feed efficiency and sustainability in beef cattle.2: Determine the abundance and function of active rumen microorganisms that are adherent to the rumen wall and their respective correlation to the epithelial gene expression. 3: Associate host genomic markers to rumen epithelial gene expression and microbial communities.
Project Methods
Objective #1: Identify cell populations, cell type-specific gene expression in the rumen epithelium, and their relation to feed efficiency and sustainability in beef cattle.Task 1 - Determine Feed Efficiency and Sustainability Measurements in Finishing Steers Management, Diet, and Feed Efficiency Trial: Steers will be managed per the University of Tennessee Plateau Research and Education Center in Crossville, TN (PREC) standards. A total of 100 Angus steers weighing approximately 450 kg will be sourced from PREC, where the study will be conducted. The feed efficiency phenotyping will be conducted where n = 100 steers will be enrolled in feed efficiency trials. To limit possible environmental impacts of the rumen microbiome, (e.g., management practices, diet, and location), steers will be kept within the same feedlot at the PREC. Steers will be fed a typical finishing beef cattle diet (Tennessee Farmer's Cooperative). After acquisition, steers will be fed a step-up ration for 21 days prior to the feed efficiency trial, where they will be acclimated to the diet, feeding bunks, and the C-Lock SmartFeed Pro (C-Lock Inc., Rapid City, SD, USA). Following, steers will be enrolled into a 70-day feed efficiency trial with feed intakes measured using the SmartFeed Pro (C-Lock, Inc., Rapid City, SD, USA). While also using C-Lock SmartScales (C-Lock Inc., Rapid City, SD, USA) for greater resolution body weight (BW) changes, BW will be taken consistently throughout the trial to determine start, mid-trial, and final weights. Blood samples will be collected from steers during BW measurement throughout the trial to determine serum metabolite profiles, host genotypes, and serum VFA concentrations. At the end of the 70-day feeding trial steers will be ranked by residual feed intake (RFI) based on performance and feed intake measured from day 0 to day 70. The average value of RFI (actual dry matter intake (DMI) vs. expected DMI) and standard deviation will be calculated for individual animals and divided into groups of low, mid, and high RFI. High RFI will be defined as RFI ≥ 1.5 SD above the mean; and Low RFI as RFI ≤ 1.5 SD below the mean.Tissue Collection: All steers (n=100) will undergo tissue collection. However, based on RFI status during the trial, approximately 50 total steers from the high and low feed efficient groups (~25 steers each) will be used for analyses. On day 70 of the trial, rumen epithelial samples will be taken. The biopsies will be taken following a procedure validated by the PIs (USDA-AFRI grant no. 2020-67015-30832). Briefly, the epimural communities and tissue present on the rumen wall will be collected using a novel and minimally-invasive surgical method to biopsy the epithelium of the ventral sac of the rumen.Task 2 - Isolate Cells for Single-cell RNAseq Rumen Epithelial Cell Dissociation: Tissues excised from rumen epithelia will be washed for rumen epithelial-associated microbiota and then immediately stored in cold MACS Tissue Storage. Live cells after filtering, disassociation, and digestion will undergo library preparation, generating a single-cell library for every animal. Samples of pooled libraries will be sequenced.Identification of Cell Types: Reads will be aligned to the ARS-UCD1.3 bovine reference genome. Downstream analyses including dimensional reduction, unsupervised clustering, and analysis will be conducted.Task 3 - Associate Single Cell-Type Gene Expression to Host Phenotypes After the filtering of gene expression counts for cell type-specific data, the counts will be used to conduct correlation analyses to production-relevant host phenotypes.Efforts. At the completion of this objective, we expect to have characterized cell types present along the rumen epithelium and cell-type respective gene expression. By identifying individual cell gene expression profiles, we anticipate discovering cell type-specific functionality which will be associated with production-relevant host phenotypes of interest, being feed efficiency in beef cattle. The expected results will be the first to describe how rumen epithelial single cell types differ in gene expression between animals with divergent efficiency phenotypes. These data are critical to understanding the roles individual types of cells perform, improving the current resolution of the biological mechanisms performed by the rumen epithelium.Objective #2: Determine the abundance and function of active rumen microorganisms that are adherent to the rumen wall and their respective correlation to the epithelial gene expression. Task 1 - Define the Metatranscriptome of the Rumen Epimural Communities Extraction and Sequencing of Microbial RNA: Objective 2 will use the same animals as described above in Objective 1. As rumen epithelial tissue is excised from the rumen and washed several times, wash-off will be collected to represent the rumen epimural microbiota. Microbial RNA will be obtained from samples using and fragments of cDNA will be sequenced.Analysis of Metatranscriptome Data: Resultant files will be checked for high-quality reads and mapped to the ARS-UCD1.3 bovine reference genome and unmapped reads will be separated to represent metatranscriptomic data of the epimural microbial communities, where microbial rRNA will be identified and used to classify microbes to the genus level, and taxonomic classification of bacteria, archaea, protozoa, and fungi can be identified. While we will identify microbial communities by their taxonomic classification, we will also perform de novo assembly to group transcripts discovered by functionality rather than taxa, permitting a deeper understanding of microorganisms performing similar metabolic activity.Task 2 - Determine Correlations between Epithelial Gene Expression and Epimural Microbiota Using the filtered and normalized single-cell rumen epithelial gene expression profiles and count data from the metatranscriptomics dataset, up and down-regulated genes will be correlated to active microbial abundances.Efforts. Once we have completed the objective, we expect to have markedly expanded knowledge of the functionality of epimural microbiota and how the microbiota can impact host phenotypes of interest. These data will improve the understanding of the epimural microorganisms in energy regulation and metabolism at the rumen wall. Following, we anticipate having correlated the presence of active microorganisms with gene expression of the ruminant host, therefore beginning to implicate specific microbiota in their capacity for host-microbiome interactions. By providing insight into how microorganisms may be impacting gene expression within the rumen epithelium, subsequent understanding of direct microbial actions on gene expression can be explored.Objective #3: Associate host genomic markers to rumen epithelial gene expression and microbial communities.Task 1 - Genotyping Steers Extraction and Sequencing of Steer Genomic DNA: Genotyping data will be generated from peripheral blood from all steers from Objective 1 using low-pass whole genome sequencing. Resultant genotypes will be imputed.Task 2 - Cis-eQTL Mapping By optimizing our single-cell experiment, we can improve power for sc-eQTL analyses. Filtered SNPs will be used to associate with normalized gene expression profiles of single cells. Identified eQTLs will be adjusted for multiple testing and false discovery rates.Efforts. Upon completion of this objective, we expect to have identified the first sc-eQTLs of cells present in rumen epithelium that represent expression profiles related to specific genotypes of steers divergent in feed efficiency phenotypes. Importantly, the research proposed within this objective will provide an unprecedented dataset of host genetic impacts on transcriptional regulation of single cell-type specific expression, which will elucidate biological mechanisms impacting feed efficiency.