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

Location: Cell Wall Biology and Utilization Research

2022 Annual Report

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.

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
This is the final report for this project which terminated in August 2022. See the report for the replacement project, 5090-31000-028-000D, “Developing Strategies to Improve Dairy Cow Performance and Nutrient Use Efficiency with Nutrition, Genetics, and Microbiology” for additional information. Objective 1: The impact of eliminating farmed animals from U.S. agriculture and converting to a plants-only system was modeled and showed that making changes to a complex system gave rise to both expected and unexpected impacts. Without animals, substantially more food would be produced, but a plants-only diet without supplementation for the U.S. population would result more nutrient deficiencies than a diet containing animal products. Greenhouse gas emissions declined, but only by approximately 60% of what animals currently produce. Requirements for synthetic fertilizer to replace animal manures, and other changes in the system counterbalanced the removal of animals. Recommendations for changes in our agricultural system requires integration of multiple disciplines to adequately evaluate potential impacts. Objective 1: Directing the developing ruminal microbial community through inoculation may improve dairy cattle performance and efficiency. Whole transcriptome sequencing was conducted in rumen and liver tissues collected from calves with or without neonatal inoculation of rumen microbial contents obtained from adult dairy cattle of differing feed efficiencies. Through comparative analysis, gene expression involved in immune and defense processes in rumen tissues was significantly elevated, which provided evidence of host transcriptome changes associated with early colonization of microbial community in neonatal calves. Likewise, this experiment showed that the effects of early-life inoculation were not limited to the rumen, with changes in microbial community and host gene expression in the abomasum. An additional experiment was conducted to explore the establishment of ruminal protists in dairy calves through direct, early-life inoculation with protist- or bacteria-enriched ruminal inoculum. During continual ruminal inoculation, microbial inoculum composition altered ruminal fermentation end product concentrations, but not calf growth. Increased bacterial species richness and diversity was also observed at three weeks after inoculation ceased. However, there was only very minor transfer of bacterial taxa directly from donor ruminal fluid. These findings provide a foundation for continued research aimed at manipulating the digestive tract microbial community to improve feed efficiency in dairy production systems. Objective 1: Using a robust, yet gentle, lysis protocol longer DNA molecules from complex, recalcitrant microbial samples could be obtained. This enables the use of long-read sequence data to be used in metagenome sequencing and assembly, which was demonstrated to improve the contiguity of rumen microbial reference genomes. Identifying new methods of screening the microbiome is of great importance, as the acquisition of even a small fragment of DNA can turn a “beneficial” bacterial host into a potential pathogen for a host species. A new sequence-based method that does not require manual labor and is high throughput was developed to identify host-virus associations using intracellular DNA-protein interactions. This method provides discriminatory power to place a viral genome within a specific bacterial cell, which is direct evidence of infection. Additionally, using cutting edge software and the latest DNA sequencing methods, 103 medium quality draft genomes from the bacteria and archaea in the cattle rumen were assembled and a total of 188 novel bacteria-virus interactions were identified from this dataset. This represents the first high-resolution glimpse at the activity of viruses in the rumen of cattle. Using the latest in high-accuracy, long-read DNA sequencing technologies, it was also demonstrated that microbial strains could be resolved down to single nucleotide variants in the population. Over 44 bacterial genomes were assembled into single, continuous chromosome genomes, which is the highest ever achieved in a single sequenced sample. Using additional DNA sequencing methods, over 400 viral- and 250 plasmid-host associations were identified in this one sample. The new methods pioneered in this work identified genes that may result in antibiotic resistance in rumen bacteria. Objective 1: A new method, called trio-binning, was developed to assemble each pair of chromosomes individually by using information derived from the parents of the sequenced individual. This method was used to assemble reference- quality genome assemblies for Yak, Gaur, and Water Buffalo in addition to several diverse breeds of European and Indicine cattle. The latest in long-read DNA sequencing technologies were applied to create the ARS- UCD1.2 reference genome for taurine cattle. This reference genome represents an order of magnitude of improved continuity compared to the prior UMD3.1 reference genome assembly and is currently the de-facto reference genome used in U.S. and international genomic evaluations for dairy and beef cattle, and several groups have reported substantial improvements in quality of these evaluations. These approaches have been adopted by interdisciplinary researchers involved in plant, animal and human genome studies, and are accepted as the current state of the art. Objective 1: Passage of digesta from the rumen is important for delivery of microbial fermentation products for digestion by the cow. Work on markers of liquid passage showed that the most commonly used marker, a cobalt salt, dissociated under ruminal conditions, meaning it was not sufficiently unreactive to use as a marker. Investigations into assays for an alternative liquid digesta marker, polyethylene glycol (PEG), resulted in refinements so that the assay was less time-sensitive and gave more reliable results and use in animal studies that assessed gram disappearance of PEG from the rumen showed that the marker was associated with both the liquid and solid digesta. This led to description of a new model of ruminal liquid flow which has major implications for our ability to predict nutrient supply to the cow and formulate diets to meet her needs. Objective 1: A new dietary starch method received final approval for use in nutritional labeling of animal feeds and pet foods. The method was meticulously refined and tested internally and then evaluated in a collaborative study with fourteen state, commercial, and research feed analysis laboratories. The method was found to be sufficiently precise and reliable across a range of diverse feedstuffs that an expert review panel appointed by the Association of Official Analytical Chemists International (AOAC) gave it Final Action approval as AOAC Official Method 2014.10. Current methods of analysis were known to have problems with specific corn-derived feeds where analyses for water- soluble carbohydrates (WSC) and starch, when summed, contributed to unrealistically high values substantially exceeding 100% of dry matter. A new method was developed for measuring soluble starch in animal feeds to avoid counting it twice as both WSC and as starch. This allowed for more accurate formulation of dairy cattle rations. Objective 1: Laboratories vary in which neutral detergent fiber (NDF) method they use, with few using the official AOAC method, preferring a more time efficient proprietary method. Laboratories also vary in how they grind the samples which may affect results. Eleven diverse feed samples were used to evaluate the effects of grinding method and NDF methods and results were compared to the AOAC methods using cutting mill-ground samples (the standard). These findings give guidance to the national associations, feed analysis laboratories, and researchers on which NDF methods are appropriate to use in feed analysis. Objective 2: Precision feeding of dietary protein throughout lactation and to cows with different genetic potential could increase nutrient use efficiency while reducing environmental nitrogen load on dairy farms. A study was conducted to evaluate the effects of feeding different levels of dietary crude protein (CP) across four stages of lactation where diets containing 16.5% CP resulted in maximized levels of milk protein secretion. Increasing CP beyond 16.5% did not result in additional increases in milk production across lactation. Additionally, a feeding study was conducted with first lactation Holstein dairy cows to evaluate the implication of milk protein genotype on response to dietary protein. Cows fed the higher protein diet produced increasing levels of milk protein according to genotype. However, under conditions of lower dietary protein, milk protein yield was blunted in higher genotype cows such that no genotype differences were observed among cows fed this diet. Data indicate that cows could be fed more precisely by changing dietary CP levels at different stages of lactation and that using cow genotype information when feeding cows could improve milk protein yield and efficiency which could lead to cost savings to dairy producers. Objective 2: Holstein and Jersey cows are two of the most prevalent breeds of dairy cows in the U.S. although most nutritional research has been conducted on Holsteins. Holstein and Jersey cows have different body types and production characteristics, which may impact their nutritional efficiency and requirements. The production efficiency of Holstein and Jersey cows was evaluated by meta-analysis of the literature in which Jersey cows were shown to be more efficient and exhibit higher nutrient digestibility. Protein and amino acid requirements for dairy cows of the Holstein and Jersey breeds were evaluated in three studies where preliminary analyses would indicate that productive response to dietary manipulation do not differ by breed.

1. Developed new methods for microbiome screening leading to record number of genomes assembled. The microbiome is the combined genetic material of all microorganisms (bacteria, fungi, protozoa, and viruses) that live in a particular environment. Because those microorganisms exist in large communities that are difficult to assess using old DNA sequencing technologies, ARS researchers in Madison, Wisconsin, led a project conducted by an international and interdisciplinary team of researchers from four countries and two private U.S. companies in developing new methods for microbiome screening. ARS researchers worked with bioinformaticians in the private sector to develop the open-source software tool, MAGPhase, which automates the process of single nucleotide polymorphisms (SNP) discovery and validation in the microbiome. The improved accuracy of newer sequencing technologies allows the MAGPhase algorithm to identify genetic variants that are representative of divergent strains of microbes that may harbor antibiotic resistance or pathogenesis genes. Using MAGPhase and improved genome assembly algorithms, 428 complete microbial genomes were assembled from that single sample, which was a record for a field that celebrated the assembly of 10 genomes from one individual. This accomplishment was of such importance that it was published in the journal, Nature Biotechnology (, with an accompanying opinion article highlighting its importance in Nature Microbiology ( These innovations have created a new benchmark for the field and are likely to serve as the foundation for new surveys in human clinical and agricultural microbial systems.

2. Identified means to measure susceptibility or resistance of dairy cattle to lipid related metabolic disorders. Energy and nutrient demands of early lactation can result in the development of lipid related metabolic disorders (LRMD) in dairy cows, such as ketosis and fatty liver resulting in reduced productivity and profitability of the dairy farm. Determining cow susceptibility to LRMD and identification of biomarkers associated with LRMD would provide an important advancement in managing cows in early lactation. To evaluate cow susceptibility and adaptability to LRDM, ARS researchers in Madison, Wisconsin, in collaboration with scientists at the University of Wisconsin-Madison, conducted an experiment to discover differences in liver gene expression that are associated with a cow’s susceptibility or resistance to metabolic disorders. Cows were retrospectively categorized into LRMD susceptibility and resistance groups. Cow grouping as susceptible or resistant to LRMD was closely associated to changes related to oxidative stress. These results suggest that responses to oxidative stress in cows in early lactation may determine susceptibility and resistance to LRMD. These results provide novel insight into the role of oxidative stress in LRMD and suggests the potential for unique control points for LRMD progression and severity for dairy cows in early lactation. This may allow researchers to evaluate early treatment options and producers to group cows to more precisely manage them according to susceptibility to LRMD.

3. Identified plasma essential amino acid concentration profiles that are associated with dairy cow performance. Dairy cows typically utilize dietary protein with low efficiency for milk protein production resulting in high feed costs and excess nitrogen excretion into the environment. Feeding cows more precisely to meet their essential amino acid (EAA) requirements would result in improved cost and nitrogen use efficiency, but knowledge of the amount and proportion of EAA required for optimizing milk protein production and nutrient use efficiency is incomplete. Whereas most studies evaluate each EAA individually, dairy cows require an adequate amount and proportion of all EAA. To better understand the association of plasma EAA concentration with dairy cow productivity, ARS researchers in Madison, Wisconsin, in collaboration with scientists from the University of Wisconsin-Madison, performed a study that allowed for an evaluation of the whole plasma EAA profile and to determine if there is a profile of EAA that is most closely associated with dairy cow production responses. Dairy cow plasma EAA concentrations were grouped into two distinct clusters that were highly associated with productivity. This information will be important for researchers and dairy cow nutritionists when evaluating the EAA feeding strategy for optimizing productivity and nitrogen use efficiency.

4. New fiber digestion kinetics evaluations support simpler, more efficient options to evaluate diets and feeds to support nutritional needs of dairy cows. Fiber digestion kinetics describe factors important to determining the value of fiber in a cow’s diet for supporting her nutritional requirements, health, and performance. Among the kinetics values, estimating the proportions of indigestible and potentially digestible pools of fiber and the rate of fermentation of the latter are among the most important. In collaborative work with a mathematical modeler, ARS researchers from Madison, Wisconsin, found that using just 2-pools of ruminal fiber fit more of the forages tested and gave more reliable and less variable values as opposed to using a more complex approach which split the digestible pool into fast and slowly fermented fiber fractions each with its own fermentation rate. When used to predict the amount of fiber that would be fermented to release nutrients to the cow, the difference between the 2-pool and 3-pool models was minimal. The work affirmed that the simpler 2-pool model was appropriate to use for estimation of kinetics values. Kinetics values are determined using in vitro fermentation time courses measuring fiber use by rumen microbes; some recommended fermentations extend as long as 10 days. By comparing 2-pool kinetics values calculated from the full 11 time point data sets to those from data sets with hour sampling points removed, we found that the fermentation time could be reduced to 72 hours – a time reduction of seven days – and that it was important to retain time points early in the fermentation. Researchers and analytical laboratories can use these more robust and simplified kinetics calculations and use of much shorter fermentation time courses to make more efficient use of laboratory resources and provide fiber digestibility information more quickly for use in designing diets to support the nutritional needs of cows.

Review Publications
Park, T., Cersosimo, L.M., Radloff, W.J., Zanton, G.I., Li, W. 2022. The rumen liquid meta-transcriptome of post-weaned dairy calves differed by pre-weaning ruminal administration of differentially-enriched, rumen-derived inocula. Animal Microbiome. 4. Article 4.
Bickhart, D.M., Kolmogorov, M., Tseng, E., Portik, D., Korobeynikov, A., Tolstoganov, I., Uritskiy, G., Liachko, I., Sullivan, S.T., Shin, S.B., Zorea, A., Andreu, V., Panke-Buisse, K., Medema, M., Mizrahi, I., Pevzner, P., Smith, T.P. 2022. Generating lineage-resolved, complete metagenome-assembled genomes from complex microbial communities. Nature Biotechnology. 40:711-719.
Bickhart, D.M., Koch, L.M., Smith, T.P., Riday, H., Sullivan, M.L. 2022. Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species. GigaByte. 42:1-13.
Davenport, K.M., Bickhart, D.M., Worley, K.C., Murali, S.C., Salavati, M., Clark, E.L., Cockett, N., Heaton, M.P., Smith, T.P., Murdoch, B.M., Rosen, B.D. 2022. An improved ovine reference genome assembly to facilitate in depth functional annotation of the sheep genome. Gigascience. 11. Article giab096.
Hall, M., Zanton, G.I. 2022. Substitution of cane molasses for corn grain at two levels of degradable protein. I. Lactating cow performance, nutrition model predictions, and potential basis for butterfat and intake responses. Journal of Dairy Science. 105(5):3939-3953.
Zanton, G.I., Hall, M. 2022. Substitution of molasses for corn grain at two levels of degradable protein. II. Effects on ruminal fermentation, digestion, and nitrogen metabolism. Journal of Dairy Science. 105(5):3954-3968.
Pralle, R., Li, W., Murphy, B., Holdorf, H.T., White, H. 2021. Novel facets of the liver transcriptome are associated with the susceptibility and resistance to lipid-related metabolic disorders in periparturient Holstein cows. Animals. 11(9). Article 2558.
Cheng, A., Li, W., Hernandez, L. 2021. Investigating the effect of positional variation on mid-lactation mammary gland transcriptomics in mice fed either a low-fat or high-fat diet. PLoS ONE. 16(8): e0255770.
Naren, G., Li, W., Gelsinger, S., Murphy, B., Li, S. 2021. Analysis of host jejunum transcriptome and associated microbial community structure variation in young calves with feed-induced acidosis. Metabolites. 11(7). Article 414.
Mezera, M., Li, W., Liu, L., Meidan, R., Penagaricano, F., Wiltbank, M. 2021. Effect of natural pre-luteolytic prostaglandin F2a pulses on the bovine luteal transcriptome during spontaneous luteal regression. Biology of Reproduction. 104(5):1016-1029.
Letelier, P., Zanton, G.I., Dorea, J.R., Wattiaux, M.A. 2022. Plasma essential amino acid concentration and profile are associated with performance of lactating dairy cows as revealed through meta-analysis and hierarchical clustering. Journal of Dairy Science. 105(6):5044-5061.
Leonard, A.S., Crysnanto, D., Fang, Z., Heaton, M.P., Vander Ley, B.L., Herrera, C., Bollwein, H., Bickhart, D.M., Kuhn, K.L., Smith, T.P.L., Rosen, B.D., Pausch, H. 2022. Structural variant-based pangenome construction has low sensitivity to variability of haplotype-resolved bovine assemblies. Nature Communications. 13. Article 3012.