Research Project: Improving Dairy Cow Feed Efficiency and Environmental Sustainability Using Genomics and Novel Technologies to Identify Physiological Contributions and Adaptations

Location: Animal Genomics and Improvement Laboratory

2023 Annual Report

Objectives
Objective 1: Develop genomic resources and molecular tools to determine limitations to nutrient use efficiency in dairy cattle. Sub-objective 1.A: Using phenotypically well-defined lines of Holstein dairy cattle selected and monitored for feed efficiency, investigate the biological and genetic bases of nutrient use efficiency and support genomic selection studies by identifying highly affected pathways against a backdrop of known RFI phenotype and methane emissions. Sub-objective 1.B: Continue to refine and validate a short-term isolated duodenal model for assessing intestinal epithelial tissue transcriptomic changes in ruminant gastrointestinal tissues in vivo in response to changes in luminal nutrient flow. Objective 2: Develop and apply novel nutritional strategies for calf rearing and weaning to reduce feed and nutritional costs to dairy cattle production through further refining the global landscape of genomic and epigenomic regulatory elements, exploring the regulatory dynamics of chromatin states in rumen development during weaning and for complex traits. Sub-objective 2.A: Characterize molecular phenotypes of the calf rumen transcriptome through strand-specific RNA sequencing (ssRNA-seq) and single-cell RNA sequencing during development. Sub-objective 2.B: 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: Using in vitro and in vivo gastrointestinal tissue responses (ruminal, duodenal, and colon) in lactating and non-lactating cows, investigate perturbations in luminal factors (changes in nutrient flow) at single-cell resolution. Sub-objective 3.A: Assess short-term (days) responses of metabolism- and transport-related genes and proteins of the intestinal epithelia in response to direct delivery of individual substrates or nutritional components (e.g., nutrients, metabolites, humoral factors). Sub-objective 3.B: Evaluate the impact of nutrient use efficiency, as defined by dairy efficiency as determined RFIlac, on total tissue, animal protein, and lipid carcass composition.

Approach
To improve feed efficiency and reduce methane emissions of dairy cattle through genetic selection and management understanding the basis for dairy cows which are divergent in feed efficiency is necessary. A database of the genetic and production information, including enteric methane emissions, has been compiled for more than 15 years and will continue to facilitate extensive analysis. Additionally, these highly phenotyed animals will be used to assess changes in tissues known to affect efficient use of nutrients. Methods to temporarily isolate regions of small intestine of live, adult cows will be established to study direct nutrient effects on gut function and gene expression. Moreover, epigenetic factors controlling 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 Report
Progress was made on all three objectives of project 8042-31310-114-000D (Improving Dairy Cow Feed Efficiency and Environmental Sustainability Using Genomics and Novel Technologies to Identify Physiological Contributions and Adaptations). Under Objective 1, an additional 35 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 the 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 400 growing heifers from the same herd to investigate the biological and genetic bases of nutrient use efficiency and to support genomic selection studies. 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 1500 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. Finally, enteric methane emissions of 200 dairy cows from 100 to 150 days of lactation have been 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. We continue the development of ruminal epithelial cell system to study dairy cattle gastrointestinal development. Using this in vitro culture system, the first global map of gene regulatory elements in cattle with a 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 previously established from rumen epithelial tissue from a 2- week-old Holstein bull calf and was used for transcriptomic analysis of REPC by both bulk and single-cell RNA sequencing techniques. Specifically, this approach 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, CTCFbinding 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. A Rumen epithelial cell differentiation and development during weaning in newborn ruminants is necessary for the efficient digestion of solid feed and optimal growth performance. Because of the ruminal wall's complex and variable physical composition, investigating the specific effects of changing ruminal environments on all aspects of rumen functions has been challenging. Recent breakthroughs in single-cell RNA-sequencing technologies enabled ARS researchers in Beltsville, Maryland, to establish an atlas of ruminal cells for dairy cattle at single-cell resolution. Scientists were able to clarify the complexity of rumen epithelial diversity and explain cell identity, fate, and function. This first effort in implementing single-cell RNA sequencing technologies in cattle opens the door for discoveries about the roles of tissue and cell types in complex traits at singlecell resolution. Under Objective 3 tissue biopsies from lactating cows at the longitudinal lactation stage with total of 118 samples have been collected and sequenced for transcriptomic analysis. Collected biopate samples were from three tissue sources (rumen, duodenum, and colon) representative of the lactation cycle to assess changes as lactational needs and rations change. Currently, RNA sequencing data analysis has been conducted and initial reports are being prepared. Using the 10X Genomics Chromium Controller, single-cell RNA-seq on Holstein ruminal epithelial cells during weaning was performed. This represents the first reported single-cell transcriptomic analysis in cattle. This study was successful in generating rumen single-cell transcriptomes, revealing major and some novel cell types. This study provides an initial example for bovine single-cell analysis and opens the door for new discoveries about tissue/cell type roles in complex traits at single-cell resolution and established first cell type profiles for cattle rumen epithelial cells at a single-cell resolution. This study characterized rumen epithelial cells’ cell cycle, component, relative timing, and regulatory networks, as well as co-expression and gene function patterns. With the proposed cell lineage development model, 6 cell types identified across their temporal and spatial distributions were revealed, which appear to be correlated with the rumen epithelium’s underlying layers, structures, and functions.

Accomplishments
1. Dynamic transcriptome in gastrointestinal tracts at different lactation stages of dairy cattle. To investigate the molecular basis for the gastrointestinal adaption of the dairy cattle to changes in nutrient delivery in response to increased nutrient requirements to support milk production, researchers at Beltsville, Maryland used RNA-seq to profile gene expression patterns in three tissues, including the colon, duodenum, and rumen at eight-time points during the transition from the dry period to lactation and during lactation (dry period, day 3, day 14, day 28, day 45, day 120, day 220, and day 305). The multi-time points sampling allowed direct comparison of expression patterns within and among tissues during different lactation periods. This resource provided comprehensive insight into nutritional efficiency of cattle during lactation and revealed the specific characteristics of gastrointestinal tract tissues for researchers to understand the complexity of genomic activities during lactation.

Review Publications
Cavani, L., Parker Gaddis, K.L., Baldwin, R.L., Santos, J.E., Koltes, J.E., Tempelman, R.J., Vandehaar, M.J., Caputo, M.J., White, H.M., Penagaricano, F., Weigel, K.A. 2023. Impact of parity differences on residual feed intake estimation in Holstein cows. Journal of Dairy Science Communications. https://doi.org/10.3168/jdsc.2022-0307.
Shadpour, S., Chud, T.C., Hailemariam, D., De Oliveira, H.R., Plastow, G., Stothard, P., Lassen, J., Baldwin, R.L., Miglior, F., Baes, C.F., Tulpan, D., Schenkel, F.S. 2022. Predicting dry matter intake in Canadian Holstein dairy cattle using milk mid-infrared reflectance spectroscopy and other commonly available predictors via artificial neural networks. Journal of Dairy Science. 105(10):8257–8271. https://doi.org/10.3168/jds.2021-21297.
Liang, Z., Prakapenka, D., Parker Gaddis, K.L., Vandehaar, M.J., Weigel, K.A., Tempelman, R.J., Koltes, J.E., Santos, J.P., White, H.M., Penagaricano, F., Baldwin, R.L., Da, Y. 2022. Impact of epistasis effects on the accuracy of predicting phenotypic values of residual feed intake in U.S. Holstein cows. Frontiers in Genetics. 13:1017490. https://doi.org/10.3389/fgene.2022.1017490.
Boschiero, C., Gao, Y., Baldwin, R.L., Ma, L., Li, C., Liu, G. 2022. Butyrate induces modifications of the CTCF-binding landscape in cattle cells. Biomolecules. 12(9):1177. https://doi.org/10.3390/biom12091177.
Marceau, A., Gao, Y., Baldwin, R.L., Li, C., Jiang, J., Ma, L., Liu, G. 2022. Investigation of rumen long noncoding RNA before and after weaning in cattle. BMC Genomics. 23:531. https://doi.org/10.1186/s12864-022-08758-4.
Boschiero, C., Gao, Y., Baldwin, R.L., Ma, L., Li, C., Liu, G. 2022. Differentially CTCF-binding sites in cattle rumen tissue during weaning. International Journal of Molecular Sciences. 23(16):9070. https://doi.org/10.3390/ijms23169070.
Liu, S., Gao, Y., Canela-Xandri, O., Wang, S., Yu, Y., Cai, W., Li, B., Pairo-Castineira, E., D'Mellow, K., Rawlik, K., Xia, C., Yao, Y., Li, X., Yan, Z., Li, C., Rosen, B.D., Van Tassell, C.P., Van Raden, P.M., Zhang, S., Ma, L., Cole, J.B., Liu, G., Tenesa, A., Fang, L. 2022. A multi-tissue atlas of regulatory variants in cattle. Nature Genetics. 54(9):1438-1447. https://doi.org/10.1038/s41588-022-01153-5.
Yao, Y., Liu, S., Xia, C., Gao, Y., Pan, Z., Canela-Xandri, O., Khamesh, A., Rawlik, K., Wang, S., Li, B., Zhang, Y., Pairo-Castineira, E., D'Mellow, K., Li, X., Yan, Z., Li, C., Yu, Y., Zhang, S., Ma, L., Cole, J.B., Ross, P.J., Zhou, H., Haley, C., Liu, G., Fang, L., Tenesa, A. 2022. Comparative transcriptome in large-scale human and cattle populations. Genome Biology. 23:176. https://doi.org/10.1186/s13059-022-02745-4.