Research Project: Investigating Microbial, Digestive, and Animal Factors to Increase Dairy Cow Performance and Nutrient Use Efficiency

Location: Cell Wall Biology and Utilization Research

2019 Annual Report

Objectives
Objective 1: Evaluate the gastrointestinal microbial and digestive factors that influence nutrient use efficiency and milk production capacity and quality in dairy cattle. • Sub-objective 1.A. Determine the relationship between the gastrointestinal microbial community composition and production capacity and efficiency; develop relevant strategies to direct rumen microbial community composition for increased milk production capacity and efficiency and improved milk quality. • Sub-objective 1.B. Evaluate dietary composition, microbial, and animal factors, and microbe-animal interactions that affect the digestion and metabolism of forage/feed by rumen microbes and the passage of digesta from the rumen to predict nutrient provisions for increased performance and nutrient use efficiency. Objective 2: Identify animal factors that affect the conversion of dietary and potentially digestible nutrients toward milk production for increased nutrient use efficiency. • Sub-objective 2.A. Evaluate the dairy cow genetic and genomic factors affecting nutrient use efficiency and their interactions with the gastrointestinal microbial community and dietary factors for increased milk production capacity and efficiency and improved milk quality. • Sub-objective 2.B. Optimize the profile of circulating nutrients and identify and improve the genetic and management related-animal factors that affect the partitioning of nutrients toward milk and away from manure and greenhouse gas emissions.

Approach
Sub-objective 1.A will develop biological resources and computational tools to enhance characterization of dairy breed-specific bovine and other genomes. Samples will initially be taken from a healthy cow in early lactation that has been exclusively fed mixed forage (alfalfa- and corn-silage based); samples will come from three separate portions of the rumen (solid, liquid, and epimural lining). Our plan is to sequence and assemble the most prevalent species/strains that occupy the solid (feed particle-associated) and liquid (planktonic) phases of rumen digesta, and the interior rumen lining (epimural community). Additionally, establishment and the potential to direct the rumen microbial community toward a feed efficient phenotype will be studied in dairy calves. Multiple doses of rumen fluid from cows having a particular milk production efficiency status will be provided to newborn and pre-weaned calves. We will evaluate if this results in the establishment of a microbial community that is more similar to that of the donor inoculum than in calves dosed with sterile rumen fluid. These heifers will be followed through their first lactation to evaluate if the dosed animals will exhibit milk production efficiency more like that of the donor cow than that of the controls. Sub-objective 1.B will consist of several in vitro studies to evaluate methods of analyzing for microbial protein, starch degradability, and microbial protein synthesis. In addition to the in vitro studies, a series of animal experiments will be conducted to evaluate within-day changes in rumen liquid volume and passage that occur in response to multiple dietary factors that alter water intake and outflow of liquid from the rumen. Water intake will also be monitored to evaluate the effect of treatment and the potential correlation with rumen liquid passage. Sub-objective 2.A will involve several studies to identify molecular markers and adaptive transcriptome changes in dairy cows in response to diet, health status, and the interaction between rumen microbiome diversification and host transcriptome and genetic profile. Host transcriptome changes will be evaluated from a diverse range of tissue and sample types. Sub-objective 2.B will use several lactation and nutrient balance studies to evaluate nutrient partitioning in response to dietary provision of different levels of protein. We will collect nitrogen balance, gaseous emission, and production measurements to determine the effects of nutritional treatment on productivity and environmental output.

Progress Report
To understand the composition of the ruminal microbiome, researchers require reference genomes for the ruminal microbial population. To facilitate this research, 172 Gigabases and 52 Gigabases of short- and long-read sequence data were generated from a rumen sample taken from a single Holstein cow (Objective 1.A). This data is currently being used in a large-scale metagenome assembly project, but has been released to the public to speed further tool development (NCBI SRA Bioproject: PRJNA507739). As an example of how useful this data is to external groups, researchers at UC Davis have already used this sequence data to validate the use of their new assembly software, metaFlye (https://www.biorxiv.org/content/10.1101/637637v1). In the evaluation of rumen microbial impact on digestibility of dietary starch (Objective 1.B.2), we determined that the degradability of starch remaining after incubation with rumen microbes increases as the amount of time to process samples increases; use of antibiotics or freezing did not alter the effect of time. An approach was developed to use rumen fluid-treated starch without enzyme as a control to account for the impact of persistent microbial enzymatic activity. Assay to be performed in September 2019. Markers for evaluating liquid passage from the rumen (Objective 1.B.3) were evaluated for their dissociation in rumen fluid. If a marker dissociates, it is not inert and is not reliable as a marker. Cobalt ethylenediaminetetraacetic acid (EDTA) dissociated extensively under reducing conditions present in the rumen, whereas chromium EDTA was not affected. Cobalt EDTA is not appropriate for use as a liquid digesta marker. An invention was developed (Objective 1.B) and submitted that altered the digestibility of the treated feedstuff and modified microbial use of carbohydrates suggesting a more efficient use of the feed. Animal studies are in planning stages. Ruminal liquid passage rate (Lkp) is used in predictions of nutrient supply to the cow. The Lkp was measured using the current standard concentration-based (CB) approach vs. gram disappearance (GD) using 8 cows and 2 diets containing more or less ruminally degradable protein (Objective 1.B). The commonly used CB estimates were 25% lower on average than those measured by GD. This underestimation appears to be due to failure to account for declining rumen liquid volume. Increasing degradable protein in diets increased liquid passage rate by 21% as measured by GD. The results indicate that the currently used CB approach grossly underestimates liquid passage rate. Molecular evaluation of the ruminant gut microbiome and gut physiological changes is hindered by the laboratory analysis techniques. To overcome this limitation, a novel analytical pipeline was developed that enables the investigation on the impact of diet and gut microbiome on gut physiological changes and their underlying mechanism in young calves (Objective 1.A). The application of this newly developed pipeline represented the first study of both the meta-transcriptome and transcriptome of cattle gut in any ruminant species. The establishment of the microbial population in dairy cattle under current commercial management conditions was evaluated (Objective 1.A). A study was conducted to determine ruminal microbial community composition changes over the first 8 months of life. This was the first study that we are aware of that followed the development of the protozoal, planktonic, and sessile microbial communities concurrently in developing dairy calves. The findings of this study suggest that rumen microbial ecology differs with heifer age and rumen phase liquid vs solid) and that these factors should be considered in future research on the development of the rumen microbial community. While the Holstein breed predominates the U.S. dairy herd, the Jersey cow population is increasing. Research on the nutrition of Jersey cows is extremely limited, however Jerseys cows are believed to be more feed efficient than Holstein cows, which is an extremely important characteristic for dairy farm sustainability. An important and unmet requirement of the dairy industry was to characterize the feed and nitrogen efficiencies and to evaluate potential differences in nutritional physiology between the genetically diverse, economically important, Holstein and Jersey breeds (Objective 2.B). Three experiments have been or are being conducted comparing the Holstein and Jersey breeds for nutritional requirements, utilization of feed nitrogen from 2 different forage sources, and ruminal microbial activity. Upon completion of the analysis of these and future experiments, results will be critical to improve our understanding of feed efficiency differences across diverse animal genotypes and enhancing dairy farm sustainability into the future.

Accomplishments
1. DNA sequences of the rumen microbial community and development of a software tool to identify candidate bacterial hosts for viruses in the rumen. Using cutting edge software and the latest DNA sequencing methods, ARS scientists in Madison, Wisconsin, were able to assemble 103 medium quality draft genomes from the bacteria and archaea in the cattle rumen. Additionally, a total of 188 novel bacteria-virus interactions were identified from this dataset, representing the first high resolution glimpse at the activity of viruses in the rumen of cattle. Finally, the new methods pioneered in this work identified genes that may result in antibiotic resistance in rumen bacteria. This dataset has already found use in the scientific community as a reference for the upcoming publication of an improved genome assembly software. This study identified more antibiotic resistance genes in the rumen microbiota than previously detected and will be of use to ongoing efforts to document the effects of antibiotics in animal feed. Also, there is the potential to design custom viral vectors with metabolic genes related to increased production.

2. Development of a method to assemble full chromosomes for an individual using data from the parents of that individual. Oftentimes, segments of chromosomes have significant structural variations that make them difficult to represent as a single DNA sequence which results in errors in a reference genome. Collaborators at the NIH with ARS scientists in Clay Center, Nebraska, and Madison, Wisconsin, developed a new method to assemble each pair of chromosomes individually by using information derived from the parents of the sequenced individual. The separation of chromosomes by parent is 99% accurate and results in more continuous assemblies. This approach is a significant advancement in genome assembly methodology and is already having an impact and is being used by research groups in India and Japan to assemble the Water Buffalo and Flowering Cherry reference genomes, respectively. The generation of haplotype resolved assemblies is likely to improve the accuracy of genomic selection of all livestock and many plant species, so this new technique is likely to impact a wide range of industries.

Review Publications
Zanton, G.I. 2019. Effect of experimental design on responses to two levels of metabolizable protein in multiparous dairy cows. Journal of Dairy Science. https://doi.org/10.3168/jds.2018-15730.
Gelsinger, S.L., Coblentz, W.K., Zanton, G.I., Ogden, R.K., Akins, M.S. 2019. Ruminal in situ disappearance and whole-tract digestion of starter feeds in calves before, during, and after weaning. Journal of Dairy Science. 102(3):2196-2206. https://doi.org/10.3168/jds.2018-15551.
Fang, L., Zhou, Y., Liu, S., Jiang, J., Bickhart, D.M., Null, D.J., Li, B., Schroeder, S.G., Rosen, B.D., Cole, J.B., Van Tassell, C.P., Ma, L., Liu, G. 2019. Integrating signals from sperm methylome analysis and genome-wide association study for a better understanding of male fertility in cattle. Epigenomes. 3(2):10. https://doi.org/10.3390/epigenomes3020010.
Hay, E.A., Utsunomiya, Y.T., Xu, L., Neves, H.H., Carvalheiro, R., Bickhart, D.M., Ma, L., Garcia, J.F., Liu, G. 2018. Genomic predictions combining SNP markers and copy number variations in Nellore cattle. BMC Genomics. 19(1):441-449. https://doi.org/10.1186/s12864-018-4787-6.
Nandolo, W., Utsunomiya, Y.T., Meszaros, G., Wurzinger, M., Khayadzadeh, N., Torrecilha, R.B., Mulindwa, H., Gondwe, T., Waldmann, P., Ferencakovic, M., Garcia, J.F., Rosen, B.D., Bickhart, D.M., Van Tassell, C.P., Curik, I., Solkner, J. 2018. Misidentification of runs of homozygosity islands in cattle caused by interference with copy number variation or large intermarker distances. Genetic Selection Evolution. 50(1):43. https://doi.org/10.1186/s12711-018-0414-x.
Liu, S., Kang, X., Catacchio, C.R., Liu, M., Fang, L., Schroeder, S.G., Li, W., Rosen, B.D., Iamartino, D., Iannuzzi, L., Sonstegard, T.S., Van Tassell, C.P., Ventura, M., Low, W., Williams, J.L., Bickhart, D.M., Liu, G. 2019. Computational detection and experimental validation of segmental duplications and associated copy number variations in water buffalo (Bubalus bubalis). Functional and Integrative Genomics. 19(3):409–419. https://doi.org/10.1007/s10142-019-00657-4.
Rexroad III, C.E., Vallet, J.L., Matukumalli, L.K., Ernst, C., Van Tassell, C.P., Cheng, H.H., Reecy, J., Fulton, J., Taylor, J., Lunney, J.K., Liu, J., Cockett, N., Smith, T.P., Van Eenennaam, A., Clutter, A., Telugu, B., Purcell, C., Bickhart, D.M., Blackburn, H.D., Neibergs, H., Wells, K., Boggess, M.V., Sonstegard, T. 2019. Genome to phenome: improving animal health, production, and well-being: a new USDA blueprint for animal genome research 2018–2027. Frontiers in Genetics. 10:327. https://doi.org/10.3389/fgene.2019.00327.
Hall, M., Van Soest, P.J. 2019. A modified method for preparing the lithium salt of chromium EDTA and comparison to the Binnerts method. Animal Feed Science And Technology. https://doi.org/10.1016/j.anifeedsci.2019.05.002.
Oliveira, R.C., Sailer, K.J., Holodorf, H.T., Seeley, C.R., Pralle, R.S., Hall, M., Bello, N.M., White, H.M. 2019. Postpartum supplementation of fermented ammoniated condensed whey improves feed efficiency and metabolic profile. Journal of Dairy Science. https://doi.org/10.3168/jds.2018-15519.
Hall, M. 2019. Physicochemical and enzymatic hydrolysis characteristics of glycogen granules isolated from ruminal protozoa that are relevant to their analysis and digestion. Journal of Dairy Science. https://doi.org/10.3168/jds.2018-15758.
Fang, L., Zhou, Y., Liu, S., Jiang, J., Bickhart, D.M., Null, D.J., Li, B., Schroeder, S.G., Rosen, B.D., Cole, J.B., Van Tassell, C.P., Ma, L., Liu, G. 2019. Comparative analyses of sperm DNA methylomes among human, mouse and cattle provide insights into epigenomic evolution and complex traits. Epigenetics. 14(3):260-276. https://doi.org/10.1080/15592294.2019.1582217.