Source: OHIO STATE UNIVERSITY submitted to NRP
AN INNOVATIVE APPROACH TO IMPROVE NITROGEN UTILIZATION IN RUMINANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024600
Grant No.
2021-67015-33393
Cumulative Award Amt.
$496,000.00
Proposal No.
2020-02704
Multistate No.
(N/A)
Project Start Date
Jan 1, 2021
Project End Date
Dec 31, 2024
Grant Year
2021
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Animal Sciences
Non Technical Summary
Ruminants, especially dairy cows and beef cattle, are an important source of high-quality protein for human consumption. They can utilize forage and grains to make animal protein like milk and beef. However, they can only utilize about 30% of the dietary nitrogen, the most important component of protein. The lower nitrogen efficiency in ruminants than in non-ruminants is attributed to some microorganisms in the stomach of the former, particularly rumen protozoa. As a unique group of microorganisms of the microbial community in the rumen, rumen protozoa engulf and degrade the microbial protein synthesized by the microbial community using the ingested feed, which creates recycling of microbial protein in the rumen and decreasing the flow of the protein to the small intestine where protein is digested and absorbed. This wasteful intraruminal recycling of microbial protein significantly decreases nitrogen utilization efficiency in all ruminants, resulting in high production cost and environmental pollution by the excreted nitrogen. Thus, there is a critical need to decrease the intraruminal recycling of microbial protein to improve nitrogen utilization efficiency in ruminants, especially in dairy cows. Recently, we found that (i) rumen protozoa depend on live bacteria for their survival and growth, (ii) the genes encoding their digestive enzymes are highly expressed, (iii) inhibition of the digestive enzymes substantially inhibits rumen protozoa and ammonia production, without adversely affecting other aspects of the rumen ecosystem, and (iv) specific inhibitors against the digestive enzymes can be predicted from the structures of the digestive enzymes. In this research, we will conduct in vitro studies to identify the best inhibitors, determine the effective doses, and investigate their effect on feed digestibility, engulfment and degradation of bacteria, activities of the digestive enzymes, and the rumen microbial community.We will then use growing lambs as a model to verify the inhibitors and their effect on the animals and the rumen microbiome. Our ultimate goals are to fill critical gaps in knowledge on the roles of protozoal digestive enzymes in rumen function and to develop an innovative approach to improve nitrogen utilization efficiency, particularly in dairy cows and beef cattle, with no adverse effect on the animals or their rumen ecosystem. This proposed research will be transformative and translational and can potentially benefit the US dairy and beef producers economically and the society environmentally in the future.
Animal Health Component
45%
Research Effort Categories
Basic
50%
Applied
45%
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3024050110370%
3024010106020%
3025230101010%
Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
4050 - Protozoa; 5230 - Feed and feed additives; 4010 - Bacteria;

Field Of Science
1103 - Other microbiology; 1060 - Biology (whole systems); 1010 - Nutrition and metabolism;
Goals / Objectives
Currently, only about30%of the dietary nitrogen is retained as milk or meat by ruminants, including dairy cows, beef cattle, and sheep. The excretion and waste of most of the dietary nitrogen not only increases production cost but also creates pollution of the environment.Improvement of NUE in ruminants without depressing animal performance is critical tothe sustainability of secure supplies of high-quality meat (lamb and beef) and milk.Predation and subsequent degradation of rumen bacteria, which are the major protein source available to cows and sheep, by rumen protozoa is one of the major causes of the low nitrogen utilization efficiency.?Our long-termgoal is to understand the metabolism, physiology, and ecology of rumen protozoa, especially thegenus Entodinium, to inform future development of new strategies to improve NUE in ruminantsby specifically inhibiting rumen protozoa and decreasing protozoa-mediated intraruminal Nrecycling without adversely affecting the ruminants or their rumen microbiome.
Project Methods
Molecular docking will be used to predict the structure of the lysozyme and peptidases and compounds that can bind to and inhibit these enzymes. Although it has never been used in animal nutrition-related research, molecular docking has been commonly used in drug development, including drugs to inhibit protozoan parasites.In vitro studies will be used to evaluate the efficacy of the potential inhibitors predicted above with respect to inhibition of rumen protozoa, predation of bacteria by rumen protozoa, and the lysozyme and peptidases. We will also evaluate if the inhibitors inhibit feed digestion or alter rumen microbiome and fermentation. The promising inhibitors determined in the in vitro studies will be tested using growing lambs. Animal growth, nitrogen utilization efficiency, feed intake, and feed digestion and fermentation will be assessed using standard methods. We will also analyze rumen microbiome (using metataxonomics); pool size of rumen protozoa, bacteria, archaea, and fungi (using qPCR); and activities of lysozyme and peptidases (using enzyme assays).

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:Dietary nitrogen utilization efficiency in ruminants is much lower than in nonruminants. One major reason is intra-ruminal microbial protein recycling, through which microbial protein, the primary protein source available to ruminants, is recycled rather than flowing to the small intestines, where it is digested and absorbed. This research project aimed to advance our understanding of intra-ruminal microbial protein recycling, informing the development of effective strategies to reduce wasteful intra-ruminal microbial protein recycling. We focus on two groups of rumen microbes: rumen protozoa and viruses. We chose to investigate these two microbial groups because the former directly drives the intra-ruminal microbial protein recycling by engulfing and degrading microbial protein, while the latter promotes intra-ruminal microbial protein recycling by lyse their hosts: rumen bacteria and other microbes. Therefore, this research project addresses an important issue of feed efficiency, specifically nitrogen efficiency in ruminants, and the outcomes of this research project shall be of interest to i) researchers who are interested in rumen microbiome research, especially rumen protozoa and viruses; ii) ruminant nutritionists who strive to improve dietary nitrogen utilization efficiency and mitigate methane emissions (rumen protozoa increase methane production in the rumen by producing hydrogen and forming a positive symbiosis with methanogens; while rumen viruses infecting methanogens may be used to mitigate methane emission by killing their methanogen hosts); and iii) feed additive companies that are developing new feed additives to improve feed efficiency and mitigate methane emissions from cattle. We found that rumen protozoa possess powerful enzymes that lyse engulfed bacteria and degrade the microbial protein. We also identified specific inhibitors that can selectively inhibit and reduce rumen protozoa that are highly active in degrading microbial protein. Additionally, we developed new analytic methods to analyze rumen protozoa, which can be used to investigate the relationship between rumen protozoa communities and dietary nitrogen utilization efficiency. With respect to rumen viruses, we developed the first global database specific to the viral communities within the rumen. Leveraging this database, we comprehensively characterized the diversity of rumen viruses, their interactions with other rumen microbes, and correlations with key animal productivity traits such as growth, milk production, and methane emissions. The research findings substantially advance our understanding of rumen protozoa and viruses and their potential impact on animal production. They can also improve the animal science curriculum for students (undergraduate and graduate) in rumen microbiology, ruminant nutrition, and herd management. Changes/Problems:The initial plan includes an animal feeding trial to evaluate the identified protozoa inhibitors in vivo using sheep. Unfortunately, purchasing these chemicals appeared cost-prohibitiveto conduct an animal feeding trial with sufficient statistical power. Therefore, the initially proposed animal feeding trial was not undertaken. We conducted an animal feeding trial to investigate the association between the rumen microbiome and diet, focusing on forage and nitrogen content. What opportunities for training and professional development has the project provided?1. One Ph.D. student (he) worked on this research project for four years. He had little background in rumen microbiome, protozoology, metataxonomics, metagenomics, or bioinformatics. He was trained in sample collection from cannulated cows, preparation of rumen samples for microscopic identification of rumen protozoa, and morphological identification of rumen protozoa at the genus level. He was also trained in protein structure modeling, molecular docking, metataxonomics, and metagenomics, all of which are important skills required to continue this line of research. He was also trained to use supercomputing to conduct large-scale bioinformatic analysis. He also learned some basic virology and bioinformatics resources (databases and software packages) to analyze virome data. Finally, he was trained to develop new algorithms and bioinformatics tools. This student has become more proficient in various bioinformatics analyses, including developing new bioinformatic analysis tools. During his graduate education, he developed analytical and creative thinking abilities, enhanced communication skills, and improved scientific writing. 2. One postdoctoral fellow (she) joined the research starting in March 2022. She has some background in freshwater ciliates and molecular biology techniques but is not familiar with rumen ciliates. She was trained in culturing rumen ciliates and performing in vitro studies using rumen ciliate cultures. She was trained to use supercomputing and perform bioinformatic analyses of the genomes of rumen protozoa, protein modeling, and molecular dock. She expanded her knowledge base, enhanced her analytical and creative thinking abilities, and improved her communication skills and scientific writing. How have the results been disseminated to communities of interest?The results of the studies of this research project have been published in several peer-reviewed articles and presented at national and international conferences (see Products). Databases and genomic sequences generated in this project are deposited in public archives. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? New genomic insights into rumen protozoa 1. We refined the genomic sequences of Entodinium caudatum and performed extensive bioinformatic analysis of the transcriptomic and genomic sequences and obtained the full length of the target genes that encode the lysozyme, serine peptidase, and cysteine peptidase in Entodinium caudatum. Using the full-length gene sequences, we repeated the structural modeling of the above enzymes and performed molecular docking analysis again to screen two small-molecule libraries to identify candidate compounds of plant origin that might potentially inhibit the lysozyme and peptidases of Entodinium caudatum. In total, we identified 33 potential inhibitors of the lysozyme and peptidases. 2. In vitro screening of the 33 potential inhibitors did not yield promising results. Then, we hypothesized that the rumen ciliate we genome-sequenced, Entodinium caudatum MZG-1, might not be representative of the rumen protozoa. Thus, in collaboration with other researchers, we sequenced 52 strains of rumen protozoa. We created a new genome-based taxonomic framework that helps improve the taxonomic classification of rumen protozoa. A vast diversity of carbohydrate-active enzymes was found, and the enzymatic activities of several of them were verified after cloning and overexpression. These protozoal genomes provided us with more complete information regarding the types of peptidases and lysozyme that rumen protozoa use. 3. We completed comprehensive analyses of the lysosomal enzymes of rumen protozoa, identifying numerous enzymes involved in the digestion of polysaccharides and proteins. Comparative analyses of the enzyme repertoires revealed protozoal species with varying capabilities in digesting feed and microbial proteins. Some of the lysosomal enzymes can be further explored for new specific inhibitors to inhibit rumen protozoa. Identifying and in vitro testing the protozoal inhibitors 1. Using the new genomic resources, particularly the genomes of the major group of rumen protozoa, the genus Entodinium, we identified potential inhibitors of peptidases using molecular docking. We sourced most of the potential inhibitors and experimentally evaluated their actual abilities to inhibit rumen protozoa. Eventually, we identified eight promising inhibitors (all are derived from plants) and then further evaluated their effect on feed digestion, fermentation, ammonia production (an indicator of protein degradation), and other rumen microbes. The data are being analyzed and one manuscript will be written to report the final results. New knowledge on rumen viruses with respect to their role in protein degradation in the rumen 1. The rumen ecosystem was recently shown to have diverse viruses that infect bacteria. These viruses infect rumen bacteria (methanogens, protozoa, and fungi) and lyse their host cells, contributing to the intra-ruminal recycling of microbial protein and decreasing the outflow of microbial protein to the small intestine of ruminants. Therefore, it is important to identify and understand the rumen virome (the totality of viruses present in the rumen ecosystem). We collected most of the published rumen metagenomes (nearly 1,000), which represent the rumen microbiomes and viromes collected from across different species of ruminants across the globe, and performed comprehensive detailed bioinformatic analyses. We created the first global rumen virome database (RVD) that documented the viruses infecting the cellular microbiomes of the rumen ecosystem (Nature Communications, 14(1): 5254). This global virome database has facilitated numerous studies to investigate the roles and associations of rumen virome in rumen functions (feed digestion, fermentation, methanogenesis) and animal performance (feed and nitrogen efficiency, methane emissions). 2. Building upon our recent study on the rumen virome and leveraging the global rumen virome database (RVD), we conducted a comprehensive investigation into the potential association between the rumen virome and key animal production traits. We found that several important production traits are associated with the diversity and abundance of rumen viruses. These traits include average daily gain, feed efficiency, lactation performance, subacute rumen acidosis, and methane emissions in beef cattle, dairy cows, and sheep. New technique to analyze rumen protozoa 1. Researchers have been using microscopy to identify and count rumen protozoa present in rumen samples. Morphologic identification and microscopic counting are time-consuming and inaccurate. Sequence-based identification and quantification with quantitative PCR (qPCR) have been used, but the marker gene (18S rRNA gene) of rumen protozoa is very conserved among rumen protozoa, not providing species-level taxonomic resolution. To address this need, we designed new qPCR primers based on marker genes that can provide better taxonomic resolution (28S rRNA gene and the internal transcribed sequence, the latter of which is between the 18S and 28S rRNA genes). The new technique will greatly facilitate comprehensive analyses of protozoal communities in the rumen of various ruminants. 2. While metagenomics has been used in most studies on the rumen microbiome and its involvement in ruminant nutrition, bacteria, and to a lesser extent archaea, are analyzed, whereas rumen protozoa and fungi are ignored due to the lack of bioinformatics tools. To address this technological gas, we developed a new software tool named GutEuk, which allows researchers to comprehensively analyze the communities of rumen protozoa and fungi in existing rumen metagenomes or future metagenomic studies. New insights into the paradigm of nitrogen metabolism in the rumen 1. The nitrogen metabolism in the rumen microbiome remains poorly understood. We completed one comprehensive study analyzing the genomes sequenced rumen bacteria and nearly 1,000 rumen metagenomes for genes encoding enzymes involved in ammonia assimilation, a critical process that converts dietary nitrogen into microbial protein, the major source protein available to ruminants. We identify new nitrogen assimilation pathways and strategies employed by prominent rumen bacteria. 2. Complement the above genomic study, we also completed one animal feeding trial to investigate the crucial rumen microbes that are involved in dietary nitrogen metabolism using sheep as a model. Using metagenomics and metatranscriptomics technologies, we analyzed the collected rumen microbiome samples and characterized the strategies of nitrogen utilization by key rumen bacteria and how they respond to dietary shifts in readily fermentable carbohydrates and levels of dietary nitrogen. These studies provide significant new insights into the nitrogen metabolism within the rumen, informing future research to improve nitrogen utilization in ruminants. One manuscript is being prepared to report the results of this integrated study. New information on protein degradation capacity of rumen bacteria 1. We came to realize that bacterial peptidases can also affect protein degradation and metabolism in the rumen. We thus analyzed the peptidases encoded by genes in sequenced genomes of Prevotella and Paraprevotella species, the most dominant rumen bacteria. That study provided new insights into the occurrence and potential capacity of proteolysis mediated by species of Prevotella and Paraprevotella.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yan M, Pratama A, Somasundaram S, Li Z, Y Jiang, Sullivan M, Yu Z. Interrogating the diversity and ecological importance of viral dark matter in the rumen ecosystem. 2024 Congress on Gastrointestinal Function, Champaign, IL. April 8-10, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Somasundaram S, Yan M, Yu Z. Peptidase distribution among rumen ciliates: A bioinformatic perspective on lysosomal enzyme profiles. 2024 Congress on Gastrointestinal Function, Champaign, IL. April 8-10, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yu Z. 2023. The Rumen Microbiome and Function - Predators within and their implication in intraruminal recycling of microbial protein. 2023 American Dairy Science Association Annual Meeting, June 25-28, Ottawa, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yu Z. 2024. The giants and the dwarfs within the rumen ecosystem - Rumen ciliates and viruses. International Symposium on Ruminant Physiology 2024. August 26-29, Chicago, IL.
  • Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2025 Citation: Yan M, Andersen TO, Pope PB, and Yu Z. 2025. Probing the eukaryotic microbes of ruminants with a deep-learning classifier and comprehensive protein databases. Genome Research, in press, https://doi.org/10.1101/gr.279825.124
  • Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2025 Citation: Yu Z, Somasundaram, and Yan M. 2025. Rumen protozoa and viruses: New insights into their diversity and potential roles through omics lensesA review. Journal of Dairy Science. In press, https://doi.org/10.3168/jds.2024-25780.
  • Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2025 Citation: Somasundaram, S. and Z. Yu. 2025. Internal transcribed spacers as phylogenetic markers enable species-level metataxonomic analysis of ciliated protozoa. ISME Communications, accepted. https://doi.org/10.1101/2024.02.02.578691
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Yan M and Yu Z. 2024. Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits. Microbiome, 12(1): 82.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Rumen protozoa and viruses: The predators within and their functionsA mini-review. JDS Communications, 5(3): 236-240.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Yan M, Pratama Akbar, Li Z, Jiang Y, Sullivan M, and Yu Z. 2023. Interrogating the viral dark matter of the rumen ecosystem with a global virome database. Nature Communications, 14(1): 5254.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2022 Citation: Patra AK and Yu Z. 2022. Genomic Insights into the Distribution of Peptidases and Proteolytic Capacity among Prevotella and Paraprevotella Species. Microbiology Spectrum. 10(2): e02185-21. https://doi.org/10.1128/spectrum.02185-21
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2021 Citation: Park T, Wijeratne S, Meulia T, Firkins J, and Yu Z. 2021. The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment. Genomics, 113(3): 1416-1427. https://doi.org/10.1016/j.ygeno.2021.03.014.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Target audience The goal of this research project was to advance our underpinning of the intra-ruminal microbial protein recycling, which has been blamed for lowering the dietary nitrogen utilization efficiency in ruminants, as well as to inform the development of effective strategies to reduce the wasteful intra-ruminal microbial protein recycling. We focus on the predators, rumen protozoa, within the rumen ecosystem and their role in driving the intra-ruminal microbial protein recycling by engulfing and degrading microbial protein, the major protein source for ruminants. Therefore, this research project addresses an important issue of feed efficiency, specifically nitrogen efficiency in ruminants, and the outcomes of this research project shall be of interest to i) researchers who are interested in rumen microbiome research, especially rumen protozoa; ii) ruminant nutritionists who strive to improve dietary nitrogen utilization efficiency and mitigate methane emissions (rumen protozoa increase methane production in the rumen by producing hydrogen and forming a positive symbiosis with methanogens); and iii) feed additive companies that are developing new feed additives to improve feed efficiency and mitigate methane emissions from cattle. The research findings can improve the animal science curriculum for students (undergraduate and graduate) in rumen microbiology, ruminant nutrition, and herd management. Changes/Problems:The initial plan includes an animal feeding trial to evaluate the identified protozoa inhibitors in vivo using sheep. Unfortunately, purchasing these chemicals is too expensive to conduct an animal feeding trial with sufficient statistical power. Therefore, the animal feeding trial will not be undertaken. What opportunities for training and professional development has the project provided?1. One Ph.D. student continued this research project. This student was trained to develop new algorithms and bioinformatics tools. This student has become more proficient in various bioinformatics analyses. 2. One postdoctoral continued to work on this project, focusing on the rumen protozoal component. This postdoc has become more proficient and experienced in culturing rumen protozoa and morphological identification, bioinformatic analyses of protozoal genomes, protein modeling, and molecular docking. How have the results been disseminated to communities of interest?Some of the results of this research project have been published in several peer-reviewed articles or presented at conferences (see Products). What do you plan to do during the next reporting period to accomplish the goals?1. Complete the evaluation of the promising inhibitors for their effects on different genera of rumen protozoa, feed digestion, fermentation, and the Microbiome. 2. Complete the analyses of the rumen microbiomes of the sheep to gain new insights into the microbes and their metabolism involved in dietary nitrogen metabolism and potentially influencing nitrogen efficiency. 3. Complete the bioinformatic investigation into the peptidases of rumen protozoa. 4. Complete the development of the new algorithm and bioinformatic tool to facilitate the analysis of eukaryotic DNA sequences within rumen metagenomic sequence data.

Impacts
What was accomplished under these goals? 1. In silico prediction found 33 compounds that could potentially inhibit the lysozyme and peptidases (the three-dimensional structures of these enzymes were predicted based on the amino acid sequences translated from our genome sequences of 53 rumen ciliates). We selected 13 of these compounds (all are derived from plants) based on their binding affinities to the target enzymes (lysozyme and peptidases) and evaluated their activities in inhibiting rumen protozoa in vitro. We found all these compounds can reduce total protozoal counts with varying degrees of reduction. 2. Following up on our effort in 2022, we have completed the design, verification, and evaluation of the new qPCR primers targeting marker genes that can provide better taxonomic resolution (28S rRNA gene and the internal transcribed sequence). One manuscript has been submitted to the journal Microbiome for publication. 3. The 53 sequenced genomes of the rumen protozoa provide us with an invaluable resource to investigate their proteolytic capability. We initiated and have made significant progress in the comprehensive bioinformatic study of the peptidases of rumen protozoa, especially the lysosomal peptidases, which are directly involved in the intra-ruminal recycling of microbial protein. 4. Building upon our recent study on the rumen virome (Nature Communications, 14(1): 5254) and leveraging the global rumen virome database (RVD), we conducted a comprehensive investigation into the potential association between the rumen virome and key animal production traits. We found that several important production traits are associated with the diversity and abundance of rumen viruses. These traits include average daily gain, feed efficiency, lactation performance, subacute rumen acidosis, and methane emissions in beef cattle, dairy cows, and sheep. One manuscript has been accepted by the journal Microbiome. 5. We completed one animal feeding trial to investigate the crucial rumen microbes that are involved in dietary nitrogen metabolism using sheep as a model. Meta-omics analyses of the collected samples are ongoing. We anticipate new information on the important microbes and their role in nitrogen metabolism within the rumen ecosystem.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Andersen, T. O., I. Altshuler, A. Vera-Ponce de Leon, J. M. Walter, E. McGovern, K. Keogh, C. Martin, L. Bernard, D. P. Morgavi, T. Park, Z. Li, Y. Jiang, J. L. Firkins, Z. Yu, T. R. Hvidsten, S. M. Waters, M. Popova, M. O. Arntzen, L. H. Hagen, and P. B. Pope. 2023. Metabolic influence of core ciliates within the rumen microbiome. ISME J 17(7):1128-1140.
  • Type: Book Chapters Status: Published Year Published: 2023 Citation: Yu, Z., A. Patra, and M. Yan. 2023. The use of bacterial probiotics and direct-fed microbials as dietary supplements in dairy cow nutrition. Pages 211-256 in Advances in sustainable dairy cattle nutrition. A. N. Hristov, ed. Burleigh Dodds Science Publishing.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Park, T. and Z. Yu. 2023. Interactions between Entodinium caudatum and an amino acid-fermenting bacterial consortium: Fermentation characteristics and protozoal population in vitro. J Anim Sci Technol 65(2):387-400.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Somasundaram, S. and Z. Yu. 2024. Internal transcribed spacers as phylogenetic markers enable species-level metataxonomic analysis of ciliated protozoa. bioRxiv:2024.2002.2002.578691.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Yan, M, and Z. Yu. 2024. Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits. Microbiome, accepted. https://doi.org/10.1101/2023.11.03.565476.
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Yan M, Pratama Akbar, Li Z, Jiang Y, Sullivan M, and Yu Z. 2023. Interrogating the viral dark matter of the rumen ecosystem with a global virome database. Nature Communications, 14(1): 5254.


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Target audience This research project aimed to advance the underpinning and mitigation of intra-ruminal protein recycling, which lowers the utilization efficiency of dietary nitrogen in ruminants. We focus on rumen protozoa and their role in driving intra-rumina protein recycling by engulfing and degrading microbial protein, the major protein source for ruminants. Thus, this research project is of interest to researchers who are interested in rumen microbiome research, especially rumen protozoa, ruminant nutritionists who want to improve dietary nitrogen utilization efficiency and mitigate methane emissions (rumen protozoa form positive symbiosis with methanogens and increase methane emissions from ruminants), feed additive companies that want to develop new feed additives to improve feed efficiency and mitigate methane emissions from cattle. The research findings can enhance and improve the curriculum of animal science students (both undergraduate and graduate), especially with regard to rumen microbiology, ruminant nutrition, and herd management. This research project may also be of interest to policy and makers who want to reduce the environmental footprint of ruminant production. Changes/Problems:1. The project was delayed by the Covid pandemic, the disruption of the supply chain by the Covid pandemic, and the need to resequence the genomes of more rumen protozoa. We may need to extend the project for one year. 2. The initial plan did not include studies on the rumen virome. However, as we realized recently, as intracellular predators of the rumen microbes, rumen viruses likely affect nitrogen utilization efficiency in ruminants. Therefore, we added the studies on the rumen virome, which entail bioinformatic analyses of metagenomic data that have been published. What opportunities for training and professional development has the project provided?1. One Ph.D. student continued on this research project. He was trained and learned to use supercomputing to conduct large-scale bioinformatic analysis. He also learned some basic virology and bioinformatics resource (databases and software packages) to analyze virome data. 2. One postdoctoral fellow joined the research starting in March. She has some background in freshwater ciliates and molecular biology techniques. She was trained in performing bioinformatic analyses of the genomes of rumen protozoa, protein modeling, molecular dock, and cultivation of rumen protozoa. How have the results been disseminated to communities of interest?Some of the results of this research project have been published in two peer-reviewed articles (see Products). The genomic sequences of 52 rumen protozoa have been deposited in the NCBI database. The rumen virome database has been archived in Zenoda. What do you plan to do during the next reporting period to accomplish the goals?1. We will in vitro screen the potential inhibitors of the protozoal lysozyme and peptidases (both cysteine and serine peptidases) that were inferred from the new genome sequences of rumen protozoa, which are more representative of the rumen protozoa of dairy and beef cattle. 2. Evaluate the inhibition of promising inhibitors to different genera of rumen protozoa using mixed rumen protozoa cultures in vitro. 3. Evaluate the effects of the inhibitors on feed digestion, fermentation, and the bacteria and methanogens in vitro. 4. Leveraging the new rumen virome database and the published metagenomic data we have collected (nearly 1,000 metagenomes), we will investigate the potential associations between the rumen virome and some of the important animal performance traits, such as methane emissions and feed efficiency. 5. All the previous studies used a gene-centric approach to investigate the association between feed/nitrogen efficiency and the rumen microbiome. With that approach, it is not possible to associate the specific microbes and their gene expression with a given animal trait. We will use a genome-centric approach to establish the direct linkage between nitrogen efficiency with specific rumen microbes and metatranscriptomics to link the gene expression to the genome of specific rumen microbes. With the information on the genome context and gene expression, we will have the opportunity to identify the rumen microbes that participate in nitrogen metabolism in the rumen and affect nitrogen efficiency.

Impacts
What was accomplished under these goals? 1. We screened the 33 compounds of potential inhibitors of the lysozyme and peptidases that were predicted based on the gene sequences of the Entodinium caudatum macronuclear genome. Of these compounds, all of plant origin, three showed inhibitions against rumen protozoa in vitro, but the potency is not as great as we hoped. 2. We hypothesized that the strain of rumen ciliate we genome-sequenced, Entodinium caudatum MZG-1, which was initially isolated from the rumen of a deer, might not be representative of the rumen protozoa of dairy cows. Thus, in collaboration with researchers from the Northwest Agricultural and Forestry University of China, we sequenced 52 strains of rumen protozoa, which were isolated from the rumen of dairy cows, dairy goats, and beef steers (that university covered all the sequencing costs). We created a new genome-based taxonomic framework that helps improve the taxonomic classification of rumen protozoa. A vast diversity of carbohydrate-active enzymes was found and the enzymatic activities of several of them were verified after cloning and overexpression. Using the new genomic sequences, we bioinformatically analyze the genes encoding lysozymes and peptidases (both serine and cysteine). Using the gene sequences that represent the major group of rumen protozoa, the genus Entodinium, we identified inhibitors of peptidases using molecular docking. We have ordered 13 potential inhibitors and will screen them for their ability and potency to inhibit rumen protozoa. 3. Researchers have been using microscopy to identify and count rumen protozoa present in rumen samples. Morphologic identification and microscopic counting are time-consuming and not accurate. Sequence-based identification and quantification with quantitative PCR (qPCR) of rumen protozoa have been used, but not the marker gene (18S rRNA gene) is very conserved among rumen protozoa, and it does not provide species-level taxonomic resolution. To address this need, we designed new qPCR primers based on marker genes that can provide better taxonomic resolution (28S rRNA gene and the internal transcribed sequence, the latter of which is between the 18S and 28S rRNA genes). We are in the process to verify the specificity of the qPCR primers and their utility to distinguish different rumen protozoa species. 4. Two recent studies showed that the rumen ecosystem has diverse viruses that infect bacteria. These bacteriophages can infect rumen bacteria (methanogens, protozoa, and fungi as well) and lyse their host cells, contributing to the intra-ruminal recycling of microbial protein and decreasing the outflow of microbial protein to the small intestine of ruminants. Therefore, it is important to identify and understand the rumen virome (the totality of viruses present in the rumen ecosystem). We collected most of the published rumen metagenomes (nearly 1,000), which represent the rumen microbiome and virome collected from across different species of ruminants across the globe, and performed comprehensive detailed bioinformatic analyses. We created the first global rumen virome database (RVD) that documented the viruses infecting the cellular microbiomes of the rumen ecosystem. This global virome database shall be useful to future research to investigate the roles and associations of rumen virome in rumen functions (feed digestion, fermentation, methanogenesis) and animal performance (feed and nitrogen efficiency, methane emissions).

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Patra A and Yu Z. 2022. Genomic insights into the distribution of peptidases and proteolytic capacity among Prevotella and Paraprevotella species. Microbiology Spectrum, 10(2):e02185-21.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Li Z, Wang X, Zhang Y, Yu Z, Zhang T, Dai X, Pan X, Jing R, Yan Y, Liu Y, Gao S, Li F, Huang Y, Tian J, Yao J, Xing X, Shi T, Ning J, Yao B, Huang H and Jiang Y. 2022. Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates. The ISME Journal, 16:2775-2787.
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Yan M, Pratama Akbar, Li Z, Jiang Y, Sullivan M, and Yu Z. 2022. Unraveling the viral dark matter of the rumen microbiome with a new global virome database. Nature Communications, submitted.


Progress 01/01/21 to 12/31/21

Outputs
Target Audience:The goal of this research project was to advance the understanding of rumen protozoa so that they could be controlled to decrease their wasteful roles, particularly in mediating intraruminal nitrogen recycling, which lowers the utilization efficiency of dietary nitrogen in ruminants, and in promoting methane emissions, which not only drains dietary energy but also contribute to global warming. Thus, this research project is of interest to researchers who are interested in rumen microbiome research, especially rumen protozoa, ruminant nutritionists who want to improve dietary nitrogen utilization efficiency, feed additive companies that want to develop new feed additives to improve feed efficiency, and probably also policy and makers who want to reduce the environmental footprint of ruminant production. Some of the results and findings of the studies conducted in 2021 have been presented to the scientific community in two peer-reviewed articles published by two international journals. Please see the Products below for detail. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Ph.D. student joined us in the fall of 2020, and he had little background in rumen microbiome, protozoology, metataxonomics, metagenomics, or bioinformatics. He was trained in sample collection from cannulated cows, preparation of rumen samples for microscopic identification of rumen protozoa, morphological identification of rumen protozoa at the genus level. This new student was also trained in protein structure modeling, molecular docking, metataxonomics, and metagenomics, all of which are important skills required to continue this line of research. How have the results been disseminated to communities of interest?Some of the results of this research project have been published in two peer-reviewed articles (see Products). The genomic sequences of Entodinium caudatum have been deposited in the NCBI GenBank so they are freely available to scientists who are interested in future research on rumen protozoa and other ciliates. What do you plan to do during the next reporting period to accomplish the goals?1. We will continue to screen the potential inhibitors of the protozoal lysozyme and peptidases (both cysteine and serine peptidases) in vitro. 2. Evaluate the inhibition of promising inhibitors to different genera of rumen protozoa using mixed rumen protozoa cultures in vitro. 3. Evaluate the effects of the inhibitors on feed digestion, fermentation, and the bacteria and methanogens in vitro.

Impacts
What was accomplished under these goals? 1. We refined the genomic sequences of Entodinium caudatum and performed extensive bioinformatic analysis of the transcriptomic and genomic sequences and obtained the full length of the target genes that encode the lysozyme, serine peptidase, and cysteine peptidase in Entodinium caudatum. Using the full-length gene sequences, we repeated the structural modeling of the above enzymes and performed molecular docking analysis again to screen two small-molecule libraries to identify candidate compounds of plant origin that might potentially inhibit the lysozyme and peptidases of Entodinium caudatum. In total, we identified a total of 33 compounds of potential inhibitors of the lysozyme and peptidases. 2. We obtained some of the identified compounds and screened them in vitro using fresh rumen microbiome with concentrated rumen protozoa. We were able to show that several of the compounds could decrease the counts of total rumen protozoa in vitro. 3. There come to our realization that bacterial peptidases can also affect protein degradation and metabolism in the rumen. We thus analyzed the peptidases represented by genes in the sequenced genomes of Prevotella and Paraprevotella species, which constitute the most dominant rumen bacteria. That study provided new insights into the occurrence and potential capacity of proteolysis mediated by species of Prevotella and Paraprevotella.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Park T, Wijeratne S, Meulia T, Firkins J, and Yu Z. 2021. The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment. Genomics, 113(3): 1416-1427. https://doi.org/10.1016/j.ygeno.2021.03.014.
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Patra A and Yu Z. 2022. Genomic insights into the distribution of peptidases and proteolytic capacity among Prevotella and Paraprevotella species. Microbiology Spectrum, accepted.