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





- Zanton, Geoffrey
- Li, Wenli
- Hall, Mary Beth
- McClure, Jennifer
- 2023
- Publications
- Food Animal Production (101)

Research Project: Developing Strategies to Improve Dairy Cow Performance and Nutrient Use Efficiency with Nutrition, Genetics, and Microbiology **Location:
                            Cell Wall Biology and Utilization Research** 2023 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**
                                

In support of Sub-objective 1.A.1 and to evaluate the most effective method to extract high molecular weight DNA from rumen liquids, methods with different commercial kits and times for bead beating (a cell disruption method) were assessed. This work was conducted because the standard extraction methods require a very long bead beating step which could overly shear DNA and prevent recovery of high molecular weight DNA from the samples. This experiment is in the data analysis phase to identify if the different methods provide different percentages of bacterial reads based on whether the bacteria are gram negative or gram positive. The hypothesis is that gram positive bacteria and the non-bacterial components such as archaea, fungi and protozoa, will have smaller DNA fragment length as the bead beating time increases and gram negative bacteria reads will be greater in the longer bead beating steps. Smaller fragment lengths make assembly of the genomes more difficult due to the inability to adequately assess sections of the genome that may repeat.

For Sub-objective 1.A.2, we initiated a study evaluating heifers raised and grazed on pasture or in barns and fed total mixed rations with assessment of vitamin B12 status, animal behavior, and hair cortisol levels. Sampling of blood, hair, and performing behavior observations has been started.

A study requested by a major stakeholder on comparison of neutral detergent fiber (NDF) methods was completed. The study compared current method variants to the official method across a range of dairy cattle feeds. The manuscript reporting these results has been accepted for publication in the Journal of Dairy Science. The research has garnered substantial interest from the feed analysis community. Once the information is released publicly, it should garner more attention. 

For addressing next year’s planned work on rumen liquid passage kinetics, we have made progress on development of the model system for in-laboratory manipulations of rumen contents. Sufficiently durable sample bags have been found and we have been working with an engineering technician to design equipment to carry out the studies. Rumen digesta volume measurements are essential for accurate estimation of liquid passage based on gram disappearance of liquid marker. Currently, rumen evacuation and weighing of the digesta, a labor-intensive procedure that is disruptive to rumen function, is the only method for obtaining rumen digesta volumes. Using data developed under the previous project plan, we have developed the calculations to estimate rumen digesta volume from external cow measurements without having to evacuate rumens. This is much less labor intensive, less disruptive, and easily done. 

For Sub-objective 2.A, meta-analysis of the core microbiome embedded in cattle liver tissue using RNA sequencing data collected from various cattle breeds was conducted. Liver transcriptome sequences were extracted from published data for several cattle breeds fed different diets (n = 441 samples). After filtering host-derived reads, bacterial RNA reads were enriched by mapping the non-cattle reads to an RNA reference library. An average of 350,000 ± 77,000 ribosomal RNA reads were obtained for each sample and five core genera were identified in the liver across the eight breeds. Significant microbial abundance differences were observed among different breeds from healthy individuals, which indicates that there is a commensal microbiome in the liver of cattle that is impacted by host genetic background. Additionally, liver microbial community analysis using metatranscriptome sequencing of liver tissues collected from young calves with or without feed-induced acidosis has been completed. We observed significant changes in the liver microbiome between the treatment groups. Most interestingly, we identified concurrent changes in microbial taxa in both the liver and rumen epithelium, indicating potential cross talk between the liver and the rumen epithelium. For each tissue type, a total of eight samples (four samples for each treatment) were included in this experiment. In addition, single-cell RNA (scRNA) sequencing library preparation, sequencing, and preliminary data analysis of calf liver tissues with or without feed-induced acidosis was completed. This analysis was limited to four samples (two samples for each treatment), which is typical for scRNA sequencing analyses due to their resource-intensive nature.

Cattle caecum transcriptome analysis was completed along with its associated microbial communities in calves artificially dosed with rumen contents extracted from adult cows. Eight calves were included in the study, with four of them receiving artificially dosed adult rumen contents and four receiving autoclaved rumen contents as a control.

For Sub-objective 2.B, the effects of early lactation milking frequency on various parameters in cows milked via an automated milking system was studied. A total of 16 cows were included in this experiment. All cows were milked three milkings per day (MPD) from 0 to 3 days in milk (DIM). From 4 to 29 DIM (experimental phase), cows were milked at frequencies of either 3 or 6 MPD. For each cow, we have collected the rumen liquid, rumen solid, blood, and urine samples at these time points: baseline (3 days), 14 days and 28-29 days. Whole transcriptome analysis will be performed on blood samples to identify host responses to different milking frequencies and metatranscriptome analysis will be performed on rumen solid and liquid samples to identify microbial community changes in response to host energy demand changes.

The transition period in dairy cows is a time of large changes in dry matter intake and a high risk for metabolic and infectious disease. The rumen microbiome was studied in rumen cannulated cows as they transitioned from the dry period to early lactation. Twelve rumen cannulated cows were enrolled 2 weeks prior to calving and rumen contents, blood, milk, feces, and urine were collected periodically until 28 DIM. These samples are currently being used for analyses of ruminal fermentation and microbiome structure, blood metabolites, rumen metatranscriptomics, and blood microbiome responses. These analyses will help identify potential biomarkers related to host metabolic changes during the transition period.

Alfalfa samples (n = 730) of differing genotypes, growing seasons, and field locations were analyzed for protein fractions and in vitro degradation. Results will be used as part of a collaborative effort to determine genome-wide association to identify multiple loci associated with alfalfa protein degradation characteristics. **Accomplishments**
                                

1. Routinely used neutral detergent fiber analysis methods agree to varying extents with reference methods. Neutral detergent fiber (NDF) is the most widely used measure of fiber in livestock diets and is used in diet formulation to keep animals healthy and productive. Additionally, NDF is used to assess the economic value of forages. ARS researchers in Madison, Wisconsin, compared currently used alternative methods to measure NDF to the established reference method using 11 diverse feeds. They found that some of the method variants were in better agreement with the reference method than others. It is highly recommended that the methods be compared by other labs and using a greater and more diverse set of feeds to assess the findings. This is important because if values from alternative NDF methods do not agree with those from the reference method for NDF, they may not be appropriate for some uses. These results are of interest to dairy farmers, consulting dairy nutritionists, and feed analysis laboratories to improve dietary formulations for optimum cow health and productivity. 2. Optimal levels of dietary protein for dairy cow productivity changes over the course of a lactation. Dairy cows convert low quality dietary crude protein (CP) to high quality milk protein, but this process can be inefficient. Feeding excess CP is costly, wasteful, and can result in detrimental impacts on the environment. For much of lactation, dairy cows are often fed a common diet with a fixed level of CP even though milk protein production changes throughout a lactation cycle, but there was little information on how these recommendations should change throughout a lactation cycle. In collaborative work with University of Wisconsin-Madison, ARS researchers in Madison, Wisconsin, determined that the optimum level of dietary CP that maximizes milk component (milk fat, protein, and lactose) output changes throughout a lactation cycle. Cows can be fed an intermediate level of CP to maintain dry matter intake and milk energy output in early and mid-early lactation, and CP can be reduced when cows reach late lactation. Feeding different diets would improve nitrogen use efficiency without affecting productivity. These results are important to dairy farmers and their consulting nutritionists when formulating diets for cows across the lactation cycle to precisely target CP levels for optimum productivity. 3. Total RNA extraction method for tissue embedded microbial communities in liver that ensures a representative measure of the microbes present. In cattle, the rumen and its microbiome have been the primary focus in investigating productive performance and nutrient use efficiency. Tissue embedded microbial communities (TEMC) may occur in multiple tissues of the body and may be correlated with various metabolic disorders and digestive diseases. Gastrointestinal tract (GIT) and liver TEMC and their relation to host health and performance remains largely unexplored in dairy cattle because methods to reliably characterize tissue microbiomes is lacking. To overcome this obstacle, ARS researchers in Madison, Wisconsin, developed an enzyme-based digestion and filtration method along with RNA extraction to minimize host RNA from cattle liver tissue. The new method results in significant improvements in recovery of microbial transcripts and identification of microbial protein coding transcripts. This new method will allow better elucidation of the involvement of TEMC in dairy cattle health and productivity and will be useful to other researchers working in this and related areas. **Review Publications**
                            *Letelier, P., Zanton, G.I., Wattiaux, M.A. 2022. Production performance of Holstein cows at 4 stages of lactation fed 4 dietary crude protein concentrations. Journal of Dairy Science. 105(12):9581-9596. https://doi.org/10.3168/jds.2022-22146.*
                                *Parian-Khajehdizaj, F., Noel, S., Johansen, M., Weisbjerg, M., Hellwing, A., Hojberg, O., Hall, M., Lund, P. 2023. Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed. Journal of Dairy Science. 106(6):4072-4091. https://doi.org/10.3168/jds.2022-22638.*
                                *Dinkins, R.D., Hancock, J.A., Bickhart, D.M., Sullivan, M.L., Zhu, H. 2022. Expression and variation of the genes involved in rhizobium nodulation in red clover. Plants. 11(21). Article 2888. https://doi.org/10.3390/plants11212888.*
                                *Zhou, Y., Yang, L., Han, X., Han, J., Hu, Y., Li, F., Xia, H., Peng, L., Boschiero, C., Rosen, B.D., Bickhart, D.M., Zhang, S., Guo, A., Van Tassell, C.P., Smith, T.P., Yang, L., Liu, G. 2022. Assembly of a pangenome for global cattle reveals missing sequences and novel structural variations, providing new insights into their diversity and evolutionary history. Genome Research. 32(8):1585-1601. https://doi.org/10.1101/gr.276550.122.*
                                *Ortega, M.S., Bickhart, D.M., Lockhart, K.N., Null, D.J., Hutchison, J.L., McClure, J.C., Cole, J.B. 2022. Truncation of IFT80 causes early embryonic loss in Holstein cattle associated with Holstein haplotype 2. Journal of Dairy Science. 105(11):9001-9011. https://doi.org/10.3168/jds.2022-21853.*
                                *Low, W.Y., Rosen, B.D., Ren, Y., Bickhart, D.M., To, T., Martin, F.J., Billis, K., Sonstegard, T.S., Sullivan, S.T., Hiendleder, S., Williams, J.L., Heaton, M.P., Smith, T.P. 2022. Gaur genome reveals expansion of sperm odorant receptors in domesticated cattle. BMC Genomics. 23. Article 344. https://doi.org/10.1186/s12864-022-08561-1.*
                                *Baldin, M., Garcia, D., Zanton, G.I., Hao, F., Patterson, A.D., and Harvatine, K.J. 2022. Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on milk fat, rumen environment and biohydrogenation, and rumen protozoa in lactating cows fed diets with increased risk for milk fat depression. Journal of Dairy Science. 105(9):7446–7461. https://doi.org/10.3168/jds.2022-21910*


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