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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Research Project #433503

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

Location: Cell Wall Biology and Utilization Research

2018 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
The rumen microbial community (Objective 1.A.1.) is a complex symbiosis consisting of a multitude of Eukaryotic, Prokaryotic and Archaeal agents, each with varying genome sizes and DNA base compositions. The recalcitrance of these microbes to cellular lysis makes accessing their DNA for sequencing quite difficult apart from the use of harsh bead beating methods, which, in turn, degrade the quality of the DNA. Long-read sequencing technologies require high quality, high molecular weight DNA as an input; however, generating a representative DNA extraction from a rumen microbial sample requires some degree of bead beating in sample preparation. We have customized a method of DNA extraction of rumen liquid and solids that produces high molecular weight (~ 7-10 kilobasepairs average molecule size) input DNA for long-read sequencing. We have sequenced these samples on nanopore-based and zero-mode waveguide sequencers and achieved a maximum read size of greater than 100 kilobases in length – which is the longest DNA sequence read for a rumen microbial sample reported to date. Whole-metagenome assembly has already commenced on the sequence data already generated by this project. The impact of ruminal microbes on animal performance (Objective 1.A.2.) is being explored in a long term, collaborative study involving ruminal inoculation of heifer calves with ruminal inoculum from high efficiency or low efficiency cows. Cohorts of calves are being inoculated and necropsy samples are being obtained from bull calves to determine effects of different inocula on growth, chemistry, rumen epithelial development and composition of the rumen microbial community. Whole transcriptome sequencing in rumen and liver tissues collected from calves was completed. Through comparative analysis, we observed significantly elevated expression in genes involved in immune and defense process in rumen tissues. Furthermore, liver transcriptome changes suggested potential increased metabolism in sphingolipids, an essential molecular signal for bacteria survival in digestive tracts. This study provided first-hand insights into host transcriptome changes associated with early colonization of microbial community in neonatal calves. An additional calf study was conducted evaluating the effects of early life inoculation on the establishment of the ruminal microbial population and subsequent phenotypic responses. Calves were ruminally inoculated with either bacterial- or protozoa-enriched inoculum from week 3 to 6 of life and samples of the ruminal contents, calf growth measurements, blood samples, and digestive tract measurements were collected. The findings of this study showed that microbial inoculation and inocula type altered the ruminal environment and animal performance of dairy calves. A follow-up study to this work is currently being conducted to evaluate ruminal microbial community composition changes over the first 8 months of life. This will be the first study that we are aware of that will follow the development of the protozoal, planktonic, and sessile microbial communities concurrently in developing dairy calves. Approaches for lysis of rumen microbes in order to measure nutrients they can provide to the cow (Objective 1.B.1) were evaluated and tested on in vitro-produced cell pellets. Efficacy of microbial cell lysis with enzymes vs. the accepted alkali method was assessed using measurement of glycogen, a storage carbohydrate that microbes store internally. We achieved 104% recovery of glycogen with enzymatic over alkaline lysis. Addition of detergent did not improve recovery. In the evaluation of rumen microbial impact on digestibility of dietary starch (Objective 1.B.2), we have 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 did not alter the effect of time. This suggests that the starch-degrading enzymes are external to the microbes, and that processing must be done as quickly as possible. 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. Both commonly used cobalt and chromium ethylenediaminetetraacetic acid markers showed signs of dissociation and so are not ideal markers. In vitro incorporation of 15N into microbial protein and fermentation effects of differing nitrogen sources on microbial protein synthesis was determined (Objective 1.B.4). Microbial non-ammonia nitrogen was progressively labeled over the 12 hour in vitro experiment. Concentration changes of volatile fatty acids, the profile of volatile fatty acids, and in vitro gas production was affected by protein source; when urea was the nitrogen source, which is a nonprotein nitrogen source, gas production was the lowest. More than 20 tissue types from more than 45 dairy cattle have been collected for transcriptomics (Objective 2.A.1). Tissues from the digestive tract represent the predominant portion of this collection. These tissue types were collected from feeding trials with various objectives: an early dosing trial in new born calves using rumen fluid collected from adult dairy cattle; individual-based feed efficiency studies using feeds differing in canola meal content; feed induced acidosis in young dairy calves. Additionally, whole transcriptome sequencing of three tissues types (rumen, liver, and Ilium) collected from 10-week old calves dosed with either rumen content obtained from adult dairy cattle or with autoclaved rumen content (control) was carried out. High-quality RNA was successfully extracted from all of the calves from five additional tissues types: omasum, abomasum, reticulum, caecum and pituitary gland. An additional analysis was conducted on lung and tonsil tissues from dairy calves with or without pneumonia. Using a comparative transcriptomic approach, host transcriptome markers are being explored to accurately detect pneumonia in young calves at an early stage. Such early detection biomarkers will most likely improve pneumonia management in dairy herds.

1. Demonstrating the impact of eliminating animal agriculture. Farmed animals provide essential nutrients in human diets, and produce greenhouse gases and use food resources that could potentially be used by people. ARS scientists in Madison, Wisconsin evaluated the impact of eliminating farmed animals from U.S. agriculture and converting to a plants only system. Without animals, substantially more food would be produced. However, with the U.S. population reliant on the crops grown in the U.S. and currently imported, a plants-only diet without supplementation would have more nutrient deficiencies than a diet containing animal products. Nutrients such as vitamin B12 and certain fatty acids are only or largely consumed from animal-derived foods, those nutrients lacking or in low concentrations in plants would not meet needs in a consumable diet. Greenhouse gas emissions declined, but only by approximately 60% of what animals currently produce. The need to produce synthetic fertilizer to replace animal manures, and other changes in the system counterbalanced the removal of animals. The study showed that making changes to a complex system gives rise to expected and unexpected impacts. Recommendations for changes in our agricultural system requires integration of multiple disciplines to adequately evaluate potential impacts.

2. New dietary starch method to improve information for consumers. Accurate information on feed composition is essential for formulating healthy diets for cows and for informing consumers about the nutritional qualities of the animal feeds and pet foods they purchase. Starch, in particular, is a carbohydrate in feeds that can provide energy to meet an animal’s requirements in properly balanced diets, but can cause health disorders if mis-fed. A new dietary starch method received final approval for use in nutritional labeling of animal feeds and pet foods. It replaces a previous method that was no longer valid. An ARS animal scientist in Madison, Wisconsin spent 6 years testing and refining the assay, and then directed and participated in a collaborative study with fourteen state, commercial, and research feed analysis laboratories to fully test that dietary starch assay. The starch 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 gave it Final Action approval as AOAC Official Method 2014.10. It can now be used for feed labeling in order to provide consumers with information they need for considering how best to feed their animals. This same method is also offered by commercial feed analysis laboratories to provide information on the starch content of feeds used by field nutritionists to formulate rations for livestock. In 2017, commercial feed analysis laboratories ran dietary starch analyses valued at $1.3 million on over 1.4 million samples; this constitutes evidence that industry and nutritionists find the analyses of value, and that the laboratories find the analysis acceptable for their use. This research provided consumers and animal agriculture with an assay to accurately determine the nutritional quality of their feeds.

3. Description of the interplay between rumen microbiome and host transcriptomic and genomic profiles. Early colonization of the microbial community in neonatal calves and the response of the calves to the microbial community is required for the calves to be a functional ruminant, but this process is poorly understood. ARS scientists in Madison, Wisconsin have finished whole transcriptome sequencing in rumen and liver tissues collected from calves with or without neonatal inoculation of rumen content obtained from adult dairy cattle. Through comparative analysis, genes involved in immune and defense processes in rumen tissues were significantly elevated. Furthermore, liver transcriptome changes suggested potential increased metabolism in sphingolipids, an essential molecular signal for bacteria survival in digestive tracts. This study provided evidence of host transcriptome changes associated with early colonization of microbial community in neonatal calves. These results will provide valuable insights into the genomic determinants of rumen microbiome diversity, help improve management of rumen microbiome diversity using information from host genomic profiles, and potentially lead to the use of this information for improved feed efficiency.

4. Development of a new method of DNA extraction that enables long-read sequencing of metagenomics samples. Long DNA molecules are required for contiguity of reference genomes, however current extraction methods do not yield DNA molecules of the appropriate length. By using a robust, yet gentle, lysis protocol, ARS scientists from Madison, Wisconsin were able to obtain longer DNA molecules from complex, recalcitrant microbial samples. This protocol will enable the use of long-read sequence data to be used in metagenome sequencing and assembly, which is likely to improve the contiguity of rumen microbial reference genomes. Beyond the sequencing and assembly of rumen microbial species, this method has applications in detecting microbial species in other samples, such as the soil, human gastrointestinal tract, and other environments. This has great potential in allowing researchers to discover new genes in complex microbial communities that may be used to improve the environment and health.

5. Cow genotype and protein nutrition interact. Cows are usually fed as a group of individual high, average, and low genetic merit cows. A feeding study was conducted by ARS researchers in Madison, Wisconsin with first lactation Holstein dairy cows to evaluate the implication of milk protein genotype on response to reduced dietary protein. Cows were blocked into four groups based on genomically informed milk protein genotype and fed either an adequate or a reduced protein diet for 12 weeks. Results indicated that milk protein yield tended to interact with genotype within the range of genotypes available for this study. 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. High genotype cows fed the low protein diet produced numerically, but not significantly, less milk than low genotype cows fed the high protein diet. More data are needed with a greater dietary diversity, however, data indicate that cows could be fed more precisely using cow genotype information to improve milk protein yield and efficiency which could lead to cost savings to dairy producers.

Review Publications
Li, W., Bickhart, D.M., Ramunno, L., Iamartino, D., Williams, J., Liu, G. 2018. Comparative sequence alignment reveals River Buffalo genomic structural differences compared with cattle. Genomics.
Schwartz, J.C., Philp, R.L., Bickhart, D.M., Smith, T.P., Hammond, J.A. 2018. The antibody loci of the domestic goat (Capra hircus). Immunogenetics. 70: 317-326. doi:
Porto-Neto, L.R., Bickhart, D.M., Landaeta-Hernandez, A.J., Utsunomiya, Y.T., Morales, M.P., Caban-Jimenez, E., Hansen, P.J., Dikmen, S., Schroeder, S.G., Sun, J., Crespo, E., Amati, N., Cole, J.B., Null, D.J., Garcia, J.F., Reverter, A., Barendse, W., Sonstegard, T.S. 2018. Convergent evolution of slick coat in cattle through truncation mutations in the prolactin receptor. Frontiers in Genetics. 9:57.
Zhou, Y., Connor, E.E., Bickhart, D.M., Li, C., Baldwin, R.L., Schroeder, S.G., Rosen, B.D., Yang, L., Van Tassell, C.P., Liu, G. 2018. Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm. Gigascience. 7(5):1-13.
White, R.R., Hall, M., Firkins, J.L., Kononoff, P.J. 2017. Physically adjusted NDF (paNDF) system for lactating dairy cow rations. I: Deriving equations that identify factors that influence effectiveness of fiber. Journal of Dairy Science. 100:9551-9568.
White, R.R., Hall, M., Firkins, J.L., Kononoff, P.J. 2017. Physically adjusted NDF (paNDF) system for lactating dairy cow rations. II: Development of feeding recommendations. Journal of Dairy Science. 100:9569-9584.
Hall, M., Mertens, D.R. 2017. A 100-year review: Carbohydrates - characterization, digestion, and utilization. Journal of Dairy Science. 100:10078-10093.
Zanton, G.I. 2017. Protein and amino acid nutrition. In: Beede, D.K., editor. Large Dairy Herd Management. 3rd edition. Champaign, IL: American Dairy Science Association. p. 625-637.
Dorea, J.R., Danes, M.A., Zanton, G.I., Armentano, L.E. 2017. Urinary purine derivatives as a tool to estimate dry matter intake in cattle: a meta-analysis. Journal of Dairy Science. 100: 8977-8994.
Heinrichs, A.J., Jones, C.M., Lascano, G.J., Zanton, G.I. 2017. Invited review: 100 years of dairy heifer research. Journal of Dairy Science. 100: 10173-10188.
Paula, E.M., Broderick, G.A., Danes, M.A., Lobos, N.E., Zanton, G.I., Faciola, A.P. 2018. Effects of replacing soybean meal with canola meal or treated canola meal on ruminal digestion, fermentation pattern, omasal nutrient flow, and performance in lactating dairy cows. Journal of Dairy Science. 101:328-339.
Baldin, M., Zanton, G.I., Harvatine, K.J. 2017. Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression. Journal of Dairy Science. 101: 376-385.
Li, W., Bickhart, D.M., Ramunno, L., Lamartino, D., Williams, J., Liu, G. 2018. Genomic structural differences between cattle and river buffalo identified through a combination and genomic and transcriptomic analysis. Data in Brief. 19:236-239.