Location: Cell Wall Biology and Utilization Research
2025 Annual Report
Objectives
Objective 1: Evaluate digestive tract function and identify gastrointestinal microorganism effects on nutrient digestibility, milk production capacity, nutrient use efficiency, and health in dairy cattle.
Sub-objective 1.A: Characterize and develop tools to evaluate the microorganisms present in the digestive tract of dairy cattle and evaluate effects of nutritional or other strategies on composition of microorganisms present and effects on performance and nutrient utilization efficiency.
Sub-objective 1.B: Evaluate functional microbiology, gastrointestinal function and digesta passage, and dietary composition effects on nutrient digestibility for increased performance, nutrient use efficiency, and health.
Objective 2: Characterize dairy cattle physiological factors contributing towards improved milk production capacity, nutrient use efficiency, and health.
Sub-objective 2.A: Utilize dairy cattle transcriptomics to inform the connections between the genotype and phenotype and the interaction of genotype and phenotype to enable improved milk production capacity, productive efficiency, milk quality, and health.
Sub-objective 2.B: Develop an understanding of the metabolic or physiological functions that determine production potential of partitioning of nutrients toward milk and away from manure and greenhouse gas emissions.
Approach
For sub-objective 1.A, we will characterize the rumen metagenomic community. We will collect rumen solids and liquids from cannulated Holstein cows and cannulated Jersey cows and sequence to a depth of 200 million paired-end reads using a circular consensus sequence protocol. These datasets will represent the finest resolution microbial references for the Holstein and Jersey breeds of cattle. We will also evaluate vitamin B12 status and behavior in heifers reared grazing on pasture or fed a total mixed ration in the barn.
For Sub-objective 1.B, we will evaluate if rumen microbial efficiency of microbial protein production is associated with the ratio of degradable protein to degradable carbohydrate through meta-analysis. We will also assess the fit of a two-pool three-exponential rate liquid passage kinetics model in vitro using two fiber sources. The fit of the in vitro data to a one-pool (free liquid) or two-pool (liquid as free liquid and associated with solids) liquid passage model will be determined. The difference between diets in in vivo rate of liquid passage and in vitro behavior of marker between liquid and solid fractions will be compared. This will provide insights related to passage of liquid-associated nutrients from the rumen as affected by physical form of the diet. In vitro rumen degradation of feedstuff protein using intrinsically labelled 15N will also be determined.
For Sub-objective 2.A, we will identify transcriptome biomarkers and adaptive transcriptome changes in dairy cows in response to different diets and during different development and lactation stages. Whole blood and milk samples will be collected prior to the administration of dietary treatments and after the cows have been adapted to the dietary treatments for downstream transcriptome biomarker analysis. We will also determine if dairy cattle with compromised ruminal health or gut barrier disfunction and healthy dairy cattle have distinct gastrointestinal tissue transcriptome and metatranscriptome profiles. Dairy bull calves will be offered calf starter diets of increased (acidosis) or typical (healthy) fermentability. Total RNA will be extracted from tissue samples, sequenced, and any reads unmapped to the cattle reference will be considered microbial and used for downstream microbial community analysis including potential microbial function analysis.
For Sub-objective 2.B, we will evaluate if milk component production is associated with greenhouse gas emissions and changes in energetic efficiency of use of absorbed nutrients through meta-analysis, which will provide a more complete description of measurable outcomes of cow performance and their relationship to greenhouse gas emissions and efficiency of diet utilization. Additionally, we will evaluate whether lactation performance, nitrogen and feed efficiency, and manure characteristics will be differentially affected by diet provided to Holstein and Jersey cows.
Progress Report
Objective 1. Sub-objective 1.A: Dairy cattle and other ruminants rely on the microbes in their rumen (the microbiome) to digest grass and other forage into protein and energy for conversion to milk. The best way to identify and understand how these microbes work is to sequence their DNA, which can give insights into their genes and how efficiently they can break down the feed provided to dairy cattle. These genomic tools can also tell us how different feeds, health conditions, animal genetics, and management affect the rumen microbiome. Together, this type of information will help dairy producers develop strategies to optimize production. Carrying out genomics analyses on microbiomes requires substantial technique optimization, computing power, and bioinformatic analysis tools. The first portion of this project was devoted to generating high quality DNA sequences from the mixture of rumen microbes making up the rumen microbiome. These DNA sequences are now being assembled into complete microbial genomes using bioinformatics approaches. The collection of microbial genomes is referred to as the metagenome. Generating the assembled metagenome is especially challenging since DNA sequence data represents a collection of thousands of different microorganisms. For this reason, we have had to develop new bioinformatics approaches to better sort the individual sequences for correct assembly of individual microbial genomes. One such approach has been to do an initial sorting of DNA sequences from similar organisms into groups. The approaches we are developing should be of benefit to other researchers working on the assembly of individual microbial genomes from complex microbiomes. Unfortunately, because some microbes are very similar and may differ only slightly at the DNA level, we continue to work on developing sorting and assembly approaches. Even with the challenges of genome assembly, we discovered many viral sequences (including bacterial viruses) in the rumen metagenome, which will be an important contribution to the final metagenomic assembled genomes. Although we currently do not know what role viruses/bacterial viruses might play in rumen function, they could be impacting population dynamics of the rumen microbiome which in turn could influence cow performance. We expect this work will eventually allow understanding of how differences in rumen microbiomes (for example between Holsteins, known for high milk production volume and Jerseys, known for production of high value milk components [butterfat and protein]) influence production parameters in dairy cows and provide producers with microbiome-based strategies to improve efficiency of their operations.
Objective 1, Sub-objective 1.B: For dairy cows to make optimal use out of their dietary forages for milk production, farmers and their nutritionists need to know how the cows digest different diets. In particular, knowledge of rumen function that contributes to digestion is required in different breeds of cows fed different forages. Research was conducted on the digestibility, ruminal kinetics and fermentation, and saliva production of Holstein and Jersey cows fed diets containing higher proportions of alfalfa or corn silages. Jersey cows had ruminal conditions that are more closely associated with fiber fermentation and milk fat production. Cows fed higher corn silage diets had ruminal conditions that are more likely to increase milk production compared to the higher alfalfa silage diets. Under the conditions of this experiment, diet and breed acted independently on the ruminal characteristics measured in this study. These results are important for farmers attempting to formulate diets for Holstien or Jersey cows to optimize production efficiency from home-grown forages such as alfalfa or corn silage.
Mixing dairy cow diets on farm can result in day-to-day variation in diet composition. Fluctuating dietary composition may result in reduced milk production or efficiency because of transient nutrient excesses or deficiencies. We have previously demonstrated that dairy cow productivity is resilient to short-term (48 hour) oscillation of dietary protein, but whether this was facilitated by the ruminal microbial community was unknown. Consistent with animal performance, the findings from this study revealed that dietary protein levels and feeding patterns did not impact rumen bacterial composition or predicted functional profiles. These results support the hypothesis that the rumen microbiome remains stable despite transient fluctuations in dietary protein indicating potential compensatory mechanisms. This knowledge can help farmers optimize microbial protein production and nitrogen utilization efficiency in the rumen.
Objective 2. Sub-objective 2.A: Under typical dairy calf management conditions, ruminal acidosis is a detrimental digestive condition that can lead to reduced growth, poor feed efficiency, and harm to animal health. To study the effects of ruminal acidosis on calf growth and health, we conducted two experiments to evaluate tissue damage, gene expression, and microbial translocation in calves fed an industry-standard control diet and a diet meant to induce ruminal acidosis. The histological analysis of tissues collected from calves after weaning indicated significant gut tissue damage in the calves fed the acidosis-inducing diet. One potential consequence of gut tissue damage is that microbes living in the gut could get transferred to body tissues resulting in gross microbial lesions such as abscesses or to otherwise poor health outcomes. We needed to be able to accurately detect microbes present in other tissues so we designed targeted, host-RNA transcript depletion panels for blood and liver RNAs. Sequencing results indicated that these targeted depletion panels can remove up to 75% of host RNA transcripts in the sequencing libraries, resulting in the ability to identify 10 times more of the microbial RNA transcripts. With this increased sensitivity in detecting microbial transcripts, we will be better able to evaluate the extent of microbial translocation when tissue damage occurs in young calves. Ultimately, this will help farmers make nutrition and management decisions to avoid gut damage and grow healthy calves.
Objective 2. Sub-objective 2.B: Amino acid nutrition is an important area of research for optimizing dairy cattle production, health, and economics. Supplementing dairy cow diets with rumen protected methionine is a common practice on many dairy farms to increase milk protein production and efficiency. Greatly increasing one amino acid such as methionine, might create an imbalance in dietary protein quality that could affect the ability of the cow to use several other amino acids. Whether this happens in dairy cows supplemented with rumen protected methionine was evaluated by meta-analysis of literature studies where the effects of supplemental methionine on the response of plasma amino acid concentrations were determined. With rumen protected methionine supplementation, there were reductions in the plasma concentrations of several essential amino acids and amino acids involved in methionine metabolism. These changes in essential blood amino acid brought about by feeding extra methionine may ultimately limit the milk protein production response to supplemental methionine. We had previously shown that blood amino acid profile in groups of dairy cows is associated with group productive performance. To further evaluate if individual cow blood amino acid profile could be associated with individual cow performance, plasma essential amino acid clustering at the individual cow level was conducted using a supervised machine learning model. The findings from this study support the results that were observed in groups of cows and highlight the potential of essential amino acid clustering as a tool for precision protein nutrition. These results will help dairy farmers, and their nutritionists formulate diets for optimum performance, cost effectiveness, and efficiency.
Accomplishments
1. Developed an improved method to evaluate impact of diet in dairy cows. Quantification of free amino acid concentrations from digested proteins in blood plasma has become an important tool in monitoring the health of individual dairy cows. Research related to formulating optimal and cost-effective diets for U.S. dairy producers greatly benefits from the added information on animal health this plasma amino acid analysis provides. Recognizing the need for a higher throughput analysis that works well with the complexity of plasma, ARS scientists in Madison, Wisconsin, developed a robust method to quantitate free amino acids in bovine plasma utilizing relatively inexpensive and available equipment found in many laboratories. The developed method is readily accessible to a wide range of researchers and should be broadly applicable to nutrition studies in ruminant and other animal production systems. This analysis has recently been used to show that levels of specific amino acids in plasma can predict individual cow performance, which will allow producers to optimize productivity of their herds.
2. Identified genetic markers associated with alfalfa forage protein and fiber digestibility. Alfalfa is an extremely important forage source for dairy cattle in the United States due to the advantageous agronomic characteristics in dairy production systems and nutritional benefits for lactating dairy cows. Selection for alfalfa varieties with favorable fiber and protein degradability characteristics could be accelerated and improved through identifying genetic markers for these traits. ARS scientists in Madison, Wisconsin, and Prosser, Washington, evaluated protein degradability and quantified genetic diversity and genetic markers for these traits in 200 alfalfa accessions planted at three different sites across two years. Twenty-two significant genetic markers associated with 12 traits related to forage digestibility were identified. The digestibility-related markers and associated genes identified in this study will benefit alfalfa breeders, dairy forage producers, and dairy farmers by providing new information about the genetic basis of forage digestibility and its interaction with environments.
Review Publications
Li, W. 2023. Genome-wide copy number variation and structural variation. A novel tool for improved livestock genomic selection. In: Mukhopadhyay, C.S., Choudhary, R.K., Panwar, H., Malik, Y.S., editors. Biotechnological interventions Augmenting Livestock Health and Production. Livestock Diseases and Management. Singapore: Springer. p.75-87. https://doi.org/10.1007/978-981-99-2209-3_5.
Erickson, M., Zanton, G.I., Wattiaux, M.A. 2023. Dynamic lactation responses to dietary crude protein oscillation in diets adequate and deficient in metabolizable protein in Holstein cows. Journal of Dairy Science. 106:8774–8786. https://doi.org/10.3168/jds.2023-23603.
Fregulia, P., Park, T., Cersosimo, L., Zanton, G.I., Li, W. 2024. Microbial inoculum effects on the rumen epithelial transcriptome and rumen epimural metatranscriptome in calves. Scientific Reports. https://doi.org/10.1038/s41598-024-65685-y.
Li, W., Larsen, A., Freguila, P. 2024. Investigating the impact of feed-induced acidosis on the rumen transcriptome and metatranscriptome in young calves at 8- and 17-weeks of age. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2024.1328539.
Zanton, G.I., Toledo, M.Z. 2024. Systematic review and meta-analysis of dairy cow responses to rumen-protected methionine supplementation before and after calving. Journal of Dairy Science Communications. 5:293-298. https://doi.org/10.3168/jdsc.2023-0512.
White, R.R., Hall, M. 2024. Estimates of optimal supplies of animal-sourced foods differ by food system goal and socioeconomic context. Proceedings of the National Academy of Sciences (PNAS). https://doi.org/10.1073/pnas.2319011121.
Smith, A., Hernandez, S., Wenner, S., Fregulia, P., Larsen, A., Li, W., Jang, Y., Duddeck, K., Petersen, T., Adkins, H.J., Yao, D., Chi, C. 2023. Dose-dependent effects of supplementing a two-strain Bacillus subtilis probiotic on growth performance, blood parameters, fecal metabolites, and microbiome in nursery pigs. Animals. https://doi.org/10.3390/ani14010109.
Lin, S., Medina, C., Patel, S.R., Xu, Z., Zanton, G.I., Combs, D., Wang, G., Shewmaker, G., Fransen, S., Llewellyn, D., Norberg, S., Yu, L. 2025. Identification of genetic loci associated with protein and fiber digestibility in alfalfa. Crop Science. 65(1). Article 70004. https://doi.org/10.1002/csc2.70004.
Svaren, L.M., Li, W. 2025. Pipeline for antimicrobial resistance gene quantification from host tissue. Current Protocols in Bioinformatics. https://doi.org/10.1002/cpz1.70116.
Barry, M.C., Hall, M. 2024. Comparison of 2-pool and 3-pool digestion kinetic model predictions of neutral detergent fiber digestibility of forages from commercially available data. Journal of Dairy Science. 108:2371-2380. https://doi.org/10.3168/jds.2024-25284.
Skarlupka, J.H., Cox, M.S., Steinberger, A.J., Sbardellati, D.L., Mcclure, J.C., Bickhart, D.M., Scheftgen, A.J., Zuniga-Chavez, I., Wolfe, L.A., Paget, E., Skadron, C., Attipetty, N., Suen, G. 2024. Oral swabs as a proxy for direct ruminal microbiome sampling in Holstein dairy cows is correlated with sample color. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2024.1466375.
Reinhardt, L.A., Svaren, L., Marathe, R., Zanton, G.I., Sullivan, M.L. 2025. Determination of free amino acid concentrations in bovine plasma using high-pressure liquid chromatography with electrospray ionization mass spectroscopy detection. Rapid Communications in Mass Spectrometry. https://doi.org/10.1002/rcm.10027.
Johnson, A., Li, W., Dittrich, B., Cole, A., Prodell, M., Fritz, S., Fregulia, P., Chen, C., Kown, C., Jang, D.Y. 2024. Effect of second iron injection on growth performance, hematological parameters, and fecal microbiome of piglets fed different dietary iron levels. Journal of Animal Science. https://doi.org/10.1093/jas/skae371.
Skarlupka, J.H., Cox, M.S., Steinberger, A.J., Sbardellati, D.L., Mcclure, J.C., Bickhart, D., Scheftgen, A.J., Zuniga-Chaves, I., Paget, E., Skadron, C., Attipetty, N., Suen, G. 2025. Correlating the oral swab microbial community with milk production metrics in holstein dairy cows. mSphere. https://doi.org/10.1128/msphere.00167-25.
Costello, M.K., Mcclure, J.C., Brown, J.A., Mantovani, H.C., Ricke, S.C. 2025. The gastrointestinal tract microbiome of Holstein × Angus cross cattle is negatively impacted by the pre-harvest process. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.02599-24.
Viquez-Umana, F., Erickson, M., Young, J.D., Zanton, G.I., Wattiaux, M., Suen, G., Mantovani, H. 2025. Assessing the impact of oscillating dietary crude protein on the stability of the rumen microbiome in dairy cattle. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2025.1568112.