Project : USDA ARS

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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

2021 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
An analysis for soluble starch in animal feeds was developed in collaboration with commercial feed analysis laboratories (Subobjective 1.B). The issue was that water-soluble starch was being detected by starch analysis as well as by analysis for water-soluble carbohydrates, resulting in gross overestimation of total carbohydrate in feeds in which soluble starch was present in appreciable quantities (e.g., bakery products). An approach for analysis was developed and released as a USDA monograph. Although other planned research has been delayed for liquid passage kinetics work, analyses of the liquid marker (polyethylene glycol; marker) were performed on pressed rumen liquid and solid digesta samples from a previous animal study Subobjective 1.B). Previously unplanned analysis of the solids which required development of extraction and analysis methods for these samples, and planned analysis of the liquid unexpectedly showed that liquid passage is best modeled as a 2-pool system with three exponential rates. Both liquid and solid fractions contained marker. The model of ruminal liquid/marker kinetics was best fit by rates describing passage of liquid/marker from the solids to the liquid pool, and rates of liquid/marker passage from the rumen with the liquid and solids pools. Milking frequency is associated with milk quality and yields (Subobjective 2.A). The most commonly used milking frequency is two times or three times daily. With the adoption of robotic milking systems, there has been an interest in studying the impact of incomplete milking on mammary gland physiology, milk quality and milk yield. We studied the impact of milking frequency and incomplete milking on the mammary gland transcriptome. Our initial transcriptome analysis of the mammary gland indicated that incomplete milking had the most impact on the mammary gland transcriptome. The highest number of differentially expressed genes were observed between completely milked and incompletely milked cows that were milked twice daily. Furthermore, for the differentially expressed genes between completely milked and incompletely milked cows, they were enriched in a distinctive set of pathways in the twice daily milked cows compared to thrice daily milked cows. Our initial results indicated that different biological molecular mechanisms are at play in the mammary gland with differing milking frequency and milking completeness. Holstein and Jersey cows are two of the most prevalent breeds of dairy cows in the United States. While they are both prevalent, most nutritional research has been conducted on Holstein cows. Holstein and Jersey cows have different body types and production characteristics, which may impact their nutritional efficiency and nutrient requirements (Subobjective 2.B). The production efficiency of Holstein and Jersey cows was evaluated by meta-analysis in which it was determined that Jersey cows are more efficient and have higher nutrient digestibility than Holstein cows. Protein and amino acid requirements for dairy cows of the Holstein and Jersey breeds were evaluated in two studies. Analyses of these studies are ongoing, but preliminary analysis would indicate that productive response to supplemental amino acids or protein sources of differing degradability do not differ by breed.

Accomplishments
1. Precision-phasing of livestock gastrointestinal microbiomes enable detection of hidden strains and mobile-element associations. All animal and plant species must co-exist with a multitude of rapidly evolving microorganisms. Collectively termed, “the microbiome,” these organisms often exist in large composite communities that are difficult to assess using older DNA sequencing technologies. Identifying new methods of screening these communities 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. Using the latest in high accuracy, long-read DNA sequencing technologies, it was 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. These discoveries represent the highest resolution image of genomic DNA prevalence and transfer within a single community, and will impact the interpretation of future microbiome sequencing results. This research led by ARS researchers at Madison, Wisconsin, was conducted by an international and interdisciplinary team of researchers, consisting of participants from four different countries (Russia, the Netherlands, Israel and the U.S.) and two private U.S. companies (Phase Genomics, Pacific Biosciences). A manuscript describing this study is currently in review and has been released to the preprint server, bioRxiv, to share results with the research community in the interim.

2. Development of a high-throughput means of assessing microbial virus-host associations. Viruses that infect bacterial species (termed “bacteriophage”) offer distinct opportunities for therapeutics and prophylactics in human medicine and agriculture. Antimicrobial treatments based on viral control of bacterial growth (“phage-therapy”) represent another weapon against the proliferation of antimicrobial resistance in bacterial pathogens. Conversely, broad-spectrum viruses could feasibly transfer new DNA to previously benign hosts, turning “beneficial microbes” into harmful pathogens through a process generally called “transduction.” In order to mine metagenomics datasets for novel phage-therapy viruses or to identify broad-spectrum transduction of pathogenic DNA, researchers previously had to rely on direct observation through sequencing or through classical microbial isolation. Both methods are prone to false-negative detections and require manual labor. A collaboration between ARS researchers at Madison, Wisconsin, and industry partners at Phase Genomics has resulted in the development of a new sequence-based method that does not require manual labor and is high throughput. This method, termed “proxiPhage” by Phase Genomics, identifies 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. The beta version of this analysis method was previously highlighted for a Federal Lab Consortium award and has already been used in clinical settings to identify putative sources for antimicrobial resistance gene alleles in the environment. A manuscript detailing this method has been submitted to the preprint server, bioRxiv, in advance of a more detailed survey on other metagenomics datasets.

3. Sustained effects of previous, early-life ruminal microbial inoculation in young dairy calves. Directing the ruminal microbial community through inoculation has been hypothesized as a means to improved dairy cattle performance and efficiency. In practice, this has not yet been achieved due to the redundancy and resiliency of the ruminal microbiome after establishment. However, the persistency of inoculating the rumen prior to establishment in early life by dosing with microbial, differentially-enriched inoculum has been incompletely studied. ARS scientists at Madison, Wisconsin, explored the sustained effects of inoculation on the ruminal microbiome in dairy calves through direct, early-life inoculation with protist- or bacteria- enriched ruminal inoculum. After four weeks of inoculation with microbial-enriched ruminal fluid before weaning, increased bacterial species richness and diversity was observed at three weeks after inoculation ceased. However, there was only very minor transfer of bacterial taxa directly from donor ruminal fluid. Overall, these results demonstrated that early-life inoculation with adult donor ruminal fluid could result in slight changes in the rumen bacteriome after weaning without large effects on ruminal fermentation or animal performance. These results will guide researchers and provide the foundation for further research or practical attempts to direct the dairy calf ruminal environment toward profitable and efficient phenotypes.

4. Early-life ruminal microbial inoculation in dairy calves affects the microbiome in and host transcriptome of the abomasum. Most of the work on the microbiome in the ruminant stomach has focused on the rumen, largely due to its relatively easy access and importance in the digestive process. The abomasum is the last stomach compartment of the ruminant stomach, and it plays an important role of nutrient digestion similar to the nonruminant, simple stomach. However, we currently have very limited knowledge about abomasal physiology and the underlying molecular mechanisms under different treatments, including early-life microbial inoculation. ARS researchers at Madison, Wisconsin, have been examining dosing of young calves with adult cattle rumen contents as a means to manipulate the gut microbiome for enhanced feed efficiency, reduced methane production, and improved health. One experiment showed effects of early-life inoculation were not limited to the rumen, with changes in microbial community and host gene expression in the abomasum. These findings provide a foundation for continued research aimed at manipulating the digestive tract microbial community to improve feed efficiency in dairy production systems.

5. Avoiding double dipping when feeding dairy cows. Double accounting is not a good idea, especially when you are trying to balance a cow’s diet. Starch is an energy rich carbohydrate and in some feeds it analyzes in the liquid and solid fractions, which makes it measure as both starch and as water-soluble carbohydrates. This double counts the starch and overestimates the amount of digestible carbohydrate present. In collaboration with commercial feed analysis laboratories, ARS researchers at Madison, Wisconsin, established recommendations for measuring soluble starch in animal feeds to avoid counting it twice as water-soluble carbohydrate and as starch. The work improved the accuracy of analyzing feeds and allows livestock nutritionists to take full advantage of the more digestible soluble-starch as they formulate diets for healthy, efficient, productive cows.

6. Agriculture recycles for society. An important message for the American public is how human society, plant agriculture, and animal agriculture recycle products among each other in our efforts to provide a sustainable food supply. That information gives people a broader understanding of how the national resources on which they depend integrate. A YouTube video “Eating Less Meat Won’t Save The Planet. Here’s Why” made substantial use of work published by ARS researchers at Madison, Wisconsin and collaborators at Virginia Tech, Blacksburg, Virginia that illustrated the cycling of products between animal agriculture and human society. In a 2-month period, the video received more than 2.4 million views and the original research paper was downloaded 15,000 times, or 7 to 11,000 more times than the 4 to 8000 downloads of all other months since publication. The interest in the video and in the research successfully introduced many more people to the workings of U.S. agriculture on which they depend for a healthful food supply.

U.S. DEPARTMENT OF AGRICULTURE

Cell Wall Biology and Utilization Research: Madison, WI