Location: Animal Genomics and Improvement Laboratory
2019 Annual Report
Objectives
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
Approach
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 Report
Relative to Objective 1, we evaluated 62 Holstein dairy heifers from the BARC herd for feed efficiency during a 91-day growth trial using an estimate called residual feed intake (RFI). Heifers with a low RFI value represent those animals that eat significantly less feed than the average animal in the herd while achieving the same rate of growth and a similar body weight. Low RFI heifers are more feed efficient, with lower associated feed costs for the dairy producer. We also measured daily enteric methane and carbon dioxide production of 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. We collected blood plasma 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 SNP genotyping using the Illumina Bovine HD 777K SNP Chip. The collected data were added to a database of 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. Using RFI information, we are classifying all heifers as either “high” or “low” and breeding them to specific Holstein sires with “high” and “low” genetic merit for RFI to develop lines of Holstein cattle divergently selected for feed efficiency. As of May 2019, a total of 106 offspring including 46 daughters were produced from these matings. The daughters will be evaluated for growth performance, feed efficiency, and plasma indicators of inflammation, metabolism, and stress. Of interest, we found that the offspring of extreme “high” RFI (less efficient) animals had significantly greater birthweights than offspring of “low” RFI (more efficient) animals.
Also, relative to Objective 1, we evaluated 86 Holstein dairy cows from the BARC herd for feed efficiency during the first 100 days of lactation using RFI estimates and associated production measures. These data were added to a database of over 1,100 lactating cows from the same herd and are being used to investigate the genetic basis of feed efficiency in dairy cattle. All data also were shared with international partners as part of multi-million-dollar grant led by investigators at the University of Guelph to improve genetic selection for feed efficient dairy cattle. A manuscript was published using these datasets showing that among heifers ranked as “high,” “middle,” or “low” RFI during growth, 43% ranked the same by RFI in early lactation, suggesting RFI during growth may be used as an indicator of future lactation RFI. We also demonstrated that a 64- to 70-day test period during mid-lactation provides the best approximation of full, 305-day lactation RFI for the least time and effort. We began measuring enteric methane emissions of dairy cows from 100 to 150 days of lactation as well as their residual feed intake during this same period of lactation to gain a better understanding of the relationship between feed efficiency during lactation and greenhouse gas emissions of dairy cows. These data are being collected as part of a $2M Foundation for Food and Agriculture grant with Michigan State University (see Incoming Agreement Log. 0000056460).
Lastly relative to Objective 1, we successfully developed and propagated intestinal organoids (enteroids) from cow intestinal biopsy samples obtained from 3 individual dairy cows. Enteroids developed from one cow were used to characterize the transcriptome of enteroids versus whole intestinal tissue using RNA-sequencing, as well as evaluate gene transcript responses of enteroids to treatment with an intestinal hormone called glucagon-like peptide 2. Analysis of the RNA-sequencing results are underway and will determine how well the cultured enteroids represent whole intestinal tissue of the live animal. This work is being funded in part by Incoming Agreement 8042-31310-078-01T with Pancosma SA, Switzerland.
Relative to Objective 2, we established a rumen epithelial primary cell (REPC) culture from 2-week-old Holstein bull calf rumen epithelial tissue for transcriptomic analysis. Ruminant forestomachs are incompletely developed at birth and must fully develop both physically and metabolically prior to weaning. Butyrate is a short-chain fatty acid produced as an end-product of fermentation and is an essential nutrient for cattle. It plays a central role in regulating genomic and epigenomic elements of the cattle genome, which influence rumen development. To study the interaction of butyrate and rumen development, we used transcriptomic analysis of REPC by both bulk and single-cell RNA sequencing techniques. We tested the direct effects of butyrate addition to the culture media on gene expression and correlated networks identified to clarify the putative roles and mechanisms of butyrate action in rumen epithelial development. The top 4 networks positively affected by butyrate treatment were gene networks WNT, JNK, NF¿B, and MAPK/ERK which are predominantly associated with epithelial tissue development. Additionally, two key upstream regulators, E2F1 and TGFB1, were identified that play critical roles in the differentiation, development, and growth of epithelial cells. Significant expression changes of CHGA, ONECUT2, OXT, PAX2, WNT1, WNT4, GRHL2, and ELF3 genes, presumably upregulated by E2F1 and TGFB1, provided further evidence that butyrate plays a specific and central role in regulating genomic and epigenomic activities influencing rumen development.
Also relative to Objective 2, we established the first global map of regulatory elements (15 chromatin states) and defined their coordinated activities in cattle through genome-wide profiling for 6 histone modifications (via ChIP-seq), RNA polymerase II (via ChIP-seq), CTCF-binding sites, DNA accessibility, DNA methylation, and transcript profiling of REPC, rumen tissues, and Madin-Darby Bovine Kidney Epithelial Cells (MDBK cells). We showed that each chromatin state exhibited specific enrichment for sequence ontology, gene expression and methylation across tissues, trait-associated variants, eQTLs, selection signatures, and evolutionarily conserved elements, implying distinct biological functions. Through treatment of REPC with butyrate, a key regulator of rumen development, we observed that the weak enhancers and flanking regions of active transcriptional start sites/enhancers were the most dynamic chromatin states. Accompanying changes in gene expression and DNA methylation were significantly associated with heifer conception rate and stature. Our functional genome annotation improves our understanding of genome regulation, complex trait variation, and adaptive evolution in livestock. Using butyrate to induce the dynamics of the epigenomic landscape, we were able to establish the correlation among nutritional elements, chromatin states, gene activities, and phenotypic outcomes.
Relative to Objective 3, we surgically prepared 8 dairy cows with rumen and duodenal cannulae and obtained biopsies via colonoscope throughout a complete lactation. Samples collected were from rumen, duodenum, and colon. This research is fundamental to establish the underlying changes that occur during lactation and putatively clarify the growth responses in these tissues during the lactation cycle. First, we evaluated the transcriptomic response of the harvested duodenal tissue to short-term perturbation by direct infusion of starch. Pathway analysis of the 1,490 genes identified as differentially expressed in response to starch infusion were association with digestive system development and function. Primary transcription regulators such as PTH, JUN, WNT, and TNFRSF11B were identified as activated by the starch infusion. Second, samples from 4 cows were used to establish the relative substrate oxidative capacity of the biopate from duodenal tissue and determine the relative rates of various oxidative substrates. Pending immunohistochemical approaches will further validate the use of duodenal biopsy for the evaluation of in vivo changes in tissue nutrient use.
Accomplishments
1. Optimal test to estimate feed efficiency in dairy cows. Feed conversion efficiency affects dairy producer profitability and is often assessed by measuring animal feed intake and production performance over a limited test period. However, the optimal test duration and production period for estimating feed efficiency in dairy cattle have been unclear. ARS scientists in Beltsville, Maryland, used an estimate called residual feed intake (RFI) to assess feed efficiency in growing heifers, those same heifers in early lactation, and cows during a whole lactation. They discovered that RFI estimated during growth was able to predict later feed efficiency during lactation and that a 64- to 70-day test period during mid-lactation provided the best approximation of RFI. This information also can be used by dairy producers concerned with monitoring herd performance, improving production profitability, and decreasing environmental impact.
2. First global map of regulatory elements (15 chromatin states) in cattle and definition of their coordinated activities. Functional annotation of the cattle genome is essential for understanding genome regulation, complex trait variation, and adaptive evolution, particularly in livestock species. Using state-of-the-art technologies such as ChIP-sequencing, ATAC-sequencing, CTCF-sequencing, and RNA-sequencing with bulk and single-cells, ARS scientists in Beltsville, Maryland, performed genome-wide profiling for six histone modifications, RNA polymerase II, CTCFbinding sites, DNA accessibility, DNA methylation, and the transcriptome of rumen epithelial primary cells, rumen tissues, and Madin-Darby Bovine Kidney Epithelial Cells (MDBK cells). By inducing changes in the epigenomic landscape, they were able to establish the correlation among nutritional elements, chromatin states, gene activities, and phenotypic outcomes. This additional functional annotation of the cattle genome is critical for understanding genome regulation, variation in complex traits, and livestock evolution and will be used by researchers worldwide to pursue further investigation of the cattle genome.
Review Publications
Cai, H., Li, M., Sun, X., Plath, M., Li, C., Lan, X., Lei, C., Huang, Y., Bai, Y., Qi, X., Lin, F., Chen, H. 2018. Global transcriptome analysis during adipogenic differentiation and involvement of Transthyretin gene in adipogenesis in cattle. Frontiers in Genetics. 9:463. https://doi.org/10.3389/fgene.2018.00463.
Lin, S., Zhang, Z., Xie, T., Hu, B., Ruan, Z., Zhang, L., Li, C., Li, C.C., Nie, Q., Zhang, X. 2019. Identification of a novel antisense RNA GHR-AS that regulates the expression of GHR in chichen. RNA Biology. 14:1-13. https://doi.org/10.1080/15476286.2019.1572440.
Oh, S., Li, C., Baldwin, R.L., Song, S., Liu, F., Li, R.W. 2019. Temporal dynamics in meta longitudinal RNA-Seq data. Scientific Reports. 9(1):763. https://doi.org/10.1038/s41598-018-37397-7.
Zhang, R., Liu, F., Hunt, P., Li, C., Zhang, L., Ingham, A., Li, R.W. 2019. Transcriptome analysis unraveled potential mechanisms of resistance to Haemonchus contortus infection in Merino sheep populations bred for parasite resistance. Veterinary Research. 50:7. https://doi.org/10.1186/s13567-019-0622-6.
Capuco, A.V., Bickhart, D.M., Li, C., Clover, C.M., Choudhary, R., Grossi, P., Bertoni, G., Trevisi, E., Aiken, G.E., Mcleod, K.R., Baldwin, R.L. 2018. Effect of consuming endophyte-infected fescue seed on transcript abundance in the mammary gland of lactating and dry cows, as assessed by RNA sequencing. Journal of Dairy Science. 101(11):10478–10494. https://doi.org/10.3168/jds.2018-14735.
Pulido, E., Fernandez, M., Prieto, N., Baldwin, R.L., Andres, S., Lopez, S., Giraldez, F.J. 2019. Effect of milking frequency and alpha-tocopherol plus selenium supplementation on sheep milk lipid composition and oxidative stability. Journal of Dairy Science. 102(4):3097-3109. https://doi.org/10.3168/jds.2018-15456.
Connor, E.E., Hutchison, J.L., Van Tassell, C.P., Cole, J.B. 2019. Defining the optimal period length and stage of growth or lactation to estimate residual feed intake in dairy cows. Journal of Dairy Science. 102(7):6131–6143. https://doi.org/10.3168/jds.2018-15407.
