Objective 1: Develop resources, tools, and selectable markers to improve nutrient use efficiency in dairy cattle. Tools and resources will be developed including: 1) genetically and phenotypically characterized lines of cattle divergently selected for feed efficiency to support genomic selection for greater efficiency and lower CH4 emissions, and identify possible negative consequences of selection on production performance; 2) whole tissue models of cattle intestine (mini-guts, or enteroids) to support tissue-specific investigations ex vivo; and 3) an ‘isolated’ small intestine model to study effects of specific nutrients on intestinal development, function, and gene expression of mature dairy cows in vivo. Sub-objective 1.A: Develop and phenotypically characterize lines of Holstein dairy cattle divergently selected for RFI during growth (RFIgrowth) to investigate the biological and genetic bases of nutrient use efficiency, and to support genomic selection studies. Sub-objective 1.B: Characterize and exploit relationships between RFI (RFIgrowth and RFIlac) and enteric CH4 emissions of dairy cattle. Sub-objective 1.C: Develop ruminant organoids to study gut health and nutrient use efficiency of dairy cattle ex vivo. Sub-objective 1.D: Develop and validate a short-term isolated duodenal model for the assessment of intestinal epithelial tissue transcriptomic response to alterations in nutrient delivery in vivo. Objective 2: Evaluate and develop novel dietary strategies to reduce feed and nutritional costs to dairy cattle production. Studies of newborn dairy calves will be conducted to characterize molecular changes controlling gene expression during rumen development and differentiation, and evaluate novel feed additives as alternatives to antibiotics to improve calf health and production performance. Sub-objective 2.A: Evaluate the effects of non-nutritive feed additives (e.g., phytochemicals) on gut health and nutrient use efficiency of dairy cattle. Sub-objective 2.B: Characterize molecular phenotypes of the calf rumen transcriptome through strand-specific RNA sequencing (ssRNA-seq) during development. Sub-objective 2.C: Functionally annotate the calf rumen epigenome and identify transcriptional cis-regulatory modules during development, including histone modification, chromatin accessibility and architecture using Chromatin Immunoprecipitation-sequencing (ChIP-Seq) technologies. Objective 3: Evaluate in vivo gastrointestinal tissue responses (ruminal and duodenal) of lactating and dry dairy cows to perturbations in luminal factors (changes in nutrient flow) and physiological stressors (transition cow and early lactation). Molecular mechanisms regulating cell proliferation and development of rumen and intestinal epithelia during critical changes in nutrient delivery and the dairy cow production cycle (transition into lactation, ration changes, stage of lactation) will be identified and characterized through transcriptomic studies of serial biopsies from live animal models. Subobjective 3A, 3B- See project plan
To improve feed efficiency and reduce methane emissions of dairy cattle through genetic selection and management, dairy cows divergent in feed efficiency will be developed, and a database of their genetic and production information, including enteric methane emissions, will be compiled for extensive analysis. Whole tissue models of intestine (mini-guts) will be developed from calves to study gut function and nutrient use, and methods to temporarily isolate regions of small intestine of live, adult cows will be established to study nutrient effects on gut function and gene expression. In addition, novel plant-derived compounds will be evaluated as alternatives to antibiotics to improve gut function, disease resistance, and feed efficiency of dairy calves. Epigenetic factors controlling calf rumen development during weaning will be investigated using state-of-the-art molecular technologies. Finally, changes in gastrointestinal cells of dairy cows related to gut growth and function during critical stages of production will be characterized by examining gene expression in gut tissues of cows under different dietary and production conditions over time.
Progress was made on all three objectives of project 8042-31310-078-00D (Improving Feed Efficiency and Environmental Sustainability of Dairy Cattle through Genomics and Novel Technologies). Under Objective 1, an additional 71 Holstein dairy heifers from the Beltsville Agricultural Research Center (BARC) herd were evaluated for feed efficiency during a 91-day growth trial using an estimate called residual feed intake (RFI). Daily enteric methane and carbon dioxide production also were measured for each heifer using an automated monitoring system called GreenFeed to determine relationships between feed efficiency of dairy cattle and their contribution to greenhouse gas emissions. Blood plasma was collected monthly from heifers during the growth trial for analysis of indicators of inflammation, metabolism, and stress. Genomic DNA was isolated from each heifer for high-density single-nucleotide polymorphism genotyping using the Illumina BovineHD Genotyping BeadChip with over 777,000 markers. The collected data were added to a database with information from over 240 growing heifers from the same herd to investigate the biological and genetic bases of nutrient use efficiency and to support genomic selection studies. All heifers are being categorized as either high- or low-RFI and being bred to specific Holstein sires with high or low genetic merit for RFI to develop lines of Holstein cattle divergently selected for feed efficiency. Offspring of animals with extremely high-RFI (less feed efficient) were found to have significantly higher birth weights than offspring of low-RFI (more efficient) animals. Holstein dairy cows from the BARC herd also were evaluated for feed efficiency during the first 100 days of lactation using RFI estimates and associated production measures. Those data were added to a database with information from over 1,200 lactating cows from the same herd and are being used to investigate the genetic basis of feed efficiency in dairy cattle. All data were shared with international partners as part of a multimillion-dollar grant led by investigators at the University of Guelph in Guelph, Canada, to improve genetic selection for feed efficient dairy cattle. Enteric methane emissions of dairy cows from 100 to 150 days of lactation were evaluated as well as their residual feed intake to gain a better understanding of the relationship between feed efficiency during lactation and greenhouse gas emissions of dairy cows as part of a $2 million Foundation for Food and Agriculture grant with Michigan State University. Development of a critical high-value experimental ruminal epithelial cell system to study dairy cattle development. An ARS scientist in Beltsville, Maryland, established a stable culture from rumen epithelial tissue of a Holstein calf. Using this in vitro culture system, the first global map of gene regulatory elements in cattle with demonstration of their coordinated activities was completed. This enables scientists to interpret how these changes can be used by the cells to modify their growth and metabolism which are required for animal health and production. Ultimately, understanding how these alterations control tissue development will enable better feeding and management strategies on farms to ensure better health and sustainability of animal production. Under Objective 2, a rumen epithelial primary cell (REPC) culture was established from rumen epithelial tissue from a 2-week-old Holstein bull calf for transcriptomic analysis. Transcriptomic analysis of REPC by both bulk and single-cell RNA sequencing techniques was used to study the interaction of butyrate and rumen development. Direct effects of butyrate addition to the culture media on gene expression and correlated networks were tested to clarify the putative roles and mechanisms of butyrate action in rumen epithelial development. The top four networks positively affected by butyrate treatment were found to be predominantly associated with epithelial tissue development. Additionally, two key upstream regulators were identified as playing critical roles in the differentiation, development, and growth of epithelial cells. Significant expression changes of eight genes presumably upregulated by those two regulators provided further evidence that butyrate plays a specific and central role in regulating genomic and epigenomic activities influencing rumen development. The first global map of regulatory elements (15 chromatin states) was established, and their coordinated activities in cattle were defined through genomewide profiling for six histone modifications, RNA polymerase II, CTCF-binding sites, DNA accessibility, DNA methylation, and transcript profiling of REPC, rumen tissues, and Madin-Darby bovine kidney epithelial cells. Each chromatin state was shown to exhibit specific enrichment for sequence ontology, gene expression, and methylation across tissues, trait-associated variants, expression quantitative trait loci, selection signatures, and evolutionarily conserved elements, which implied distinct biological functions. Weak enhancers and flanking regions of active transcriptional start sites/enhancers were observed to be the most dynamic chromatin states through treatment of REPC with butyrate, a key regulator of rumen development. Accompanying changes in gene expression and DNA methylation were significantly associated with heifer conception rate and stature. Understanding of genome regulation, complex trait variation, and adaptive evolution in livestock was improved through functional genome annotation. Correlations among nutritional elements, chromatin states, gene activities, and phenotypic outcomes were established using butyrate to induce the dynamics of the epigenomic landscape. Under Objective 3, using previously surgically prepared dairy cows with both rumen and duodenal cannula, biopsies were obtained via colonoscope throughout a complete lactation. All samples for physiological states of interest (dry, transition, early lactation, and late lactation) have been collected from rumen, duodenum, and colon and prepared for RNA sequencing to facilitate identification of regulatory elements of interest. Transcriptomic response of harvested duodenal tissue to short-term perturbation by direct infusion of starch was evaluated as the first step in validating this approach for application of functional in vivo analysis of nutrient gene interactions during short- and long-term perturbation with direct infusion. Pathway analysis of 1,490 genes identified as differentially expressed in response to starch infusion were associated with digestive system development and function. Primary transcription regulators were identified as activated by starch infusion. Samples from eight cows were used to establish the relative substrate oxidative capacity of the biopsies from duodenal tissue and determine the relative rates of various oxidative substrates.
Kang, X., Liu, S., Fang, L., Lin, S., Liu, M., Baldwin, R.L., Liu, G., Li, C. 2020. Epigenomic profiling of histone marks and CTCF binding sites in bovine rumen epithelial primary cells before and after butyrate treatment. Data in Brief. 28:104983. https://doi.org/10.1016/j.dib.2019.104983.
Fang, L., Liu, S., Liu, M., Kang, X., Lin, S., Li, B., Connor, E.E., Baldwin, R.L., Tenesa, A., Ma, L., Liu, G., Li, C. 2019. Functional annotation of the cattle genome through systematic discovery and characterization of chromatin states and butyrate-induced variations. BMC Biology. 17(1):68. https://doi.org/10.1186/s12915-019-0687-8.
Lin, S., Fang, L., Kang, X., Liu, S., Liu, M., Connor, E.E., Baldwin, R.L., Liu, G., Li, C. 2020. Establishment and transcriptomic analyses of a cattle rumen epithelial primary cells (REPC) culture by bulk and single-cell RNA sequencing to elucidate interactions of butyrate and rumen development. Heliyon. 6(6):e041123. https://doi.org/10.1016/j.heliyon.2020.e04112.